Metformin and Cancer

**113**

**Chapter 7**

**Abstract**

**1. Introduction**

Cancer Therapy

Metformin and Its Implication in

Metformin has been used for almost half a century as the first line of treatment for type 2 diabetes. Mechanisms of action are still incompletely known, recent studies have shown that metformin exerts its effects through several mechanisms, including the stimulation of AMP-activated protein kinase, decreasing production of cyclic AMP, inhibition of mitochondrial complex I of the electron transport chain, targeting glycerophosphate dehydrogenase and altering gut microbiota. In recent years, studies have shown that patients with type 2 diabetes mellitus have a lower risk of developing cancer, and patients with cancer and type 2 diabetes have a lower mortality. Experimental studies have demonstrated that metformin has anti-tumor activity by inhibiting mTORC1 signaling pathway and mitochondrial complex, inhibiting tumor growth and proliferation, and inducing cellular apoptosis. There are multiple studies showing that combination of metformin with different types of anti-cancer therapies may reduce toxicities and tumor resistance. This chapter is focused on the progress made in understanding the anti-tumor effect of

Guanidine derivatives, metformin, buformin and phenoformin, were discovered in the 1920s, extracted from the isoamylene plant [1]. Metformin it is a biguanide extracted from herb *Galega officinalis*, and it was first proposed by Emile Werner and James Bell in 1922, when they found that metformin is reducing the amount of glucose in rabbits and does not affect heart and blood pressure [2, 3]. Due to the increased risk of lactic acidosis and of cardiac death, buformin and phenoformin were withdrawn from the market in 1970 [4]. Due to the good safety profile of metformin, the use of this drug was extended beyond type 2 diabetes to ovarian polycystic disease, gestational diabetes, diabetic nephropathy and cardiovascular

The association between cancer and diabetes was first proven in 1930 by Marble [6]. Over the past 20 years, numerous studies have shown that diabetic patients have a higher incidence of cancers, increased mortality [7, 8], and the fact that patients with diabetes and cancer are less sensitive to chemotherapy [9–11].

*Laura Mazilu, Dana Stanculeanu, Andreea Gheorghe,* 

*Adrian-Paul Suceveanu, Irinel Parepa, Felix Voinea,* 

*Doina Catrinoiu and Andra-Iulia Suceveanu*

metformin and its association with cancer therapy.

complications associated with type 2 diabetes [5].

**Keywords:** metformin, cancer, chemotherapy, targeted therapy

### **Chapter 7**

## Metformin and Its Implication in Cancer Therapy

*Laura Mazilu, Dana Stanculeanu, Andreea Gheorghe, Adrian-Paul Suceveanu, Irinel Parepa, Felix Voinea, Doina Catrinoiu and Andra-Iulia Suceveanu*

### **Abstract**

Metformin has been used for almost half a century as the first line of treatment for type 2 diabetes. Mechanisms of action are still incompletely known, recent studies have shown that metformin exerts its effects through several mechanisms, including the stimulation of AMP-activated protein kinase, decreasing production of cyclic AMP, inhibition of mitochondrial complex I of the electron transport chain, targeting glycerophosphate dehydrogenase and altering gut microbiota. In recent years, studies have shown that patients with type 2 diabetes mellitus have a lower risk of developing cancer, and patients with cancer and type 2 diabetes have a lower mortality. Experimental studies have demonstrated that metformin has anti-tumor activity by inhibiting mTORC1 signaling pathway and mitochondrial complex, inhibiting tumor growth and proliferation, and inducing cellular apoptosis. There are multiple studies showing that combination of metformin with different types of anti-cancer therapies may reduce toxicities and tumor resistance. This chapter is focused on the progress made in understanding the anti-tumor effect of metformin and its association with cancer therapy.

**Keywords:** metformin, cancer, chemotherapy, targeted therapy

### **1. Introduction**

Guanidine derivatives, metformin, buformin and phenoformin, were discovered in the 1920s, extracted from the isoamylene plant [1]. Metformin it is a biguanide extracted from herb *Galega officinalis*, and it was first proposed by Emile Werner and James Bell in 1922, when they found that metformin is reducing the amount of glucose in rabbits and does not affect heart and blood pressure [2, 3]. Due to the increased risk of lactic acidosis and of cardiac death, buformin and phenoformin were withdrawn from the market in 1970 [4]. Due to the good safety profile of metformin, the use of this drug was extended beyond type 2 diabetes to ovarian polycystic disease, gestational diabetes, diabetic nephropathy and cardiovascular complications associated with type 2 diabetes [5].

The association between cancer and diabetes was first proven in 1930 by Marble [6]. Over the past 20 years, numerous studies have shown that diabetic patients have a higher incidence of cancers, increased mortality [7, 8], and the fact that patients with diabetes and cancer are less sensitive to chemotherapy [9–11].

### *Metformin*

Regarding the anti-tumor effect of metformin, numerous studies have shown that metformin-treated diabetes patients have a low incidence of cancers and low mortality compared with patients treated with other types of anti-diabetics such as sulfonylureas or insulin [9, 12, 13].

In vivo and in vitro studies have demonstrated that metformin has an antitumoral effect both directly and indirectly, which translates into inhibition of tumor cell proliferation, induction of apoptosis, and cell cycle arrest [14–16].

Taking all these into consideration, metformin appears to be useful as an adjuvant to cancer treatment.

### **2. Anti-tumor mechanism of action of metformin**

Metformin's mechanisms of action and its anti-tumor effects are multiple and have been described over the years in numerous studies, both in vivo and in vitro, but they are not yet completely understood. The main mechanisms of actions are activation of liver kinase B1 (LKB1) and AMP-activated kinase (AMPK), and inhibition of mammalian target of rapamycin (mTOR). Other mechanisms described in literature are inhibition of protein synthesis, activation of apoptosis by p21 and p53, inhibition of unfolded protein response (UPR), activation of immune system, prevention of angiogenesis, reduction of blood insulin levels and reduction of hyperlipidemia [17, 18].

Metformin is entering the cells with the help of organic cation transporter 1 and 3, and as a result is blocking the complex I of electron transfer chain (ETC) and an enzyme named mitochondrial glycerophosphate 3 dehydrogenase (mGDP). Introduction of Metformin into the cell results in reduced activity of adenosine triphosphate (ATP) and reduced oxygen consumption, which further increase the levels of adenosine monophosphate within the cells and activate AMPK, and in the end this will put the cells under stressful conditions [19, 20].

Metformin inhibits mTOR pathway by activating LKB1 and AMPK, resulting in reduction of protein synthesis and inhibition of angiogenesis. AKPK inhibits mTOR pathway by activation of tuberculous sclerosis complex (TSC2) and by direct phosphorylation of co-signaling molecules that will attached to mTOR molecules [21, 22]. Metformin is also inhibiting mTOR by reducing phosphorylation of ribosomal protein S6 kinase (S6Ks) [23].

Ataxia teleangectasia mutated (ATM) and LKB1 are proteins with an important role in cell cycle. Both ATM and LKB1 are tumor suppressors. The response of ATM to metformin is phosphorylation of LKB and in the end the activation of AMPK [24].

Inhibition of unfolded protein response (UPR) is another mechanism by which metformin exerts its anti-tumor effect. UPR activity is vital for cell survival of under stress conditions. Metformin inhibits the activity of UPR and determine cells to undergo apoptosis [25].

Insulin and insulin growth-like factor (IGF) promote mitosis and cell growth and inhibit apoptosis. All this processes are very important in carcinogenesis and the relation between hyperinsulinemia, insulin resistance and cancer promotion are well known [26]. Metformin inactivates I/IGF pathway by reducing blood insulin levels and by inhibiting glucose absorption by intestinal cells [27, 28].

### **3. Metformin: epidemiologic evidence of its anti-tumor effect**

Metformin was approved by Food and Drug Administration (FDA) in 1957 for type 2 diabetes and became the first line treatment due to its superior safety profile and hypoglycemic and cardiovascular protective effect [29].

**115**

*Metformin and Its Implication in Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.88803*

patients with type 2 diabetes [32].

the control group [35].

disease [36–39].

with non-users [41].

related biases [42, 43].

mainly due to life style.

**3.2 Metformin in colorectal cancer**

reduce the risk of breast or prostate cancer [33].

sulfonylurea, comparing with those using metformin [34].

**3.1 Metformin in hepatocellular carcinoma and pancreatic cancer**

fatty liver disease and type 2 diabetes are newly described risk factors.

The effect of metformin on cancer risk reduction was first observed in a study published in 2005 by Evans et al., which included 11,776 patients with type 2 diabetes; this observation was reiterated in another trial in 2009, involving more than 4,000 patients with diabetes treated with metformin, the risk of developing cancer being 7.3% for patients receiving metformin vs. 11.6% in the control group [30, 31]. In 2009, a study conducted at the MD Anderson Cancer Center by Li et al., showed that metformin use is associated with a low risk of pancreatic cancer in

A very large retrospective study that evaluated more than 62.000 patients with diabetes showed that metformin treatment reduces the risk of cancer compared to other antidiabetic therapies (insulin, sulfonylureas), and also showed that the combination of metformin with insulin or sulfonylureas reduces the risk of cancer associated with these therapies. This study showed that the risk of developing colorectal and pancreatic cancer is higher in patients with diabetes treated with insulin, compared to patients treated with metformin, and that metformin does not

In terms of mortality, in 2006 a study conducted by Bowker el al, retrospectively reported that mortality is higher in patients with type 2 diabetes using insulin and

In 2010, a prospective study, ZODIAC-16, evaluating the influence of metformin on cancer mortality in 1353 patients with type 2 diabetes showed that metformintreated patients had a lower mortality rate (with a median of 9.6 years) compared to

Hepatocellular carcinoma is one of the leading causes of death in cancer patients.

Well known risk factors implicated in etiology of hepatocellular carcinoma are chronic hepatitis B and C and hepatic cirrhosis. In the last years, due to the rising incidence of obesity and diabetes worldwide, non-alcoholic steatosis, non-alcoholic

Donadon has focused his studies on patients with hepatocarcinoma and has shown that metformin significantly reduces the risk of hepatocarcinoma in diabetic patients, compared to patients treated with sulfonylureas or insulin, and also reduces the risk of hepatocarcinoma in patients with diabetes and chronic liver

There are several meta-analyses supporting this data, for example a 31% incidence reduction of pancreatic and hepatocellular carcinoma for patients using metformin was reported by a meta-analyses of 11 trials [40]. Another meta-analysis evaluating 37 trials of patients with colorectal, pancreatic, breast and hepatic cancer, reported a reduced incidence of cancer in patients using metformin, comparing

One meta-analysis stated that metformin does not significantly reduce the risk of hepatocellular carcinoma. This meta-analysis excluded all the studies with time-

Colorectal cancer is increasing in incidence and mortality worldwide, especially

The first data that reported the relationship between metformin and colorectal cancer risk emerged in 2004 and since then numerous studies have evaluated this

in countries with low and middle income, but also in high developed countries

*Metformin and Its Implication in Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.88803*

*Metformin*

sulfonylureas or insulin [9, 12, 13].

**2. Anti-tumor mechanism of action of metformin**

end this will put the cells under stressful conditions [19, 20].

vant to cancer treatment.

protein S6 kinase (S6Ks) [23].

to undergo apoptosis [25].

Regarding the anti-tumor effect of metformin, numerous studies have shown that metformin-treated diabetes patients have a low incidence of cancers and low mortality compared with patients treated with other types of anti-diabetics such as

In vivo and in vitro studies have demonstrated that metformin has an antitumoral effect both directly and indirectly, which translates into inhibition of tumor cell proliferation, induction of apoptosis, and cell cycle arrest [14–16].

Taking all these into consideration, metformin appears to be useful as an adju-

Metformin's mechanisms of action and its anti-tumor effects are multiple and have been described over the years in numerous studies, both in vivo and in vitro, but they are not yet completely understood. The main mechanisms of actions are activation of liver kinase B1 (LKB1) and AMP-activated kinase (AMPK), and inhibition of mammalian target of rapamycin (mTOR). Other mechanisms described in literature are inhibition of protein synthesis, activation of apoptosis by p21 and p53, inhibition of unfolded protein response (UPR), activation of immune system, prevention of angiogenesis, reduction of blood insulin levels and reduction of hyperlipidemia [17, 18]. Metformin is entering the cells with the help of organic cation transporter 1 and 3, and as a result is blocking the complex I of electron transfer chain (ETC) and an enzyme named mitochondrial glycerophosphate 3 dehydrogenase (mGDP). Introduction of Metformin into the cell results in reduced activity of adenosine triphosphate (ATP) and reduced oxygen consumption, which further increase the levels of adenosine monophosphate within the cells and activate AMPK, and in the

Metformin inhibits mTOR pathway by activating LKB1 and AMPK, resulting in reduction of protein synthesis and inhibition of angiogenesis. AKPK inhibits mTOR pathway by activation of tuberculous sclerosis complex (TSC2) and by direct phosphorylation of co-signaling molecules that will attached to mTOR molecules [21, 22]. Metformin is also inhibiting mTOR by reducing phosphorylation of ribosomal

Ataxia teleangectasia mutated (ATM) and LKB1 are proteins with an important role in cell cycle. Both ATM and LKB1 are tumor suppressors. The response of ATM to metformin is phosphorylation of LKB and in the end the activation of AMPK [24]. Inhibition of unfolded protein response (UPR) is another mechanism by which metformin exerts its anti-tumor effect. UPR activity is vital for cell survival of under stress conditions. Metformin inhibits the activity of UPR and determine cells

Insulin and insulin growth-like factor (IGF) promote mitosis and cell growth and inhibit apoptosis. All this processes are very important in carcinogenesis and the relation between hyperinsulinemia, insulin resistance and cancer promotion are well known [26]. Metformin inactivates I/IGF pathway by reducing blood insulin

Metformin was approved by Food and Drug Administration (FDA) in 1957 for type 2 diabetes and became the first line treatment due to its superior safety profile

levels and by inhibiting glucose absorption by intestinal cells [27, 28].

**3. Metformin: epidemiologic evidence of its anti-tumor effect**

and hypoglycemic and cardiovascular protective effect [29].

**114**

The effect of metformin on cancer risk reduction was first observed in a study published in 2005 by Evans et al., which included 11,776 patients with type 2 diabetes; this observation was reiterated in another trial in 2009, involving more than 4,000 patients with diabetes treated with metformin, the risk of developing cancer being 7.3% for patients receiving metformin vs. 11.6% in the control group [30, 31].

In 2009, a study conducted at the MD Anderson Cancer Center by Li et al., showed that metformin use is associated with a low risk of pancreatic cancer in patients with type 2 diabetes [32].

A very large retrospective study that evaluated more than 62.000 patients with diabetes showed that metformin treatment reduces the risk of cancer compared to other antidiabetic therapies (insulin, sulfonylureas), and also showed that the combination of metformin with insulin or sulfonylureas reduces the risk of cancer associated with these therapies. This study showed that the risk of developing colorectal and pancreatic cancer is higher in patients with diabetes treated with insulin, compared to patients treated with metformin, and that metformin does not reduce the risk of breast or prostate cancer [33].

In terms of mortality, in 2006 a study conducted by Bowker el al, retrospectively reported that mortality is higher in patients with type 2 diabetes using insulin and sulfonylurea, comparing with those using metformin [34].

In 2010, a prospective study, ZODIAC-16, evaluating the influence of metformin on cancer mortality in 1353 patients with type 2 diabetes showed that metformintreated patients had a lower mortality rate (with a median of 9.6 years) compared to the control group [35].

### **3.1 Metformin in hepatocellular carcinoma and pancreatic cancer**

Hepatocellular carcinoma is one of the leading causes of death in cancer patients. Well known risk factors implicated in etiology of hepatocellular carcinoma are chronic hepatitis B and C and hepatic cirrhosis. In the last years, due to the rising incidence of obesity and diabetes worldwide, non-alcoholic steatosis, non-alcoholic fatty liver disease and type 2 diabetes are newly described risk factors.

Donadon has focused his studies on patients with hepatocarcinoma and has shown that metformin significantly reduces the risk of hepatocarcinoma in diabetic patients, compared to patients treated with sulfonylureas or insulin, and also reduces the risk of hepatocarcinoma in patients with diabetes and chronic liver disease [36–39].

There are several meta-analyses supporting this data, for example a 31% incidence reduction of pancreatic and hepatocellular carcinoma for patients using metformin was reported by a meta-analyses of 11 trials [40]. Another meta-analysis evaluating 37 trials of patients with colorectal, pancreatic, breast and hepatic cancer, reported a reduced incidence of cancer in patients using metformin, comparing with non-users [41].

One meta-analysis stated that metformin does not significantly reduce the risk of hepatocellular carcinoma. This meta-analysis excluded all the studies with timerelated biases [42, 43].

### **3.2 Metformin in colorectal cancer**

Colorectal cancer is increasing in incidence and mortality worldwide, especially in countries with low and middle income, but also in high developed countries mainly due to life style.

The first data that reported the relationship between metformin and colorectal cancer risk emerged in 2004 and since then numerous studies have evaluated this

association and had different outcomes, reporting a decrease risk, an increased risk or no association [43, 44].

The first clinical trial that examine the chemopreventive effect of low-dose metformin on metachronous colorectal adenoma/polyp formation, was conducted in 2016, and the observation was that Metformin suppress the formation of metachronous colorectal adenoma/polyp [45].

Another study investigating the use of Metformin as chemopreventive therapy was performed in 2018 on a small number of patients without diabetes, and showed that metformin is reducing the risk of developing polyps. The adverse events were mild and with no differences between groups [46].

### **3.3 Metformin in breast cancer**

A meta-analysis that included 11 clinical trials of patients with breast cancer, reported a 65% improvement in overall survival for patients with breast cancer and diabetes that are treated with metformin [47].

There are also studies suggesting that the use of metformin is changing the type of cancers diagnosed in patients with diabetes. For example, a study conducted by Berstein reported that in patients using metformin, breast cancer is much more frequent, especially the progesterone receptor positive-type [46], and another study reported that triple negative-type is less common [47]. Other data suggests that response rate is higher in diabetic patients with breast cancer receiving neo-adjuvant chemotherapy and metformin, comparing with those not receiving metformin [48].

### **3.4 Metformin in renal cancer**

Kidney cancers incidence is increasing mainly due to the increasing rates of hypertension and due to the improvement of imaging techniques, because kidney cancers are most often asymptomatic. Renal cell carcinoma is the most common type of kidney cancer.

There are several studies reporting that patients with renal cancer and diabetes have a poor prognosis, and that diabetes has a negative impact on survival of these patients [49]. Also some articles suggest that type 2 diabetes may be an independent risk factor for renal cancers [50].

A meta-analysis performed in 2017 which included 8 publications on kidney cancer showed that metformin could improve the survival of renal cancer patients, especially for patients with localized renal cell carcinoma, and concluded that further investigation is needed regarding the effect of metformin on patients with localized and metastatic renal carcinoma in order to exclude disease heterogeneity [51].

### **3.5 Metformin in lung cancer**

Lung cancer is the leading cause of death all over the world in both sexes and despite the recent advances in therapy, the prognosis of these patients is still no satisfactory.

Regarding lung cancer, Mazzone et al. and Tan et al. reported that in patients receiving metformin the incidence of adenocarcinomas is higher comparing with other histopathological types, and that patients receiving metformin had a better response to chemotherapy [52, 53].

A meta-analysis conducted in 2017 reported that metformin demonstrates a significant improvement of overall survival and progression free survival of patients with lung cancer [54].

**117**

*Metformin and Its Implication in Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.88803*

colorectal cancers.

nous adenoma or polyp [45].

pathological response [63].

Although, numerous trials reported a reduction in cancer incidence in patients receiving metformin, there are recent studies on diabetic patients with breast, endometrial, prostate and renal cancer receiving metformin that suggests no

For prostate cancer, studies and meta-analysis showed that diabetes may reduce the risk of prostate cancer [56], but also showed that patients with diabetes and prostate cancer have higher rates of mortality and relapse after prostatectomy [57, 58]. All these results are in conflict with other studies that reported that metformin may

Taking in consideration all the information available stating that metformin has a positive effect on cancer incidence and mortality, over the years numerous trial have evaluated or are underway to evaluate the combination of metformin with different antineoplastic drugs in breast, endometrial, prostate, lung, pancreatic and

There are numerous chemotherapeutic drugs evaluated in combination with metformin. For example doxorubicin, cyclophosphamide, docetaxel, trastuzumab,

Combination of 5-fluorouracyl and metformin showed a modest activity in patients with colorectal cancer [61], but when used as chemopreventive treatment in monotherapy, metformin showed a reduced incidence of colorectal metachro-

Metformin in combination with medroxyprogesterone acetate in endometrial cancer and atypical endometrial hyperplasia, showed a complete response rate of 14% in endometrial cancer and 81% in atypical endometrial hyperplasia and a good

For patients with diabetes and breast cancer receiving neo-adjuvant chemotherapy and metformin, Jiralerspong et al. reported a superior rate of complete

In patients with prostate cancer the combination of bicalutamide and metformin may reduce cancer cells growth rate; in androgen receptor positive cells (AR) the reduction of cell growth appear to be mediated by anti-proliferative effect, and in

Targeted therapies are used with success in the treatment of many cancer types,

First targeted therapy approved by the FDA, was Gefitinib, a molecule targeting

but usually the disease becomes unresponsive to treatment and shows acquired resistance, and this is a challenge for clinicians. Preclinical and clinical data showed that the combination of metformin with targeted therapies have good results. Targeted therapies comprise mostly of kinase inhibitors. At present more than 35

epidermal growth factor receptor (EGFR) in 2003 for the treatment of patients with locally advanced and metastatic non-small cell lung cancer (NSCLC) after failure of platinum and docetaxel chemotherapy [66]. A high percent of patients receiving gefitinib have high response rate, but despite this, patients rapidly develop

association between the use of metformin and cancer incidence [55].

reduce the risk for prostate cancer and may improve survival [59, 60].

**4. Metformin: combination with antineoplastic drugs**

**4.1 Metformin: combination with chemotherapy**

exemestane, letrozole, carboplatin, 5-flurouracyl.

clinical profile with no severe adverse events [62].

androgen receptor negative cells by pro-apoptotic effect [64].

different types of kinase inhibitors are approved by FDA [65].

**4.2 Metformin: combination with targeted therapies**

*Metformin and Its Implication in Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.88803*

*Metformin*

or no association [43, 44].

chronous colorectal adenoma/polyp [45].

**3.3 Metformin in breast cancer**

**3.4 Metformin in renal cancer**

risk factor for renal cancers [50].

**3.5 Metformin in lung cancer**

response to chemotherapy [52, 53].

with lung cancer [54].

metformin [48].

type of kidney cancer.

mild and with no differences between groups [46].

diabetes that are treated with metformin [47].

association and had different outcomes, reporting a decrease risk, an increased risk

The first clinical trial that examine the chemopreventive effect of low-dose metformin on metachronous colorectal adenoma/polyp formation, was conducted in 2016, and the observation was that Metformin suppress the formation of meta-

Another study investigating the use of Metformin as chemopreventive therapy was performed in 2018 on a small number of patients without diabetes, and showed that metformin is reducing the risk of developing polyps. The adverse events were

A meta-analysis that included 11 clinical trials of patients with breast cancer, reported a 65% improvement in overall survival for patients with breast cancer and

Kidney cancers incidence is increasing mainly due to the increasing rates of hypertension and due to the improvement of imaging techniques, because kidney cancers are most often asymptomatic. Renal cell carcinoma is the most common

There are several studies reporting that patients with renal cancer and diabetes have a poor prognosis, and that diabetes has a negative impact on survival of these patients [49]. Also some articles suggest that type 2 diabetes may be an independent

A meta-analysis performed in 2017 which included 8 publications on kidney cancer showed that metformin could improve the survival of renal cancer patients, especially for patients with localized renal cell carcinoma, and concluded that further investigation is needed regarding the effect of metformin on patients with localized and metastatic renal carcinoma in order to exclude disease heterogeneity [51].

Lung cancer is the leading cause of death all over the world in both sexes and despite the recent advances in therapy, the prognosis of these patients is still no

Regarding lung cancer, Mazzone et al. and Tan et al. reported that in patients receiving metformin the incidence of adenocarcinomas is higher comparing with other histopathological types, and that patients receiving metformin had a better

A meta-analysis conducted in 2017 reported that metformin demonstrates a significant improvement of overall survival and progression free survival of patients

There are also studies suggesting that the use of metformin is changing the type of cancers diagnosed in patients with diabetes. For example, a study conducted by Berstein reported that in patients using metformin, breast cancer is much more frequent, especially the progesterone receptor positive-type [46], and another study reported that triple negative-type is less common [47]. Other data suggests that response rate is higher in diabetic patients with breast cancer receiving neo-adjuvant chemotherapy and metformin, comparing with those not receiving

**116**

satisfactory.

Although, numerous trials reported a reduction in cancer incidence in patients receiving metformin, there are recent studies on diabetic patients with breast, endometrial, prostate and renal cancer receiving metformin that suggests no association between the use of metformin and cancer incidence [55].

For prostate cancer, studies and meta-analysis showed that diabetes may reduce the risk of prostate cancer [56], but also showed that patients with diabetes and prostate cancer have higher rates of mortality and relapse after prostatectomy [57, 58]. All these results are in conflict with other studies that reported that metformin may reduce the risk for prostate cancer and may improve survival [59, 60].

### **4. Metformin: combination with antineoplastic drugs**

Taking in consideration all the information available stating that metformin has a positive effect on cancer incidence and mortality, over the years numerous trial have evaluated or are underway to evaluate the combination of metformin with different antineoplastic drugs in breast, endometrial, prostate, lung, pancreatic and colorectal cancers.

### **4.1 Metformin: combination with chemotherapy**

There are numerous chemotherapeutic drugs evaluated in combination with metformin. For example doxorubicin, cyclophosphamide, docetaxel, trastuzumab, exemestane, letrozole, carboplatin, 5-flurouracyl.

Combination of 5-fluorouracyl and metformin showed a modest activity in patients with colorectal cancer [61], but when used as chemopreventive treatment in monotherapy, metformin showed a reduced incidence of colorectal metachronous adenoma or polyp [45].

Metformin in combination with medroxyprogesterone acetate in endometrial cancer and atypical endometrial hyperplasia, showed a complete response rate of 14% in endometrial cancer and 81% in atypical endometrial hyperplasia and a good clinical profile with no severe adverse events [62].

For patients with diabetes and breast cancer receiving neo-adjuvant chemotherapy and metformin, Jiralerspong et al. reported a superior rate of complete pathological response [63].

In patients with prostate cancer the combination of bicalutamide and metformin may reduce cancer cells growth rate; in androgen receptor positive cells (AR) the reduction of cell growth appear to be mediated by anti-proliferative effect, and in androgen receptor negative cells by pro-apoptotic effect [64].

### **4.2 Metformin: combination with targeted therapies**

Targeted therapies are used with success in the treatment of many cancer types, but usually the disease becomes unresponsive to treatment and shows acquired resistance, and this is a challenge for clinicians. Preclinical and clinical data showed that the combination of metformin with targeted therapies have good results. Targeted therapies comprise mostly of kinase inhibitors. At present more than 35 different types of kinase inhibitors are approved by FDA [65].

First targeted therapy approved by the FDA, was Gefitinib, a molecule targeting epidermal growth factor receptor (EGFR) in 2003 for the treatment of patients with locally advanced and metastatic non-small cell lung cancer (NSCLC) after failure of platinum and docetaxel chemotherapy [66]. A high percent of patients receiving gefitinib have high response rate, but despite this, patients rapidly develop resistance. Mechanisms involved in resistance to Gefitinib are activation of mTOR pathway and upregulation of insulin-like growth factor-1 receptor (IGF-1R), and taking into consideration the effect of metformin on mTOR pathway inhibition and IGF-1R pathway suppression, multiple studies started to evaluate this relationship. The result were that the addition to metformin to Gefitinib reduce proliferation and can revert resistance to gefitinib [67, 68]. Combination of metformin and Gefitinib also improve prognosis of patients with NSCLC, by increasing survival and by delaying resistance to targeted therapy [69]. At this moment, a phase II multicenter double blind trial evaluating gefitinib in combination with metformin as first-line treatment for patients with locally advanced NSCLC, is ongoing [70].

Sorafenib was approved in 2007 for treatment of advanced hepatocellular carcinoma, but showed low response rate and serious adverse events [71]. Combination of Sorafenib and other drugs was necessary in order to improve treatment efficacy. So far, data showed that metformin has the capability to increase sorafenib efficacy by reducing lung metastasis in patients with hepatocellular carcinoma. The mechanism of action of this combination is targeting the mTOR pathway [72].

Trastuzumab was approved in 1998 for the treatment of HER2-positive breast cancer. Combination of Trastuzumab and metformin in clinical trials conducted over the years, showed that metformin suppresses the proliferation of trastuzumabresistant breast cancer cells and also have a cardio-protective effect, against cardiac events related to trastuzumab [73, 74].

Bevacizumab, inhibits VEGF-A, the result being inhibition of angiogenesis and regression of tumor vascularization, thereby inhibiting cancer growth. It was approved in 2004 in combination with chemotherapy for metastatic colorectal cancer and now it is used in the treatment of numerous cancer types-metastatic breast cancer, renal cell carcinoma, advanced epithelial ovarian cancer, non-squamous NSCLC [75]. Combination of metformin with bevacizumab was found to be effective in the treatment of ovarian cancer and metastatic non-squamous NSCLC in combination with chemotherapy [76, 77].

### **4.3 Metformin: combination with radiotherapy**

Metformin in combination with radiotherapy may increase cancer response to treatment. As already mentioned, one of the mechanism of action of metformin is affecting complex I in the electron transfer chain, reducing the oxygen consumption and increasing the reactive oxygen species (ROS) within the cells, resulting in DNA damage [78]. Another proposed mechanism is activation of p53 by activating AMPK, and as a result cell cycle arrest. Both, metformin and radiotherapy can activate p53 and stop cell proliferation [79]. There are several articles and case reports, showing a better response for patients receiving radiotherapy and metformin, comparing with those without metformin in: esophageal cancer, rectal cancer and head and neck carcinomas [80].

### **5. Conclusions**

Many studies reported a reduced incidence of cancer in patients receiving metformin in standard dose, but also these trials have limitations: most of the trials were retrospective, others included both patients with invasive and non-invasive neoplasms, others trials did not exclude patients exposed to other antidiabetic treatments, all these findings being responsible for potential biases.

In general, chemopreventive agents are used as long term therapies. Metformin meets all necessary criteria as a long term chemopreventive agent, because it is safe,

**119**

*Metformin and Its Implication in Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.88803*

Authors report no conflicts of interest.

\*, Dana Stanculeanu<sup>2</sup>

\*Address all correspondence to: lauragrigorov@gmail.com

, Felix Voinea1

provided the original work is properly cited.

, Andreea Gheorghe1

, Doina Catrinoiu<sup>2</sup>

2 Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

1 Ovidius University of Constanta, Faculty of Medicine, Constanta, Romania

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Adrian-Paul Suceveanu1

and Andra-Iulia Suceveanu1

,

and it is cost effective.

used in cancer prevention.

population.

**Conflict of interest**

**Author details**

Laura Mazilu1

Irinel Parepa1

has a well-known mechanism of action, it is well tolerated with few adverse effects

Clinical trials which evaluated the effect of metformin in combination with different types of antineoplastic treatment included only patients with diabetes, therefore clinical trials evaluating the effect of metformin in non-diabetic population are needed in order to explore the benefit of metformin and also to evaluate the adverse events of combinations compared with monotherapy in this particular

Based on the available information, we can conclude that metformin is reducing cancer incidence and mortality, is increasing tumor response when used in combination with different types of cancer therapies, either chemotherapy, targeted therapies or radiotherapy, is improving the outcome of cancer patients, and can be

*Metformin and Its Implication in Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.88803*

has a well-known mechanism of action, it is well tolerated with few adverse effects and it is cost effective.

Based on the available information, we can conclude that metformin is reducing cancer incidence and mortality, is increasing tumor response when used in combination with different types of cancer therapies, either chemotherapy, targeted therapies or radiotherapy, is improving the outcome of cancer patients, and can be used in cancer prevention.

Clinical trials which evaluated the effect of metformin in combination with different types of antineoplastic treatment included only patients with diabetes, therefore clinical trials evaluating the effect of metformin in non-diabetic population are needed in order to explore the benefit of metformin and also to evaluate the adverse events of combinations compared with monotherapy in this particular population.

### **Conflict of interest**

*Metformin*

resistance. Mechanisms involved in resistance to Gefitinib are activation of mTOR pathway and upregulation of insulin-like growth factor-1 receptor (IGF-1R), and taking into consideration the effect of metformin on mTOR pathway inhibition and IGF-1R pathway suppression, multiple studies started to evaluate this relationship. The result were that the addition to metformin to Gefitinib reduce proliferation and can revert resistance to gefitinib [67, 68]. Combination of metformin and Gefitinib also improve prognosis of patients with NSCLC, by increasing survival and by delaying resistance to targeted therapy [69]. At this moment, a phase II multicenter double blind trial evaluating gefitinib in combination with metformin as first-line

Sorafenib was approved in 2007 for treatment of advanced hepatocellular carcinoma, but showed low response rate and serious adverse events [71]. Combination of Sorafenib and other drugs was necessary in order to improve treatment efficacy. So far, data showed that metformin has the capability to increase sorafenib efficacy by reducing lung metastasis in patients with hepatocellular carcinoma. The mecha-

Trastuzumab was approved in 1998 for the treatment of HER2-positive breast cancer. Combination of Trastuzumab and metformin in clinical trials conducted over the years, showed that metformin suppresses the proliferation of trastuzumabresistant breast cancer cells and also have a cardio-protective effect, against cardiac

Bevacizumab, inhibits VEGF-A, the result being inhibition of angiogenesis and regression of tumor vascularization, thereby inhibiting cancer growth. It was approved in 2004 in combination with chemotherapy for metastatic colorectal cancer and now it is used in the treatment of numerous cancer types-metastatic breast cancer, renal cell carcinoma, advanced epithelial ovarian cancer, non-squamous NSCLC [75]. Combination of metformin with bevacizumab was found to be effective in the treatment of ovarian cancer and metastatic non-squamous NSCLC

Metformin in combination with radiotherapy may increase cancer response to treatment. As already mentioned, one of the mechanism of action of metformin is affecting complex I in the electron transfer chain, reducing the oxygen consumption and increasing the reactive oxygen species (ROS) within the cells, resulting in DNA damage [78]. Another proposed mechanism is activation of p53 by activating AMPK, and as a result cell cycle arrest. Both, metformin and radiotherapy can activate p53 and stop cell proliferation [79]. There are several articles and case reports, showing a better response for patients receiving radiotherapy and metformin, comparing with those without metformin in: esophageal cancer, rectal cancer and

Many studies reported a reduced incidence of cancer in patients receiving metformin in standard dose, but also these trials have limitations: most of the trials were retrospective, others included both patients with invasive and non-invasive neoplasms, others trials did not exclude patients exposed to other antidiabetic treat-

In general, chemopreventive agents are used as long term therapies. Metformin meets all necessary criteria as a long term chemopreventive agent, because it is safe,

ments, all these findings being responsible for potential biases.

treatment for patients with locally advanced NSCLC, is ongoing [70].

nism of action of this combination is targeting the mTOR pathway [72].

events related to trastuzumab [73, 74].

in combination with chemotherapy [76, 77].

head and neck carcinomas [80].

**5. Conclusions**

**4.3 Metformin: combination with radiotherapy**

**118**

Authors report no conflicts of interest.

### **Author details**

Laura Mazilu1 \*, Dana Stanculeanu<sup>2</sup> , Andreea Gheorghe1 , Adrian-Paul Suceveanu1 , Irinel Parepa1 , Felix Voinea1 , Doina Catrinoiu<sup>2</sup> and Andra-Iulia Suceveanu1

1 Ovidius University of Constanta, Faculty of Medicine, Constanta, Romania

2 Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

\*Address all correspondence to: lauragrigorov@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[49] Chen L, Li H, Gu L, Ma X, Li X, et al. The impact of diabetes mellitus on renal cell carcinoma prognosis: A metaanalysis of cohort studies. Medicine (Baltimore). 2015;**94**:e1055. DOI: 10.1097/MD.0000000000001055

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[51] Li Y, Hu L, Xia Q, Yuan Y, Mi Y. The impact of metformin use on survival in kidney cancer patients with diabetes: A meta-analysis. International Urology and Nephrology. 2017;**49**(6):975-981. DOI: 10.1007/ s11255-017-1548-4

[52] Mazzone PJ, Rai H, Beukemann M, Xu M, Jain A, Sasidhar M. The effect of metformin and thiazolidinedione use on lung cancer in diabetics. BMC Cancer. 2012;**12**:410. DOI: 10.1186/1471-2407-12-410

[53] Tan BX et al. Prognostic influence of metformin as first-line chemotherapy for advanced nonsmall cell lung cancer in patients with type 2 diabetes. Cancer. 2011;**117**:5103-5111. DOI: 10.1002/ cncr.26151

[54] Cao X, Wen ZS, Wang XD, Li Y, Liu KY, Wang X. The clinical effect of metformin on the survival of lung cancer patients with diabetes: A comprehensive systematic review and meta-analysis of retrospective studies. Journal of Cancer. 2017;**8**(13):2532-2541. DOI: 10.7150/jca.19750

[55] Coperchini F, Leporati P, Rotondi M, et al. Expanding the therapeutic spectrum of metformin: From diabetes to cancer. Journal of Endocrinological Investigation. 2015;**38**:1047. DOI: 10.1007/ s40618-015-0370-z

[56] Kasper JS, Giovannucci E. A meta-analysis of diabetes mellitus and the risk of prostate cancer. Cancer Epidemiology, Biomarkers & Prevention. 2006;**15**:2056-2062. DOI: 10.1158/1055-9965.EPI-06-0410

[57] Snyder CF, Stein KB, Barone BB, Peairs KS, Yeh HC, Derr RL, et al. Does pre-existing diabetes affect prostate cancer prognosis? A systematic review. Prostate Cancer and Prostatic Diseases. 2010;**13**:58-64. DOI: 10.1038/ pcan.2009.39

[58] Patel T, Hruby G, Badani K, Abate-Shen C, McKiernan JM. Clinical outcomes after radical prostatectomy in diabetic patients treated with metformin. Urology. 2010;**76**:1240-1244. DOI: 10.1016/j.urology.2010.03.059

[59] Wright JL, Stanford JL. Metformin use and prostate cancer in Caucasian men: Results from a population-based case-control study. Cancer Causes & Control. 2009;**20**:1617-1622. DOI: 10.1007/s10552-009-9407-y

[60] He XX, Tu SM, Lee MH, Yeung SC. Thiazolidinediones and metformin associated with improved survival of diabetic prostate cancer patients. Annals of Oncology. 2011;**22**:2640-2645. DOI: 10.1093/ annonc/mdr020

[61] Miranda VC, Braghiroli MI, Faria LD, et al. Phase 2 trial of metformin combined with 5-fluorouracil in patients with refractory metastatic colorectal cancer. Clinical Colorectal Cancer. 2016;**15**(4):321-328. DOI: 10.1016/j.clcc.2016.04.011

[62] Mitsuhashi A, Sato Y, Kiyokawa T, Koshizaka M, Hanaoka H, Shozu M. Phase II study of medroxyprogesterone acetate plus metformin as a fertility-sparing treatment for atypical endometrial hyperplasia and endometrial cancer. Annals of Oncology. 2016;**27**(2):262-266. DOI: 10.1093/annonc/mdv539

[63] Jiralerspong S, Palla SL, Giordano SH, et al. Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. Journal of Clinical Oncology. 2009;**27**(20):3297- 3302. DOI: 10.1200/JCO.2009.19.6410

[64] Colquhoun AJ, Venier NA, Vandersluis AD, Besla R, Sugar LM, Kiss A, et al. Metformin enhances the antiproliferative and apoptotic effect of bicalutamide in prostate cancer. Prostate Cancer and Prostatic Diseases. 2012;**15**:346-352. DOI: 10.1038/ pcan.2012.16

[65] Ferguson FM, Gray NS. Kinase inhibitors: The road ahead. Nature Reviews. Drug Discovery. 2018;**17**(5):353-377. DOI: 10.1038/ nrd.2018.21

[66] Cohen MH, Williams GA, Sridhara R, et al. FDA drug approval summary: Gefitinib (ZD1839) (Iressa) tablets. The Oncologist.

2003;**8**(4):303-306. DOI: 10.1634/ theoncologist.8-4-303

[67] Wheeler DL, Dunn EF, Harari PM. Understanding resistance to EGFR inhibitors-impact on future treatment strategies. Nature Reviews. Clinical Oncology. 2010;**7**(9):493-507. DOI: 10.1038/nrclinonc.2010.97

[68] Pan YH, Jiao L, Lin CY, et al. Combined treatment with metformin and gefitinib overcomes primary resistance to EGFR-TKIs with EGFR mutation via targeting IGF-1R signaling pathway. Biologics. 2018;**12**:75-86. DOI: 10.2147/BTT.S166867

[69] Chen H, Yao W, Chu Q, et al. Synergistic effects of metformin in combination with EGFR-TKI in the treatment of patients with advanced non-small cell lung cancer and type 2 diabetes. Cancer Letters. 2015;**369**(1):97-102. DOI: 10.1016/j. canlet.2015.08.024

[70] Li KL, Li L, Zhang P, et al. A multicenter double-blind phase II study of metformin with gefitinib as first-line therapy of locally advanced non-smallcell lung cancer. Clinical Lung Cancer. 2017;**18**(3):340-343. DOI: 10.1016/j. cllc.2016.12.003

[71] Guan YS, He Q. Sorafenib: Activity and clinical application in patients with hepatocellular carcinoma. Expert Opinion on Pharmacotherapy. 2011;**12**(2):303-313. DOI: 10.1517/14656566.2011.546346

[72] Ling S, Song L, Fan N, et al. Combination of metformin and sorafenib suppresses proliferation and induces autophagy of hepatocellular carcinoma via targeting the mTOR pathway. International Journal of Oncology. 2017;**50**(1):297-309. DOI: 10.3892/ijo.2016.3799

[73] Vazquez-Martin A, Oliveras-Ferraros C, Del Barco S, et al.

**125**

*Metformin and Its Implication in Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.88803*

> [80] Rao M, Gao C, Guo M, Law BYK, Xu Y. Effects of metformin treatment on radiotherapy efficacy in patients

> systematic review and meta-analysis. Cancer Management and Research. 2018;**10**:4881-4890. DOI: 10.2147/

with cancer and diabetes: A

CMAR.S174535

The anti-diabetic drug metformin suppresses self-renewal and

10.1007/s10549-010-0924-x

[75] Yamazaki K, Nagase M,

[76] Markowska A, Sajdak S,

Markowska J, et al. Angiogenesis and cancer stem cells: New perspectives on therapy of ovarian cancer. European Journal of Medicinal Chemistry. 2017;**142**:87-94. DOI: 10.1016/j.

[77] Marrone KA, Zhou X, Forde PM, et al. A randomized phase II study of metformin plus paclitaxel/carboplatin/

[78] Samsuri NAB, Leech M, Marignol L. Metformin and improved treatment outcomes in radiation therapy–A review. Cancer Treatment Reviews. 2017;**55**:150- 162. DOI: 10.1016/j.ctrv.2017.03.005

[79] Baskar R, Dai J, Wenlong N, Yeo R, Yeoh K-W. Biological response of cancer cells to radiation treatment. Frontiers in Molecular Biosciences. 2014;**1**:24. DOI:

bevacizumab in patients with chemotherapy-naïve advanced or metastatic nonsquamous non-small cell lung cancer. The Oncologist. 2018;**23**(7):859-865. DOI: 10.1634/

theoncologist.2017-0465

10.3389/fmolb.2014.00024

2016;**36**(1):87-93

annonc/mdw206

ejmech.2017.06.030

proliferation of trastuzumab-resistant tumor-initiating breast cancer stem cells. Breast Cancer Research and Treatment. 2011;**126**(2):355-364. DOI:

[74] Smith TA, Phyu SM, Akabuogu EU. Effects of administered cardioprotective drugs on treatment response of breast cancer cells. Anticancer Research.

Tamagawa H, et al. Randomized phase III study of bevacizumab plus FOLFIRI and bevacizumab plus mFOLFOX6 as first-line treatment for patients with metastatic colorectal cancer (WJOG4407G). Annals of Oncology. 2016;**27**(8):1539-1546. DOI: 10.1093/

*Metformin and Its Implication in Cancer Therapy DOI: http://dx.doi.org/10.5772/intechopen.88803*

*Metformin*

annonc/mdr020

[60] He XX, Tu SM, Lee MH, Yeung SC. Thiazolidinediones and metformin associated with improved survival of diabetic prostate cancer patients. Annals of Oncology. 2011;**22**:2640-2645. DOI: 10.1093/

2003;**8**(4):303-306. DOI: 10.1634/

[67] Wheeler DL, Dunn EF, Harari PM. Understanding resistance to EGFR inhibitors-impact on future treatment strategies. Nature Reviews. Clinical Oncology. 2010;**7**(9):493-507. DOI:

theoncologist.8-4-303

10.1038/nrclinonc.2010.97

10.2147/BTT.S166867

canlet.2015.08.024

cllc.2016.12.003

2011;**12**(2):303-313. DOI: 10.1517/14656566.2011.546346

10.3892/ijo.2016.3799

[73] Vazquez-Martin A,

Oliveras-Ferraros C, Del Barco S, et al.

[72] Ling S, Song L, Fan N, et al. Combination of metformin and sorafenib suppresses proliferation and induces autophagy of hepatocellular carcinoma via targeting the mTOR pathway. International Journal of Oncology. 2017;**50**(1):297-309. DOI:

[68] Pan YH, Jiao L, Lin CY, et al. Combined treatment with metformin and gefitinib overcomes primary resistance to EGFR-TKIs with EGFR mutation via targeting IGF-1R signaling pathway. Biologics. 2018;**12**:75-86. DOI:

[69] Chen H, Yao W, Chu Q, et al. Synergistic effects of metformin in combination with EGFR-TKI in the treatment of patients with advanced non-small cell lung cancer and type 2 diabetes. Cancer Letters. 2015;**369**(1):97-102. DOI: 10.1016/j.

[70] Li KL, Li L, Zhang P, et al. A multicenter double-blind phase II study of metformin with gefitinib as first-line therapy of locally advanced non-smallcell lung cancer. Clinical Lung Cancer. 2017;**18**(3):340-343. DOI: 10.1016/j.

[71] Guan YS, He Q. Sorafenib: Activity and clinical application in patients with hepatocellular carcinoma. Expert Opinion on Pharmacotherapy.

[61] Miranda VC, Braghiroli MI, Faria LD, et al. Phase 2 trial of metformin combined with

DOI: 10.1016/j.clcc.2016.04.011

acetate plus metformin as a fertility-sparing treatment for atypical endometrial hyperplasia and endometrial cancer. Annals of Oncology. 2016;**27**(2):262-266. DOI:

10.1093/annonc/mdv539

[63] Jiralerspong S, Palla SL, Giordano SH, et al. Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. Journal of Clinical Oncology. 2009;**27**(20):3297- 3302. DOI: 10.1200/JCO.2009.19.6410

[64] Colquhoun AJ, Venier NA, Vandersluis AD, Besla R, Sugar LM, Kiss A, et al. Metformin enhances the antiproliferative and apoptotic effect of bicalutamide in prostate cancer. Prostate Cancer and Prostatic Diseases.

2012;**15**:346-352. DOI: 10.1038/

inhibitors: The road ahead. Nature Reviews. Drug Discovery. 2018;**17**(5):353-377. DOI: 10.1038/

[66] Cohen MH, Williams GA, Sridhara R, et al. FDA drug approval summary: Gefitinib (ZD1839) (Iressa) tablets. The Oncologist.

[65] Ferguson FM, Gray NS. Kinase

pcan.2012.16

nrd.2018.21

5-fluorouracil in patients with refractory metastatic colorectal cancer. Clinical Colorectal Cancer. 2016;**15**(4):321-328.

[62] Mitsuhashi A, Sato Y, Kiyokawa T, Koshizaka M, Hanaoka H, Shozu M. Phase II study of medroxyprogesterone

**124**

The anti-diabetic drug metformin suppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiating breast cancer stem cells. Breast Cancer Research and Treatment. 2011;**126**(2):355-364. DOI: 10.1007/s10549-010-0924-x

[74] Smith TA, Phyu SM, Akabuogu EU. Effects of administered cardioprotective drugs on treatment response of breast cancer cells. Anticancer Research. 2016;**36**(1):87-93

[75] Yamazaki K, Nagase M, Tamagawa H, et al. Randomized phase III study of bevacizumab plus FOLFIRI and bevacizumab plus mFOLFOX6 as first-line treatment for patients with metastatic colorectal cancer (WJOG4407G). Annals of Oncology. 2016;**27**(8):1539-1546. DOI: 10.1093/ annonc/mdw206

[76] Markowska A, Sajdak S, Markowska J, et al. Angiogenesis and cancer stem cells: New perspectives on therapy of ovarian cancer. European Journal of Medicinal Chemistry. 2017;**142**:87-94. DOI: 10.1016/j. ejmech.2017.06.030

[77] Marrone KA, Zhou X, Forde PM, et al. A randomized phase II study of metformin plus paclitaxel/carboplatin/ bevacizumab in patients with chemotherapy-naïve advanced or metastatic nonsquamous non-small cell lung cancer. The Oncologist. 2018;**23**(7):859-865. DOI: 10.1634/ theoncologist.2017-0465

[78] Samsuri NAB, Leech M, Marignol L. Metformin and improved treatment outcomes in radiation therapy–A review. Cancer Treatment Reviews. 2017;**55**:150- 162. DOI: 10.1016/j.ctrv.2017.03.005

[79] Baskar R, Dai J, Wenlong N, Yeo R, Yeoh K-W. Biological response of cancer cells to radiation treatment. Frontiers in Molecular Biosciences. 2014;**1**:24. DOI: 10.3389/fmolb.2014.00024

[80] Rao M, Gao C, Guo M, Law BYK, Xu Y. Effects of metformin treatment on radiotherapy efficacy in patients with cancer and diabetes: A systematic review and meta-analysis. Cancer Management and Research. 2018;**10**:4881-4890. DOI: 10.2147/ CMAR.S174535

**Chapter 8**

**Abstract**

on Cancer

Preventive and (Neo)Adjuvant

*Yile Jiao, Xiaochen Wang and Zhijun Luo*

of metformin in prevention and treatment of various cancer types.

prevention and therapy, clinical trials

**1. Introduction**

**127**

**Keywords:** metformin, AMPK, mTORC1, diabetes, lipogenesis, cancer

Metformin is derived from *Galega officinalis*, a natural herbal medicine. The herb

was first used to relieve polyuria, a symptom of diabetes in ancient Egypt and medieval Europe [1]. Metformin is a widely used frontline drug for type 2 diabetes mellitus (T2DM). The major function of metformin is to decrease hepatic gluconeogenesis and enhance insulin sensitivity by increasing glucose uptake in muscle and adipose [2]. In addition to antidiabetes, metformin has proved to be beneficial to metabolic syndrome and nonalcoholic fatty liver disease [3, 4]. Cancer is characteristic of a metabolic disorder, inasmuch as metabolism is reprogrammed by switching oxidative phosphorylation into aerobic glycolysis, and thus, many of key molecules in these two routes are altered in their expression or posttranslational modification [5]. The incidence of cancer is higher in patients with T2DM than those without diabetes, indicating that diabetes is a risk factor of cancer [6]. Since Evan et al. reported in 2005 lower cancer incidence in patients with T2DM taking metformin than those with other antidiabetic drugs, great efforts have been made to

Therapeutic Effects of Metformin

Metformin, the first-line antidiabetic drug, has become an attractive candidate in cancer therapy since retrospective clinical investigations reported that patients with type 2 diabetes receiving metformin had lower incidence of cancer than those with other glucose lowering drugs. In line with this, preclinical studies have demonstrated that the antitumor activity of metformin could proceed through several mechanisms. Thus far, metformin has been used in cancer prevention with reduced risk as consequence and treatment of various cancers as an adjuvant or neoadjuvant drug. Thus, existing data support the beneficial effects of metformin on many types of cancers such as reducing metastasis and mortality and improving pathological responses and survival rates. However, some reports do not support this and even show adverse effects. The discrepancy may be attributed to expression levels of its transporters or genetic background. Hence, this chapter briefly reviews information on the mechanism of metformin action and summarizes both completed and ongoing clinical trials in an attempt to evaluate the value

### **Chapter 8**

## Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer

*Yile Jiao, Xiaochen Wang and Zhijun Luo*

### **Abstract**

Metformin, the first-line antidiabetic drug, has become an attractive candidate in cancer therapy since retrospective clinical investigations reported that patients with type 2 diabetes receiving metformin had lower incidence of cancer than those with other glucose lowering drugs. In line with this, preclinical studies have demonstrated that the antitumor activity of metformin could proceed through several mechanisms. Thus far, metformin has been used in cancer prevention with reduced risk as consequence and treatment of various cancers as an adjuvant or neoadjuvant drug. Thus, existing data support the beneficial effects of metformin on many types of cancers such as reducing metastasis and mortality and improving pathological responses and survival rates. However, some reports do not support this and even show adverse effects. The discrepancy may be attributed to expression levels of its transporters or genetic background. Hence, this chapter briefly reviews information on the mechanism of metformin action and summarizes both completed and ongoing clinical trials in an attempt to evaluate the value of metformin in prevention and treatment of various cancer types.

**Keywords:** metformin, AMPK, mTORC1, diabetes, lipogenesis, cancer prevention and therapy, clinical trials

### **1. Introduction**

Metformin is derived from *Galega officinalis*, a natural herbal medicine. The herb was first used to relieve polyuria, a symptom of diabetes in ancient Egypt and medieval Europe [1]. Metformin is a widely used frontline drug for type 2 diabetes mellitus (T2DM). The major function of metformin is to decrease hepatic gluconeogenesis and enhance insulin sensitivity by increasing glucose uptake in muscle and adipose [2]. In addition to antidiabetes, metformin has proved to be beneficial to metabolic syndrome and nonalcoholic fatty liver disease [3, 4]. Cancer is characteristic of a metabolic disorder, inasmuch as metabolism is reprogrammed by switching oxidative phosphorylation into aerobic glycolysis, and thus, many of key molecules in these two routes are altered in their expression or posttranslational modification [5]. The incidence of cancer is higher in patients with T2DM than those without diabetes, indicating that diabetes is a risk factor of cancer [6]. Since Evan et al. reported in 2005 lower cancer incidence in patients with T2DM taking metformin than those with other antidiabetic drugs, great efforts have been made to elucidate the antitumor activity of metformin [7]. A considerable number of preclinical and clinical investigations support the beneficial effects of metformin on both prevention and treatment of various cancers. At the same time, some of mechanisms underlying metformin action on cancer cells have been unraveled, although much of them is still incomplete. Thus far, more than 300 clinical trials using metformin as a single or adjuvant agent in combination with other chemotherapies have been initiated in the treatment of various types of cancer in the world (www.clinicaltrials.gov).

### **2. Targets of metformin**

Many functions of metformin are mediated by adenosine monophosphateactivated protein kinase (AMPK). Metformin at high doses leads to elevation of AMP, which binds to and allosterically activates AMPK, while at low doses, it engages lysosomes in the absence of AMP [8, 9]. The upstream kinases that phosphorylate AMPK α subunits at Thr172 include liver kinase B1 (LKB1), calmodulindependent kinase beta, and TGF-β-activated protein kinase [10–12].

AMPK plays important roles in regulating lipid and protein metabolisms by phosphorylating a series of target proteins. Thus, LKB1-AMPK pathway is critically important for metabolic adaption under stress condition, which aims to protect cells in the beginning [13]. However, persistent activation of AMPK by metformin can also cause cytostatic and even cytotoxic effects. Mounting evidence shows that metabolic syndrome and diabetes increase the risk of cancer, and correction of metabolic abnormalities alleviates cancer burdens and improves survival [14–16]. Drugs that target AMPK or downstream molecules are research focus nowadays for cancer prevention and treatment. Some of pathways downstream of AMPK essential for tumorigenesis and cancer progression are depicted in **Figure 1**.

these transcription factors, so as to inhibit transcription of target genes for lipogen-

*AMPK activation and its biological functions. AMPK is activated by increased AMP:ATP ratio induced by metabolic stress and metformin. In addition, metformin can activate AMPK through lysosomal pathway, where v-ATPase-regulator-AXIN/LKB1-AMPK complex is formed. After activation, AMPK acts on multiple molecules/pathways, including inhibition of mTORC1, lipogenesis and IGF-1 expression, and activation of p53 and FOXO3a [17, 22, 87–89]. As such, AMPK regulates cell proliferation, autophagy, and apoptosis of*

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.91291*

Decreases from 20 to 94% in cancer risk among patients with T2DM after the use of metformin have been reported since 2005 [24]. A large population study conducted by Taiwan National Health Insurance Data Survey evaluated 16,602 individuals treated with metformin or other antidiabetic drug between 2000 and 2007 and concluded a 88% reduction in the risk of various cancer types after metformin treatment [25, 26]. In line with this, numerous investigations provided supporting data that metformin reduced incidence of various cancers. For example, DeCenci et al. have found a 30% decrease in cancer incidence in patients with T2DM treated with metformin compared to those with other drugs [27, 28]. Currie et al. conducted a large cohort study with around 60,000 patients from the UK database and revealed that metformin alone decreased the incidence of colorectal and pancreatic cancer compared with insulin and sulfonylureas monotherapy after the adjustment of confound bias, but this was not seen in breast cancer (BC) and prostate cancer [29]. It is noteworthy that metformin plus insulin could alleviate the progression of cancer [hazard ratio (HR) = 0.54, 95% confidence interval (CI) 0.43– 0.66] [29]. With respect to mortality, ZODIAC trial with a 10-year follow up has indicated a lower death rate of cancer among metformin users with T2DM [30]. According to Noto et al. meta-analysis, diabetic patients taking metformin showed significant reduction of incidence of multiple types of cancer [risk ratio (RR) = 0.67, 0.53–0.85], including colorectal cancer (CRC) (RR = 0.68, 0.53–0.88) and cancer

esis, including those encoding ACC and fatty acid synthase (FASN) [23].

**3. Clinical investigations**

**Figure 1.**

*cancer cells.*

**129**

PI3K-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway is well received as the target of AMPK. Mammalian target of rapamycin complex 1 (mTORC1) consists of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal with SEC13 protein 8, proline-rich AKT substrate 40 kDa, and DEP domain-containing mTOR-interacting protein [17]. Tuberous sclerosis complex 2 (TSC2) is a GTPase-activating protein that forms a complex with TSC1 to stimulate GTPase activity of Ras homolog enriched in brain (Rheb) and thus inhibits mammalian target of rapamycin complex 1 (mTORC1) activation. TSC2 is subjected to inhibition by AKT and activation by AMPK via phosphorylation at different sites. In addition, AMPK phosphorylates and inhibits Raptor, a scaffold of mTORC1. A plethora of cellular events, such as protein translation, lipogenesis, cell cycle progression, and autophagy, are regulated by the activated mTOR pathway, which are counteracted by AMPK [18]. Thus, control of mTORC1 activity is crucial for prevention and treatment of cancer.

Cancer cells always require large amount of building blocks for dividing progenitor cells. Thus, synthesis of fatty acid and cholesterol is very active [19]. Acutely, AMPK inhibits acetyl CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), which are rate-limiting enzymes for de novo synthesis of fatty acid and cholesterol, respectively [20]. In addition, AMPK activates malonyl-CoA decarboxylase (MAD) that converts malonyl-CoA to acetyl CoA. As cytosolic malonyl-CoA decreases, fatty acid synthesis is attenuated [17, 21]. AMPK also influences de novo synthesis of glycerolipid by inhibiting the rate-limiting enzyme glycerol phosphate acyltransferase (PAT) [17, 22]. Chronically, AMPK phosphorylates sterol regulatory element-binding protein-1c (SREBP-1c) and its related protein carbohydrateresponse-element-binding protein (ChREBP), restricting the nuclear localization of *Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer DOI: http://dx.doi.org/10.5772/intechopen.91291*

### **Figure 1.**

elucidate the antitumor activity of metformin [7]. A considerable number of preclinical and clinical investigations support the beneficial effects of metformin on both prevention and treatment of various cancers. At the same time, some of mechanisms underlying metformin action on cancer cells have been unraveled, although much of them is still incomplete. Thus far, more than 300 clinical trials using metformin as a single or adjuvant agent in combination with other chemotherapies have been initiated in the treatment of various types of cancer in the

Many functions of metformin are mediated by adenosine monophosphateactivated protein kinase (AMPK). Metformin at high doses leads to elevation of AMP, which binds to and allosterically activates AMPK, while at low doses, it engages lysosomes in the absence of AMP [8, 9]. The upstream kinases that phosphorylate AMPK α subunits at Thr172 include liver kinase B1 (LKB1), calmodulin-

AMPK plays important roles in regulating lipid and protein metabolisms by phosphorylating a series of target proteins. Thus, LKB1-AMPK pathway is critically important for metabolic adaption under stress condition, which aims to protect cells in the beginning [13]. However, persistent activation of AMPK by metformin can also cause cytostatic and even cytotoxic effects. Mounting evidence shows that metabolic syndrome and diabetes increase the risk of cancer, and correction of metabolic abnormalities alleviates cancer burdens and improves survival [14–16]. Drugs that target AMPK or downstream molecules are research focus nowadays for cancer prevention and treatment. Some of pathways downstream of AMPK essen-

PI3K-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway is well received as the target of AMPK. Mammalian target of rapamycin complex 1 (mTORC1) consists of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal with SEC13 protein 8, proline-rich AKT substrate 40 kDa, and DEP domain-containing mTOR-interacting protein [17]. Tuberous sclerosis complex 2 (TSC2) is a GTPase-activating protein that forms a complex with TSC1 to stimulate GTPase activity of Ras homolog enriched in brain (Rheb) and thus inhibits mammalian target of rapamycin complex 1 (mTORC1) activation. TSC2 is subjected to inhibition by AKT and activation by AMPK via phosphorylation at different sites. In addition, AMPK phosphorylates and inhibits Raptor, a scaffold of mTORC1. A plethora of cellular events, such as protein translation, lipogenesis, cell cycle progression, and autophagy, are regulated by the activated mTOR pathway, which are counteracted by AMPK [18]. Thus, control of mTORC1

Cancer cells always require large amount of building blocks for dividing progenitor cells. Thus, synthesis of fatty acid and cholesterol is very active [19]. Acutely, AMPK inhibits acetyl CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), which are rate-limiting enzymes for de novo synthesis of fatty acid and cholesterol, respectively [20]. In addition, AMPK activates malonyl-CoA decarboxylase (MAD) that converts malonyl-CoA to acetyl CoA. As cytosolic malonyl-CoA decreases, fatty acid synthesis is attenuated [17, 21]. AMPK also influences de novo synthesis of glycerolipid by inhibiting the rate-limiting enzyme glycerol phosphate acyltransferase (PAT) [17, 22]. Chronically, AMPK phosphorylates sterol regulatory element-binding protein-1c (SREBP-1c) and its related protein carbohydrateresponse-element-binding protein (ChREBP), restricting the nuclear localization of

dependent kinase beta, and TGF-β-activated protein kinase [10–12].

tial for tumorigenesis and cancer progression are depicted in **Figure 1**.

activity is crucial for prevention and treatment of cancer.

**128**

world (www.clinicaltrials.gov).

**2. Targets of metformin**

*Metformin*

*AMPK activation and its biological functions. AMPK is activated by increased AMP:ATP ratio induced by metabolic stress and metformin. In addition, metformin can activate AMPK through lysosomal pathway, where v-ATPase-regulator-AXIN/LKB1-AMPK complex is formed. After activation, AMPK acts on multiple molecules/pathways, including inhibition of mTORC1, lipogenesis and IGF-1 expression, and activation of p53 and FOXO3a [17, 22, 87–89]. As such, AMPK regulates cell proliferation, autophagy, and apoptosis of cancer cells.*

these transcription factors, so as to inhibit transcription of target genes for lipogenesis, including those encoding ACC and fatty acid synthase (FASN) [23].

### **3. Clinical investigations**

Decreases from 20 to 94% in cancer risk among patients with T2DM after the use of metformin have been reported since 2005 [24]. A large population study conducted by Taiwan National Health Insurance Data Survey evaluated 16,602 individuals treated with metformin or other antidiabetic drug between 2000 and 2007 and concluded a 88% reduction in the risk of various cancer types after metformin treatment [25, 26]. In line with this, numerous investigations provided supporting data that metformin reduced incidence of various cancers. For example, DeCenci et al. have found a 30% decrease in cancer incidence in patients with T2DM treated with metformin compared to those with other drugs [27, 28]. Currie et al. conducted a large cohort study with around 60,000 patients from the UK database and revealed that metformin alone decreased the incidence of colorectal and pancreatic cancer compared with insulin and sulfonylureas monotherapy after the adjustment of confound bias, but this was not seen in breast cancer (BC) and prostate cancer [29]. It is noteworthy that metformin plus insulin could alleviate the progression of cancer [hazard ratio (HR) = 0.54, 95% confidence interval (CI) 0.43– 0.66] [29]. With respect to mortality, ZODIAC trial with a 10-year follow up has indicated a lower death rate of cancer among metformin users with T2DM [30]. According to Noto et al. meta-analysis, diabetic patients taking metformin showed significant reduction of incidence of multiple types of cancer [risk ratio (RR) = 0.67, 0.53–0.85], including colorectal cancer (CRC) (RR = 0.68, 0.53–0.88) and cancer

mortality (RR = 0.66, 95% CI = 0.49–0.88) [31]. A study of Bowker et al. reported that metformin decreased cancer mortality in T2DM, as compared with insulin and/ or sulfonylurea groups [32]. After 1-year observation, the cancer death rate of metformin, insulin, and sulfonylurea users is 3.5, 8.8, and 4.9 per 1000 patients, respectively.

**Cancer type Intervention Outcome**

*DOI: http://dx.doi.org/10.5772/intechopen.91291*

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer*

Metformin or other antidiabetic drugs

Metformin A significant decrease in the risk of developing colon

antidiabetics users

Metformin A total of 151 nondiabetic patients with CRC after

Metformin A total of 40 women with atypical endometrial

treated group

Metformin 20 nondiabetic women with EC and obesity

surgery

Metformin + MPA 17 AEH and 19 noninvasive EC patients received

Adjuvant metformin Metformin significantly improved OS (HR = 0.54, 95%

nonusers with EC

Metformin + prednisone A total of 102 nondiabetic patients with ALL was

high levels of ABCB1

neoplasia [RRs (95% CI) = 0.75 (0.65–0.87), *Z* = 3.95, *P* < 0.001], including the reduction of colon cancer [0.79 (0.69–0.91), *Z* = 3.34, *P* < 0.001] and colon polyps [0.58 (0.42–0.80), *Z* = 3.30, *P* < 0.001] among patients with T2DB after metformin treatment

After adjustment of cofound variates, a 30% increase in OS was demonstrated among 424/4758 patients who were diagnosed of T2DM and CRC and administrated to metformin as compared with that in other

polypectomy was randomized to metformin-treated arm (250 mg daily over 1 year) or placebo control arm with 1-year endoscopy reports. The incidence of total polyps and adenomas decreased in metformin-treated group by 18.5% [RR = 0.67, 95% CI (0.47–0.97), *P* = 0.034] and 21% [RR = 0.60, 95% CI (0.39–0.92), *P* = 0.16], compared with that in control group

hyperplasia (AEH) or EC was assigned to receive metformin 850 mg b.i.d. over average 20 day, or no treatment before hysterectomy. Ki67 was reduced by 17.2% (95% CI 27.4–7.0, *P* < 0.002) in metformin-

(BMI ≥ 30) were administrated with metformin 850 mg daily for 1–4 weeks before surgery. The levels of Ki67 and p-S6 were reduced between pretreatment and postsurgery by 11.75% (*P* = 0.008) and 51.2% (*P* = 0.0002), respectively. Besides, the levels of p-AMPK (*P* = 0.00001), p-Akt (*P* = 0.0002), p-4EBP1 (*P* = 0.001), and ER (*P* = 0.0002) also decreased after

metformin (escalating from 750 to 2250 mg daily) after complete response treated by MPA and other drugs. Relapse rate among patients was 10%, and

CI 0.30–0.97, *P* < 0.04) in diabetic patients with nonendometrioid EC when compared with that in

enrolled, 26 received metformin (850 mg t.i.d.) for 6 days during preinduction stage, and 76 were treated with traditional chemotherapy without metformin. The use of metformin prevented therapy failure and early relapse (*P* = 0.025) in patients bearing relative to

Metformin users along with CRT resulted in higher pCR (34.5%) than nonmetformin cohort (4.8%,

estimated 3-year RFS rate was 89%

Rokkas and Portincasa [55]

Garrett et al. [58]

Higurashi et al. [59]

**Endometrial cancer**

Sivalingam et al. [60]

Schuler et al. [61]

Mitsuhashi et al. [63]

Nevadunsky et al. [66]

Ramos-Peñafiel et al. [67]

**131**

**Esophageal Cancer** Skinner et al. [68]

**Acute lymphoblastic leukemia**

Neoadjunvant metformin + CRT

Regarding tumor types, dosage of metformin, study setting, and period of intervention associated with the treatment outcomes, examples are listed in **Table 1**.



*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer DOI: http://dx.doi.org/10.5772/intechopen.91291*

mortality (RR = 0.66, 95% CI = 0.49–0.88) [31]. A study of Bowker et al. reported that metformin decreased cancer mortality in T2DM, as compared with insulin and/ or sulfonylurea groups [32]. After 1-year observation, the cancer death rate of metformin, insulin, and sulfonylurea users is 3.5, 8.8, and 4.9 per 1000 patients,

Regarding tumor types, dosage of metformin, study setting, and period of intervention associated with the treatment outcomes, examples are listed in

Metformin + chemotherapy The pCR rate in 68 diabetic patients treated with

Metformin Reduction of cancer cell proliferation (Ki67) by 3%

(*P* = 0.02)

Hou et al. [51] Metformin + chemotherapy 1013 BC patients with diabetes and 4621 BC patients

*P*< 0.001)

metformin

Metformin Significant benefit of RFS (*n* = 623 patients in two

nonmetformin using group

Diabetic patients treated with metformin ≧ 5 years had a lower incidence of cancer, compared with nonusers or short-term (<5 years treatment) metformin users

metformin, 87 diabetic patients without metformin, and 2374 nondiabetic patients was 24, 8, and 16%

(*P* = 0.016) and increases in apoptosis by 0.49% (*P* = 0.004) was compared between pre- and postsurgery, despite minor change of fasting insulin level

without diabetes were analyzed. Nondiabetic group had higher 5-year survival rate than diabetic group (82 vs. 79%, *P* < 0.001). In diabetic subgroup, metformintreated group had significant higher 5-year survival rates than nonmetformin-treated group (88 vs. 73%,

Non-diabetic women with newly diagnosed BC (68/ 129) were prescribed with metformin (860 mg b.i.d.) along with chemotherapy or hormone therapy compared to nonmetformin-treated control arm over 6 or 12 months. A 3.27-fold decrease (*P* = 0.023, 95% CI 1.17–9.06) at the time of developing metastasis and an increase in average DFS by 2.137 (*P* = 0.044) in the metformin-treated group. Also, the levels of IGF-1, the ratio of IGF-1 to IGFBP-3, insulin, fasting blood glucose, HOMA-IR index notably decease, while IGFBP-3 levels significantly increase after using of

A cohort study evaluated a total of 1983 women with stage ≧ 2 Her2 positive BC. Among 154/1983 diabetic patients who had already responded to previous chemotherapy. Metformin users had prolonged OS (HR = 0.52, 95% CI 0.28–0.97, *P* = 0.041) and reduced cancer-specific mortality of BC (HR = 0.47, 95% CI 0.24–0.90, *P* = 0.023), compared with nonusers

studies), OS (*n* = 1936 patients in five studies), and CSS (*n* = 535 patients in two studies) was observed in metformin-treated patients from 3094 patients with early stage CRC in nine studies, compared with that in

**Cancer type Intervention Outcome**

Metformin + chemotherapy or +hormone therapy,

tamoxifen

He et al. [53] Metformin or other

antidiabetic drugs

Metformin or other antidiabetic drugs

respectively.

*Metformin*

**Breast cancer** Bodmer et al. [39]

Jiralerspong et al. [45]

Niraula et al. [46]

El-Haggar et al. [42]

**Colon cancer** Coyle et al. [33]

**130**

**Table 1**.


A previous study has shown that metformin increases radiosensitivity of luminal BC by influencing expression of thioredoxin and intracellular redox homeostasis [34]. A high level of AMPKα expression correlates with the increased radiosensitivity and better prognosis. A systemic review and meta-analysis conducted in 2018 summarized the impact of metformin on the efficacy of radiotherapy in 17 studies, including prostate cancer, head and neck cancer, rectal cancer, lung cancer, esophageal cancer, and liver cancer [35]. The study compared diabetic patients with metformin (D + M) and diabetic or nondiabetic cohort without metformin (D M or N M) after radiotherapy. An improved pathologic complete response (pCR), 2y-OS, and 5y-OS vary in different cancer types when analyzing D + M and D M groups, supporting that metformin is beneficial to OS of diabetic patients while distant metastasis-free survival (DMFS) and 5-year OS were not significantly different between D + M and N M groups. With respect to the possible mechanisms by which metformin enhances radiosensitivity, studies have indicated that p53 and AMPKα are involved [36, 37]. Despite the increased sensitivity to radiotherapy and chemotherapy, cumulative side effects and toxicity concur with the use of metformin. For example, a study has shown that combination of metformin with radio-

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.91291*

chemotherapy can lead to less tolerance to cisplatin and radiotherapy and

risk was found in colon and pancreas cancer [29].

with hormone therapy [53, 54].

**3.2 Breast cancer**

static effect [47].

**133**

exacerbate gastrointestinal adverse effects such as grade ≥ 3 nausea/vomiting [38].

Several lines of clinical investigations have been conducted to assess the beneficial effects of metformin on BC [39–52]. Two retrospective studies revealed that long-term use of metformin (>5 years) reduced the risk of BC in T2DM women as compared with other antidiabetic drugs [39, 40]. However, Currie et al. reported that metformin use did not affect risk of breast and prostate cancer, but the reduced

He et al. have shown improvement of disease-free survival (DFS), DMFS, and OS in diabetic women who well-responded to previous hormone therapy and then received metformin treatment. The results demonstrated that metformin synergizes

Metformin was used as neoadjuvant chemotherapy of BC to improve pathological conditions prior to surgery [45–48]. The increased pCR in 2529 women with BC

has been demonstrated in metformin-treated diabetic patients, compared to nonmetformin-treated patients with or without diabetes [45]. Another study by Niraula et al. evaluated the effect of metformin on serum biomarkers in nondiabetic BC patients before surgery [46]. The patients were treated with metformin for 2 weeks, and serum biomarkers were assessed. A notably reduction of Ki67 and elevation of apoptosis were observed in invasive tumor after the use of metformin. The significant decrease of homeostatic model assessment of insulin resistance (- HOMA-IR) was also observed, while insulin and leptin displayed a modest change. However, a study showed that metformin increased phospho-AMPK (p-AMPK) and decreased p-Akt and Ki67 without induction of apoptosis, suggesting a cyto-

The long-term use of metformin has been shown to reduce risk of distant metastasis and mortality of BC patients with type 2 diabetes [49–51]. Furthermore, metformin use as adjuvant therapy can also improve outcomes of BC in nondiabetic patients [41, 42, 52]. For example, a single-arm phase II trial enrolled nondiabetic women with M0 stage BC. After receiving metformin of 500 mg t.i.d. for 6 months, the result showed that fasting insulin level and HOMA-IR were significantly reduced. Total cholesterol, low density lipoprotein, and leptin also similarly declined [52]. Another study focused on the optimal dose of metformin that

**Table 1.**

*Examples of clinical investigations of metformin used as a neoadjuvant and adjuvant agent in cancer therapy.*

### **3.1 The role of metformin in radiotherapy and chemotherapy**

Metformin has been reported to be a useful adjuvant drug to radiotherapy or chemotherapy for different cancers, especially prostate and colon cancers [33]. The effects of metformin on overall survival (OS), relapse-free survival (RFS), and cancer-specific survival (CSS) after concurrent chemotherapy and/or radiotherapy vary on cancer types.

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer DOI: http://dx.doi.org/10.5772/intechopen.91291*

A previous study has shown that metformin increases radiosensitivity of luminal BC by influencing expression of thioredoxin and intracellular redox homeostasis [34]. A high level of AMPKα expression correlates with the increased radiosensitivity and better prognosis. A systemic review and meta-analysis conducted in 2018 summarized the impact of metformin on the efficacy of radiotherapy in 17 studies, including prostate cancer, head and neck cancer, rectal cancer, lung cancer, esophageal cancer, and liver cancer [35]. The study compared diabetic patients with metformin (D + M) and diabetic or nondiabetic cohort without metformin (D M or N M) after radiotherapy. An improved pathologic complete response (pCR), 2y-OS, and 5y-OS vary in different cancer types when analyzing D + M and D M groups, supporting that metformin is beneficial to OS of diabetic patients while distant metastasis-free survival (DMFS) and 5-year OS were not significantly different between D + M and N M groups. With respect to the possible mechanisms by which metformin enhances radiosensitivity, studies have indicated that p53 and AMPKα are involved [36, 37]. Despite the increased sensitivity to radiotherapy and chemotherapy, cumulative side effects and toxicity concur with the use of metformin. For example, a study has shown that combination of metformin with radiochemotherapy can lead to less tolerance to cisplatin and radiotherapy and exacerbate gastrointestinal adverse effects such as grade ≥ 3 nausea/vomiting [38].

### **3.2 Breast cancer**

Several lines of clinical investigations have been conducted to assess the beneficial effects of metformin on BC [39–52]. Two retrospective studies revealed that long-term use of metformin (>5 years) reduced the risk of BC in T2DM women as compared with other antidiabetic drugs [39, 40]. However, Currie et al. reported that metformin use did not affect risk of breast and prostate cancer, but the reduced risk was found in colon and pancreas cancer [29].

He et al. have shown improvement of disease-free survival (DFS), DMFS, and OS in diabetic women who well-responded to previous hormone therapy and then received metformin treatment. The results demonstrated that metformin synergizes with hormone therapy [53, 54].

Metformin was used as neoadjuvant chemotherapy of BC to improve pathological conditions prior to surgery [45–48]. The increased pCR in 2529 women with BC has been demonstrated in metformin-treated diabetic patients, compared to nonmetformin-treated patients with or without diabetes [45]. Another study by Niraula et al. evaluated the effect of metformin on serum biomarkers in nondiabetic BC patients before surgery [46]. The patients were treated with metformin for 2 weeks, and serum biomarkers were assessed. A notably reduction of Ki67 and elevation of apoptosis were observed in invasive tumor after the use of metformin. The significant decrease of homeostatic model assessment of insulin resistance (- HOMA-IR) was also observed, while insulin and leptin displayed a modest change. However, a study showed that metformin increased phospho-AMPK (p-AMPK) and decreased p-Akt and Ki67 without induction of apoptosis, suggesting a cytostatic effect [47].

The long-term use of metformin has been shown to reduce risk of distant metastasis and mortality of BC patients with type 2 diabetes [49–51]. Furthermore, metformin use as adjuvant therapy can also improve outcomes of BC in nondiabetic patients [41, 42, 52]. For example, a single-arm phase II trial enrolled nondiabetic women with M0 stage BC. After receiving metformin of 500 mg t.i.d. for 6 months, the result showed that fasting insulin level and HOMA-IR were significantly reduced. Total cholesterol, low density lipoprotein, and leptin also similarly declined [52]. Another study focused on the optimal dose of metformin that

**3.1 The role of metformin in radiotherapy and chemotherapy**

**Cancer type Intervention Outcome**

Metformin + neoadjuvant chemo(radio)therapy

Leamm et al. [69]

*Metformin*

**Prostate cancer** Wright et al. [70]

Rothermundt et al. [74]

Joshua et al. [75]

Rieken et al. [77]

Spratt et al. [78]

**Table 1.**

**132**

*P* = 0.01) and nondiabetic patients (19.6%, *P* = 0.05). Higher pCR rate was found to be associated with higher metformin dose (≥1500 mg/d). Post-CRT maximum SUV decreased significantly in patients

gastroenterological cancers including CRC, HCC, and so on, among which the CID of esophageal cancer decreased in diabetic groups taking adjuvant metformin in comparison to non-DM groups. Metformin dosage giving rise to a significant decrease

taking metformin (*P* = 0.05)

in cancer incidence was ≤500 mg/day

as reported by a case-control study

Metformin A reduced risk of prostate cancer was showed among

Metformin A total of 44 men with castration-resistant prostate

significant

Metformin Metformin 500 mg t.i.d. was prescribed to 24 men

Metformin Metformin users with prostate cancer exhibited a

Metformin A retrospective study examined 2901 noninvasive

postprostatectomy sections

No statistically significant difference between metformin users and nonmetformin users for median overall survival (43.6 vs. 42.8 months, *P* = 0.66) or for median DFS (31.1 vs.47.0 months, *P* = 0.68)

white men at age of 35–74 after the use of metformin,

cancer was assigned to receive metformin 500 mg b.i. d. until progression. After initial metformin treatment, changes in IGF and IGBP3 and improvement of insulin sensitivity from baseline were observed but without correlation with progression. At week 4, only four patients did not have progression (95% CI, 3–22). Average PFS was 2.8 months (95% CI, 2.8–3.2) and PSA double time declined in 23 patients but not

with operable prostate cancer before prostatectomy. In a per patient and per tumor analyses, Ki67 was reduced by 29.5% (*P* = 0.0064) and 28.6%

(*P* = 0.0042) in comparison with the initial biopsy and

prostate cancer patients through radiation therapy. In 157 patients treated with metformin, PSA-RFS and DMFS were improved and the castration-resistant prostate cancer progression was alleviated

minor improvement of RFS after prostatectomy

Lee et al. [25] Adjuvant metformin Reduction of total CID and incidence of some

vary on cancer types.

Metformin has been reported to be a useful adjuvant drug to radiotherapy or chemotherapy for different cancers, especially prostate and colon cancers [33]. The effects of metformin on overall survival (OS), relapse-free survival (RFS), and cancer-specific survival (CSS) after concurrent chemotherapy and/or radiotherapy

*Examples of clinical investigations of metformin used as a neoadjuvant and adjuvant agent in cancer therapy.*

achieves favorable effects on BC by comparing dose between 1500 and 1000 mg daily [41]. For postmenopausal women with basal testosterone levels≧0.28 ng/mL, it seemed that metformin of 1500 mg/d was better than 1000 mg/d in reduction of insulin and testosterone levels, which were associated with cancer incidence and prognosis. Combination of metformin with other chemotherapy usually generates better outcomes in nondiabetic BC patients with the higher HOMA-IR (>2.8), and HOMA-IR can be improved by metformin [42–44, 48].

shown a remarkable reduction after metformin use at 850 mg b.i.d. for average 20 days [60]. A significant reduction in phospho-4E-binding protein 1 (p-4EFBP1) downstream of mTOR was also observed by immunohistochemistry, while indirect serum markers of insulin resistance (fasting glucose, insulin, and HOMA-IR) and leptin only showed a decrease trend but not significant after adjusting difference. Another preoperative clinical trial was done in nondiabetic women with body mass index (BMI) ≧ 30 [61]. After taking metformin 850 mg daily for 1–4 weeks prior to surgery, Ki67, p-AMPK, p-Akt, phospho-S6 Ribosomal Protein (p-S6), and p-4EBP were significantly lower in resected specimens than in pretreatment. The reduction of p-AMPK is inconsistent with purported positive effect of metformin. This study also showed a decrease in estrogen receptor (ER) but not progesterone receptor. According to a study evaluating the effect of metformin on EC of diabetic patients (*n* = 114) as compared with diabetic (*n* = 136) and nondiabetic (*n* = 735) patients without metformin from 1999 to 2009, metformin-treated group exhibits prolonged OS than nonusers before and after the adjustment of confound bias [66]. A phase II study has examined the effects of long-term metformin (2250 mg daily until recurrence) on RFS after a complete response to medroxyprogesterone acetate (MPA) in 17 individuals with atypical endometrial hyperplasia and 19 with EC [63]. The 3-year estimated RFS was 89%, and the 3-year recurrence rate showed a 4.7 fold decrease in this study compared with a previous study [64]. In contrast to short-term treatment, the other randomized factorial study does not have a significant change in PFS/OS after metformin treatment (1700 mg/d for 16 weeks and

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.91291*

A single study randomized to assign 102 patients with nondiabetic acute lymphoid leukemia (ALL) into a group of 26 with metformin at 850 mg t.i.d. for 10 days and the rest to the group without metformin before remission therapy [67]. Metformin displayed a beneficial effect on OS in the patients with high levels of ABCB1 expression, the gene encoding multidrug resistant protein-1. The failure rate of therapy was significantly reduced and early relapse after remission prevented

Oesophagal cancer is deadly cancer with poor prognosis, and patients usually do survive or die no longer than 30 months after chemoradiation and surgery [68]. A prospective cohort study by Taiwan National Health Insurance revealed a positive effect of metformin as an adjunct to standard chemotherapies on the cancer incidence density (CID) of gastroenterological cancers [25]. In this study, a decrease in total CID including esophageal cancer was found in diabetic groups taking adjuvant metformin in comparison to nondiabetic groups. Another study reported that metformin enhanced the efficacy of radiochemotherapy in patients with T2DM

resulting in superior pCR and low postconcurrent chemoradiation (CRT) maximum SUV compared to patients with T2DM without metformin and non-DM patients [68]. Additionally, higher pCR rate was correlated with higher metformin dose (≥1500 mg/d). However, a report in 2015 demonstrated inconsistent results, in

which no difference in pCR was found between metformin users and nonmetformin users [69]. Furthermore, it was shown that together with neoadjunvant chemoradiation, metformin did not improve the median OS or

median DFS in diabetic patients with esophageal cancer.

1-year follow up) [65].

**3.6 Oesophagal cancer**

**135**

**3.5 Acute lymphoid leukemia**

by metformin, as compared with nonusers.

In summary, studies showing beneficial effects of metformin are more than those without effects. Metformin as an adjuvant agent can suppress BC at various doses ranging from 500 to 1500 mg. The outcomes mainly include reduced risk of BC, decreases in cancer-promoting markers and metastatic events, increases in apoptotic markers, and improvement of progression-free survival (PFS) and OS.

### **3.3 Colon cancer**

The role of metformin in preventing colon cancer has been documented in the following studies conducted in both diabetic and nondiabetic patients. A metaanalysis was carried out in 709,980 individuals with T2DM from 17 studies showing a significant decrease in the risk of colon neoplasia among metformin-treated patients compared to those without metformin, with respective reduction for either cancer or polys [55]. A randomized study enrolled a total of 26 nondiabetic individuals with aberrant crypt foci (ACF) (biomarker of CRC development) and assigned them to either receive metformin 250 mg daily for 1 month or control group [56]. Significant decreases in the average number of ACF by a 3.67-fold (*P* = 0.007) and in proliferating cell nuclear antigen index were discovered in metformin arm. This indicates that metformin prevents CRC by attenuating cell proliferation and ACF development.

Metformin has been used as an adjuvant agent in the treatment of CRC. First, a single-arm study has demonstrated a median PFS of 1.8 months and an OS of 7.9 months in metastatic CRC with combination of metformin (850 mg b.i.d.) and 5-fluorouracil treatment. Surprisingly, the improvement in median survival was more obvious in obese patients [57]. Second, Coyle et al. have evaluated 3092 patients with early stage of CRC [33]. It was found that the use of metformin significantly improved RFS (HR = 0.63, 95% CI 0.47–0.85), OS (0.69, 95% CI 0.58– 0.83), and CSS (0.58, 95% CI 0.39–0.86) in patients with T2DM, compared with other antidiabetic drugs. Likewise, progression of CRC is also inhibited by metformin. A similar study showed prolonged OS in patients with T2DM with CRC receiving metformin, as compared with nonmetformin users (79.6 vs. 56.9 months, *P* = 0.048) [58]. The last randomized trial used metformin (250 mg daily) for a year in nondiabetic patients with high-risk adenoma recurrence and no colorectal polyps after polypectomy [59]. The results showed that polyps and adenomas are noticeably fewer in the metformin arm than in the control arm. The study also showed that average HOMA-IR status was significantly reduced in nonrecurrent patients by metformin, while the value remained stable in recurrent patients, indicating that insulin resistance is associated with chemoprevention outcome.

### **3.4 Endometrial cancer**

Clinical investigations support that metformin could serve as a potential drug for protection against endometrial cancer (EC) [60–65]. Several studies have evaluated the effects of short-term use of metformin as a neoadjuvant therapy between initial recruitment and hysterectomy surgery in nondiabetic women with EC [60–62]. The first nonrandomized trial has examined the change of Ki67 and

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer DOI: http://dx.doi.org/10.5772/intechopen.91291*

shown a remarkable reduction after metformin use at 850 mg b.i.d. for average 20 days [60]. A significant reduction in phospho-4E-binding protein 1 (p-4EFBP1) downstream of mTOR was also observed by immunohistochemistry, while indirect serum markers of insulin resistance (fasting glucose, insulin, and HOMA-IR) and leptin only showed a decrease trend but not significant after adjusting difference. Another preoperative clinical trial was done in nondiabetic women with body mass index (BMI) ≧ 30 [61]. After taking metformin 850 mg daily for 1–4 weeks prior to surgery, Ki67, p-AMPK, p-Akt, phospho-S6 Ribosomal Protein (p-S6), and p-4EBP were significantly lower in resected specimens than in pretreatment. The reduction of p-AMPK is inconsistent with purported positive effect of metformin. This study also showed a decrease in estrogen receptor (ER) but not progesterone receptor.

According to a study evaluating the effect of metformin on EC of diabetic patients (*n* = 114) as compared with diabetic (*n* = 136) and nondiabetic (*n* = 735) patients without metformin from 1999 to 2009, metformin-treated group exhibits prolonged OS than nonusers before and after the adjustment of confound bias [66]. A phase II study has examined the effects of long-term metformin (2250 mg daily until recurrence) on RFS after a complete response to medroxyprogesterone acetate (MPA) in 17 individuals with atypical endometrial hyperplasia and 19 with EC [63]. The 3-year estimated RFS was 89%, and the 3-year recurrence rate showed a 4.7 fold decrease in this study compared with a previous study [64]. In contrast to short-term treatment, the other randomized factorial study does not have a significant change in PFS/OS after metformin treatment (1700 mg/d for 16 weeks and 1-year follow up) [65].

### **3.5 Acute lymphoid leukemia**

achieves favorable effects on BC by comparing dose between 1500 and 1000 mg daily [41]. For postmenopausal women with basal testosterone levels≧0.28 ng/mL, it seemed that metformin of 1500 mg/d was better than 1000 mg/d in reduction of insulin and testosterone levels, which were associated with cancer incidence and prognosis. Combination of metformin with other chemotherapy usually generates better outcomes in nondiabetic BC patients with the higher HOMA-IR (>2.8), and

In summary, studies showing beneficial effects of metformin are more than those without effects. Metformin as an adjuvant agent can suppress BC at various doses ranging from 500 to 1500 mg. The outcomes mainly include reduced risk of BC, decreases in cancer-promoting markers and metastatic events, increases in apoptotic markers, and improvement of progression-free survival (PFS) and OS.

The role of metformin in preventing colon cancer has been documented in the following studies conducted in both diabetic and nondiabetic patients. A metaanalysis was carried out in 709,980 individuals with T2DM from 17 studies showing a significant decrease in the risk of colon neoplasia among metformin-treated patients compared to those without metformin, with respective reduction for either cancer or polys [55]. A randomized study enrolled a total of 26 nondiabetic individuals with aberrant crypt foci (ACF) (biomarker of CRC development) and assigned them to either receive metformin 250 mg daily for 1 month or control group [56]. Significant decreases in the average number of ACF by a 3.67-fold (*P* = 0.007) and in proliferating cell nuclear antigen index were discovered in metformin arm. This indicates that metformin prevents CRC by attenuating cell proliferation and ACF

Metformin has been used as an adjuvant agent in the treatment of CRC. First, a single-arm study has demonstrated a median PFS of 1.8 months and an OS of 7.9 months in metastatic CRC with combination of metformin (850 mg b.i.d.) and 5-fluorouracil treatment. Surprisingly, the improvement in median survival was more obvious in obese patients [57]. Second, Coyle et al. have evaluated 3092 patients with early stage of CRC [33]. It was found that the use of metformin significantly improved RFS (HR = 0.63, 95% CI 0.47–0.85), OS (0.69, 95% CI 0.58– 0.83), and CSS (0.58, 95% CI 0.39–0.86) in patients with T2DM, compared with other antidiabetic drugs. Likewise, progression of CRC is also inhibited by metformin. A similar study showed prolonged OS in patients with T2DM with CRC receiving metformin, as compared with nonmetformin users (79.6 vs. 56.9 months, *P* = 0.048) [58]. The last randomized trial used metformin (250 mg daily) for a year in nondiabetic patients with high-risk adenoma recurrence and no colorectal polyps after polypectomy [59]. The results showed that polyps and adenomas are noticeably fewer in the metformin arm than in the control arm. The study also showed that average HOMA-IR status was significantly reduced in nonrecurrent patients by metformin, while the value remained stable in recurrent patients, indicating that

Clinical investigations support that metformin could serve as a potential drug for protection against endometrial cancer (EC) [60–65]. Several studies have evaluated the effects of short-term use of metformin as a neoadjuvant therapy between initial recruitment and hysterectomy surgery in nondiabetic women with EC [60–62]. The first nonrandomized trial has examined the change of Ki67 and

insulin resistance is associated with chemoprevention outcome.

HOMA-IR can be improved by metformin [42–44, 48].

**3.3 Colon cancer**

*Metformin*

development.

**3.4 Endometrial cancer**

**134**

A single study randomized to assign 102 patients with nondiabetic acute lymphoid leukemia (ALL) into a group of 26 with metformin at 850 mg t.i.d. for 10 days and the rest to the group without metformin before remission therapy [67]. Metformin displayed a beneficial effect on OS in the patients with high levels of ABCB1 expression, the gene encoding multidrug resistant protein-1. The failure rate of therapy was significantly reduced and early relapse after remission prevented by metformin, as compared with nonusers.

### **3.6 Oesophagal cancer**

Oesophagal cancer is deadly cancer with poor prognosis, and patients usually do survive or die no longer than 30 months after chemoradiation and surgery [68]. A prospective cohort study by Taiwan National Health Insurance revealed a positive effect of metformin as an adjunct to standard chemotherapies on the cancer incidence density (CID) of gastroenterological cancers [25]. In this study, a decrease in total CID including esophageal cancer was found in diabetic groups taking adjuvant metformin in comparison to nondiabetic groups. Another study reported that metformin enhanced the efficacy of radiochemotherapy in patients with T2DM resulting in superior pCR and low postconcurrent chemoradiation (CRT) maximum SUV compared to patients with T2DM without metformin and non-DM patients [68]. Additionally, higher pCR rate was correlated with higher metformin dose (≥1500 mg/d). However, a report in 2015 demonstrated inconsistent results, in which no difference in pCR was found between metformin users and nonmetformin users [69]. Furthermore, it was shown that together with neoadjunvant chemoradiation, metformin did not improve the median OS or median DFS in diabetic patients with esophageal cancer.

### **3.7 Prostate cancer**

The effect of metformin on prostate cancer is ambiguous. Studies of Wright and Stanford have provided a 44% decrease in the risk of prostate cancer among Caucasian men with diabetes [70]. However, investigations by others could not obtain the same conclusion on the incidence of prostate cancer in diabetic patients treated with metformin, but the mortality might be reduced [71–73]. A single-arm clinical trial has revealed a decrease in insulin-like growth factor-1 (IGF-1) and an increase in insulin-like growth factor-binding protein-3 (IGFBP-3), alongside lowering prostate-specific antigen (PSA), after giving metformin 500 mg b.i.d. over 12 weeks to patients with castration-resistant prostate cancer [74]. In a single-arm study on men with biopsy-proven localized prostate cancer, 22 patients were selected to receive metformin at 500 mg/d or b.i.d., followed by t.i.d. for 28–84 days preceding their prostatectomy. The results revealed that Ki67 index was reduced by comparing the initial biopsy with postprostatectomy sections [75]. However, the changes were not recapitulated by another study, although metformin in the prostate tissue was detected after a median of 34 days prior to prostatectomy [76]. In a retrospective study, metformin-treated diabetic individuals gained the improvement of RFS among 6863 patients after radical prostatectomy [77]. Study of Spratt et al. also demonstrated the significantly elevated PSA-RFS, DFS, and lower cancer mortality in localized prostate cancer with metformin treatment compared with that of nonusers [78].

**NCT number Status Participants Period Intervention Cancer**

June 10, 2016 to not indicated

March 7, 2014 to June 30, 2020

May 1, 2013 to September 1, 2022

May 1, 2013 to September 1, 2022

May 1, 2013 to October 1, 2020

Match 17, 2014 to

March 15, 2017 to March 15, 2020

August 2013 to

December 2015 to July 2021

May 4, 2018 to June 1, 2020

October 1, 2018 to September 30, 2020

Drug: metformin hydrochloride

Drug: metformin &

Drug: metformin &

Drug: metformin &

Drug: metformin, letrozole, & everolimus

Drug: carboplatin, metformin hydrochloride, paclitaxel, & placebo

Drug: aspirin (ASA) + metformin (MET)|Drug: ASA|Drug: MET|Drug: placebos

Drug: metformin hydrochloride & placebo

Drug: metformin &

Drug: metformin & acetylsalicylic acid & drug: olaparib & drug:

Drug: metformin hydrochloride & placebo

placebo

letrozole

placebo

placebo

placebo

Active (a) 26, (b)18 years and older (adult, older adult), (c) all sex

> 21 years to 54 years (adult), (c) female

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer*

50 years to 65 years (adult, older adult), (c) female

50 years to 65 years (adult, older adult), (c) female

18 years to older (adult, older adult), (c) female

18 years to 80 years (adult, older adult), (c) all sex

25 years to 55 years (adult), (c) female

20 years to 80 years (adult, older adult), (c) all sex

18 years and older (adult, older adult), (c) female

to 70 years (adult, older adult), (c) all sex

Active (a) 151, (b)

*DOI: http://dx.doi.org/10.5772/intechopen.91291*

Active (a) 100, (b)

Active (a) 100, (b)

Active (a) 540, (b)

Recruiting (a) 160, (b)

Recruiting (a) 128, (b)

Recruiting (a) 593, (b)

Recruiting (a) 143, (b)

Recruiting (a) 62, (b)20 years

Active (a) 62, (b) 18 years and older (adult, older adult), (c) all sex

NCT02581137 https://Clinica lTrials.gov/ show/ NCT02581137

NCT02028221 https://Clinica lTrials.gov/ show/ NCT02028221

NCT02431676 https://Clinica lTrials.gov/ show/ NCT02431676

NCT01697566 https://Clinica lTrials.gov/ show/ NCT01697566

NCT01797523 https://Clinica lTrials.gov/ show/ NCT01797523

NCT02065687 https://Clinica lTrials.gov/ show/ NCT02065687

NCT03047837 https://Clinica lTrials.gov/sh ow/ NCT03047837

NCT01905046 https://Clinica lTrials.gov/ show/ NCT01905046

NCT02614339 https://Clinica lTrials.gov/ show/ NCT02614339

NCT03378297 https://Clinica lTrials.gov/ show/ NCT03378297

NCT03685409 https://Clinica lTrials.gov/ show/ NCT03685409

**137**

**type**

BC

EC

EC

EC

EC

Tertiary prevention in colon cancer

BC

CRC

Ovarian cancer

Oral cancer

Oral cancer

### **4. Ongoing clinical trials**

Previous studies of metformin use as neoadjuvant or adjuvant therapy for various types of cancer provide strong rationale of clinical trials in more vigorous settings. Thus far, more than 300 clinical trials have initiated in the world despite some are somehow either terminated or withdrawn. **Table 2** lists some of them. For example, NCT02065687 is a randomized, metformin-placebo, phase II/III study that enrolls a total of 540 participants and examines the effect of adjuvant metformin together with paclitaxel and carboplatin in treatment of stages III–IV or recurrent EC. Patients receive metformin twice a day in a 5-year follow up until disease progression or undesirable adverse effects appear. According to this trial, prolonged PFS and OS will be observed after the use of metformin together with other chemotherapeutic drugs. One of the ongoing phase II trials carrying out in 151 premenopausal BC patients with BMI ≧ 25 kg/m<sup>2</sup> evaluates treatment effect with 850 mg metformin b.i.d. vs. placebo for a year, by examining the primary outcome changes of breast density at time points of 6 and 12 months. This study spanning from March 7, 2014 to June 30, 2020 also identifies biomarkers associated with metabolic effects of metformin and attempts to find prediction factors of BC risk (NCT02028221). Also, a trial (NCT02614339) is undergoing to follow-up 3-year DFS and 5-year OS in nondiabetic patients with stage II high-risk/III CRC treated with metformin (1000 mg/day) for 48 months. This study has enrolled 593 participants and is still recruiting and expected to complete in July 2021.

The trial of double-blinded 2 2 factorial (aspirin metformin) design registers 160 patients with stages I–III colon cancer who undertake a completed polypectomy within recent 24 months (NCT03047837). After randomized allocation, patients will receive metformin at 850 mg b.i.d. or aspirin at 100 mg daily or two drugs together vs. placebo over 1 year. Immunohistochemistry for NF-κB, glucose metabolism, pS6K, and other biomarker will be compared pre- and postintervention (ClinicalTrials.gov Identifier: NCT03047837).


*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer DOI: http://dx.doi.org/10.5772/intechopen.91291*

**3.7 Prostate cancer**

*Metformin*

nonusers [78].

**4. Ongoing clinical trials**

The effect of metformin on prostate cancer is ambiguous. Studies of Wright and Stanford have provided a 44% decrease in the risk of prostate cancer among Caucasian men with diabetes [70]. However, investigations by others could not obtain the same conclusion on the incidence of prostate cancer in diabetic patients treated with metformin, but the mortality might be reduced [71–73]. A single-arm clinical trial has revealed a decrease in insulin-like growth factor-1 (IGF-1) and an increase in insulin-like growth factor-binding protein-3 (IGFBP-3), alongside lowering prostate-specific antigen (PSA), after giving metformin 500 mg b.i.d. over 12 weeks to patients with castration-resistant prostate cancer [74]. In a single-arm study on men with biopsy-proven localized prostate cancer, 22 patients were selected to receive metformin at 500 mg/d or b.i.d., followed by t.i.d. for 28–84 days preceding their prostatectomy. The results revealed that Ki67 index was reduced by comparing the initial biopsy with postprostatectomy sections [75]. However, the changes were not recapitulated by another study, although metformin in the prostate tissue was detected after a median of 34 days prior to prostatectomy [76]. In a retrospective study, metformin-treated diabetic individuals gained the improvement of RFS among 6863 patients after radical prostatectomy [77]. Study of Spratt et al. also demonstrated the significantly elevated PSA-RFS, DFS, and lower cancer mortality in localized prostate cancer with metformin treatment compared with that of

Previous studies of metformin use as neoadjuvant or adjuvant therapy for vari-

ous types of cancer provide strong rationale of clinical trials in more vigorous settings. Thus far, more than 300 clinical trials have initiated in the world despite some are somehow either terminated or withdrawn. **Table 2** lists some of them. For example, NCT02065687 is a randomized, metformin-placebo, phase II/III study that enrolls a total of 540 participants and examines the effect of adjuvant metformin together with paclitaxel and carboplatin in treatment of stages III–IV or recurrent EC. Patients receive metformin twice a day in a 5-year follow up until disease progression or undesirable adverse effects appear. According to this trial, prolonged PFS and OS will be observed after the use of metformin together with other chemotherapeutic drugs. One of the ongoing phase II trials carrying out in 151 premenopausal BC patients with BMI ≧ 25 kg/m<sup>2</sup> evaluates treatment effect with 850 mg metformin b.i.d. vs. placebo for a year, by examining the primary outcome changes of breast density at time points of 6 and 12 months. This study spanning from March 7, 2014 to June 30, 2020 also identifies biomarkers associated with metabolic effects of metformin and attempts to find prediction factors of BC risk (NCT02028221). Also, a trial (NCT02614339) is undergoing to follow-up 3-year DFS and 5-year OS in nondiabetic patients with stage II high-risk/III CRC treated with metformin (1000 mg/day) for 48 months. This study has enrolled 593 partic-

ipants and is still recruiting and expected to complete in July 2021.

(ClinicalTrials.gov Identifier: NCT03047837).

**136**

The trial of double-blinded 2 2 factorial (aspirin metformin) design registers 160 patients with stages I–III colon cancer who undertake a completed polypectomy within recent 24 months (NCT03047837). After randomized allocation, patients will receive metformin at 850 mg b.i.d. or aspirin at 100 mg daily or two drugs together vs. placebo over 1 year. Immunohistochemistry for NF-κB, glucose metabolism, pS6K, and other biomarker will be compared pre- and postintervention


than nonstem cancer cells [81]. This finding suggests that metformin could effectively prevent metastasis. It is especially meaningful in the case of surgically resected cancer when local metastasis in lymph nodes is cytologically tested negative, but a few CSCs may escape to circulation. At this time, metformin can be used

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer*

Previous studies have demonstrated that metformin selectively targets CSCs via regulation of different pathways in various cancer types including breast, pancreatic, prostate, and colon cancer [82, 83]. For example, Zhu et al. have shown that metformin inhibits CD61high/CD49fhigh subpopulation, markers of tumor initiating cells, by inactivating epidermal growth factor receptor/ErbB2 signaling. Similarly, CD133+, aldehyde dehydrogenases 1+, and other molecules are inhibited in pancreatic and colon cancer through inhibition of the Akt/mTOR pathway [84, 85]. However, a recent study using head and neck squamous cell carcinoma has shown that metformin protects CSCs against the cisplatin-induced cell death when combining these two, which discord with previous studies [86]. Thus, it should be cautious to ascertain if metformin exerts inhibitory or protective effects on specifically

Metformin is a cheap and nontoxic first-line antidiabetic medicine. It is an attractive drug that is being repurposed for multiple usages in treatment of other diseases in addition to diabetes. Metformin implements its function through AMPKdependent and independent mechanisms. Preclinical and retrospective clinical investigations have inspired clinical trials of metformin use in various cancer therapies. It is a promising drug in neoadjuvant and adjuvant therapies. We hope these trials will come to end with positive or negative results in the next few years. In considering genetic heterogeneity of cancer, responses of different cancer types and subpopulations in the same cancer might be different. Therefore, we still have long

ZL is supported by the National Natural Science Foundation of China (81572753, 31660332) and the Innovation and Entrepreneurship grant from Jiangxi Provincial

way to go and loads of questions to be addressed.

The authors declare no conflict of interest.

as preventive measure.

*DOI: http://dx.doi.org/10.5772/intechopen.91291*

originated CSCs.

**6. Conclusion**

**Acknowledgements**

**Conflict of interest**

**139**

Bureau of Foreign Experts, China.

**Table 2.**

*Summary of ongoing clinical trials approved by FDA.*

### **5. Cautions to be considered**

### **5.1 Cancer type-specific effects**

Whether a cancer type is sensitive to metformin depends on expression level of OCT1 in the cell membrane. Thus far, majority of previous studies have demonstrated that metformin exerts beneficial effects on different types of cancer, while some do not respond. On contrary, in some cases, for example, in glioma and leukemia cancer cells, metformin reduces cisplatin-induced apoptosis, suggesting that metformin exerts a protective effect on cytotoxic agents in some cells [79]. Hence, before going to clinical trials, preclinical tests should be undertaken to ascertain if metformin enhances the inhibitory effect of other drugs. This is feasible when PDX animal models or organoid culture techniques are available.

### **5.2 Genetic background of cancer**

Responses of cancer cells with and without LKB1 to metformin are different. Metformin exerts cytostatic effect on cancer cells with wild-type LKB1, while it causes cytotoxicity in cells lacking LKB1. If metformin is used together with most of chemotherapeutic drugs that are cytotoxic in cancer containing wild-type LKB1, the cooperative effects might not be achieved. The reason is that more rapidly dividing cells are more sensitive to cytotoxic drugs, while cytostatic drugs slow down speed of cell growth, which might compromise the efficacy of cytotoxic chemotherapy. In this scenario, it might be a good idea to take metformin and cytotoxic drug alternately. For example, patients take a couple of cycles of cytotoxic chemotherapy and then have rest for period of time during which metformin is alternately used. The purpose is to restrain cancer in dormancy and allow the patients to restore healthy condition. In addition, Birsoy et al. have delineated that the most metforminsensitive cells contain mutations of genes responsible for upregulation of mitochondrial oxidative phosphorylation, for example, complex I components, or glucose utilization [80]. Thus, these genes may serve as biomarkers for metformin use. Altogether, these studies point to importance of personalized medicine to determine the efficacy of metformin in cancer therapy.

### **5.3 Sensitivity of cancer stem cells**

Cancer stem cells (CSCs) are refractory to chemotherapy, leading to the relapse of cancer. These cells metastasize to distant organs after flowing in circulation, resulting in poor prognosis. Thus, CSCs have become an important target for anticancer therapies. Hirsch et al. have reported that the CSCs derived from BC are preferentially sensitive to metformin that is used from 10 to 100 times less dosage

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer DOI: http://dx.doi.org/10.5772/intechopen.91291*

than nonstem cancer cells [81]. This finding suggests that metformin could effectively prevent metastasis. It is especially meaningful in the case of surgically resected cancer when local metastasis in lymph nodes is cytologically tested negative, but a few CSCs may escape to circulation. At this time, metformin can be used as preventive measure.

Previous studies have demonstrated that metformin selectively targets CSCs via regulation of different pathways in various cancer types including breast, pancreatic, prostate, and colon cancer [82, 83]. For example, Zhu et al. have shown that metformin inhibits CD61high/CD49fhigh subpopulation, markers of tumor initiating cells, by inactivating epidermal growth factor receptor/ErbB2 signaling. Similarly, CD133+, aldehyde dehydrogenases 1+, and other molecules are inhibited in pancreatic and colon cancer through inhibition of the Akt/mTOR pathway [84, 85]. However, a recent study using head and neck squamous cell carcinoma has shown that metformin protects CSCs against the cisplatin-induced cell death when combining these two, which discord with previous studies [86]. Thus, it should be cautious to ascertain if metformin exerts inhibitory or protective effects on specifically originated CSCs.

### **6. Conclusion**

**5. Cautions to be considered**

*Summary of ongoing clinical trials approved by FDA.*

Recruiting (a) 408, (b)

18 years to 79 years (adult, older adult), (c) male

NCT01864096 https://Clinica lTrials.gov/ show/ NCT01864096

**Table 2.**

*Metformin*

**5.1 Cancer type-specific effects**

**5.2 Genetic background of cancer**

mine the efficacy of metformin in cancer therapy.

**5.3 Sensitivity of cancer stem cells**

**138**

Whether a cancer type is sensitive to metformin depends on expression level of OCT1 in the cell membrane. Thus far, majority of previous studies have demonstrated that metformin exerts beneficial effects on different types of cancer, while some do not respond. On contrary, in some cases, for example, in glioma and leukemia cancer cells, metformin reduces cisplatin-induced apoptosis, suggesting that metformin exerts a protective effect on cytotoxic agents in some cells [79]. Hence, before going to clinical trials, preclinical tests should be undertaken to ascertain if metformin enhances the inhibitory effect of other drugs. This is feasible

**NCT number Status Participants Period Intervention Cancer**

October 1, 2013 to August 1, 2024

Drug: metformin &

placebo

**type**

Prostate cancer

Responses of cancer cells with and without LKB1 to metformin are different. Metformin exerts cytostatic effect on cancer cells with wild-type LKB1, while it causes cytotoxicity in cells lacking LKB1. If metformin is used together with most of chemotherapeutic drugs that are cytotoxic in cancer containing wild-type LKB1, the cooperative effects might not be achieved. The reason is that more rapidly dividing cells are more sensitive to cytotoxic drugs, while cytostatic drugs slow down speed of cell growth, which might compromise the efficacy of cytotoxic chemotherapy. In this scenario, it might be a good idea to take metformin and cytotoxic drug alternately. For example, patients take a couple of cycles of cytotoxic chemotherapy and then have rest for period of time during which metformin is alternately used. The purpose is to restrain cancer in dormancy and allow the patients to restore healthy condition. In addition, Birsoy et al. have delineated that the most metforminsensitive cells contain mutations of genes responsible for upregulation of mitochondrial oxidative phosphorylation, for example, complex I components, or glucose utilization [80]. Thus, these genes may serve as biomarkers for metformin use. Altogether, these studies point to importance of personalized medicine to deter-

Cancer stem cells (CSCs) are refractory to chemotherapy, leading to the relapse

of cancer. These cells metastasize to distant organs after flowing in circulation, resulting in poor prognosis. Thus, CSCs have become an important target for anticancer therapies. Hirsch et al. have reported that the CSCs derived from BC are preferentially sensitive to metformin that is used from 10 to 100 times less dosage

when PDX animal models or organoid culture techniques are available.

Metformin is a cheap and nontoxic first-line antidiabetic medicine. It is an attractive drug that is being repurposed for multiple usages in treatment of other diseases in addition to diabetes. Metformin implements its function through AMPKdependent and independent mechanisms. Preclinical and retrospective clinical investigations have inspired clinical trials of metformin use in various cancer therapies. It is a promising drug in neoadjuvant and adjuvant therapies. We hope these trials will come to end with positive or negative results in the next few years. In considering genetic heterogeneity of cancer, responses of different cancer types and subpopulations in the same cancer might be different. Therefore, we still have long way to go and loads of questions to be addressed.

### **Acknowledgements**

ZL is supported by the National Natural Science Foundation of China (81572753, 31660332) and the Innovation and Entrepreneurship grant from Jiangxi Provincial Bureau of Foreign Experts, China.

### **Conflict of interest**

The authors declare no conflict of interest.

*Metformin*

### **Author details**

Yile Jiao†, Xiaochen Wang† and Zhijun Luo\* Queen Mary School, Nanchang University, Nanchang, China

\*Address all correspondence to: zluo559914@ncu.edu.cn

† Equal contribution to this work.

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**References**

[1] Rena G, Hardie DG, Pearson ER. The mechanisms of action of metformin. Diabetologia. 2017;**60**(9):1577-1585. Available from: http://www.ncbi.nlm.

*DOI: http://dx.doi.org/10.5772/intechopen.91291*

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer*

from: https://linkinghub.elsevier.com/re

[8] Zhang C-S, Hawley SA, Zong Y, Li M, Wang Z, Gray A, et al. Fructose-1,6 bisphosphate and aldolase mediate glucose sensing by AMPK. Nature. 2017; **548**(7665):112-116. Available from: http:// www.nature.com/articles/nature23275

[9] Zhang C-S, Li M, Ma T, Zong Y, Cui J, Feng J-W, et al. Metformin activates AMPK through the lysosomal pathway. Cell Metabolism. 2016;**24**(4): 521-522. Available from: https://linkingh

[10] Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: From mechanisms of action to therapies. Cell Metabolism. 2014;**20**(6):953-966. Available from: https://linkinghub.else vier.com/retrieve/pii/S155041311400

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CD-12-0263

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[12] Pollak MN. Investigating metformin for cancer prevention and treatment: The end of the beginning. Cancer Discovery. 2012;**2**(9):778-790. Available from: http://cancerdiscovery.aacrjourna ls.org/lookup/doi/10.1158/2159-8290.

[13] Jeon S-M, Chandel NS, Hay N. AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature. 2012;**485**(7400): 661-665. Available from: http://www.na

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nih.gov/pubmed/28776086

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om/journals/jdr/2012/716404/

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ub.elsevier.com/retrieve/pii/

metabolic disease. Nutrition & Metabolism. 2010;**7**(1):7. Available from: http://nutritionandmetabolism.b iomedcentral.com/articles/10.1186/

[6] Kasznicki J, Sliwinska A, Drzewoski J. Metformin in cancer prevention and therapy. Annals of Translational Medicine. 2014;**2**(6):1-11

[7] Ikhlas S, Ahmad M. Metformin: Insights into its anticancer potential with special reference to AMPK dependent and independent pathways. Life Sciences. 2017;**185**:53-62. Available

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management of NAFLD. Experimental Diabetes Research. 2012;**2012**:1-13. Available from: http://www.hindawi.c

Bhadada SV, Panchal SJ. Investigation of the potential effects of metformin on atherothrombotic risk factors in hyperlipidemic rats. European Journal of Pharmacology. 2011;**659**(2–3): 213-223. Available from: https://linkingh

[5] Seyfried TN, Shelton LM. Cancer as a

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer DOI: http://dx.doi.org/10.5772/intechopen.91291*

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[2] Zhou M, Xia L, Wang J. Metformin transport by a newly cloned protonstimulated organic cation transporter (plasma membrane monoamine transporter) expressed in human intestine. Drug Metabolism & Disposition. 2007;**35**(10):1956-1962. Available from: http://www.ncbi.nlm. nih.gov/pubmed/27011019

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[6] Kasznicki J, Sliwinska A, Drzewoski J. Metformin in cancer prevention and therapy. Annals of Translational Medicine. 2014;**2**(6):1-11

[7] Ikhlas S, Ahmad M. Metformin: Insights into its anticancer potential with special reference to AMPK dependent and independent pathways. Life Sciences. 2017;**185**:53-62. Available from: https://linkinghub.elsevier.com/re trieve/pii/S0024320517303685

[8] Zhang C-S, Hawley SA, Zong Y, Li M, Wang Z, Gray A, et al. Fructose-1,6 bisphosphate and aldolase mediate glucose sensing by AMPK. Nature. 2017; **548**(7665):112-116. Available from: http:// www.nature.com/articles/nature23275

[9] Zhang C-S, Li M, Ma T, Zong Y, Cui J, Feng J-W, et al. Metformin activates AMPK through the lysosomal pathway. Cell Metabolism. 2016;**24**(4): 521-522. Available from: https://linkingh ub.elsevier.com/retrieve/pii/ S1550413116304818

[10] Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: From mechanisms of action to therapies. Cell Metabolism. 2014;**20**(6):953-966. Available from: https://linkinghub.else vier.com/retrieve/pii/S155041311400 4410

[11] Muaddi H, Chowdhury S, Vellanki R, Zamiara P, Koritzinsky M. Contributions of AMPK and p53 dependent signaling to radiation response in the presence of metformin. Radiotherapy and Oncology. 2013; **108**(3):446-450. Available from: https:// linkinghub.elsevier.com/retrieve/pii/ S0167814013002922

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**Author details**

*Metformin*

**140**

Yile Jiao†, Xiaochen Wang† and Zhijun Luo\*

† Equal contribution to this work.

provided the original work is properly cited.

Queen Mary School, Nanchang University, Nanchang, China

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: zluo559914@ncu.edu.cn

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10.1055/s-0029-1241797

10.1055/s-0029-1241870

301824

320-344.2004

**142**

2008-1081488

*Metformin*

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6410

821-830

i/doi/10.2337/dc09-1791

10-0817

*Metformin*

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Sorensen HT, Pedersen L, Lash TL. Metformin and incident breast cancer among diabetic women: A populationbased case-control study in Denmark. Cancer Epidemiology, Biomarkers & Prevention. 2011;**20**(1):101-111.

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2016;**33**(4):339-357

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05070-2

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[57] Miranda VC, Braghiroli MI, Faria LD, Bariani G, Alex A, Bezerra Neto JE, et al. Phase 2 trial of metformin combined with 5-fluorouracil in patients with refractory metastatic colorectal cancer. Clinical Colorectal Cancer. 2016; **15**(4):321-328.e1. Available from: https://linkinghub.elsevier.com/retrieve/ pii/S1533002816300597

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[62] Laskov I, Drudi L, Beauchamp M-C, Yasmeen A, Ferenczy A, Pollak M, et al. Anti-diabetic doses of metformin decrease proliferation markers in tumors of patients with endometrial

cancer. Gynecologic Oncology. 2014; **134**(3):607-614. Available from: https:// linkinghub.elsevier.com/retrieve/pii/ S0090825814010543

[63] Mitsuhashi A, Sato Y, Kiyokawa T, Koshizaka M, Hanaoka H, Shozu M. Phase II study of medroxyprogesterone acetate plus metformin as a fertilitysparing treatment for atypical endometrial hyperplasia and endometrial cancer. Annals of Oncology. 2016;**27**(2):262-266. Available from: https://academic.oup. com/annonc/article-lookup/doi/ 10.1093/annonc/mdv539

[64] Ushijima K, Yahata H, Yoshikawa H, Konishi I, Yasugi T, Saito T, et al. Multicenter phase II study of fertility-sparing treatment with medroxyprogesterone acetate for endometrial carcinoma and atypical hyperplasia in young women. Journal of Clinical Oncology. 2007;**25**(19): 2798-2803. Available from: http://asc opubs.org/doi/10.1200/ JCO.2006.08.8344

[65] Yates MS, Coletta AM, Zhang Q, Schmandt RE, Medepalli M, Nebgen D, et al. Prospective randomized biomarker study of metformin and lifestyle intervention for prevention in obese women at increased risk for endometrial cancer. Cancer Prevention Research. 2018;**11**(8):477-490. Available from: http://cancerpreventionresearch.aac rjournals.org/lookup/doi/10.1158/ 1940-6207.CAPR-17-0398

[66] Nevadunsky NS, Van Arsdale A, Strickler HD, Moadel A, Kaur G, Frimer M, et al. Metformin use and endometrial cancer survival. Gynecologic Oncology. 2014;**132**(1): 236-240. Available from: https:// linkinghub.elsevier.com/retrieve/pii/ S0090825813012754

[67] Ramos-Peñafiel C, Olarte-Carrillo I, Cerón-Maldonado R, Rozen-Fuller E,

Kassack-Ipiña JJ, Meléndez-Mier G, et al. Effect of metformin on the survival of patients with ALL who express high levels of the ABCB1 drug resistance gene. Journal of Translational Medicine. 2018;**16**(1):245. DOI: 10.1186/ s12967-018-1620-6

[73] Feng T, Sun X, Howard LE,

[74] Rothermundt C, Hayoz S, Templeton AJ, Winterhalder R,

trieve/pii/S0302283813014826

[75] Joshua AM, Zannella VE, Downes MR, Bowes B, Hersey K, Koritzinsky M, et al. A pilot 'window of opportunity' neoadjuvant study of metformin in localised prostate cancer. Prostate Cancer and Prostatic Diseases. 2014;**17**(3):252-258. DOI: 10.1038/

pcan.2014.20

1171-7

**147**

Strebel RT, Bärtschi D, et al. Metformin in chemotherapy-naive castrationresistant prostate cancer: A multicenter phase 2 trial (SAKK 08/09). European Urology. 2014;**66**(3):468-474. Available from: https://linkinghub.elsevier.com/re

[76] Nguyen MM, Martinez JA, Hsu CH, Sokoloff M, Krouse RS, Gibson BA, et al.

Bioactivity and prostate tissue distribution of metformin in a preprostatectomy prostate cancer cohort. European Journal of Cancer Prevention. 2018;**27**(6):557-562

[77] Rieken M, Kluth LA, Xylinas E, Fajkovic H, Becker A, Karakiewicz PI, et al. Association of diabetes mellitus and metformin use with biochemical recurrence in patients treated with radical prostatectomy for prostate cancer. World Journal of Urology. 2014; **32**(4):999-1005. Available from: http:// link.springer.com/10.1007/s00345-013-

[78] Spratt DE, Zhang C, Zumsteg ZS, Pei X, Zhang Z, Zelefsky MJ. Metformin

and prostate cancer: Reduced development of castration-resistant

Vidal AC, Gaines AR, Moreira DM, et al. Metformin use and risk of prostate cancer: Results from the REDUCE study. Cancer Prevention Research. 2015;**8**(11):1055-1060. Available from: http://cancerpreventionresearch.aac rjournals.org/cgi/doi/10.1158/ 1940-6207.CAPR-15-0141

*DOI: http://dx.doi.org/10.5772/intechopen.91291*

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer*

disease and prostate cancer mortality. European Urology. 2013;**63**(4):709-716. Available from: https://linkinghub.else vier.com/retrieve/pii/S03022838120

Metformin reduces cisplatin-mediated apoptotic death of cancer cells through AMPK-independent activation of Akt. European Journal of Pharmacology. 2011;**651**(1–3):41-50. Available from: https://linkinghub.elsevier.com/retrie

[80] Solano ME, Sander V, Wald MR, Motta AB. Dehydroepiandrosterone and metformin regulate proliferation of murine T lymphocytes. Clinical and Experimental Immunology. 2008; **153**(2):289-296. Available from: http:// doi.wiley.com/10.1111/j.1365-2249.

[79] Janjetovic K, Vucicevic L, Misirkic M, Vilimanovich U, Tovilovic G, Zogovic N, et al.

ve/pii/S001429991001126X

[81] Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission. Cancer Research. 2009; **69**(19):7507-7511. Available

from: http://cancerres.aacrjournals.org/ cgi/doi/10.1158/0008-5472.CAN-

[82] Zhu P, Davis M, Blackwelder AJ, Bachman N, Liu B, Edgerton S, et al. Metformin selectively targets tumorinitiating cells in ErbB2-overexpressing

breast cancer models. Cancer Prevention Research. 2014;**7**(2): 199-210. Available from: http://cance rpreventionresearch.aacrjournals.org/cg i/doi/10.1158/1940-6207.CAPR-13-0181

[83] Zhang Y, Guan M, Zheng Z, Zhang Q, Gao F, Xue Y. Effects of metformin on CD133+ colorectal cancer cells in diabetic patients. PLoS One. 2013;**8**(11):e81264. Available from:

2008.03696.x

09-2994

14728

[68] Skinner HD, McCurdy MR, Echeverria AE, Lin SH, Welsh JW, O'Reilly MS, et al. Metformin use and improved response to therapy in esophageal adenocarcinoma. Acta Oncologica. 2013;**52**(5):1002-1009

[69] Leamm S, Lagarde SM, van Oijen MGH, Gisbertz SS, Wilmink JW, Hulshof MCCM, et al. Metformin use during treatment of potentially curable esophageal cancer patients is not associated with better outcomes. Annals of Surgical Oncology. 2015;**22**(S3): 766-771. Available from: http://link. springer.com/10.1245/s10434-015- 4850-3

[70] Wright JL, Stanford JL. Metformin use and prostate cancer in Caucasian men: Results from a population-based case-control study. Cancer Causes & Control. 2009;**20**(9):1617-1622. Available from: http://link.springer. com/10.1007/s10552-009-9407-y

[71] Azoulay L, Dell'Aniello S, Gagnon B, Pollak M, Suissa S. Metformin and the incidence of prostate cancer in patients with type 2 diabetes. Cancer Epidemiology, Biomarkers & Prevention. 2011;**20**(2):337-344. Available from: http://cebp.aacrjournals. org/cgi/doi/10.1158/1055-9965.EPI-10-0940

[72] He XX, Tu SM, Lee MH, Yeung S-CJ. Thiazolidinediones and metformin associated with improved survival of diabetic prostate cancer patients. Annals of Oncology. 2011;**22**(12):2640-2645. Available from: https://academic.oup.c om/annonc/article-lookup/doi/10.1093/ annonc/mdr020

*Preventive and (Neo)Adjuvant Therapeutic Effects of Metformin on Cancer DOI: http://dx.doi.org/10.5772/intechopen.91291*

[73] Feng T, Sun X, Howard LE, Vidal AC, Gaines AR, Moreira DM, et al. Metformin use and risk of prostate cancer: Results from the REDUCE study. Cancer Prevention Research. 2015;**8**(11):1055-1060. Available from: http://cancerpreventionresearch.aac rjournals.org/cgi/doi/10.1158/ 1940-6207.CAPR-15-0141

cancer. Gynecologic Oncology. 2014; **134**(3):607-614. Available from: https:// linkinghub.elsevier.com/retrieve/pii/

Kassack-Ipiña JJ, Meléndez-Mier G, et al. Effect of metformin on the survival of patients with ALL who express high levels of the ABCB1 drug resistance gene. Journal of Translational Medicine. 2018;**16**(1):245. DOI: 10.1186/

[68] Skinner HD, McCurdy MR, Echeverria AE, Lin SH, Welsh JW, O'Reilly MS, et al. Metformin use and improved response to therapy in esophageal adenocarcinoma. Acta Oncologica. 2013;**52**(5):1002-1009

[69] Leamm S, Lagarde SM, van Oijen MGH, Gisbertz SS, Wilmink JW, Hulshof MCCM, et al. Metformin use during treatment of potentially curable esophageal cancer patients is not

associated with better outcomes. Annals of Surgical Oncology. 2015;**22**(S3): 766-771. Available from: http://link. springer.com/10.1245/s10434-015-

[70] Wright JL, Stanford JL. Metformin use and prostate cancer in Caucasian men: Results from a population-based case-control study. Cancer Causes & Control. 2009;**20**(9):1617-1622. Available from: http://link.springer. com/10.1007/s10552-009-9407-y

[71] Azoulay L, Dell'Aniello S, Gagnon B, Pollak M, Suissa S. Metformin and the incidence of prostate cancer in patients

Available from: http://cebp.aacrjournals. org/cgi/doi/10.1158/1055-9965.EPI-

[72] He XX, Tu SM, Lee MH, Yeung S-CJ. Thiazolidinediones and metformin associated with improved survival of diabetic prostate cancer patients. Annals of Oncology. 2011;**22**(12):2640-2645. Available from: https://academic.oup.c om/annonc/article-lookup/doi/10.1093/

with type 2 diabetes. Cancer Epidemiology, Biomarkers & Prevention. 2011;**20**(2):337-344.

s12967-018-1620-6

4850-3

10-0940

annonc/mdr020

[63] Mitsuhashi A, Sato Y, Kiyokawa T, Koshizaka M, Hanaoka H, Shozu M. Phase II study of medroxyprogesterone acetate plus metformin as a fertilitysparing treatment for atypical endometrial hyperplasia and endometrial cancer. Annals of Oncology. 2016;**27**(2):262-266. Available from: https://academic.oup. com/annonc/article-lookup/doi/ 10.1093/annonc/mdv539

S0090825814010543

*Metformin*

[64] Ushijima K, Yahata H,

Yoshikawa H, Konishi I, Yasugi T, Saito T, et al. Multicenter phase II study of fertility-sparing treatment with medroxyprogesterone acetate for endometrial carcinoma and atypical hyperplasia in young women. Journal of

Clinical Oncology. 2007;**25**(19): 2798-2803. Available from: http://asc

[65] Yates MS, Coletta AM, Zhang Q, Schmandt RE, Medepalli M, Nebgen D, et al. Prospective randomized biomarker

study of metformin and lifestyle intervention for prevention in obese women at increased risk for endometrial cancer. Cancer Prevention Research. 2018;**11**(8):477-490. Available from: http://cancerpreventionresearch.aac rjournals.org/lookup/doi/10.1158/

1940-6207.CAPR-17-0398

S0090825813012754

**146**

[66] Nevadunsky NS, Van Arsdale A, Strickler HD, Moadel A, Kaur G, Frimer M, et al. Metformin use and endometrial cancer survival.

Gynecologic Oncology. 2014;**132**(1): 236-240. Available from: https:// linkinghub.elsevier.com/retrieve/pii/

[67] Ramos-Peñafiel C, Olarte-Carrillo I, Cerón-Maldonado R, Rozen-Fuller E,

opubs.org/doi/10.1200/ JCO.2006.08.8344

[74] Rothermundt C, Hayoz S, Templeton AJ, Winterhalder R, Strebel RT, Bärtschi D, et al. Metformin in chemotherapy-naive castrationresistant prostate cancer: A multicenter phase 2 trial (SAKK 08/09). European Urology. 2014;**66**(3):468-474. Available from: https://linkinghub.elsevier.com/re trieve/pii/S0302283813014826

[75] Joshua AM, Zannella VE, Downes MR, Bowes B, Hersey K, Koritzinsky M, et al. A pilot 'window of opportunity' neoadjuvant study of metformin in localised prostate cancer. Prostate Cancer and Prostatic Diseases. 2014;**17**(3):252-258. DOI: 10.1038/ pcan.2014.20

[76] Nguyen MM, Martinez JA, Hsu CH, Sokoloff M, Krouse RS, Gibson BA, et al. Bioactivity and prostate tissue distribution of metformin in a preprostatectomy prostate cancer cohort. European Journal of Cancer Prevention. 2018;**27**(6):557-562

[77] Rieken M, Kluth LA, Xylinas E, Fajkovic H, Becker A, Karakiewicz PI, et al. Association of diabetes mellitus and metformin use with biochemical recurrence in patients treated with radical prostatectomy for prostate cancer. World Journal of Urology. 2014; **32**(4):999-1005. Available from: http:// link.springer.com/10.1007/s00345-013- 1171-7

[78] Spratt DE, Zhang C, Zumsteg ZS, Pei X, Zhang Z, Zelefsky MJ. Metformin and prostate cancer: Reduced development of castration-resistant

disease and prostate cancer mortality. European Urology. 2013;**63**(4):709-716. Available from: https://linkinghub.else vier.com/retrieve/pii/S03022838120 14728

[79] Janjetovic K, Vucicevic L, Misirkic M, Vilimanovich U, Tovilovic G, Zogovic N, et al. Metformin reduces cisplatin-mediated apoptotic death of cancer cells through AMPK-independent activation of Akt. European Journal of Pharmacology. 2011;**651**(1–3):41-50. Available from: https://linkinghub.elsevier.com/retrie ve/pii/S001429991001126X

[80] Solano ME, Sander V, Wald MR, Motta AB. Dehydroepiandrosterone and metformin regulate proliferation of murine T lymphocytes. Clinical and Experimental Immunology. 2008; **153**(2):289-296. Available from: http:// doi.wiley.com/10.1111/j.1365-2249. 2008.03696.x

[81] Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission. Cancer Research. 2009; **69**(19):7507-7511. Available from: http://cancerres.aacrjournals.org/ cgi/doi/10.1158/0008-5472.CAN-09-2994

[82] Zhu P, Davis M, Blackwelder AJ, Bachman N, Liu B, Edgerton S, et al. Metformin selectively targets tumorinitiating cells in ErbB2-overexpressing breast cancer models. Cancer Prevention Research. 2014;**7**(2): 199-210. Available from: http://cance rpreventionresearch.aacrjournals.org/cg i/doi/10.1158/1940-6207.CAPR-13-0181

[83] Zhang Y, Guan M, Zheng Z, Zhang Q, Gao F, Xue Y. Effects of metformin on CD133+ colorectal cancer cells in diabetic patients. PLoS One. 2013;**8**(11):e81264. Available from:

https://dx.plos.org/10.1371/journal. pone.0081264

[84] Mohammed A, Janakiram NB, Brewer M, Ritchie RL, Marya A, Lightfoot S, et al. Antidiabetic drug metformin prevents progression of pancreatic cancer by targeting in part cancer stem cells and mTOR signaling. Translational Oncology. 2013;**6**(6):649- IN7. Available from: https://linkinghub. elsevier.com/retrieve/pii/S1936523313 800046

[85] Montales MTE, Simmen RCM, Ferreira ES, Neves VA, Simmen FA. Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells. Genes and Nutrition. 2015;**10**(6):49. Available from: http://link.springer. com/10.1007/s12263-015-0499-6

[86] Kuo SZ, Honda CO, Li WT, Honda TK, Kim E, Altuna X, et al. Metformin results in diametrically opposed effects by targeting non-stem cancer cells but protecting cancer stem cells in head and neck squamous cell carcinoma. International Journal of Molecular Sciences. 2019;**20**(1):193. Available from: https://www.mdpi.com/ 1422-0067/20/1/193

[87] Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H. Cell cycle regulation via p53 phosphorylation by a 5<sup>0</sup> -AMP activated protein kinase activator, 5-aminoimidazole-4 carboxamide-1-β–ribofuranoside, in a human hepatocellular carcinoma cell line. Biochemical and Biophysical Research Communications. 2001; **287**(2):562-567. Available from: https:// linkinghub.elsevier.com/retrieve/pii/ S0006291X0195627X

[88] Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, et al. AMPactivated protein kinase induces a p53 dependent metabolic checkpoint.

Molecular Cell. 2005;**18**(3):283-293. Available from: https://linkinghub.else vier.com/retrieve/pii/S1097276 505012207

[89] Zhou J, Huang W, Tao R, Ibaragi S, Lan F, Ido Y, et al. Inactivation of AMPK alters gene expression and promotes growth of prostate cancer cells. Oncogene. 2009;**28**(18): 1993-2002. Available from: http://www. nature.com/articles/onc200963

**149**

**1. Introduction**

malignant cells [3].

the body.

**Chapter 9**

**Abstract**

Metformin in Cervical Cancer:

The reprogrammed metabolism plays a crucial role in intensively proliferating tumor cells to meet high energetic demands and adapt to metastasis and invasion. Metformin may counteract flexible metabolic phenotype of cervical cancer cells by restraining aerobic glycolysis (*Warburg effect*) and promoting mitochondrial-based metabolism. Metformin inhibits master oncogene c-Myc as well as hypoxia-inducible factor 1 (HIF-1α) and suppresses its downstream glycolytic regulatory enzymes and glucose transporters. Metformin targets bioenergetics of cervical cancer cells with aggressive phenotype and regulates the expression of enzymes controlling tricarboxylic acid cycle (TCA cycle) supplementation with substrates, glucose, and glutamine. The exposition of cervical tumor cells to Metformin alleviates their migratory capacity, restrains epithelial-to-mesenchymal transition (EMT) program implementation, and elucidates oxidative stress, which results in massive cell death due to apoptosis. The metabolic alterations caused by Metformin are specific to cancer cells. In summary, Metformin exerts antitumor effect in cervical cancer cells by regulating specific molecular targets in reprogrammed metabolism. Metformin selectively modulates metabolic pathways and thus may be potentially used in new

Metabolic Reprogramming

*Malgorzata Tyszka-Czochara and Marcin Majka*

precisely targeted therapeutic strategies for cervical cancer.

epithelial-mesenchymal transition, targeted anticancer therapy

**Keywords:** Metformin, cancer, metabolism, metabolic reprogramming, *Warburg effect*, mitochondria, apoptosis, oncogenes, reactive oxygen species,

The malignant transformation results in a specific rearrangement of metabolic processes called metabolic reprogramming of tumor cell. The altered metabolism causes a selective advantage to a transformed cell by facilitating its survival in a harsh environment and promoting the spread of tumor cells within

Malignant cells very effectively adapt to high proliferation rate, metastasis, and invasion. Several molecular mechanisms were pointed out to drive such metabolic adaptation of cancer cells. The critical aspects of metabolic reprogramming in tumor cells substantially contribute to the *Warburg effect* [1], an increased catabolism of glucose to lactate in the presence of oxygen [2]. The altered metabolism of tumors results in elevated biosynthesis of macromolecules such as proteins, carbohydrates, and lipids and, in consequence, supports high proliferation rate of

### **Chapter 9**

https://dx.plos.org/10.1371/journal.

Molecular Cell. 2005;**18**(3):283-293. Available from: https://linkinghub.else

[89] Zhou J, Huang W, Tao R, Ibaragi S, Lan F, Ido Y, et al. Inactivation of AMPK alters gene expression and promotes growth of prostate cancer cells. Oncogene. 2009;**28**(18):

1993-2002. Available from: http://www.

nature.com/articles/onc200963

vier.com/retrieve/pii/S1097276

505012207

[84] Mohammed A, Janakiram NB, Brewer M, Ritchie RL, Marya A, Lightfoot S, et al. Antidiabetic drug metformin prevents progression of pancreatic cancer by targeting in part cancer stem cells and mTOR signaling. Translational Oncology. 2013;**6**(6):649- IN7. Available from: https://linkinghub. elsevier.com/retrieve/pii/S1936523313

[85] Montales MTE, Simmen RCM, Ferreira ES, Neves VA, Simmen FA. Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells. Genes and Nutrition. 2015;**10**(6):49. Available from: http://link.springer. com/10.1007/s12263-015-0499-6

[86] Kuo SZ, Honda CO, Li WT, Honda TK, Kim E, Altuna X, et al. Metformin results in diametrically opposed effects by targeting non-stem cancer cells but protecting cancer stem cells in head and neck squamous cell carcinoma. International Journal of Molecular Sciences. 2019;**20**(1):193. Available from: https://www.mdpi.com/

[87] Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H. Cell cycle regulation via p53 phosphorylation by


1422-0067/20/1/193

S0006291X0195627X

[88] Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, et al. AMPactivated protein kinase induces a p53 dependent metabolic checkpoint.

a 5<sup>0</sup>

**148**

pone.0081264

*Metformin*

800046

## Metformin in Cervical Cancer: Metabolic Reprogramming

*Malgorzata Tyszka-Czochara and Marcin Majka*

### **Abstract**

The reprogrammed metabolism plays a crucial role in intensively proliferating tumor cells to meet high energetic demands and adapt to metastasis and invasion. Metformin may counteract flexible metabolic phenotype of cervical cancer cells by restraining aerobic glycolysis (*Warburg effect*) and promoting mitochondrial-based metabolism. Metformin inhibits master oncogene c-Myc as well as hypoxia-inducible factor 1 (HIF-1α) and suppresses its downstream glycolytic regulatory enzymes and glucose transporters. Metformin targets bioenergetics of cervical cancer cells with aggressive phenotype and regulates the expression of enzymes controlling tricarboxylic acid cycle (TCA cycle) supplementation with substrates, glucose, and glutamine. The exposition of cervical tumor cells to Metformin alleviates their migratory capacity, restrains epithelial-to-mesenchymal transition (EMT) program implementation, and elucidates oxidative stress, which results in massive cell death due to apoptosis. The metabolic alterations caused by Metformin are specific to cancer cells. In summary, Metformin exerts antitumor effect in cervical cancer cells by regulating specific molecular targets in reprogrammed metabolism. Metformin selectively modulates metabolic pathways and thus may be potentially used in new precisely targeted therapeutic strategies for cervical cancer.

**Keywords:** Metformin, cancer, metabolism, metabolic reprogramming, *Warburg effect*, mitochondria, apoptosis, oncogenes, reactive oxygen species, epithelial-mesenchymal transition, targeted anticancer therapy

### **1. Introduction**

The malignant transformation results in a specific rearrangement of metabolic processes called metabolic reprogramming of tumor cell. The altered metabolism causes a selective advantage to a transformed cell by facilitating its survival in a harsh environment and promoting the spread of tumor cells within the body.

Malignant cells very effectively adapt to high proliferation rate, metastasis, and invasion. Several molecular mechanisms were pointed out to drive such metabolic adaptation of cancer cells. The critical aspects of metabolic reprogramming in tumor cells substantially contribute to the *Warburg effect* [1], an increased catabolism of glucose to lactate in the presence of oxygen [2]. The altered metabolism of tumors results in elevated biosynthesis of macromolecules such as proteins, carbohydrates, and lipids and, in consequence, supports high proliferation rate of malignant cells [3].

### *Metformin*

In particular, the regulation of mitochondrial processes in cancer cells differs from normal counterparts, and it may be specific to the stage of tumor [4]. Therefore, cancer cells are sensitive to drugs that disrupt energy homeostasis, such as Metformin (1,1-dimethylbiguanide, Met) [5].

A generic drug, Metformin, has been widely used for treatment of *diabetes mellitus* in humans. However, it exerts pleiotropic effect in human organism. In particular, a great interest has been paid to Met, since retrospective analyses demonstrated that it significantly decreased the relative risk of cancer incidence in diabetic patients when compared with patients treated with other drugs. Clinical trials confirmed the epidemiological observations that Met exerted anticancer effects in humans [6]. It has been established that Met inhibits proliferation of various neoplastic cell lines in vitro, including breast, prostatic, colon, gastric, and cervical cancers [7, 8]. Currently, there is an intense ongoing research focused on molecular mechanisms behind these effects, since the implications of Met action in tumor cell are not completely understood [9].

To date, several molecular mechanisms were reported to play critical role in anticancer activity of Met. In particular, it was established that Met may affect energy metabolism of cancer cells by inhibition of complex I of mitochondrial electron transport chain (ETC) in mitochondria, which results in adenosine-5′ triphosphate (ATP) depletion and remodeling of the network of biosynthetic processes within the cell [9]. Met may act as an anticancer drug through the activation of the main energy regulator within the cell, adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) [7], and inhibition of mechanistic target of rapamycin complex-1 (mTORC1) [10] in tumor cells. Some of the pharmacological effects of Met seem to be independent of its action on glycemia homeostasis. Several reports demonstrated that treatment of tumor cells with Met results in cell cycle perturbations and apoptosis [11, 12]. The intracellular targets affected by Met were comprehensively reviewed by Ikhlas and Ahmad [9] and Pierotti et al. [13].

Along with the advent of human papillomavirus (HPV) vaccines, the primary prevention of cervical cancer has become more successful, but cervical malignancy still remains the significant cause of cancer mortality in women worldwide. Currently, chemotherapy using cytostatic drugs (mainly cisplatin, cis-dichlorodiammineplatinum (II)) is still the primal regimen, despite low specificity and substantial toxicity in patients [14].

Aerobic glycolysis has been recognized as the most common metabolic feature of malignant cells. The alterations in metabolism of cancer cells combined with the overexpression of oncogenes (c-Myc) and transcription factors (hypoxiainducible factor 1a, HIF 1a) confer a great advantage to malignant cells to avoid apoptosis induced by reactive oxygen species (ROS). In this study we focused on the effects of Met on metabolism of metastatic cervical tumor cells. Based on recent data, we reported that Met inhibited glycolytic phenotype of aggressive cervical cancer cells by regulation of expression of oncogenes and their downstream proteins, which led to cellular death. Furthermore, Met regulated mitochondrial metabolism, especially via supplementation of tricarboxylic acid cycle (TCA cycle, Krebs cycle) with pyruvate and glutamine. Met, by targeting epithelial and mesenchymal markers of tumor cells, alleviated invasive properties of cervical cancer cells.

This review summarizes recent findings on Met and cervical cancer underscoring new implications of this drug in regulation of peculiar metabolism of tumor cells. We discuss new perspectives about targeting specific alterations in cervical tumor metabolic pathways using Met.

**151**

*Metformin in Cervical Cancer: Metabolic Reprogramming*

**2. Metformin regulates metabolism of metastatic cervical cancer** 

A growing evidence suggests that the screening for molecular targets for anticancer therapeutic treatments should take into account the existing differences in tumor cell phenotypes. Therefore, the metabolic effects exerted by Met were studied using SiHa cells (American Type Culture Collection, ATCC designation HTB-35) originating from aggressive cervical tumor, which acquired malignant characteristics [15]. The regulation of apoptosis pathways in HTB-35 (SiHa) cells highly reflects the specificity of cervical tumor in vivo [16]. HTB-35 cells, even unstimulated with cytokines, have mesenchymal-like characteristics, especially high vimentin expression, along with enhancement of cell scattering and ability to move [17]. Another cell line, C-4I cells (ATCC, designation CRL1594) with epithelial phenotype, was derived from primary in situ tumor [18]. HTB-34 cells (ATCC designation MS751) were isolated from metastatic site in lymph node [19]. HTB-35, C-4I and HTB-34 are human squamous cell cervical carcinoma lines and it is worth noting that squamous cell cancer is the most common cervical cancer and accounts for almost 80% of cervical carcinomas in patients [14]. HeLa human cervical cancer cells (ATCC designation CCL2), which have been extensively used in mechanistic studies, expressed epithelial traits and were derived from *adenocarcinoma* [8].

**2.1 Metformin hampers the expression of oncogenes controlling glycolytic phenotype of cervical cancer cells under hypoxic and normoxic conditions** 

The reliance on glucose supply is linked to the aggressiveness of malignant cells. Such reprogrammed metabolism makes migrating cancer cells more robust and independent of environmental conditions. The dysregulation of glucose metabolism is caused by alterations in functioning of several oncogenes. Malignant cells may gain metabolic plasticity by upregulation of only few oncogenes, such as c-Myc, p53, phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) [20]. Additionally, the activation of transcription factors, such as HIF-1α, makes malignant cells more resistant to hypoxia (decreased oxygen level in microenvironment), which is one of the main factors affecting tumor growth [20]. The activation of HIF-1α is one of the crucial processes that promote glycolysis to generate ATP along with the decrease of mitochondrial pathways' activity in aggressive tumors. What is more, the migrating tumor cells may avoid oxidative stress by relying on glucose catabolism. As a result, tumor cells have higher chance to survive detachment from extracellular matrix (ECM), whereas normal cells undergo programmed death due to anoikis in the absence of attachment to ECM [21]. Following detachment from primary tumor bed and transportation to plasma and lymph, malignant cells may spread within the body and form secondary tumors. Therefore, the reprogrammed metabolism plays a crucial role in facilitating tumor metastasis. We found that Met may regulate glycolysis in aggressive cervical cancer cells. The glycolytic phenotype of tumor cells is triggered mainly by a master regulator HIF-1α and its downstream proteins. Our study showed that Met alleviated the hypoxia-induced activation of HIF-1α, which was followed by decreased expression of HIF-1α downstream protein effectors in HTB-35 cells, as demonstrated in [22]. In particular, Met downregulated GLUT transporters (solute carrier family 2 member receptors, SLC2A), specifically GLUT1 and GLUT3. Additionally, Met inhibited the regulatory enzymes of the glycolytic pathway, hexokinase 2 (HK2), bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4),

*DOI: http://dx.doi.org/10.5772/intechopen.88930*

**cells in vitro study**

**and promotes apoptosis**

*Metformin*

In particular, the regulation of mitochondrial processes in cancer cells differs from normal counterparts, and it may be specific to the stage of tumor [4]. Therefore, cancer cells are sensitive to drugs that disrupt energy homeostasis, such

A generic drug, Metformin, has been widely used for treatment of *diabetes mellitus* in humans. However, it exerts pleiotropic effect in human organism. In particular, a great interest has been paid to Met, since retrospective analyses demonstrated that it significantly decreased the relative risk of cancer incidence in diabetic patients when compared with patients treated with other drugs. Clinical trials confirmed the epidemiological observations that Met exerted anticancer effects in humans [6]. It has been established that Met inhibits proliferation of various neoplastic cell lines in vitro, including breast, prostatic, colon, gastric, and cervical cancers [7, 8]. Currently, there is an intense ongoing research focused on molecular mechanisms behind these effects, since the implications of Met action in

To date, several molecular mechanisms were reported to play critical role in anticancer activity of Met. In particular, it was established that Met may affect energy metabolism of cancer cells by inhibition of complex I of mitochondrial electron transport chain (ETC) in mitochondria, which results in adenosine-5′ triphosphate (ATP) depletion and remodeling of the network of biosynthetic processes within the cell [9]. Met may act as an anticancer drug through the activation of the main energy regulator within the cell, adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) [7], and inhibition of mechanistic target of rapamycin complex-1 (mTORC1) [10] in tumor cells. Some of the pharmacological effects of Met seem to be independent of its action on glycemia homeostasis. Several reports demonstrated that treatment of tumor cells with Met results in cell cycle perturbations and apoptosis [11, 12]. The intracellular targets affected by Met were comprehensively reviewed by Ikhlas and Ahmad [9] and

Along with the advent of human papillomavirus (HPV) vaccines, the primary prevention of cervical cancer has become more successful, but cervical malignancy still remains the significant cause of cancer mortality in women worldwide. Currently, chemotherapy using cytostatic drugs (mainly cisplatin, cis-dichlorodiammineplatinum (II)) is still the primal regimen, despite low specificity and

Aerobic glycolysis has been recognized as the most common metabolic feature of malignant cells. The alterations in metabolism of cancer cells combined with the overexpression of oncogenes (c-Myc) and transcription factors (hypoxiainducible factor 1a, HIF 1a) confer a great advantage to malignant cells to avoid apoptosis induced by reactive oxygen species (ROS). In this study we focused on the effects of Met on metabolism of metastatic cervical tumor cells. Based on recent data, we reported that Met inhibited glycolytic phenotype of aggressive cervical cancer cells by regulation of expression of oncogenes and their downstream proteins, which led to cellular death. Furthermore, Met regulated mitochondrial metabolism, especially via supplementation of tricarboxylic acid cycle (TCA cycle, Krebs cycle) with pyruvate and glutamine. Met, by targeting epithelial and mesenchymal markers of tumor cells, alleviated invasive properties

This review summarizes recent findings on Met and cervical cancer underscoring new implications of this drug in regulation of peculiar metabolism of tumor cells. We discuss new perspectives about targeting specific alterations in cervical

as Metformin (1,1-dimethylbiguanide, Met) [5].

tumor cell are not completely understood [9].

Pierotti et al. [13].

substantial toxicity in patients [14].

of cervical cancer cells.

tumor metabolic pathways using Met.

**150**

### **2. Metformin regulates metabolism of metastatic cervical cancer cells in vitro study**

A growing evidence suggests that the screening for molecular targets for anticancer therapeutic treatments should take into account the existing differences in tumor cell phenotypes. Therefore, the metabolic effects exerted by Met were studied using SiHa cells (American Type Culture Collection, ATCC designation HTB-35) originating from aggressive cervical tumor, which acquired malignant characteristics [15]. The regulation of apoptosis pathways in HTB-35 (SiHa) cells highly reflects the specificity of cervical tumor in vivo [16]. HTB-35 cells, even unstimulated with cytokines, have mesenchymal-like characteristics, especially high vimentin expression, along with enhancement of cell scattering and ability to move [17]. Another cell line, C-4I cells (ATCC, designation CRL1594) with epithelial phenotype, was derived from primary in situ tumor [18]. HTB-34 cells (ATCC designation MS751) were isolated from metastatic site in lymph node [19]. HTB-35, C-4I and HTB-34 are human squamous cell cervical carcinoma lines and it is worth noting that squamous cell cancer is the most common cervical cancer and accounts for almost 80% of cervical carcinomas in patients [14]. HeLa human cervical cancer cells (ATCC designation CCL2), which have been extensively used in mechanistic studies, expressed epithelial traits and were derived from *adenocarcinoma* [8].

### **2.1 Metformin hampers the expression of oncogenes controlling glycolytic phenotype of cervical cancer cells under hypoxic and normoxic conditions and promotes apoptosis**

The reliance on glucose supply is linked to the aggressiveness of malignant cells. Such reprogrammed metabolism makes migrating cancer cells more robust and independent of environmental conditions. The dysregulation of glucose metabolism is caused by alterations in functioning of several oncogenes. Malignant cells may gain metabolic plasticity by upregulation of only few oncogenes, such as c-Myc, p53, phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) [20]. Additionally, the activation of transcription factors, such as HIF-1α, makes malignant cells more resistant to hypoxia (decreased oxygen level in microenvironment), which is one of the main factors affecting tumor growth [20]. The activation of HIF-1α is one of the crucial processes that promote glycolysis to generate ATP along with the decrease of mitochondrial pathways' activity in aggressive tumors. What is more, the migrating tumor cells may avoid oxidative stress by relying on glucose catabolism. As a result, tumor cells have higher chance to survive detachment from extracellular matrix (ECM), whereas normal cells undergo programmed death due to anoikis in the absence of attachment to ECM [21]. Following detachment from primary tumor bed and transportation to plasma and lymph, malignant cells may spread within the body and form secondary tumors. Therefore, the reprogrammed metabolism plays a crucial role in facilitating tumor metastasis.

We found that Met may regulate glycolysis in aggressive cervical cancer cells. The glycolytic phenotype of tumor cells is triggered mainly by a master regulator HIF-1α and its downstream proteins. Our study showed that Met alleviated the hypoxia-induced activation of HIF-1α, which was followed by decreased expression of HIF-1α downstream protein effectors in HTB-35 cells, as demonstrated in [22]. In particular, Met downregulated GLUT transporters (solute carrier family 2 member receptors, SLC2A), specifically GLUT1 and GLUT3. Additionally, Met inhibited the regulatory enzymes of the glycolytic pathway, hexokinase 2 (HK2), bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4),

pyruvate kinase (PKM), and lactate dehydrogenase (LDH) (**Figure 1**). Met exerted greater effect on regulatory proteins in HTB-35 cells exposed to decreased oxygen level in the air than normal conditions.

Recent studies have reported that overexpression of c-Myc oncogene plays a significant role in the formation of cervical cancer. The enhanced expression of c-Myc is also of particular relevance to promoting invasive phenotype of cancer cells. What is more, the upregulated c-Myc may collaborate with HIF to effectively induce glucose and glutamine consumption in tumor cells. As a result, mitochondrial oxidative phosphorylation decreases. In particular, the upregulated c-Myc enhances glutamine catabolism in tumor cells, since the oncogene controls glutaminase (GLS) expression [23]. As measured using qPCR analysis, Met decreased *c-MYC* transcript level in HTB-35 cells [22], which was in compliance with inhibition of GLS protein expression [11]. The treatment of cervical tumor cells with Met decreased mRNA level for another c-Myc downstream protein, *CCND1* (cyclin D1), which regulates cell cycle progression [22]. Zhang et al. [24] reported that Met caused a substantial decrease of cyclin D1 expression in bladder cancer cells. The overexpression of oncogene cyclin D1 is positively correlated with chemotherapeutic resistance and apoptosis avoidance in squamous cell cancers [23]. The inhibition of *CCND1* expression in aggressive cervical tumor cells resulted in enhanced apoptosis [22].

Met triggered another pro-apoptotic mechanism in cervical carcinoma cells via regulation of Bcl-2 (B-cell lymphoma 2) protein family members' expression [22]. Bcl-2 proteins are key players in the regulation of mitochondrial-dependent programmed cell death. The activation of BAX protein leads to disruption of mitochondrial membrane potential and apoptosis, whereas Bcl-2 acts as an apoptotic suppressor. The counterbalancing pro- and anti-apoptotic effectors of Bcl-2 protein family play a crucial role in the regulation of the mitochondrial apoptotic cascade within the cell and constitute another important apoptotic checkpoint [25]. However, the disturbance of BAX/Bcl-2 pathway may result in the resistance to apoptosis by inducing compensatory mechanisms, thereby influencing the efficacy of some therapeutic regimens [26]. The exposition of cervical tumor cells to Met

**Figure 1.** *Metformin inhibits glycolytic phenotype of cervical carcinoma cells (*↑*—activation,* Ⱶ*—inhibition) [11, 12, 21, 22].*

**153**

*Metformin in Cervical Cancer: Metabolic Reprogramming*

significantly upregulated *BAX* transcript. It was found that the expression of *BAX* under hypoxic conditions was greater than in normoxia [22]. Additionally, Met downregulated transcript for *BCL-2* in HTB-35 cells in both, normoxic and hypoxic

The study using cervical cancer cells with metastatic phenotype cells showed that the downregulation of oncogenes/downstream regulatory proteins, together with the upregulation of pro-apoptotic BAX/Bcl-2, elucidated mitochondrialdependent apoptosis in tumor cells. The obtained data suggest that Met was highly effective in facilitating cell death in cervical tumor cells [22], since it exerted its effect targeting independent events controlling mitochondrial apoptosis including the induction of ROS [11], the regulation of Bcl-2 protein family expression, and downregulation of cyclin D1. It should be emphasized that Met induced cell death solely in tumor cells, without causing detrimental effects to normal cells [11].

**2.2 Metformin regulates TCA cycle supplementation in cervical cancer cells via pyruvate dehydrogenase (PDH) complex and generates oxidative stress in** 

The reprogrammed metabolism of tumor cells not only meets high energetic demands but also provides intermediates for intensive proliferation. Therefore, glycolysis and mitochondrial oxidative phosphorylation may operate simultaneously in cancer cells. Many tumors may even switch between these pathways accordingly to the current requirements. Recent studies showed that most cancer cells have metabolically efficient mitochondria to provide intermediates for biosynthesis, generate reductive power (nicotinamide adenine dinucleotide phosphate, NADPH), and restore cofactor pool (e.g., nicotinamide adenine dinucleotide, NADH). In highly proliferating cancer cells, mitochondrial TCA cycle is active enough to sustain the biochemical reactions. Currently, the precise regulation of anabolic pathways and keeping their activities at adequate level is thought to play a key role in determination of "flexible" metabolic phenotype of cancer cells that enables their rapid division. Moreover, oxidative phosphorylation (OXPHOS) may represent a significant contribution to energy generation within malignant cell. On the other hand, inevitable products of OXPHOS are ROS and oxidative stress due to ROS

It was demonstrated that the process of detachment of migrating squamous cancer cells from extracellular matrix (ECM) results in reprogramed metabolism toward glycolysis, particularly by PDH complex inhibition and following suppression of glucose respiration in mitochondria. Such metabolic phenotype of tumor cell enables efficient production of energy without excessive ROS generation. On the other hand, the stimulation of PDH activity may lead to increased anoikis

We found that Met may precisely regulate PDH metabolic checkpoint in cervical tumor cells (**Figure 2**). Met had great potency to activate oxidative decarboxylation of pyruvate to acetyl-CoA in HTB-35 cells expressing invasive phenotype, and it occurred via activation of PDH complex [11]. PDH complex plays a determinant role in the overall glucose disposal within the cell, since it funnels mitochondrial TCA cycle instead of lactate formation in cytosol. PDH activity is precisely regulated via covalent modification by the action of specific enzyme pyruvate dehydrogenase kinase (PDK). Several PDK activators were found to expand potent antitumor effect, also in cervical tumor HeLa cells [29]. We showed in aggressive cervical cancer HTB-35 cells that Met suppressed both PDK activity and the expression of gene encoding tumor-specific isoenzyme PDK1 [22]. This finding may have practical implications, since the screening strategy for PDK inhibitors should

sensitivity and attenuation of metastatic potential of cancer cells [28].

*DOI: http://dx.doi.org/10.5772/intechopen.88930*

conditions.

**mitochondria**

overproduction may kill tumor cells [27].

*Metformin in Cervical Cancer: Metabolic Reprogramming DOI: http://dx.doi.org/10.5772/intechopen.88930*

*Metformin*

level in the air than normal conditions.

pyruvate kinase (PKM), and lactate dehydrogenase (LDH) (**Figure 1**). Met exerted greater effect on regulatory proteins in HTB-35 cells exposed to decreased oxygen

Recent studies have reported that overexpression of c-Myc oncogene plays a significant role in the formation of cervical cancer. The enhanced expression of c-Myc is also of particular relevance to promoting invasive phenotype of cancer cells. What is more, the upregulated c-Myc may collaborate with HIF to effectively induce glucose and glutamine consumption in tumor cells. As a result, mitochondrial oxidative phosphorylation decreases. In particular, the upregulated c-Myc enhances glutamine catabolism in tumor cells, since the oncogene controls glutaminase (GLS) expression [23]. As measured using qPCR analysis, Met decreased *c-MYC* transcript level in HTB-35 cells [22], which was in compliance with inhibition of GLS protein expression [11]. The treatment of cervical tumor cells with Met decreased mRNA level for another c-Myc downstream protein, *CCND1* (cyclin D1), which regulates cell cycle progression [22]. Zhang et al. [24] reported that Met caused a substantial decrease of cyclin D1 expression in bladder cancer cells. The overexpression of oncogene cyclin D1 is positively correlated with chemotherapeutic resistance and apoptosis avoidance in squamous cell cancers [23]. The inhibition of *CCND1* expression in aggressive cervical tumor cells resulted in enhanced apoptosis [22]. Met triggered another pro-apoptotic mechanism in cervical carcinoma cells via regulation of Bcl-2 (B-cell lymphoma 2) protein family members' expression [22]. Bcl-2 proteins are key players in the regulation of mitochondrial-dependent programmed cell death. The activation of BAX protein leads to disruption of mitochondrial membrane potential and apoptosis, whereas Bcl-2 acts as an apoptotic suppressor. The counterbalancing pro- and anti-apoptotic effectors of Bcl-2 protein family play a crucial role in the regulation of the mitochondrial apoptotic cascade within the cell and constitute another important apoptotic checkpoint [25]. However, the disturbance of BAX/Bcl-2 pathway may result in the resistance to apoptosis by inducing compensatory mechanisms, thereby influencing the efficacy of some therapeutic regimens [26]. The exposition of cervical tumor cells to Met

*Metformin inhibits glycolytic phenotype of cervical carcinoma cells (*↑*—activation,* Ⱶ*—inhibition) [11, 12, 21, 22].*

**152**

**Figure 1.**

significantly upregulated *BAX* transcript. It was found that the expression of *BAX* under hypoxic conditions was greater than in normoxia [22]. Additionally, Met downregulated transcript for *BCL-2* in HTB-35 cells in both, normoxic and hypoxic conditions.

The study using cervical cancer cells with metastatic phenotype cells showed that the downregulation of oncogenes/downstream regulatory proteins, together with the upregulation of pro-apoptotic BAX/Bcl-2, elucidated mitochondrialdependent apoptosis in tumor cells. The obtained data suggest that Met was highly effective in facilitating cell death in cervical tumor cells [22], since it exerted its effect targeting independent events controlling mitochondrial apoptosis including the induction of ROS [11], the regulation of Bcl-2 protein family expression, and downregulation of cyclin D1. It should be emphasized that Met induced cell death solely in tumor cells, without causing detrimental effects to normal cells [11].

### **2.2 Metformin regulates TCA cycle supplementation in cervical cancer cells via pyruvate dehydrogenase (PDH) complex and generates oxidative stress in mitochondria**

The reprogrammed metabolism of tumor cells not only meets high energetic demands but also provides intermediates for intensive proliferation. Therefore, glycolysis and mitochondrial oxidative phosphorylation may operate simultaneously in cancer cells. Many tumors may even switch between these pathways accordingly to the current requirements. Recent studies showed that most cancer cells have metabolically efficient mitochondria to provide intermediates for biosynthesis, generate reductive power (nicotinamide adenine dinucleotide phosphate, NADPH), and restore cofactor pool (e.g., nicotinamide adenine dinucleotide, NADH). In highly proliferating cancer cells, mitochondrial TCA cycle is active enough to sustain the biochemical reactions. Currently, the precise regulation of anabolic pathways and keeping their activities at adequate level is thought to play a key role in determination of "flexible" metabolic phenotype of cancer cells that enables their rapid division. Moreover, oxidative phosphorylation (OXPHOS) may represent a significant contribution to energy generation within malignant cell. On the other hand, inevitable products of OXPHOS are ROS and oxidative stress due to ROS overproduction may kill tumor cells [27].

It was demonstrated that the process of detachment of migrating squamous cancer cells from extracellular matrix (ECM) results in reprogramed metabolism toward glycolysis, particularly by PDH complex inhibition and following suppression of glucose respiration in mitochondria. Such metabolic phenotype of tumor cell enables efficient production of energy without excessive ROS generation. On the other hand, the stimulation of PDH activity may lead to increased anoikis sensitivity and attenuation of metastatic potential of cancer cells [28].

We found that Met may precisely regulate PDH metabolic checkpoint in cervical tumor cells (**Figure 2**). Met had great potency to activate oxidative decarboxylation of pyruvate to acetyl-CoA in HTB-35 cells expressing invasive phenotype, and it occurred via activation of PDH complex [11]. PDH complex plays a determinant role in the overall glucose disposal within the cell, since it funnels mitochondrial TCA cycle instead of lactate formation in cytosol. PDH activity is precisely regulated via covalent modification by the action of specific enzyme pyruvate dehydrogenase kinase (PDK). Several PDK activators were found to expand potent antitumor effect, also in cervical tumor HeLa cells [29]. We showed in aggressive cervical cancer HTB-35 cells that Met suppressed both PDK activity and the expression of gene encoding tumor-specific isoenzyme PDK1 [22]. This finding may have practical implications, since the screening strategy for PDK inhibitors should

### **Figure 2.**

*Metformin regulates mitochondrial metabolism of cervical carcinoma cells (*↑*—activation,* Ⱶ*—inhibition) [11, 13, 22, 27, 30].*

recognize the specificity among the PDK isoenzymes in order to avoid side effects in vivo [30]. Under hypoxic conditions inside tumors, the activation of HIF-1α decreases mitochondrial metabolism, which prevents the cell from oxidative stress and helps cancer cells avoid apoptosis [20, 23]. Our study showed that in aggressive cervical cancer cells Met counteracted these metabolic alterations by inhibiting PDK1, which is at the same time HIF-1α prime downstream effector. Furthermore, Met downregulated PDK1 gene expression also in normoxia [22].

In tumor cells that have functional mitochondria, the generation of oxidative stress may become an important therapeutic target [27, 30]. The imbalance of metabolic regulation and the resulting overproduction of ROS in mitochondrial ETC cause oxidative stress, which, at some point, becomes toxic to cancer cells, and that escalation of ROS elicits apoptosis-inducing factors and triggers death program through multiple mechanisms. In compliance, it has been newly reported that Met significantly increased ROS level, altered apoptosis-associated signaling, and induced cell death in human gastric adenocarcinoma cells [31] and human cervical cancer HeLa cells [32]. We found that in HTB-35 cervical cancer cells, Met caused excessive generation of mitochondrial ROS and elicited apoptosis [11, 22]. As shown in [22], the effect of Met was specific to tumor cells, and the formation of mitochondrial ROS was not affected in normal cells exposed to Met.

Met concomitantly targeted cytosolic glycolysis and mitochondrial pathways in HTB-35 cells, which increased apoptosis and suppressed survival of cervical tumor cells under normoxic and hypoxic conditions [22].

### **2.3 Met restrains glutamine entry into TCA cycle and inhibits cervical tumor cell proliferation**

Glutamine may provide precursors to feed TCA cycle under limited flux of pyruvate from cytosolic glycolysis within tumor cells. The facilitated use of glutamine is a significant metabolic adaptation of cancer cell, besides enhanced glucose catabolism, and it provides intermediates sufficient for intensive biosynthesis and

**155**

*Metformin in Cervical Cancer: Metabolic Reprogramming*

energy production [20]. Glutaminase (GLS) is a key regulator of glutamine entry to TCA [33], and the inhibition of the enzyme may suppress tumor cell growth [25]. As shown in [11], the exposition of cervical cancer cells with invasive phenotype to Met downregulated the expression of GLS, thereby protecting mitochondrial anabolism from additional carbon supply for synthesis of macromolecules. Additionally, the effect of Met on GLS expression was specific toward cervical cancer cells, and in normal cells drug did not change the expression of the

Glutamine entry to tumor cell not only improves carbon supply for macromolecules buildup, but it also replenishes the pool of cellular NADPH, since the conversion of malate to pyruvate catalyzed by malic enzyme 1 (ME1) is accompanied by

plays a significant role in the antioxidant protection of tumor cell by reducing glutathione molecule. Met downregulated expression of ME1 and alleviated generation of NADPH in cells, which, in conditions of limited supplementation of HTB-35 cells with glucose (suppressed expression of GLUTs), resulted in hampering of

Furthermore, Met treatment caused acute drop in ATP concentration in HTB-35 cells. This is in compliance with data obtained by Parker et al. [34] who demonstrated that non-small cell lung cancer (NSCLC) cells may be uniquely sensitized to metabolic stresses by the action of other biguanide, phenformin (1-(diaminomethylidene)-2-(2-phenylethyl)guanidine). The inhibition of ATP generation may block biosynthesis in cervical tumor cells which results in restrain-

**2.4 Alterations of fatty acid (FA) de novo synthesis in cervical tumor cells upon** 

The facilitated fatty acid (FA) de novo synthesis together with upregulated glycolysis was recognized as one of the prime metabolic alterations in such tumor cells [35]. The enhanced FA biosynthesis meets high demands of rapidly proliferating malignant cells (generating components for cell membranes and signaling molecules). We found that Met decreased unsaturated lipid content in aggressive cervical cancer cells (**Figure 2**). The mechanism of Met action included downregulation of regulatory enzyme elongase 6 (ELOVL6), which catalyzes elongation of fatty acid molecule. Met also suppressed stearoyl-CoA desaturase (SCD1), which controls desaturation of FA. It was shown by Fritz et al. [36] that pharmacologic inhibition of SCD1 activity impaired unsaturated FA synthesis, which resulted in decreased proliferation of both androgen-sensitive and androgen-resistant prostate cancer cells. The treatment of cervical cancer cell lines [22, 37] with Met decreased cervical tumor cell proliferation, but Met did not affect the growth of

**2.5 Metformin inhibits epithelial-to-mesenchymal transition (EMT) process** 

Emerging data indicate that the enhanced activity of enzymes regulating lipid de novo synthesis may contribute to activation of EMT process in tumor cells [36]. The activation of EMT program in epithelial cancer cells facilitates tumor progression, invasion, and metastasis. It has been shown in independent studies that Met inhibits EMT in various cancer cell lines [8, 37]. Recently, it has been reported that Met reversed EMT phenotype induced with *transforming growth factor beta 1* (TGF-β1) in breast, lung, and cervical cancer cells by targeting the mechanisms regulating the

biosynthesis and alleviation of ROS detoxification [11, 22].

**exposition to Metformin affect cell proliferation**

**and migration properties of cervical cancer cells**

(**Figure 2**). NADPH is used for biosynthesis, but it also

*DOI: http://dx.doi.org/10.5772/intechopen.88930*

enzyme [11].

the reduction of NADP+

ing of cell proliferation.

normal cells [11].

### *Metformin in Cervical Cancer: Metabolic Reprogramming DOI: http://dx.doi.org/10.5772/intechopen.88930*

*Metformin*

**Figure 2.**

*[11, 13, 22, 27, 30].*

recognize the specificity among the PDK isoenzymes in order to avoid side effects in vivo [30]. Under hypoxic conditions inside tumors, the activation of HIF-1α decreases mitochondrial metabolism, which prevents the cell from oxidative stress and helps cancer cells avoid apoptosis [20, 23]. Our study showed that in aggressive cervical cancer cells Met counteracted these metabolic alterations by inhibiting PDK1, which is at the same time HIF-1α prime downstream effector. Furthermore,

*Metformin regulates mitochondrial metabolism of cervical carcinoma cells (*↑*—activation,* Ⱶ*—inhibition)* 

In tumor cells that have functional mitochondria, the generation of oxidative stress may become an important therapeutic target [27, 30]. The imbalance of metabolic regulation and the resulting overproduction of ROS in mitochondrial ETC cause oxidative stress, which, at some point, becomes toxic to cancer cells, and that escalation of ROS elicits apoptosis-inducing factors and triggers death program through multiple mechanisms. In compliance, it has been newly reported that Met significantly increased ROS level, altered apoptosis-associated signaling, and induced cell death in human gastric adenocarcinoma cells [31] and human cervical cancer HeLa cells [32]. We found that in HTB-35 cervical cancer cells, Met caused excessive generation of mitochondrial ROS and elicited apoptosis [11, 22]. As shown in [22], the effect of Met was specific to tumor cells, and the formation of

Met concomitantly targeted cytosolic glycolysis and mitochondrial pathways in HTB-35 cells, which increased apoptosis and suppressed survival of cervical tumor

**2.3 Met restrains glutamine entry into TCA cycle and inhibits cervical tumor** 

Glutamine may provide precursors to feed TCA cycle under limited flux of pyruvate from cytosolic glycolysis within tumor cells. The facilitated use of glutamine is a significant metabolic adaptation of cancer cell, besides enhanced glucose catabolism, and it provides intermediates sufficient for intensive biosynthesis and

Met downregulated PDK1 gene expression also in normoxia [22].

mitochondrial ROS was not affected in normal cells exposed to Met.

cells under normoxic and hypoxic conditions [22].

**cell proliferation**

**154**

energy production [20]. Glutaminase (GLS) is a key regulator of glutamine entry to TCA [33], and the inhibition of the enzyme may suppress tumor cell growth [25].

As shown in [11], the exposition of cervical cancer cells with invasive phenotype to Met downregulated the expression of GLS, thereby protecting mitochondrial anabolism from additional carbon supply for synthesis of macromolecules. Additionally, the effect of Met on GLS expression was specific toward cervical cancer cells, and in normal cells drug did not change the expression of the enzyme [11].

Glutamine entry to tumor cell not only improves carbon supply for macromolecules buildup, but it also replenishes the pool of cellular NADPH, since the conversion of malate to pyruvate catalyzed by malic enzyme 1 (ME1) is accompanied by the reduction of NADP+ (**Figure 2**). NADPH is used for biosynthesis, but it also plays a significant role in the antioxidant protection of tumor cell by reducing glutathione molecule. Met downregulated expression of ME1 and alleviated generation of NADPH in cells, which, in conditions of limited supplementation of HTB-35 cells with glucose (suppressed expression of GLUTs), resulted in hampering of biosynthesis and alleviation of ROS detoxification [11, 22].

Furthermore, Met treatment caused acute drop in ATP concentration in HTB-35 cells. This is in compliance with data obtained by Parker et al. [34] who demonstrated that non-small cell lung cancer (NSCLC) cells may be uniquely sensitized to metabolic stresses by the action of other biguanide, phenformin (1-(diaminomethylidene)-2-(2-phenylethyl)guanidine). The inhibition of ATP generation may block biosynthesis in cervical tumor cells which results in restraining of cell proliferation.

### **2.4 Alterations of fatty acid (FA) de novo synthesis in cervical tumor cells upon exposition to Metformin affect cell proliferation**

The facilitated fatty acid (FA) de novo synthesis together with upregulated glycolysis was recognized as one of the prime metabolic alterations in such tumor cells [35]. The enhanced FA biosynthesis meets high demands of rapidly proliferating malignant cells (generating components for cell membranes and signaling molecules). We found that Met decreased unsaturated lipid content in aggressive cervical cancer cells (**Figure 2**). The mechanism of Met action included downregulation of regulatory enzyme elongase 6 (ELOVL6), which catalyzes elongation of fatty acid molecule. Met also suppressed stearoyl-CoA desaturase (SCD1), which controls desaturation of FA. It was shown by Fritz et al. [36] that pharmacologic inhibition of SCD1 activity impaired unsaturated FA synthesis, which resulted in decreased proliferation of both androgen-sensitive and androgen-resistant prostate cancer cells. The treatment of cervical cancer cell lines [22, 37] with Met decreased cervical tumor cell proliferation, but Met did not affect the growth of normal cells [11].

### **2.5 Metformin inhibits epithelial-to-mesenchymal transition (EMT) process and migration properties of cervical cancer cells**

Emerging data indicate that the enhanced activity of enzymes regulating lipid de novo synthesis may contribute to activation of EMT process in tumor cells [36]. The activation of EMT program in epithelial cancer cells facilitates tumor progression, invasion, and metastasis. It has been shown in independent studies that Met inhibits EMT in various cancer cell lines [8, 37]. Recently, it has been reported that Met reversed EMT phenotype induced with *transforming growth factor beta 1* (TGF-β1) in breast, lung, and cervical cancer cells by targeting the mechanisms regulating the

expression of E-cadherin. The exposition of tumor cells to Met resulted in suppression of their metastatic properties [8, 38].

In our study, EMT process was induced upon 48 h incubation of cervical cancer cells with 10 ng/mL of cytokine TGF-β1, as described in detail in [17]. HTB-35 cells, even unstimulated, expressed mesenchymal-like characteristics, and the incubation with TGF-β further enforced expression of mesenchymal marker, vimentin, along with enhancement of cell scattering and ability to move [17]. The study showed that Met was an effective suppressor of mesenchymal phenotype and, in particular, downregulated vimentin in HTB-35 cells (**Figure 3**). Recently, it was reported by Laskov et al. [39] that Met downregulated the expression of vimentin in endometrial cancers in vitro and in vivo in diabetic patients. The incubation of cervical cancer cell lines with Met reduced cells' ability to move, as shown using functional scratch test in C4-I and HTB-35 cells stimulated with TGF-β1 [17]. Mechanistic study revealed that Met inhibited the expression of transcription factors Snail-1, ZEB-1, and Twist-1. These mesenchymal markers facilitate EMT progress in cervical cancer cells.

Cheng and Hao [8] proposed another mechanism of Met action in cervical carcinoma cells via inhibition of mTOR/p70s6k signaling pathway and downregulation of glycolytic regulatory protein pyruvate kinase, isozyme M2 (PKM2), in HeLa cell line.

In order to clarify the molecular action of Met in cervical tumor cells with aggressive characteristics, the effect of the drug was tested in the hypoxic conditions. In cervical cancers, hypoxia and concomitant enhanced lactate formation result in acidification of microenvironment, which may promote the ability of metastatic cells to rapidly spread in tissue [41]. In such conditions, the activation of HIF1α induces its downstream protein carbonic anhydrase IX (CAIX). By regulation of tumor milieu pH, CAIX acts as a survival factor protecting malignant cells

### **Figure 3.**

*Metformin inhibits TGF-β1-induced EMT phenotype of cervical carcinoma cells (*↑*—activation,*  Ⱶ*—inhibition) [8, 17, 40].*

**157**

*Metformin in Cervical Cancer: Metabolic Reprogramming*

**3. In vivo findings related to the effect of Metformin**

against enhanced acidification of microenvironment. As a result, lactate damages adjacent normal cells and does not harm tumor cells [42]. Due to its relevant role in cell invasion, CAIX was proposed as a potential therapeutic target, also in cervical cancers [41, 42]. We showed that the exposition of HTB-35 cells to Met under hypoxia suppressed HIF-1α, which resulted in decreased transcription of *CAIX* gene, thereby alleviating invasive properties of cervical malignant cells [17].

Recently, numerous beneficial activities of Met were reported. Met was shown to improve cardiovascular outcomes in humans [43], and the ability of Met to extend life-span in mammals has attracted great attention [44]. Emerging data indicate that Met may be applied as adjuvant in therapies aiming at combating diseases with high mortality rate, also in cervical cancer [45]. The clinical benefits of the use of Met in gynecologic oncology in humans were reviewed by Irie et al. [46] and Imai et al. [47]. Met also reduced the incidence of endometrial tumors and improved survival of patients with diagnosed local or advanced endometrial cancer [48]. Several clinical trials showed the potential of Met to elicit apoptosis in the uterus

The potential pathological effects of Met have been well studied in long term in human population. One of the most undesirable effects in the context of peculiar metabolic alterations of cancer cell is the enhanced generation of lactic acid caused by biguanides. In fact, the application of phenformin (1-(diaminomethylidene)-2-(2-phenylethyl)guanidine) was associated with a much higher risk of lactic acidosis in patients, than Metformin. Therefore, the former drug was withdrawn from clinical use. Currently, the contraindication for the use of Met in patients is renal failure, since this group has greater risk of lactic acidosis. However, the concerns over lactic acidosis were shown to be largely unfounded, unless kidney disease was advanced. Yet, based on the recent data, Met can be safely used in patients with mild renal dysfunction, provided that

The exposition of aggressive cervical cancer cells to Met restrained the function

Met precisely regulated PDH and GLS metabolic checkpoints in cervical tumor cells. In particular, in tumor cells Met targeted supplementation of mitochondrial pathways in pyruvate by downregulation of PDK1 gene expression and decreasing PDK activity. As a result, Met effectively enhanced TCA cycle flux in normoxic and hypoxic conditions. The downregulation of GLS and ME1 resulted in decreased regeneration of NADPH, the factor essential both for biosynthesis and cell protection against oxidative stress. The metabolic alterations of mitochondrial pathways caused by Met caused excessive generation of ROS which led to apoptosis. In cervical cancer cells, Met additionally induced apoptosis via upregulation of proapoptotic BAX protein expression and by downregulation of cyclin D1, oncogene *c-MYC* downstream protein. Met exerted its pro-apoptotic effect both in normal and decreased oxygen availability. This aspect of Met action may be important

of HIF-1α master regulator and downregulated HIF-1α downstream glycolytic genes. Met also downregulated glycolytic phenotype of HTB-35 cells through inhibition of oncogene *c-MYC* expression, which resulted in impairment of metabolic

plasticity of cervical tumor cells, especially via downregulation of GLS.

*DOI: http://dx.doi.org/10.5772/intechopen.88930*

and prostate cancers in humans [49].

patients are monitored appropriately [43, 50].

**4. Conclusions**

*Metformin*

cal cancer cells.

cell line.

sion of their metastatic properties [8, 38].

expression of E-cadherin. The exposition of tumor cells to Met resulted in suppres-

Cheng and Hao [8] proposed another mechanism of Met action in cervical carcinoma cells via inhibition of mTOR/p70s6k signaling pathway and downregulation of glycolytic regulatory protein pyruvate kinase, isozyme M2 (PKM2), in HeLa

In order to clarify the molecular action of Met in cervical tumor cells with aggressive characteristics, the effect of the drug was tested in the hypoxic conditions. In cervical cancers, hypoxia and concomitant enhanced lactate formation result in acidification of microenvironment, which may promote the ability of metastatic cells to rapidly spread in tissue [41]. In such conditions, the activation of HIF1α induces its downstream protein carbonic anhydrase IX (CAIX). By regulation of tumor milieu pH, CAIX acts as a survival factor protecting malignant cells

*Metformin inhibits TGF-β1-induced EMT phenotype of cervical carcinoma cells (*↑*—activation,* 

In our study, EMT process was induced upon 48 h incubation of cervical cancer cells with 10 ng/mL of cytokine TGF-β1, as described in detail in [17]. HTB-35 cells, even unstimulated, expressed mesenchymal-like characteristics, and the incubation with TGF-β further enforced expression of mesenchymal marker, vimentin, along with enhancement of cell scattering and ability to move [17]. The study showed that Met was an effective suppressor of mesenchymal phenotype and, in particular, downregulated vimentin in HTB-35 cells (**Figure 3**). Recently, it was reported by Laskov et al. [39] that Met downregulated the expression of vimentin in endometrial cancers in vitro and in vivo in diabetic patients. The incubation of cervical cancer cell lines with Met reduced cells' ability to move, as shown using functional scratch test in C4-I and HTB-35 cells stimulated with TGF-β1 [17]. Mechanistic study revealed that Met inhibited the expression of transcription factors Snail-1, ZEB-1, and Twist-1. These mesenchymal markers facilitate EMT progress in cervi-

**156**

**Figure 3.**

Ⱶ*—inhibition) [8, 17, 40].*

against enhanced acidification of microenvironment. As a result, lactate damages adjacent normal cells and does not harm tumor cells [42]. Due to its relevant role in cell invasion, CAIX was proposed as a potential therapeutic target, also in cervical cancers [41, 42]. We showed that the exposition of HTB-35 cells to Met under hypoxia suppressed HIF-1α, which resulted in decreased transcription of *CAIX* gene, thereby alleviating invasive properties of cervical malignant cells [17].

### **3. In vivo findings related to the effect of Metformin**

Recently, numerous beneficial activities of Met were reported. Met was shown to improve cardiovascular outcomes in humans [43], and the ability of Met to extend life-span in mammals has attracted great attention [44]. Emerging data indicate that Met may be applied as adjuvant in therapies aiming at combating diseases with high mortality rate, also in cervical cancer [45]. The clinical benefits of the use of Met in gynecologic oncology in humans were reviewed by Irie et al. [46] and Imai et al. [47]. Met also reduced the incidence of endometrial tumors and improved survival of patients with diagnosed local or advanced endometrial cancer [48]. Several clinical trials showed the potential of Met to elicit apoptosis in the uterus and prostate cancers in humans [49].

The potential pathological effects of Met have been well studied in long term in human population. One of the most undesirable effects in the context of peculiar metabolic alterations of cancer cell is the enhanced generation of lactic acid caused by biguanides. In fact, the application of phenformin (1-(diaminomethylidene)-2-(2-phenylethyl)guanidine) was associated with a much higher risk of lactic acidosis in patients, than Metformin. Therefore, the former drug was withdrawn from clinical use. Currently, the contraindication for the use of Met in patients is renal failure, since this group has greater risk of lactic acidosis. However, the concerns over lactic acidosis were shown to be largely unfounded, unless kidney disease was advanced. Yet, based on the recent data, Met can be safely used in patients with mild renal dysfunction, provided that patients are monitored appropriately [43, 50].

### **4. Conclusions**

The exposition of aggressive cervical cancer cells to Met restrained the function of HIF-1α master regulator and downregulated HIF-1α downstream glycolytic genes. Met also downregulated glycolytic phenotype of HTB-35 cells through inhibition of oncogene *c-MYC* expression, which resulted in impairment of metabolic plasticity of cervical tumor cells, especially via downregulation of GLS.

Met precisely regulated PDH and GLS metabolic checkpoints in cervical tumor cells. In particular, in tumor cells Met targeted supplementation of mitochondrial pathways in pyruvate by downregulation of PDK1 gene expression and decreasing PDK activity. As a result, Met effectively enhanced TCA cycle flux in normoxic and hypoxic conditions. The downregulation of GLS and ME1 resulted in decreased regeneration of NADPH, the factor essential both for biosynthesis and cell protection against oxidative stress. The metabolic alterations of mitochondrial pathways caused by Met caused excessive generation of ROS which led to apoptosis. In cervical cancer cells, Met additionally induced apoptosis via upregulation of proapoptotic BAX protein expression and by downregulation of cyclin D1, oncogene *c-MYC* downstream protein. Met exerted its pro-apoptotic effect both in normal and decreased oxygen availability. This aspect of Met action may be important

when designing anticancer therapies targeting cells in hypoxic milieu inside solid tumors.

It is also important to highlight another cellular mechanism of Met action, namely, the suppression of EMT process in cervical tumor cells. EMT seems implicated into invasiveness and metastasis of cancer, and Met was able to inhibit EMT pathways. In cervical tumor cells stimulated with TGF-β1 as well as in unstimulated ones, Met decreased the expression of the main mesenchymal marker vimentin and reduced motility of cells. In addition, Met downregulated adaptive enzyme *CAIX* in tumor cells under hypoxia. CAIX promoted migration of malignant cells and acted as an important survival factor, and thus it has recently been proposed as therapeutic target in cervical cancers. Met might be considered as a potential factor targeting CAIX to hamper cervical tumor invasiveness.

These findings provide a new insight into regulation of glycolysis and mitochondrial pathways in cervical tumor cells using nontoxic and well-studied drug, Metformin, indicating the future prospect about utilization of this molecule in clinical oncological routine. The identification and targeting of specific alterations in tumor metabolic pathways may constitute a sole basis to design new precise therapeutic strategies in cervical malignancy. To date, very few innovative therapies against cervical malignancy are being tested in clinical trials; thus more specific and effective intervention is highly required.

The artworks were prepared using elements from Servier Medical Art.

### **Author details**

Malgorzata Tyszka-Czochara1 \* and Marcin Majka<sup>2</sup> \*

1 Department of Food Chemistry and Nutrition, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland

2 Department of Transplantation, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland

\*Address all correspondence to: malgorzata.tyszka-czochara@uj.edu.pl and mmajka@cm-uj.krakow.pl

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**159**

*Metformin in Cervical Cancer: Metabolic Reprogramming*

International Journal of Molecular Sciences. 2016;**17**:e2000. DOI: 10.3390/

[9] Ikhlas S, Metformin AM. Insights into its anticancer potential with special reference to AMPK dependent and independent pathways. Life Sciences.

[10] Barrière G, Tartary M, Rigaud M. Metformin: A rising star to fight the epithelial mesenchymal transition in oncology. Anti-Cancer Agents in Medicinal Chemistry. 2013;**13**:333-340

[11] Tyszka-Czochara M,

2017;**106**:260-272

[12] Sacco F, Calderone A,

10.1038/onc.2012.181

Cancer. 2017;**123**:2404-2412

Castagnoli L, Cesareni G. The cellautonomous mechanisms underlying the activity of metformin as an anticancer drug. British Journal of Cancer. 2016;**115**:1451-1456

[13] Pierotti MA, Berrino F, Gariboldi M, Melani C, Mogavero A, Negri T, et al. Targeting metabolism for cancer treatment and prevention: Metformin, an old drug with multi-faceted effects. Oncogene. 2013;**32**:1475-1487. DOI:

[14] Small W Jr, Baco MA, Bajaj A, et al. Cervical cancer: A global health crisis.

[15] Miekus K, Pawlowska M, Sekuła M, Drabik G, Madeja Z, Adamek D, et al. MET receptor is a potential therapeutic target in high grade cervical cancer. Oncotarget. 2015;**12**:10086-10101

Bukowska-Strakova K, Majka M. Metformin and caffeic acid regulate metabolic reprogramming in human cervical carcinoma SiHa/HTB-35 cells and augment anticancer activity of cisplatin via cell cycle regulation. Food and Chemical Toxicology.

ijms17122000

2017;**185**:53-62

*DOI: http://dx.doi.org/10.5772/intechopen.88930*

[2] Wilde L, Roche M, Domingo-Vidal M,

[1] Liberti MV, Locasale JW. The Warburg effect: How does it benefit cancer cells? Trends in Biochemical Sciences. 2016;**41**:211-218. DOI: 10.1016/j.tibs.2015.12.001

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[3] Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nature Reviews. Cancer. 2011;**11**:85-95

Carreño-Fuentes L, Gallardo-Pérez JC, Saavedra E, Quezada H, Vega A, et al. Oxidative phosphorylation is impaired by prolonged hypoxia in breast and possibly in cervix carcinoma. The International Journal of Biochemistry &

[4] Rodríguez-Enríquez S,

Cell Biology. 2010;**42**:1744-1751

2016;**5**:1-8

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[6] Kim HJ, Lee S, Chun KH, Jeon JY, Han SJ, Kim DJ, et al. Metformin reduces the risk of cancer in patients with type 2 diabetes: An analysis based on the Korean National Diabetes Program Cohort. Medicine (Baltimore).

[7] Lin SC, Hardie DGAMPK. Sensing glucose as well as cellular energy status. Cell Metabolism. 2018;**27**:299-313. DOI:

10.1016/j.cmet.2017.10.009

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*Metformin in Cervical Cancer: Metabolic Reprogramming DOI: http://dx.doi.org/10.5772/intechopen.88930*

### **References**

*Metformin*

tumors.

when designing anticancer therapies targeting cells in hypoxic milieu inside solid

It is also important to highlight another cellular mechanism of Met action, namely, the suppression of EMT process in cervical tumor cells. EMT seems implicated into invasiveness and metastasis of cancer, and Met was able to inhibit EMT pathways. In cervical tumor cells stimulated with TGF-β1 as well as in unstimulated ones, Met decreased the expression of the main mesenchymal marker vimentin and reduced motility of cells. In addition, Met downregulated adaptive enzyme *CAIX* in tumor cells under hypoxia. CAIX promoted migration of malignant cells and acted as an important survival factor, and thus it has recently been proposed as therapeutic target in cervical cancers. Met might be considered as a potential factor targeting

These findings provide a new insight into regulation of glycolysis and mitochondrial pathways in cervical tumor cells using nontoxic and well-studied drug, Metformin, indicating the future prospect about utilization of this molecule in clinical oncological routine. The identification and targeting of specific alterations in tumor metabolic pathways may constitute a sole basis to design new precise therapeutic strategies in cervical malignancy. To date, very few innovative therapies against cervical malignancy are being tested in clinical trials; thus more specific and

The artworks were prepared using elements from Servier Medical Art.

\* and Marcin Majka<sup>2</sup>

2 Department of Transplantation, Faculty of Medicine, Jagiellonian University

\*Address all correspondence to: malgorzata.tyszka-czochara@uj.edu.pl

1 Department of Food Chemistry and Nutrition, Faculty of Pharmacy, Jagiellonian

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*

CAIX to hamper cervical tumor invasiveness.

effective intervention is highly required.

**158**

**Author details**

Malgorzata Tyszka-Czochara1

Medical College, Krakow, Poland

and mmajka@cm-uj.krakow.pl

University Medical College, Krakow, Poland

provided the original work is properly cited.

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signaling pathway. European Review for Medical and Pharmacological Sciences. 2018;**22**:8104-8112

*Metformin*

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to inhibit the growth of pancreatic cancer in vitro and in vivo. Oncology

[25] Green DR, Galluzzi L, Kroemer G. Cell biology. Metabolic control of cell death. Science. 2014;**345**:1250256

[27] Zhao Y, Butler EB, Tan M. Targeting cellular metabolism to improve cancer therapeutics. Cell Death & Disease.

[28] Kamarajugadda L, Stemboroski Q, Cai NE, Simpson S, Nayak M, Tan JL. Glucose oxidation modulates anoikis and tumor metastasis. Molecular and Cellular Biology. 2012;**32**:1893-1907

[29] Choi YW, Lim IK. Sensitization of metformin-cytotoxicity by dichloroacetate via reprogramming glucose metabolism in cancer cells. Cancer Letters. 2014;**346**:300-308

[30] Luengo A, Gui DY, Vander

cells by activating AMPK and suppressing mTOR/AKT signaling. International Journal of Oncology. 2019;**54**;1271-1281. DOI: 10.3892/

[32] Tang ZY, Sheng MJ, Qi YX, Wang LY, He DY. Metformin enhances inhibitive effects of carboplatin on HeLa cell proliferation and increases sensitivity to carboplatin by activating mitochondrial associated apoptosis

chembiol.2017.08.028

ijo.2019.4704

Heiden MG. Targeting metabolism for cancer therapy. Cell Chemical Biology. 2017;**24**:1161-1180. DOI: 10.1016/j.

[31] Lu CC, Chiang JH, Tsai FJ, Hsu YM, Juan YN, Yang JS, et al. Metformin triggers the intrinsic apoptotic response in human AGS gastric adenocarcinoma

[26] Indran IR, Tufo G, Pervaiz S, Brenner C. Recent advances in apoptosis, mitochondria and drug resistance in cancer cells. Biochimica et Biophysica Acta. 1807;**2011**:735-745A

Letters. 2018;**15**:1811-1816

2013;**4**:e532

BMC Genomics. 2007;**10**:2-13

[17] Tyszka-Czochara M, Lasota M, Majka M. Caffeic acid and Metformin inhibit invasive phenotype induced by TGF-β1 in C-4I and HTB-35/SiHa human cervical squamous carcinoma cells by acting on different molecular targets. International Journal of Molecular Sciences. 2018;**19**:e266

[18] Auersperg N. Histogenetic behavior of tumors. I. Morphologic variation in vitro and in vivo of two related human carcinoma cell lines. Journal of the National Cancer Institute.

[19] Available from: https://www.

[20] Pavlova NN, Thompson CB. The emerging hallmarks of cancer metabolism. Cell Metabolism.

[21] Paoli P, Giannoni E, Chiarugi P. Anoikis molecular pathways and its role in cancer progression. Biochimica et Biophysica Acta. 1833;**2013**:3481-3498

[22] Tyszka-Czochara M, Bukowska-Strakova K, Kocemba-Pilarczyk KA, Majka M. Caffeic acid targets AMPK signaling and regulates tricarboxylic acid cycle anaplerosis while Metformin

downregulates HIF-1α-induced glycolytic enzymes in human cervical squamous cell carcinoma lines. Nutrients. 2018;**10**:pii: E841

[23] Dang CVA. Time for MYC: Metabolism and therapy. Cold Spring Harbor Symposia on Quantitative Biology. 2016;**81**:79-83. DOI: 10.1101/

[24] Zhang JW, Zhao F, Sun Q.

Metformin synergizes with rapamycin

sqb.2016.81.031153

1969;**43**:151-173

2016;**23**:27-47

lgcstandards-atcc.org

**160**

[33] Li Y, Erickson JW, Stalnecker CA, Katt WP, Huang Q, Cerione RA, et al. Mechanistic basis of glutaminase activation: A key enzyme that promotes glutamine metabolism in cancer cells. The Journal of Biological Chemistry. 2016;**291**:20900-20910

[34] Parker SJ, Svensson RU, Divakaruni AS, Lefebvre AE, Murphy AN, Shaw RJ, et al. LKB1 promotes metabolic flexibility in response to energy stress. Metabolic Engineering. 2017;**43**(Pt B):208-217

[35] Currie A, Schulze A, Zechner R, Walther TC, Farese R Jr. Cellular fatty acid metabolism and cancer. Cell Metabolism. 2013;**18**:153-161

[36] Fritz V, Benfodda Z, Rodier G, Henriquet C, Iborra F, Avancès C, et al. Abrogation of de novo lipogenesis by stearoyl-CoA desaturase 1 inhibition interferes with oncogenic signaling and blocks prostate cancer progression in mice. Molecular Cancer Therapeutics. 2010;**9**:1740-1754

[37] Tyszka-Czochara M, Konieczny P, Majka M. Caffeic acid expands antitumor effect of metformin in human metastatic cervical carcinoma HTB-34 cells: Implications of AMPK activation and impairment of fatty acids de novo biosynthesis. International Journal of Molecular Sciences. 2017;**18**:E462

[38] Koeck S, Amann A, Huber JM, Gamerith G, Hilbe W, Zwierzina H. The impact of Metformin and salinomycin on transforming growth factor β-induced epithelial-to-mesenchymal transition in non-small cell lung cancer cell lines. Oncology Letters. 2016;**11**:2946-2952

[39] Laskov I, Abou-Nader P, Amin O, Philip CA, Beauchamp MC, Yasmeen A, et al. Metformin increases E-cadherin in tumors of diabetic patients with endometrial cancer and suppresses epithelial-mesenchymal transition in endometrial cancer cell lines. International Journal of Gynecological Cancer. 2016;**26**:1213-1221

[40] Lee MY, Shen MR. Epithelialmesenchymal transition in cervical carcinoma. American Journal of Translational Research. 2012;**4**:1-13

[41] Svastova E, Pastorekova S. Carbonic anhydrase IX: A hypoxia-controlled "catalyst" of cell migration. Cell Adhesion & Migration. 2013;**7**:226-231

[42] Pastorek J, Pastorekova S. Hypoxiainduced carbonic anhydrase IX as a target for cancer therapy: From biology to clinical use. Seminars in Cancer Biology. 2015;**31**:52-64

[43] Lipska KJ, Flory JH, Hennessy S, Inzucchi SE. Citizen petition to the US Food and Drug Administration to change prescribing guidelines: The Metformin experience. Circulation. 2016;**134**:1405-1408

[44] Martin-Montalvo A, Mercken EM, Mitchell SJ, Palacios HH, Mote PL, Scheibye-Knudsen M, et al. Metformin improves healthspan and lifespan in mice. Nature Communications. 2013;**4**:2192

[45] Uehara T, Mitsuhashi A, Tsuruoka N, Shozu M. Metformin potentiates the anticancer effects of cisplatin under normoxic conditions in vitro. Oncology Reports. 2015;**33**:744- 750. DOI: 10.3892/or.2014.3611

[46] Irie H, Banno K, Yanokura M, Iida M, Adachi M, Nakamura K, et al. Metformin: A candidate for the treatment of gynecological tumors based on drug repositioning. Oncology Letters. 2016;**11**:1287-1293

[47] Imai A, Ichigo S, Matsunami K, Takagi H, Yasuda K. Clinical benefits of Metformin in gynecologic oncology. Oncology Letters. 2015;**10**:577-582

[48] Tang YL, Zhu LY, Li Y, Yu J, Wang J, Zeng XX, et al. Metformin use is associated with reduced incidence and improved survival of endometrial cancer: A meta-analysis. BioMed Research International. 2017;**2017**:5905384. DOI: 10.1155/2017/5905384

[49] Vancura A, Bu P, Bhagwat M, Zeng J, Vancurova I. Metformin as an anticancer agent. Trends in Pharmacological Sciences. 2018;**39**:867-878. DOI: 10.1016/j.tips.2018.07.006

[50] Imam TH. Changes in metformin use in chronic kidney disease. Clinical Kidney Journal. 2017;**10**:301-304

**163**

**Chapter 10**

**Abstract**

factors, AMPK

**1. Introduction**

patients [8].

components [14–19].

in Ovarian Cancer

with focus on epithelial ovarian cancer.

endometrial physiology in these patients [7].

Antitumoral Effects of Metformin

*Maritza P. Garrido, Margarita Vega and Carmen Romero*

In the last years, the antidiabetic drug metformin has received considerable attention in pursuing new drugs for anticancer treatments. Several reports have shown that metformin would have antitumor effects, not only attributable to its systemic effects but also due to direct effects on tumor cells. It has been proposed that metformin could be a suitable alternative for the treatment of gynecological cancers, such as ovarian cancer. This disease is characterized by high cell proliferation and angiogenesis potential, because ovarian cancer cells overexpress most oncogenic molecules including growth factors. The aim of the present chapter is to discuss the molecular mechanism by which metformin would affect tumor cells,

**Keywords:** metformin, ovarian cancer, cell proliferation, angiogenesis, growth

Metformin or 1,1-dimethylbiguanide is a derivate of isoamylene guanidine, a substance found in the plant *Galega officinalis* [1]. This drug is widely used in metabolic disorders as type 2 diabetes mellitus, metabolic syndrome, and gestational diabetes [2, 3]. Besides, metformin is used as a treatment for polycystic ovarian syndrome [4], which is characterized by the dysfunction of reproductive tissues such as the ovary and endometrium. In this context, metformin improves ovarian follicle dynamics and frequency of ovulation [5, 6], and it increases the expression of endometrial GLUT4 (insulin-regulated glucose transporter), which may improve

In the last decades, metformin has been studied in the context of cancer, especially after an initial report by Evans et al., performed with a Scottish database, who found that metformin intake reduces the risk of cancer in type 2 diabetic

Type 2 diabetes and obesity affect a significant percentage of the world population [9, 10] whose food habits and lifestyle have been changing in the last decades. Both obesity and type 2 diabetes are pathologies associated with increased incidence and poor prognosis of ovarian cancer by several authors [11–13]. These observations could be explained because obesity and type 2 diabetes are characterized by molecular changes that could encourage tumoral transformation and progression, such as hyperinsulinemia, hyperglycemia, dyslipidemia, increased insulin-like growth factors (IGF), adipose tissue factors, and inflammatory

### **Chapter 10**

*Metformin*

of Metformin in gynecologic oncology. Oncology Letters. 2015;**10**:577-582

[49] Vancura A, Bu P, Bhagwat M, Zeng J, Vancurova I. Metformin as an anticancer agent. Trends in Pharmacological Sciences. 2018;**39**:867-878. DOI: 10.1016/j.tips.2018.07.006

[50] Imam TH. Changes in metformin use in chronic kidney disease. Clinical Kidney Journal. 2017;**10**:301-304

[48] Tang YL, Zhu LY, Li Y, Yu J, Wang J, Zeng XX, et al. Metformin use is associated with reduced incidence and improved survival of endometrial cancer: A meta-analysis. BioMed Research International. 2017;**2017**:5905384. DOI: 10.1155/2017/5905384

**162**

## Antitumoral Effects of Metformin in Ovarian Cancer

*Maritza P. Garrido, Margarita Vega and Carmen Romero*

### **Abstract**

In the last years, the antidiabetic drug metformin has received considerable attention in pursuing new drugs for anticancer treatments. Several reports have shown that metformin would have antitumor effects, not only attributable to its systemic effects but also due to direct effects on tumor cells. It has been proposed that metformin could be a suitable alternative for the treatment of gynecological cancers, such as ovarian cancer. This disease is characterized by high cell proliferation and angiogenesis potential, because ovarian cancer cells overexpress most oncogenic molecules including growth factors. The aim of the present chapter is to discuss the molecular mechanism by which metformin would affect tumor cells, with focus on epithelial ovarian cancer.

**Keywords:** metformin, ovarian cancer, cell proliferation, angiogenesis, growth factors, AMPK

### **1. Introduction**

Metformin or 1,1-dimethylbiguanide is a derivate of isoamylene guanidine, a substance found in the plant *Galega officinalis* [1]. This drug is widely used in metabolic disorders as type 2 diabetes mellitus, metabolic syndrome, and gestational diabetes [2, 3]. Besides, metformin is used as a treatment for polycystic ovarian syndrome [4], which is characterized by the dysfunction of reproductive tissues such as the ovary and endometrium. In this context, metformin improves ovarian follicle dynamics and frequency of ovulation [5, 6], and it increases the expression of endometrial GLUT4 (insulin-regulated glucose transporter), which may improve endometrial physiology in these patients [7].

In the last decades, metformin has been studied in the context of cancer, especially after an initial report by Evans et al., performed with a Scottish database, who found that metformin intake reduces the risk of cancer in type 2 diabetic patients [8].

Type 2 diabetes and obesity affect a significant percentage of the world population [9, 10] whose food habits and lifestyle have been changing in the last decades. Both obesity and type 2 diabetes are pathologies associated with increased incidence and poor prognosis of ovarian cancer by several authors [11–13]. These observations could be explained because obesity and type 2 diabetes are characterized by molecular changes that could encourage tumoral transformation and progression, such as hyperinsulinemia, hyperglycemia, dyslipidemia, increased insulin-like growth factors (IGF), adipose tissue factors, and inflammatory components [14–19].

By its chemical nature, metformin gets into the cell through organic cation transporters (OCTs) and multidrug and toxin extrusion transporters [20]. Because metformin cannot be metabolized, almost its entirety is excreted by the kidneys; the plasmatic levels of this drug do not reflect its intracellular concentration, mainly by its high apparent volume of distribution and prolonged half-life [21, 22]. Therefore, metformin is accumulated in tissues, and its plasmatic concentration is probably lower than of organs that express OCT transporters. This observation supports most *in vitro* studies that use high concentrations of metformin to study its antitumoral properties. Importantly, these transporters are present in the ovary [23, 24], so ovarian cancer cells could be a target for metformin action.

### **2. Indirect antitumoral effects of metformin in cancer**

It is discussed that metformin could display direct and indirect antitumoral effects. The systemic effects of this drug include the decrease of blood glucose and insulin levels by action in its classical target organs: liver, muscle, and fat tissues. In humans, metformin decreases the hepatic gluconeogenesis and the release of glucose from hepatic reserves, which produces an increase in the peripheral uptake of glucose and its metabolism, decreasing patients' hyperglycemia and hyperinsulinemia [1, 2, 25]. These conditions (hyperglycemia and hyperinsulinemia) favor tumoral growth and are associated with cancer incidence, by two possible mechanisms: (1) high availability of glucose for cancer cells and (2) high levels of insulin, which could act in insulin-like growth factor (IGF) receptors [14–16]. IGF/ IGF receptors display an important role in the ovary, because 100% of the ovarian carcinomas express IGF receptors [26].

In fat tissue, metformin decreases the activity of lipogenic enzymes such as HMG-CoA reductase, acetyl-CoA carboxylase (ACC), and fatty acid synthase, decreasing the endogen production of cholesterol and the fatty acid synthesis [1, 27, 28]. This produces a decrease in the plasma levels of lipids in patients using metformin [29–32], which in addition to metformin-hypoglycemic properties, decreases the readiness of energy substrates of tumoral cells.

All these metformin-mediated changes impair survival and mitogenic signaling and decrease nutrient availability for ovarian cancer cells.

### **3. Effects of metformin in ovarian cancer**

### **3.1 Direct effects of metformin in ovarian cancer cells: role of AMPK**

Several studies have shown that metformin displays direct antitumoral effects. Most of these studies have been performed in ovarian cancer cell lines, where metformin impairs cell proliferation, migration, and angiogenesis potential and enhances the chemotherapy sensibility [33–36].

The direct antitumoral effects of metformin are commanded by metabolic changes in cancer cells. Because metformin is a drug with pleiotropic effects, several molecular targets at different levels of the tumoral cell have been described. One of the most studied targets for metformin is the adenosine monophosphate-activated protein kinase (AMPK), a key sensor of the energetic status of the cell [37], and it was described that metformin treatment can activate AMPK in *in vitro* and *in vivo* experiments of ovarian cancer models [33, 38]. The activation of AMPK occurs by increasing the AMP/ATP ratio [39] which exposes the activation loop of AMPK to be phosphorylated in the residue threonine 172 by serine/threonine kinases such as

**165**

cancer cells [65].

*Antitumoral Effects of Metformin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.88911*

this drug is more complex.

proliferation in these cells [44–46].

signaling in uterine serous carcinoma [52].

liver kinase B1 (LKB1) [40]. Activated AMPK phosphorylates several proteins; the phosphorylation can either activate or repress protein function at the cellular level [41, 42]. Despite that an important part of the studies indicates that the antitumoral effect of metformin could be AMPK-dependent; in the absence of AMPK, metformin preserves most of its antitumoral effects [43], indicating that the mechanism of

One of the characteristic hallmarks of cancer cells is an increased cell proliferation. To do so, ovarian cancer cells overexpress several growth factors and its receptors, which produce an enhanced cell signaling related with survival and

In ovarian cancer, growth factors can activate protein kinase B (AKT) and the extracellular signal-regulated kinase (ERK) signaling pathways, among others [47–49]. These signaling pathways are associated with an increase of cell proliferation in most kinds of cancer cells [50, 51]. Some studies have shown that metformin treatment decreases IGF-1 and insulin levels, in a mice model with ovarian cancer [51], and also metformin treatment blocks the pro-tumoral effects of the nerve growth factor (NGF) in epithelial ovarian cancer cells [35] or the insulin/IGF-I

The activation by growth factors of AKT and ERK signaling in ovarian cancer cells induces the activation of mechanistic target of rapamycin complex 1 (mTORC1), which controls protein translation and cell growth [53–55]. It is described that metformin-activated AMPK inhibits mTORC1 signaling in ovarian cancer cells [56, 57], which could impair its cell potential to proliferate and fend it in unfavorable conditions. Additionally, one key point in the antitumoral effect of metformin is that AMPK decreases the signaling pathways mediated by AKT and ERK in several types of cells, including cancer cells [38, 57, 58]. These signaling pathways are associated with the increase of most oncoproteins, for example, the transcription factor c-MYC and the inhibitory apoptotic protein survivin (BIRC5) [59–62]. c-MYC is a proto-oncogene that controls several genes related with cell growth and cell proliferation, and some reports show that metformin decreases c-MYC protein levels in ovarian cancer cell lines [63, 64]. In addition, metformin decreases the mRNA levels of survivin in metastatic ovarian

According to current evidences, c-MYC controls the transcription and cell cycle inhibitors [66]. In agreement with the metformin-depending decrease of c-MYC in ovarian cancer cells, metformin induces the degradation of cyclin D1 [33, 38], a protein required for progression from G1 to S phase of the cell cycle, and increases p21 expression (a negative regulator of cell cycle) [67]. These results are consistent with experiments performed in primary ovarian cancer cell cultures and ovarian cancer cell lines, which show that metformin induces cell cycle arrest in the G0/G1 phase and decreases the percentage of cells in S phase of the cellular cycle [35, 68, 69]. These findings highly suggest that metformin

Even more, several authors have shown that metformin can elicit cytostatic or cytotoxic effects in ovarian cancer cells. A key point for a better understanding of these differences is that metformin inhibits tumor cell proliferation in the presence of glucose (with a cytostatic effect) but induces apoptosis in low-glucose conditions [70]. For example, ovarian cancer cells are more sensitive to metformin at concentrations of 2.5 millimolar than in 25 millimolar of glucose (found in culture conditions). This is a consequence of reactive oxygen species accumulation, which

decreases the progression of the cell cycle in ovarian cancer cells.

**3.2 Antiproliferative mechanism of metformin in ovarian cancer cells**

*Metformin*

By its chemical nature, metformin gets into the cell through organic cation transporters (OCTs) and multidrug and toxin extrusion transporters [20]. Because metformin cannot be metabolized, almost its entirety is excreted by the kidneys; the plasmatic levels of this drug do not reflect its intracellular concentration, mainly by its high apparent volume of distribution and prolonged half-life [21, 22]. Therefore, metformin is accumulated in tissues, and its plasmatic concentration is probably lower than of organs that express OCT transporters. This observation supports most *in vitro* studies that use high concentrations of metformin to study its antitumoral properties. Importantly, these transporters are present in the ovary [23, 24],

It is discussed that metformin could display direct and indirect antitumoral effects. The systemic effects of this drug include the decrease of blood glucose and insulin levels by action in its classical target organs: liver, muscle, and fat tissues. In humans, metformin decreases the hepatic gluconeogenesis and the release of glucose from hepatic reserves, which produces an increase in the peripheral uptake of glucose and its metabolism, decreasing patients' hyperglycemia and hyperinsulinemia [1, 2, 25]. These conditions (hyperglycemia and hyperinsulinemia) favor tumoral growth and are associated with cancer incidence, by two possible mechanisms: (1) high availability of glucose for cancer cells and (2) high levels of insulin, which could act in insulin-like growth factor (IGF) receptors [14–16]. IGF/ IGF receptors display an important role in the ovary, because 100% of the ovarian

In fat tissue, metformin decreases the activity of lipogenic enzymes such as HMG-CoA reductase, acetyl-CoA carboxylase (ACC), and fatty acid synthase, decreasing the endogen production of cholesterol and the fatty acid synthesis [1, 27, 28]. This produces a decrease in the plasma levels of lipids in patients using metformin [29–32], which in addition to metformin-hypoglycemic properties, decreases

All these metformin-mediated changes impair survival and mitogenic signaling

Several studies have shown that metformin displays direct antitumoral effects.

so ovarian cancer cells could be a target for metformin action.

**2. Indirect antitumoral effects of metformin in cancer**

carcinomas express IGF receptors [26].

the readiness of energy substrates of tumoral cells.

**3. Effects of metformin in ovarian cancer**

enhances the chemotherapy sensibility [33–36].

and decrease nutrient availability for ovarian cancer cells.

**3.1 Direct effects of metformin in ovarian cancer cells: role of AMPK**

Most of these studies have been performed in ovarian cancer cell lines, where metformin impairs cell proliferation, migration, and angiogenesis potential and

The direct antitumoral effects of metformin are commanded by metabolic changes in cancer cells. Because metformin is a drug with pleiotropic effects, several molecular targets at different levels of the tumoral cell have been described. One of the most studied targets for metformin is the adenosine monophosphate-activated protein kinase (AMPK), a key sensor of the energetic status of the cell [37], and it was described that metformin treatment can activate AMPK in *in vitro* and *in vivo* experiments of ovarian cancer models [33, 38]. The activation of AMPK occurs by increasing the AMP/ATP ratio [39] which exposes the activation loop of AMPK to be phosphorylated in the residue threonine 172 by serine/threonine kinases such as

**164**

liver kinase B1 (LKB1) [40]. Activated AMPK phosphorylates several proteins; the phosphorylation can either activate or repress protein function at the cellular level [41, 42]. Despite that an important part of the studies indicates that the antitumoral effect of metformin could be AMPK-dependent; in the absence of AMPK, metformin preserves most of its antitumoral effects [43], indicating that the mechanism of this drug is more complex.

### **3.2 Antiproliferative mechanism of metformin in ovarian cancer cells**

One of the characteristic hallmarks of cancer cells is an increased cell proliferation. To do so, ovarian cancer cells overexpress several growth factors and its receptors, which produce an enhanced cell signaling related with survival and proliferation in these cells [44–46].

In ovarian cancer, growth factors can activate protein kinase B (AKT) and the extracellular signal-regulated kinase (ERK) signaling pathways, among others [47–49]. These signaling pathways are associated with an increase of cell proliferation in most kinds of cancer cells [50, 51]. Some studies have shown that metformin treatment decreases IGF-1 and insulin levels, in a mice model with ovarian cancer [51], and also metformin treatment blocks the pro-tumoral effects of the nerve growth factor (NGF) in epithelial ovarian cancer cells [35] or the insulin/IGF-I signaling in uterine serous carcinoma [52].

The activation by growth factors of AKT and ERK signaling in ovarian cancer cells induces the activation of mechanistic target of rapamycin complex 1 (mTORC1), which controls protein translation and cell growth [53–55]. It is described that metformin-activated AMPK inhibits mTORC1 signaling in ovarian cancer cells [56, 57], which could impair its cell potential to proliferate and fend it in unfavorable conditions. Additionally, one key point in the antitumoral effect of metformin is that AMPK decreases the signaling pathways mediated by AKT and ERK in several types of cells, including cancer cells [38, 57, 58]. These signaling pathways are associated with the increase of most oncoproteins, for example, the transcription factor c-MYC and the inhibitory apoptotic protein survivin (BIRC5) [59–62]. c-MYC is a proto-oncogene that controls several genes related with cell growth and cell proliferation, and some reports show that metformin decreases c-MYC protein levels in ovarian cancer cell lines [63, 64]. In addition, metformin decreases the mRNA levels of survivin in metastatic ovarian cancer cells [65].

According to current evidences, c-MYC controls the transcription and cell cycle inhibitors [66]. In agreement with the metformin-depending decrease of c-MYC in ovarian cancer cells, metformin induces the degradation of cyclin D1 [33, 38], a protein required for progression from G1 to S phase of the cell cycle, and increases p21 expression (a negative regulator of cell cycle) [67]. These results are consistent with experiments performed in primary ovarian cancer cell cultures and ovarian cancer cell lines, which show that metformin induces cell cycle arrest in the G0/G1 phase and decreases the percentage of cells in S phase of the cellular cycle [35, 68, 69]. These findings highly suggest that metformin decreases the progression of the cell cycle in ovarian cancer cells.

Even more, several authors have shown that metformin can elicit cytostatic or cytotoxic effects in ovarian cancer cells. A key point for a better understanding of these differences is that metformin inhibits tumor cell proliferation in the presence of glucose (with a cytostatic effect) but induces apoptosis in low-glucose conditions [70]. For example, ovarian cancer cells are more sensitive to metformin at concentrations of 2.5 millimolar than in 25 millimolar of glucose (found in culture conditions). This is a consequence of reactive oxygen species accumulation, which

increase cell apoptosis and endoplasmic reticulum stress and decrease of c-MYC protein levels [63, 70].

### **3.3 Effect of metformin in lipid metabolism of ovarian cancer cells**

For cell proliferation, the cancer cell has high requirements of substrates for synthesis of structural components and signaling. One target of AMPK is the sterol regulatory element-binding protein 1 (SREBP1), a lipogenic transcription factor [71], which increases cellular biosynthesis of fatty acids and cholesterol by transcription of the enzymes ACC, HMG-CoA reductase, and fatty acid synthase [72], not only in fat tissue but also in ovarian cancer cells [73]. Because ACC is involved in the taxol-mediated cytotoxic effect of ovarian cancer cells [74], besides the fact that the inhibition of ACC suppresses ovarian cancer cell growth *in vivo* and *in vitro* [75], it is possible to conclude that ACC inhibition could contribute to an important part of the antitumoral effects of metformin.

### **3.4 Anti-angiogenic activity of metformin in ovarian cancer**

Angiogenesis, defined as the generation of new blood vessels from preexisting ones [76], is an essential process to supply oxygen and nutrients to normal and tumoral ovarian cells. Unfortunately, this process is exacerbated in ovarian cancer cells, which overexpress some growth factors, such as vascular endothelial growth factor (VEGF) or NGF [77, 78] which promotes angiogenesis.

The relevance of metformin in the vascular context is recognized; however, its action depends on the cell type, metabolic status, and nutrient availability. For example, some pro-angiogenic properties have been attributed to metformin under hypoxia and hyperglycemia, similar characteristics to myocardial infarction in diabetic patients. In this context, metformin enhances endothelial cell survival, migration, and apoptosis inhibition [79, 80]; this strongly suggests that the use of metformin could be beneficial in the context of cardiovascular diseases in diabetic patients. On the other hand, metformin could have an opposite effect in endothelial cells under hypoglycemic conditions (as tumor endothelial cells), where metformin produces an inhibition of its cell proliferation and angiogenesis potential, as will be discussed later.

In the ovary, the correct formation and regression of blood vessels during each ovarian cycle is indispensable for proper follicular development, ovulation, and corpus luteum formation, so that angiogenesis displays a key role in ovarian homeostasis and pathogenesis [81]. In patients with polycystic ovary syndrome, an increased expression of VEGF is described, and it is hypothesized that part of the beneficial metformin-associated effects will be mediated by a decrease or normalization of its VEGF levels. For example, it is described that in a rat model with dehydroepiandrosterone-induced polycystic ovaries, metformin administration restores the ovarian-increased levels of VEGF and angiopoietin 1, both angiogenic factors [82]. In addition, women with polycystic ovarian syndrome who take metformin have decreased their levels of plasmatic endothelin 1 and plasminogen activator inhibitor-1 [83, 84], molecules that also promote angiogenesis.

The angioprotection is an antitumoral mechanism that has been explored in ovarian cancer. Considering that the most studied angiogenic factor is VEGF, a monoclonal antibody against VEGF called bevacizumab has been developed and was approved for the use in advanced stages of ovarian cancer [85, 86]. In ovarian cancer models, the main knowledge of anti-angiogenic characteristics of metformin comes from VEGF modulation. Several *in vitro* models have shown that metformin

**167**

**Figure 1.**

*(NGF 100 ng/ml). Magnification bar: 50 μm.*

*Antitumoral Effects of Metformin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.88911*

ovarian cancer treatment.

decreases both VEGF mRNA and protein levels in ovarian cancer cell lines and then, its angiogenic potential [33, 64]. In a mice model with ovarian cancer, metformin decreases VEGF levels in plasma and ascitic fluid, with a consistent decrease of the ovarian tumor growth [51]. Interestingly, metformin reduces the vascular density (showed by CD31 staining) of ovarian cancer xenografts in mice, and metformin-/ cisplatin-treated mice have significantly less vascular density than either metformin or cisplatin alone [33]. Because cisplatin/carboplatin and paclitaxel are drugs used in the first-line chemotherapy in ovarian cancer [87, 88], these results suggest that metformin could potentiate the anti-angiogenic effects of chemotherapy during

On the other hand, metformin treatment (in millimolar concentrations) displays direct effects in the endothelial cells, by reducing cell proliferation in human umbilical vein endothelial cells (HUVEC) and endothelial progenitor cells [89, 90]. Similar results were replicated by our group where metformin decreases cell proliferation of the endothelial cell line EA.hy926, in a dose-dependent manner [35], as well as, the endothelial cell differentiation (**Figure 1**). These results suggest that metformin affects in a direct manner the angiogenesis potential of endothelial cells.

**3.5 Posttranscriptional regulation by metformin in ovarian cancer cells**

23-b and miR-145 in the epithelial ovarian cells [96].

In the ovarian cell, posttranscriptional regulations control gene expression at RNA level [91]. The micro-RNAs (miRs) are short non-codificant RNAs that regulate the expression of approximately 60% of protein-coding genes of the human genome [92]. miRs bind to a messenger RNA target, producing its degradation or translational repression depending of complementary degree [93]. The machinery for expression, processing, and exportation of miRs depends on several proteins as RNAse III DICER and exportins [93]. It is described that DICER downregulation is an oncogenic event that enhances epithelial-mesenchymal transition (EMT) and metastatic dissemination in cancer cells [94]. An important antecedent is that metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC in breast cancer cells, increasing oncosuppressor miRs [95]. These mechanisms have not been investigated in ovarian cancer cells; nevertheless, preliminary results from our group show that metformin increases the oncosuppressor miRs

As already mentioned in point 3.3, the activation of AMPK by metformin produces an inhibitory phosphorylation of acetyl-CoA carboxylase, an enzyme that regulates lipid metabolism. Importantly, intermediaries of lipid metabolism participate in cell signaling and chromatin structure, modulating processes as cell histone acetylation that depends on cytosolic acetyl-CoA [97]. The decrease of the

*Effect of metformin on the differentiation of endothelial cells. Metformin reduces the multicellular junctions and polygonal structures of endothelial cells EA.hy926 in a matrigel assay (4 h). Upper insert: positive control*  *Metformin*

protein levels [63, 70].

increase cell apoptosis and endoplasmic reticulum stress and decrease of c-MYC

For cell proliferation, the cancer cell has high requirements of substrates for synthesis of structural components and signaling. One target of AMPK is the sterol regulatory element-binding protein 1 (SREBP1), a lipogenic transcription factor [71], which increases cellular biosynthesis of fatty acids and cholesterol by transcription of the enzymes ACC, HMG-CoA reductase, and fatty acid synthase [72], not only in fat tissue but also in ovarian cancer cells [73]. Because ACC is involved in the taxol-mediated cytotoxic effect of ovarian cancer cells [74], besides the fact that the inhibition of ACC suppresses ovarian cancer cell growth *in vivo* and *in vitro* [75], it is possible to conclude that ACC inhibition could contribute to an important

Angiogenesis, defined as the generation of new blood vessels from preexisting ones [76], is an essential process to supply oxygen and nutrients to normal and tumoral ovarian cells. Unfortunately, this process is exacerbated in ovarian cancer cells, which overexpress some growth factors, such as vascular endothelial growth

The relevance of metformin in the vascular context is recognized; however, its action depends on the cell type, metabolic status, and nutrient availability. For example, some pro-angiogenic properties have been attributed to metformin under hypoxia and hyperglycemia, similar characteristics to myocardial infarction in diabetic patients. In this context, metformin enhances endothelial cell survival, migration, and apoptosis inhibition [79, 80]; this strongly suggests that the use of metformin could be beneficial in the context of cardiovascular diseases in diabetic patients. On the other hand, metformin could have an opposite effect in endothelial cells under hypoglycemic conditions (as tumor endothelial cells), where metformin produces an inhibition of its cell proliferation and angiogenesis potential, as will be

In the ovary, the correct formation and regression of blood vessels during each ovarian cycle is indispensable for proper follicular development, ovulation, and corpus luteum formation, so that angiogenesis displays a key role in ovarian homeostasis and pathogenesis [81]. In patients with polycystic ovary syndrome, an increased expression of VEGF is described, and it is hypothesized that part of the beneficial metformin-associated effects will be mediated by a decrease or normalization of its VEGF levels. For example, it is described that in a rat model with dehydroepiandrosterone-induced polycystic ovaries, metformin administration restores the ovarian-increased levels of VEGF and angiopoietin 1, both angiogenic factors [82]. In addition, women with polycystic ovarian syndrome who take metformin have decreased their levels of plasmatic endothelin 1 and plasminogen

activator inhibitor-1 [83, 84], molecules that also promote angiogenesis.

The angioprotection is an antitumoral mechanism that has been explored in ovarian cancer. Considering that the most studied angiogenic factor is VEGF, a monoclonal antibody against VEGF called bevacizumab has been developed and was approved for the use in advanced stages of ovarian cancer [85, 86]. In ovarian cancer models, the main knowledge of anti-angiogenic characteristics of metformin comes from VEGF modulation. Several *in vitro* models have shown that metformin

**3.3 Effect of metformin in lipid metabolism of ovarian cancer cells**

part of the antitumoral effects of metformin.

**3.4 Anti-angiogenic activity of metformin in ovarian cancer**

factor (VEGF) or NGF [77, 78] which promotes angiogenesis.

**166**

discussed later.

decreases both VEGF mRNA and protein levels in ovarian cancer cell lines and then, its angiogenic potential [33, 64]. In a mice model with ovarian cancer, metformin decreases VEGF levels in plasma and ascitic fluid, with a consistent decrease of the ovarian tumor growth [51]. Interestingly, metformin reduces the vascular density (showed by CD31 staining) of ovarian cancer xenografts in mice, and metformin-/ cisplatin-treated mice have significantly less vascular density than either metformin or cisplatin alone [33]. Because cisplatin/carboplatin and paclitaxel are drugs used in the first-line chemotherapy in ovarian cancer [87, 88], these results suggest that metformin could potentiate the anti-angiogenic effects of chemotherapy during ovarian cancer treatment.

On the other hand, metformin treatment (in millimolar concentrations) displays direct effects in the endothelial cells, by reducing cell proliferation in human umbilical vein endothelial cells (HUVEC) and endothelial progenitor cells [89, 90]. Similar results were replicated by our group where metformin decreases cell proliferation of the endothelial cell line EA.hy926, in a dose-dependent manner [35], as well as, the endothelial cell differentiation (**Figure 1**). These results suggest that metformin affects in a direct manner the angiogenesis potential of endothelial cells.

### **3.5 Posttranscriptional regulation by metformin in ovarian cancer cells**

In the ovarian cell, posttranscriptional regulations control gene expression at RNA level [91]. The micro-RNAs (miRs) are short non-codificant RNAs that regulate the expression of approximately 60% of protein-coding genes of the human genome [92]. miRs bind to a messenger RNA target, producing its degradation or translational repression depending of complementary degree [93]. The machinery for expression, processing, and exportation of miRs depends on several proteins as RNAse III DICER and exportins [93]. It is described that DICER downregulation is an oncogenic event that enhances epithelial-mesenchymal transition (EMT) and metastatic dissemination in cancer cells [94]. An important antecedent is that metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC in breast cancer cells, increasing oncosuppressor miRs [95]. These mechanisms have not been investigated in ovarian cancer cells; nevertheless, preliminary results from our group show that metformin increases the oncosuppressor miRs 23-b and miR-145 in the epithelial ovarian cells [96].

As already mentioned in point 3.3, the activation of AMPK by metformin produces an inhibitory phosphorylation of acetyl-CoA carboxylase, an enzyme that regulates lipid metabolism. Importantly, intermediaries of lipid metabolism participate in cell signaling and chromatin structure, modulating processes as cell histone acetylation that depends on cytosolic acetyl-CoA [97]. The decrease of the

### **Figure 1.**

*Effect of metformin on the differentiation of endothelial cells. Metformin reduces the multicellular junctions and polygonal structures of endothelial cells EA.hy926 in a matrigel assay (4 h). Upper insert: positive control (NGF 100 ng/ml). Magnification bar: 50 μm.*

conversion of acetyl-CoA to malonyl-CoA leads to an increase in the acetylation of histones in the chromatin and altered gene expression in ovarian cancer cells [67]. Because acetylation of nucleosomal histones is linked to nuclear processes as transcription, replication, and repair among other functions [98], it is possible that several antitumoral effects of metformin could be regulated by protein acetylation and transcriptional regulation of several oncosuppressor proteins.

The summary of the main studied antitumoral effects of metformin is shown in **Figure 2**.

### **3.6 Studies of metformin in diabetic patients with ovarian cancer**

A recent meta-analysis shows that among available studies of relationship between metformin intake with ovarian cancer incidence and prognosis in diabetic patients, the majority of the studies indicate a negative correlation between the use of metformin and the incidence of ovarian cancer, as well as, a positive correlation with better prognosis [99]. The same study shows that metformin treatment in diabetic patients has a reduction of 24% risk of ovarian cancer occurrence and also a 42% of reduction in mortality [99]. The main studies that showed metformin benefits in the context of ovarian cancer diabetic patients are summarized in **Table 1**.

### **Figure 2.**

*Main antitumoral mechanism of metformin in ovarian cancer cells. Metformin enters the cell through organic cationic transporters (OCT) and produces the activation of liver kinase B1 (LKB1) and an increase of AMP/ ATP ratio, which results in the activation of AMPK. This kinase has several targets as sterol regulatory element-binding protein 1 (SREBP) and acetyl-CoA carboxylase (ACC); the mechanistic target of rapamycin complex 1 (mTORC1) and AKT/ERK signaling; key proteins in the fatty acid synthesis and cell growth, survival, proliferation, and migration; and the processes of epithelial-mesenchymal transition (EMT). On the other hand, metformin can block the growth factor (GF) signaling dependent or independent of AMPK activation. Also metformin decreases the angiogenic potential of ovarian cancer cells, impairs the expression of vascular endothelial growth factor (VEGF), or acts directly on the endothelial cells.*

**169**

should be interpreted with caution.

metformin in the following phases of the study.

*Antitumoral Effects of Metformin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.88911*

Retrospective cohort study

Retrospective cohort study

Retrospective cohort study N = 479,475, China

Case-control study 72 cases (OvCa, metformin users), 142 controls (OvCa, non-metformin)

Retrospective cohort study

Case-control study 1611 cases (OvCa) and 9170 controls (non-

N = 568, China

N = 143, Israel

USA

N = 341, USA

OvCa), UK

*disease but it does not get worse).*

*a defined period of time).*

Wang et al. [12]

Bar et al. [114]

Tseng et al. [115]

Kumar et al. [116]

Romero et al. [102]

Bodmer et al. [117]

*\**

**Table 1.**

*using metformin.*

**Research Study and population Main finding**

• Metformin group of OvCa patients had longer

and metformin-discontinued groups

recurrence of OvCa (lower PFS\*

100,000 person-years)

of OvCa

survival

patients

recurrence

of OvCa

OvCa patients

tion was stronger in diabetic patients

than non-metformin, nondiabetic,

), and this associa-

and overall sur-

in dose (500 or 1000 mg of

• Metformin treatment must be continuous to obtain

• Metformin was associated with a reduced risk of

• 601 metformin ever-users and 2600 never-users developed OvCa (incidence of 49.4 and 146.4 per

• Metformin use was associated with a decreased risk

• Metformin was associated with a better survival in

vival of OvCa compared to nonusers or nondiabetic

Metformin use was associated with a decreased of risk

• Metformin group decreased hazard for disease

• 5-year DSS\*\* was higher in metformin group • Metformin was an independent predictor of

• Metformin group had a longer PFS\*

median PFS\*

• Similar PFS\*

metformin)

beneficial effects

Although several observational studies show positive effects of metformin in diabetic patients, it has not yet been elucidated if metformin could be beneficial in nondiabetic patients. In addition, ovarian cancer has a low incidence, and the number of participants in some of the available studies is low; therefore, the evidence

*Summary of studies that evaluated incidence and prognosis of ovarian cancer (OvCa) patients using and not* 

*PFS: progression-free survival (length of time during and after the treatment of OvCa that a patient lives with the* 

*\*\*DSS: disease-specific survival (percentage of people in a study or treatment group who have not died from OvCa in* 

Because of the increased interest in the possible use of metformin in nondiabetic patients, there are currently six clinical trials inscribed in NIH ClinicalTrials.gov database to study metformin intake in association with carboplatin and paclitaxel (first-line chemotherapy) in nondiabetic woman with ovarian cancer (NCT02312661, NCT02437812, NCT03378297, NCT02122185, NCT01579812, and NCT02201381) from phase 0 to phase III of the study. The results of one of these trials show that metformin was well tolerated and the outcome results were favorable, because tumors from metformin-treated women have a threefold decrease in specific subpopulations of ovarian cancer stem cells with an increased sensitivity to cisplatin *in vitro* [100], supporting the use of


*Antitumoral Effects of Metformin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.88911*

*Metformin*

**Figure 2**.

summarized in **Table 1**.

conversion of acetyl-CoA to malonyl-CoA leads to an increase in the acetylation of histones in the chromatin and altered gene expression in ovarian cancer cells [67]. Because acetylation of nucleosomal histones is linked to nuclear processes as transcription, replication, and repair among other functions [98], it is possible that several antitumoral effects of metformin could be regulated by protein acetylation

The summary of the main studied antitumoral effects of metformin is shown in

A recent meta-analysis shows that among available studies of relationship between metformin intake with ovarian cancer incidence and prognosis in diabetic patients, the majority of the studies indicate a negative correlation between the use of metformin and the incidence of ovarian cancer, as well as, a positive correlation with better prognosis [99]. The same study shows that metformin treatment in diabetic patients has a reduction of 24% risk of ovarian cancer occurrence and also a 42% of reduction in mortality [99]. The main studies that showed metformin benefits in the context of ovarian cancer diabetic patients are

*Main antitumoral mechanism of metformin in ovarian cancer cells. Metformin enters the cell through organic cationic transporters (OCT) and produces the activation of liver kinase B1 (LKB1) and an increase of AMP/ ATP ratio, which results in the activation of AMPK. This kinase has several targets as sterol regulatory element-binding protein 1 (SREBP) and acetyl-CoA carboxylase (ACC); the mechanistic target of rapamycin complex 1 (mTORC1) and AKT/ERK signaling; key proteins in the fatty acid synthesis and cell growth, survival, proliferation, and migration; and the processes of epithelial-mesenchymal transition (EMT). On the other hand, metformin can block the growth factor (GF) signaling dependent or independent of AMPK activation. Also metformin decreases the angiogenic potential of ovarian cancer cells, impairs the expression of* 

*vascular endothelial growth factor (VEGF), or acts directly on the endothelial cells.*

and transcriptional regulation of several oncosuppressor proteins.

**3.6 Studies of metformin in diabetic patients with ovarian cancer**

**168**

**Figure 2.**

*\* PFS: progression-free survival (length of time during and after the treatment of OvCa that a patient lives with the disease but it does not get worse).*

*\*\*DSS: disease-specific survival (percentage of people in a study or treatment group who have not died from OvCa in a defined period of time).*

### **Table 1.**

*Summary of studies that evaluated incidence and prognosis of ovarian cancer (OvCa) patients using and not using metformin.*

Although several observational studies show positive effects of metformin in diabetic patients, it has not yet been elucidated if metformin could be beneficial in nondiabetic patients. In addition, ovarian cancer has a low incidence, and the number of participants in some of the available studies is low; therefore, the evidence should be interpreted with caution.

Because of the increased interest in the possible use of metformin in nondiabetic patients, there are currently six clinical trials inscribed in NIH ClinicalTrials.gov database to study metformin intake in association with carboplatin and paclitaxel (first-line chemotherapy) in nondiabetic woman with ovarian cancer (NCT02312661, NCT02437812, NCT03378297, NCT02122185, NCT01579812, and NCT02201381) from phase 0 to phase III of the study. The results of one of these trials show that metformin was well tolerated and the outcome results were favorable, because tumors from metformin-treated women have a threefold decrease in specific subpopulations of ovarian cancer stem cells with an increased sensitivity to cisplatin *in vitro* [100], supporting the use of metformin in the following phases of the study.

### **3.7 Role of metformin in metastasis and chemoresistance**

Besides the abovementioned benefits, metformin treatment has a relevant role in the metastasis and chemoresistance prevention of several ovarian cancer models. For example, *in vitro* experiments have shown that metformin decreases the adhesion capacity, invasion, and migration of ovarian cancer cell lines [101]. In rodents, metformin treatment inhibits the growth of metastatic nodules in the lung product of ovarian cancer [33], and importantly, the use of metformin in diabetic women decreases the probability of disease recurrence [102].

The cancer stem cells, recently called "tumor-initiating cells," are a tumoral cell subpopulation with critical role in therapy resistance and metastasis [103–105]. There are several markers to identify them, as lactate dehydrogenase (LDH), aldehyde dehydrogenase (ALDH), or cell-surface antigens as CD44, CD133, or CD117 [106–108]. Metformin treatment decreases the abundance of ovarian cancer LDH+ and decreases its ability to form tumor spheres, an attachment-independent growth characteristic of these kinds of cells [109]. At the same time, a low dose of metformin (micromolar concentration) decreases the abundance of CD44+/ CD117+ ovarian cancer cells selectively, whereas CD133+ or ALDH+ cell subpopulation were more sensitive to millimolar concentration of this drug [109, 110].

Another key point is that metformin decreases the expression of classical markers related with EMT. This process is necessary to confer an increased migratory capacity to tumor cells, participating in the intra-/extravasation and hence, in the tumor cell dissemination. In CD44+/CD117+ ovarian cancer cells, metformin treatment decreases snail2, twist, and vimentin protein levels (these are mesenchymal markers), increasing E-cadherin protein levels (a known epithelial marker) [110]. These observations are related with a study performed in diabetic patients with endometrial cancer, where in the biopsies of these patients using metformin were found increased levels of E-cadherin [111]. These findings suggest that metformin decreases the process of EMT in ovarian cancer cells, affecting preferentially tumor-initiating cells, which constitutes a relevant advantage, because this type of cells is not affected by traditional chemotherapy.

One important aspect in ovarian cancer treatment is the high percentage of chemoresistance developed by patients. In this context, metformin stands as a promising drug, since several studies showed that it could increase the susceptibility of ovarian cancer cells to chemotherapy and revert its acquired chemoresistance [34, 112, 113]. One recent study performed in ovarian cancer cell lines treated for 6 months with cisplatin and paclitaxel (for the acquirement of chemoresistance phenotype) shows that metformin treatment increases drug sensitivity and reduces migratory abilities of these ovarian cancer cells. In addition, the same study shows that metformin decrease the ovarian cancer stem cell population and the expression of specific biomarkers of pluripotent genes [112].

### **3.8 Main conclusions**

Metformin is an antidiabetic drug that displays antitumoral effects in several *in vivo* and *in vitro* models of cancer, including ovarian cancer. The mechanism of its antitumoral effects could be either dependent or independent of AMPK, a key sensor of the cell energetic status. Metformin has several cell targets which include transcription factors and cell cycle regulators; wherewith it impairs cell proliferation by the arrest of the cell cycle. In addition, metformin modulates enzymes of metabolic pathways and lipid metabolism, as well as epigenetic and posttranscriptional regulation of the ovarian cancer cells, which can explain its pleiotropic actions. Another important point is that metformin regulates angiogenesis in

**171**

*Antitumoral Effects of Metformin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.88911*

complement its anti-angiogenic effect.

**Acknowledgements**

**Conflict of interest**

the ovarian cancer cells, mainly decreasing VEGF expression, which impairs the angiogenic potential of these cells. On the other hand, metformin acts directly in endothelial cells, decreasing its proliferation, migration and differentiation, which

nondiabetic women with ovarian cancer should be considered with caution.

The authors would like to thank the National Fund for Scientific and

AMPK adenosine monophosphate-activated protein kinase

as a coadjuvant alternative in the treatment of ovarian cancer.

Technological Development (FONDECYT) #1160139.

The authors declare no conflict of interest.

**Appendices and nomenclature**

ACC acetyl-CoA carboxylase AKT activate protein kinase B

ALDH aldehyde dehydrogenase

IGF insulin-like growth factor LDH lactate dehydrogenase

OCTs organic cationic transporters

VEGF vascular endothelial growth factor

LKB1 liver kinase B1

miRs micro-RNAs NGF nerve growth factor

EMT epithelial-mesenchymal transition ERK extracellular signal-regulated kinase HUVEC human umbilical vein endothelial cells

mTORC1 mechanistic target of rapamycin complex 1

SREBP1 sterol regulatory element-binding protein 1

Currently, there are several clinical trials performed in women with ovarian cancer. These trials are studying the effect of metformin treatment together with standard chemotherapy in the ovarian cancer prognosis and clinic-pathological markers, which could be helpful to elucidate whether this drug could be considered

An important niche for metformin treatment could be its selective effect in ovarian cancer cells with stem cell phenotype, which are responsible for ovarian cancer dissemination and chemotherapy resistance. Several studies show that metformin reduces ovarian cancer stem cells abundance and that it could have a chemosensitivity role when used in combination with first-line chemotherapy agents. This opens the possibility to the potential use of metformin as a coadjuvant agent in ovarian cancer treatment. Finally, there are several observational studies in diabetic women with ovarian cancer which show that metformin is associated with less ovarian cancer incidence and better prognosis. However, it is important to consider that the number of participants using metformin in some of these studies is low and that several *in vitro* experiments have shown that metformin action depends on the metabolic context and nutrient and oxygen availability of ovarian cancer cells. For these reasons, the use of metformin in

### *Antitumoral Effects of Metformin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.88911*

*Metformin*

**3.7 Role of metformin in metastasis and chemoresistance**

decreases the probability of disease recurrence [102].

cells is not affected by traditional chemotherapy.

of specific biomarkers of pluripotent genes [112].

**3.8 Main conclusions**

Besides the abovementioned benefits, metformin treatment has a relevant role in the metastasis and chemoresistance prevention of several ovarian cancer models. For example, *in vitro* experiments have shown that metformin decreases the adhesion capacity, invasion, and migration of ovarian cancer cell lines [101]. In rodents, metformin treatment inhibits the growth of metastatic nodules in the lung product of ovarian cancer [33], and importantly, the use of metformin in diabetic women

The cancer stem cells, recently called "tumor-initiating cells," are a tumoral cell subpopulation with critical role in therapy resistance and metastasis [103–105]. There are several markers to identify them, as lactate dehydrogenase (LDH), aldehyde dehydrogenase (ALDH), or cell-surface antigens as CD44, CD133, or CD117 [106–108]. Metformin treatment decreases the abundance of ovarian cancer LDH+ and decreases its ability to form tumor spheres, an attachment-independent growth characteristic of these kinds of cells [109]. At the same time, a low dose of metformin (micromolar concentration) decreases the abundance of CD44+/ CD117+ ovarian cancer cells selectively, whereas CD133+ or ALDH+ cell subpopulation were more sensitive to millimolar concentration of this drug [109, 110].

Another key point is that metformin decreases the expression of classical markers related with EMT. This process is necessary to confer an increased migratory capacity to tumor cells, participating in the intra-/extravasation and hence, in the tumor cell dissemination. In CD44+/CD117+ ovarian cancer cells, metformin treatment decreases snail2, twist, and vimentin protein levels (these are mesenchymal markers), increasing E-cadherin protein levels (a known epithelial marker) [110]. These observations are related with a study performed in diabetic patients with endometrial cancer, where in the biopsies of these patients using metformin were found increased levels of E-cadherin [111]. These findings suggest that metformin decreases the process of EMT in ovarian cancer cells, affecting preferentially tumor-initiating cells, which constitutes a relevant advantage, because this type of

One important aspect in ovarian cancer treatment is the high percentage of chemoresistance developed by patients. In this context, metformin stands as a promising drug, since several studies showed that it could increase the susceptibility of ovarian cancer cells to chemotherapy and revert its acquired chemoresistance [34, 112, 113]. One recent study performed in ovarian cancer cell lines treated for 6 months with cisplatin and paclitaxel (for the acquirement of chemoresistance phenotype) shows that metformin treatment increases drug sensitivity and reduces migratory abilities of these ovarian cancer cells. In addition, the same study shows that metformin decrease the ovarian cancer stem cell population and the expression

Metformin is an antidiabetic drug that displays antitumoral effects in several *in vivo* and *in vitro* models of cancer, including ovarian cancer. The mechanism of its antitumoral effects could be either dependent or independent of AMPK, a key sensor of the cell energetic status. Metformin has several cell targets which include transcription factors and cell cycle regulators; wherewith it impairs cell proliferation by the arrest of the cell cycle. In addition, metformin modulates enzymes of metabolic pathways and lipid metabolism, as well as epigenetic and posttranscriptional regulation of the ovarian cancer cells, which can explain its pleiotropic actions. Another important point is that metformin regulates angiogenesis in

**170**

the ovarian cancer cells, mainly decreasing VEGF expression, which impairs the angiogenic potential of these cells. On the other hand, metformin acts directly in endothelial cells, decreasing its proliferation, migration and differentiation, which complement its anti-angiogenic effect.

An important niche for metformin treatment could be its selective effect in ovarian cancer cells with stem cell phenotype, which are responsible for ovarian cancer dissemination and chemotherapy resistance. Several studies show that metformin reduces ovarian cancer stem cells abundance and that it could have a chemosensitivity role when used in combination with first-line chemotherapy agents. This opens the possibility to the potential use of metformin as a coadjuvant agent in ovarian cancer treatment.

Finally, there are several observational studies in diabetic women with ovarian cancer which show that metformin is associated with less ovarian cancer incidence and better prognosis. However, it is important to consider that the number of participants using metformin in some of these studies is low and that several *in vitro* experiments have shown that metformin action depends on the metabolic context and nutrient and oxygen availability of ovarian cancer cells. For these reasons, the use of metformin in nondiabetic women with ovarian cancer should be considered with caution.

Currently, there are several clinical trials performed in women with ovarian cancer. These trials are studying the effect of metformin treatment together with standard chemotherapy in the ovarian cancer prognosis and clinic-pathological markers, which could be helpful to elucidate whether this drug could be considered as a coadjuvant alternative in the treatment of ovarian cancer.

### **Acknowledgements**

The authors would like to thank the National Fund for Scientific and Technological Development (FONDECYT) #1160139.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Appendices and nomenclature**


*Metformin*

### **Author details**

Maritza P. Garrido, Margarita Vega and Carmen Romero\* Laboratory of Endocrinology and Reproduction Biology, Clinical Hospital University of Chile, Obstetrics and Gynecology Department, Faculty of Medicine, University of Chile, Santiago, Chile

\*Address all correspondence to: cromero@hcuch.cl

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**173**

*Antitumoral Effects of Metformin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.88911*

[1] Rena G, Hardie DG, Pearson ER. The mechanisms of action of metformin. Diabetologia. 2017;**60**(9):1577-1585

risk of cancer in diabetic patients. BMJ.

[9] NCD\_Risk\_Factor\_Collaboration\_ (NCD-RisC). Worldwide trends in body-mass index, underweight, overweight, and obesity from 1975 to 2016: A pooled analysis of 2416 population-based measurement studies in 128.9 million children, adolescents, and adults. Lancet. 2017;**390**(10113):2627-2642

[10] Unnikrishnan R, Pradeepa R, Joshi SR, Mohan V. Type 2 diabetes: Demystifying the global epidemic. Diabetes. 2017;**66**(6):1432-1442

[11] Beral V, Hermon C, Peto R, Reeves G, Brinton L, Marchbanks P, et al. Ovarian cancer and body size: Individual participant meta-analysis including 25,157 women with ovarian cancer from 47 epidemiological studies. PLoS Medicine. 2012;**9**(4):e1001200

[12] Wang SB, Lei KJ, Liu JP, Jia YM. Continuous use of metformin can improve survival in type 2 diabetic patients with ovarian cancer: A retrospective study. Medicine (Baltimore). 2017;**96**(29):e7605

[13] Akhavan S, Ghahghaei-Nezamabadi A,

Modaresgilani M, Mousavi AS, Sepidarkish M, Tehranian A, et al. Impact of diabetes mellitus on epithelial ovarian cancer survival. BMC Cancer.

[14] Perseghin G, Calori G,

Lattuada G, Ragogna F, Dugnani E, Garancini MP, et al. Insulin resistance/ hyperinsulinemia and cancer mortality: The Cremona study at the 15th year of follow-up. Acta Diabetologica.

[15] Tsujimoto T, Kajio H, Sugiyama T. Association between hyperinsulinemia and increased risk of cancer death

2018;**18**(1):1246

2012;**49**(6):421-428

2005;**330**(7503):1304-1305

[2] Scarpello JH, Howlett HC. Metformin therapy and clinical uses. Diabetes & Vascular Disease Research.

[3] Kelley KW, Carroll DG, Meyer A. A review of current treatment strategies for gestational diabetes mellitus. Drugs

[4] Mathur R, Alexander CJ, Yano J, Trivax B, Azziz R. Use of metformin in polycystic ovary syndrome. American Journal of Obstetrics and Gynecology.

[5] Pirwany IR, Yates RW, Cameron IT, Fleming R. Effects of the insulin sensitizing drug metformin on ovarian function, follicular growth and ovulation rate in obese women with oligomenorrhoea. Human Reproduction. 1999;**14**(12):2963-2968

[6] Mahamed RR, Maganhin CC, Sasso GRS, de Jesus Simoes M, Baracat MCP, Baracat EC, et al. Metformin improves ovarian follicle dynamics by reducing theca cell proliferation and CYP-17 expression in an androgenized rat model. Journal of Ovarian Research. 2018;**11**(1):18

[7] Carvajal R, Rosas C, Kohan K, Gabler F, Vantman D, Romero C, et al. Metformin augments the levels of molecules that regulate the expression of the insulin-dependent glucose transporter GLUT4 in the endometria

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*Antitumoral Effects of Metformin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.88911*

### **References**

*Metformin*

**172**

**Author details**

University of Chile, Santiago, Chile

provided the original work is properly cited.

Maritza P. Garrido, Margarita Vega and Carmen Romero\*

\*Address all correspondence to: cromero@hcuch.cl

Laboratory of Endocrinology and Reproduction Biology, Clinical Hospital

University of Chile, Obstetrics and Gynecology Department, Faculty of Medicine,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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[95] Blandino G, Valerio M, Cioce M, Mori F, Casadei L, Pulito C, et al. Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC. Nature Communications. 2012;**3**:865

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Chauvin C, Batandier C, Fontaine E, Wiernsperger N, et al. Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-

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[77] Folkman J. Role of angiogenesis in tumour growth and metastasis. Seminars in Oncology. 2002;**29**(6 Suppl. endothelin-1 levels in women with polycystic ovary syndrome and the beneficial effect of metformin therapy. The Journal of Clinical Endocrinology and Metabolism.

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mTOR/autophagy pathway. International Journal of Molecular Medicine. 2017;**39**(5):1262-1268

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[90] Li WD, Li NP, Song DD, Rong JJ, Qian AM, Li XQ. Metformin inhibits endothelial progenitor cell migration by decreasing matrix metalloproteinases, MMP-2 and MMP-9, via the AMPK/

Huang H, et al. Incorporation of bevacizumab in the primary treatment of ovarian cancer. The New England Journal of Medicine.

[78] Tapia V, Gabler F, Munoz M, Yazigi R, Paredes A, Selman A, et al. Tyrosine kinase A receptor (trkA): A potential marker in epithelial ovarian cancer. Gynecologic Oncology.

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[94] Martello G, Rosato A, Ferrari F, Manfrin A, Cordenonsi M, Dupont S, et al. A microRNA targeting dicer for metastasis control. Cell. 2010;**141**(7):1195-1207

[95] Blandino G, Valerio M, Cioce M, Mori F, Casadei L, Pulito C, et al. Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC. Nature Communications. 2012;**3**:865

[96] Garrido MP, Valenzuela M, Vallejos C, Salvatierra R, Hernández A, Vega M, et al, editors. Metformin decreases NGF-induced cell proliferation of ovarian cancer cells by modulation of c-MYC and β-catenin/ TCF-Lef transcriptional activity and oncosuppressors micro-RNAs. In: 23rd International Symposium on Molecular Medicine; 28-30 March 2019; Bangkok, Thailand; 2019

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[99] Shi J, Liu B, Wang H, Zhang T, Yang L. Association of metformin use with ovarian cancer incidence and prognosis: A systematic review and meta-analysis. International Journal of Gynecological Cancer. 2019;**29**(1):140-146

[100] Buckanovich RJ, Brown J, Shank J, Griffith K, Reynolds K, Johnston C, et al. A phase II clinical trial of metformin as a cancer stem cell targeting agent in stage IIc/III/IV ovarian, fallopian tube, and primary peritoneal cancer. Journal of Clinical Oncology. 2017;**35**(15\_suppl):5556

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[103] Prieto-Vila M, Takahashi RU, Usuba W, Kohama I, Ochiya T. Drug resistance driven by cancer stem cells and their niche. International Journal of Molecular Sciences. 2017;**18**(12):2574

[104] Lu W, Kang Y. Epithelialmesenchymal plasticity in cancer progression and metastasis. Developmental Cell. 2019;**49**(3):361-374

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[107] Kryczek I, Liu S, Roh M, Vatan L, Szeliga W, Wei S, et al. Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells. International Journal of Cancer. 2012;**130**(1):29-39

[108] Parte SC, Batra SK, Kakar SS. Characterization of stem cell and cancer stem cell populations in ovary and ovarian tumours. Journal of Ovarian Research. 2018;**11**(1):69

[109] Shank JJ, Yang K, Ghannam J, Cabrera L, Johnston CJ, Reynolds RK, et al. Metformin targets ovarian cancer stem cells *in vitro* and *in vivo*. Gynecologic Oncology. 2012;**127**(2):390-397

[110] Zhang R, Zhang P, Wang H, Hou D, Li W, Xiao G, et al. Inhibitory effects of metformin at low concentration on epithelial-mesenchymal transition of CD44(+)CD117(+) ovarian cancer stem cells. Stem Cell Research & Therapy. 2015;**6**:262

[111] Laskov I, Abou-Nader P, Amin O, Philip CA, Beauchamp MC, Yasmeen A, et al. Metformin increases E-cadherin in tumours of diabetic patients with endometrial cancer and suppresses epithelial-mesenchymal transition in endometrial cancer cell lines. International Journal of Gynecological Cancer. 2016;**26**(7):1213-1221

[112] Bishnu A, Sakpal A, Ghosh N, Choudhury P, Chaudhury K, Ray P. Long term treatment of metformin impedes development of chemoresistance by regulating cancer stem cell differentiation through taurine generation in ovarian cancer cells. The International Journal of Biochemistry & Cell Biology. 2019;**107**:116-127

[113] Kim NY, Lee HY, Lee C. Metformin targets Axl and Tyro3 receptor tyrosine kinases to inhibit cell proliferation and overcome chemoresistance in ovarian cancer cells. International Journal of Oncology. 2015;**47**(1):353-360

[114] Bar D, Lavie O, Stein N, Feferkorn I, Shai A. The effect of metabolic comorbidities and commonly used drugs on the prognosis of patients with ovarian cancer. European Journal of Obstetrics, Gynecology, and Reproductive Biology. 2016;**207**:227-231

[115] Tseng CH. Metformin reduces ovarian cancer risk in Taiwanese women with type 2 diabetes mellitus. Diabetes/ Metabolism Research and Reviews. 2015;**31**(6):619-626

[116] Kumar S, Meuter A, Thapa P, Langstraat C, Giri S, Chien J, et al. Metformin intake is associated with better survival in ovarian cancer: A case-control study. Cancer. 2013;**119**(3):555-562

[117] Bodmer M, Becker C, Meier C, Jick SS, Meier CR. Use of metformin and the risk of ovarian cancer: A casecontrol analysis. Gynecologic Oncology. 2011;**123**(2):200-204

**181**

**1. Introduction**

**Chapter 11**

**Abstract**

Metformin Activity against Breast

Obesity and type 2 diabetes increase the risk of and reduce survival in breast cancer (BC) patients. Metformin is the only anti-diabetic drug that alters this risk, with a reduction in BC incidence and improved outcomes. Metformin has AMP-kinase (AMPK) dependent and independent mechanisms of action, most notably affecting the liver and skeletal muscle. We and others have shown that metformin also downregulates protein and lipid synthesis; deactivates various receptor tyrosine kinases; alters cell cycle transcription/translation; modulates mitochondrial respiration and miRNA activation; targets key metabolic molecules; induces stem cell death and may induce apoptosis or autophagy in BC cells. Many of these anti-cancer effects are molecular subtype-specific. Metformin is most potent against triple negative (basal), followed by luminal BCs. The efficacy of metformin, as well as dose needed for the activity, is also modulated by the extracellular glucose concentration, cellular expression of the glucose transporter protein 1 (GLUT1), and the organic cation transporter protein 1 (OCT1, which transports metformin into cells). This chapter summarizes the diverse clinical and preclinical data related

to the anti-cancer effects of metformin, focused against breast cancer.

MiR-193b, cancer stem cells, EGFR, cholesterol, glucose

**Keywords:** metformin, breast cancer, TGF-β, STAT3, and PI3K/AKT/mTOR, FASN,

Metabolic dysregulation of carbohydrate and lipid metabolism is frequent in cancer cells, facilitating growth and survival through adaptive mechanisms. Otto Warburg was the first to recognize that cancer cells favor glycolysis as compared to oxidative phosphorylation for the generation of energy (ATP) [1]. While the former is less efficient in terms of energy production per molecule of glucose, it also generates precursor molecules (amino acids, fatty acids, etc.) for replication and facilitates survival under oxidative stress [2]. This is in contrast to normal cells, which typically use oxidative metabolism to derive more energy (ATP) per molecule of glucose [3, 4]. Nearly a century later, we now recognize that cancer cells may utilize either aerobic or anaerobic respiration. The majority of cancer cells also have alterations of mitochondrial respiration, further providing a selective advantage to

Cancer: Mechanistic Differences

by Molecular Subtype and

*Reema S. Wahdan-Alaswad and Ann D. Thor*

Metabolic Conditions

### **Chapter 11**

*Metformin*

2012;**130**(1):29-39

Research. 2018;**11**(1):69

2015;**6**:262

[106] Suraneni MV, Badeaux MD. Tumour-initiating cells, cancer

metastasis and therapeutic implications. In: Madame Curie Bioscience Database. Austin: Landes Bioscience; 2000-2013

[113] Kim NY, Lee HY, Lee C. Metformin targets Axl and Tyro3 receptor tyrosine kinases to inhibit cell proliferation and overcome chemoresistance in ovarian cancer cells. International Journal of Oncology. 2015;**47**(1):353-360

Reproductive Biology. 2016;**207**:227-231

[115] Tseng CH. Metformin reduces ovarian cancer risk in Taiwanese women with type 2 diabetes mellitus. Diabetes/ Metabolism Research and Reviews.

[116] Kumar S, Meuter A, Thapa P, Langstraat C, Giri S, Chien J, et al. Metformin intake is associated with better survival in ovarian cancer: A case-control study. Cancer.

[117] Bodmer M, Becker C, Meier C, Jick SS, Meier CR. Use of metformin and the risk of ovarian cancer: A casecontrol analysis. Gynecologic Oncology.

2015;**31**(6):619-626

2013;**119**(3):555-562

2011;**123**(2):200-204

[114] Bar D, Lavie O, Stein N, Feferkorn I, Shai A. The effect of metabolic comorbidities and commonly used drugs on the prognosis of patients with ovarian cancer. European Journal of Obstetrics, Gynecology, and

[107] Kryczek I, Liu S, Roh M, Vatan L, Szeliga W, Wei S, et al. Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells. International Journal of Cancer.

[108] Parte SC, Batra SK, Kakar SS. Characterization of stem cell and cancer stem cell populations in ovary and ovarian tumours. Journal of Ovarian

[109] Shank JJ, Yang K, Ghannam J, Cabrera L, Johnston CJ, Reynolds RK, et al. Metformin targets ovarian cancer stem cells *in vitro* and *in vivo*. Gynecologic

Oncology. 2012;**127**(2):390-397

[110] Zhang R, Zhang P, Wang H, Hou D, Li W, Xiao G, et al. Inhibitory effects of metformin at low concentration on epithelial-mesenchymal transition of CD44(+)CD117(+) ovarian cancer stem cells. Stem Cell Research & Therapy.

[111] Laskov I, Abou-Nader P, Amin O, Philip CA, Beauchamp MC, Yasmeen A, et al. Metformin increases E-cadherin in tumours of diabetic patients with endometrial cancer and suppresses epithelial-mesenchymal transition in endometrial cancer cell lines. International Journal of Gynecological

Cancer. 2016;**26**(7):1213-1221

Cell Biology. 2019;**107**:116-127

[112] Bishnu A, Sakpal A, Ghosh N, Choudhury P, Chaudhury K, Ray P. Long term treatment of metformin impedes development of chemoresistance by regulating cancer stem cell differentiation through taurine generation in ovarian cancer cells. The International Journal of Biochemistry &

**180**

## Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype and Metabolic Conditions

*Reema S. Wahdan-Alaswad and Ann D. Thor*

### **Abstract**

Obesity and type 2 diabetes increase the risk of and reduce survival in breast cancer (BC) patients. Metformin is the only anti-diabetic drug that alters this risk, with a reduction in BC incidence and improved outcomes. Metformin has AMP-kinase (AMPK) dependent and independent mechanisms of action, most notably affecting the liver and skeletal muscle. We and others have shown that metformin also downregulates protein and lipid synthesis; deactivates various receptor tyrosine kinases; alters cell cycle transcription/translation; modulates mitochondrial respiration and miRNA activation; targets key metabolic molecules; induces stem cell death and may induce apoptosis or autophagy in BC cells. Many of these anti-cancer effects are molecular subtype-specific. Metformin is most potent against triple negative (basal), followed by luminal BCs. The efficacy of metformin, as well as dose needed for the activity, is also modulated by the extracellular glucose concentration, cellular expression of the glucose transporter protein 1 (GLUT1), and the organic cation transporter protein 1 (OCT1, which transports metformin into cells). This chapter summarizes the diverse clinical and preclinical data related to the anti-cancer effects of metformin, focused against breast cancer.

**Keywords:** metformin, breast cancer, TGF-β, STAT3, and PI3K/AKT/mTOR, FASN, MiR-193b, cancer stem cells, EGFR, cholesterol, glucose

### **1. Introduction**

Metabolic dysregulation of carbohydrate and lipid metabolism is frequent in cancer cells, facilitating growth and survival through adaptive mechanisms. Otto Warburg was the first to recognize that cancer cells favor glycolysis as compared to oxidative phosphorylation for the generation of energy (ATP) [1]. While the former is less efficient in terms of energy production per molecule of glucose, it also generates precursor molecules (amino acids, fatty acids, etc.) for replication and facilitates survival under oxidative stress [2]. This is in contrast to normal cells, which typically use oxidative metabolism to derive more energy (ATP) per molecule of glucose [3, 4]. Nearly a century later, we now recognize that cancer cells may utilize either aerobic or anaerobic respiration. The majority of cancer cells also have alterations of mitochondrial respiration, further providing a selective advantage to

facilitate cancer growth and survival [5]. More specifically, it may increase intracellular reactive oxygen species by disruption of the mitochondrial electron transport chain to reduce the mitochondrial membrane potential in BC or act directly to inhibit the mitochondrial respiratory-chain complex 1 (MRCC1) [6–8].

Chronic energy excess and physical inactivity lead to systemic alterations of carbohydrate and fatty acid metabolism characterized by systemic hyperglycemia, hyperinsulinemia with insulin resistance followed by hypoinsulinemia, an increase in inflammatory cytokines and adipokines, alterations of steroid and growth hormones, and downregulation of immune surveillance and tissue oxygenation [3, 9, 10]. These changes are frequent but variable in patients with obesity and type 2 diabetes and can be modified by drugs, exercise, body weight, socioeconomic factors, access to healthcare, genetic risk, and other factors. Patients with these disorders are at an increased risk of cardiovascular disease, cancer, and other diseases associated with significant morbidity and mortality. In the U.S., there are ~13.8 million type 2 diabetics, 5 million undiagnosed diabetics, and 41 million persons with prediabetes/metabolic syndrome [11–13]. Obesity is a frequent comorbidity, often proceeding diabetes by years or decades.

Energy-sensing systems are integral to maintaining homeostasis in normal and transformed cells. Energy deprivation is frequent in cancer cells due to an inadequate vascular supply to meet the needs of increased cell replication. In energy-stressed cells, AMPK is allosterically modified by binding to AMP and ADP, rendering them targetable by AMPK kinases. AMPK activation induces signaling, upregulates energy production, and inhibits energy programming for cell growth and motility. In cancerous cells, this shift often fails to occur even with stress. As a result, cancer cells typically prioritize replication and motility to favor cancer growth and metastasis. Drugs that activate AMPK, most notably metformin, reengage the AMPK failsafe to inhibit proliferation and motility. Thus, metformin provides a unique and generally less-toxic approach to combat the emergence or growth of cancers through inhibition of cell replication. This is particularly important for patients with obesity and type 2 diabetes, who lack homeostasis and experience wide swings in systemic glucose, insulin, and other energy precursors and growth factors that contribute to systemic energy stress.

### **2. Metabolic dysregulation, breast cancer, and metformin**

Abundant epidemiologic and clinical data have shown that obesity and type 2 diabetes increase the risk and severity of cardiovascular disease and human cancer. Each of these chronic metabolic disorders as a single variable significantly increases the risk of breast cancer (BC) [10, 14]. In combination, the risk is increased by 20–50%, depending on the severity of disease and other variables. It is highest in women with abdominal (central) obesity in the postmenopausal setting, in women of all ethnic backgrounds [15–17]. Obesity also promotes BC in premenopausal women of color, especially African Americans and Latinos [18–23]. In patients with obesity and diabetes, BC also presents at a higher disease stage and is more resistant to treatment, resulting in a shorter disease-free interval and a significantly higher mortality rate [24, 25].

Steroid receptor-positive BC (luminal A) and basal (triple negative) BC cells are the most responsive to extracellular glucose at or above 7 mM of glucose to promote cell replication, tumor growth, and motility. In contrast, steroid receptor-positive BC cells that also express high HER2 (luminal B) and steroid receptor-negative, HER2 positive (the HER2 subtype) are less responsive to hyperglycemia, even at levels associated with untreated type 2 diabetes (10 mM glucose or higher) [26].

**183**

BC patients.

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

Glucose directly promotes signaling in epithelial cells or can act indirectly by interacting with molecular signaling proteins, such as the insulin-like growth factor (IGF-1), sex hormones, and adipokines [3, 27, 28]. Insulin and insulin-like growth factors are frequently increased in newly diagnosed BC patients [28–31]. These potent growth factors promote BC growth and are associated with a worse prognosis, both in overweight and 'normal' weight women [24, 29, 32–34]. Epidemiological and clinical data show that obesity and type 2 diabetes are particularly associated with luminal A (estrogen and progesterone responsive) as well as triple negative

Metformin (N′, N′-dimethylbiguanide) is the most frequently used drug to treat patients with metabolic syndrome (prediabetes) and type 2 diabetes worldwide. It has been used successfully for over six decades and has a very favorable benefitrisk profile [37]. Metformin is stable at room temperature with a long shelf life, is inexpensive and orally administered, and has low rates of significant toxicity or drug-drug interaction. Metformin is best known for its effects on liver and skeletal muscle cells, where it downregulates insulin resistance, lowers serum insulin, stimulates insulin receptor tyrosine kinase activity, inhibits hepatic glucose output (thus lowering A1C), increases glucose uptake by skeletal muscle cells, and can alter

Epidemiologic data show a significant lowering of cancer risk in patients with metabolic dysregulation (obesity, diabetes, or metabolic syndrome) who take metformin [29, 34, 38, 39]. Metformin use by BC patients has also been associated with improved treatment response and survival. In one meta-analytic study of BC patients with diabetes, metformin use was associated with a 65% improvement in BC-specific survival as compared to nonusers [40]. The anticancer properties of metformin are in contrast to other antidiabetic agents, including sulfonylureas and

It is also taken for its 'antiaging' properties in individuals without obesity or

Numerous clinical trials are currently underway in BC patients to evaluate the benefit of metformin combined with or following the administration of other therapeutic agents [29, 32, 33, 43–46]. Studies designed to test the benefit of metformin in patients in only specific molecular subtypes of BC have not been performed, although some have looked at molecular cohort interactions as a secondary goal [30, 47–51]. There are limited data on the use of metformin in metabolically 'normal' BC patients. However, our preclinical data suggest that metformin is most active in all molecular subtypes with physiological levels of extracellular glucose [26]. This evidence provides a rationale for testing metformin in otherwise healthy

Cellular uptake of metformin requires expression and functionality of the organic

metabolic dysregulation, particularly outside of the US [10, 41, 42].

**3. AMPK-dependent mechanisms of metformin action in BC**

cation transporter 1 (OCT1) protein, which in some individuals or BCs may be altered (more or less effective in transporting metformin into the cell) by polymorphism or genetic error [52]. Polymorphisms have also been associated with a decrease in metformin efficacy in diabetic patients [53–55]. In BC cells, we have demonstrated that OCT1 expression is associated with the anticancer activity *in vivo* [44]. Once inside the cell, metformin may directly interact with the metabolic sensor AMPK to induce activation, restoring homeostasis and blocking cellular replication and motility under low energy (stress) conditions. The AMPK 'switch' is also influenced by the intracellular AMP:ATP ratio, which in turn is influenced by fatty acid oxidation and

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

BCs [19, 21, 27, 35, 36].

fatty acid metabolism.

insulin, which promote cancer growth [9].

### *Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*

Glucose directly promotes signaling in epithelial cells or can act indirectly by interacting with molecular signaling proteins, such as the insulin-like growth factor (IGF-1), sex hormones, and adipokines [3, 27, 28]. Insulin and insulin-like growth factors are frequently increased in newly diagnosed BC patients [28–31]. These potent growth factors promote BC growth and are associated with a worse prognosis, both in overweight and 'normal' weight women [24, 29, 32–34]. Epidemiological and clinical data show that obesity and type 2 diabetes are particularly associated with luminal A (estrogen and progesterone responsive) as well as triple negative BCs [19, 21, 27, 35, 36].

Metformin (N′, N′-dimethylbiguanide) is the most frequently used drug to treat patients with metabolic syndrome (prediabetes) and type 2 diabetes worldwide. It has been used successfully for over six decades and has a very favorable benefitrisk profile [37]. Metformin is stable at room temperature with a long shelf life, is inexpensive and orally administered, and has low rates of significant toxicity or drug-drug interaction. Metformin is best known for its effects on liver and skeletal muscle cells, where it downregulates insulin resistance, lowers serum insulin, stimulates insulin receptor tyrosine kinase activity, inhibits hepatic glucose output (thus lowering A1C), increases glucose uptake by skeletal muscle cells, and can alter fatty acid metabolism.

Epidemiologic data show a significant lowering of cancer risk in patients with metabolic dysregulation (obesity, diabetes, or metabolic syndrome) who take metformin [29, 34, 38, 39]. Metformin use by BC patients has also been associated with improved treatment response and survival. In one meta-analytic study of BC patients with diabetes, metformin use was associated with a 65% improvement in BC-specific survival as compared to nonusers [40]. The anticancer properties of metformin are in contrast to other antidiabetic agents, including sulfonylureas and insulin, which promote cancer growth [9].

It is also taken for its 'antiaging' properties in individuals without obesity or metabolic dysregulation, particularly outside of the US [10, 41, 42].

Numerous clinical trials are currently underway in BC patients to evaluate the benefit of metformin combined with or following the administration of other therapeutic agents [29, 32, 33, 43–46]. Studies designed to test the benefit of metformin in patients in only specific molecular subtypes of BC have not been performed, although some have looked at molecular cohort interactions as a secondary goal [30, 47–51]. There are limited data on the use of metformin in metabolically 'normal' BC patients. However, our preclinical data suggest that metformin is most active in all molecular subtypes with physiological levels of extracellular glucose [26]. This evidence provides a rationale for testing metformin in otherwise healthy BC patients.

### **3. AMPK-dependent mechanisms of metformin action in BC**

Cellular uptake of metformin requires expression and functionality of the organic cation transporter 1 (OCT1) protein, which in some individuals or BCs may be altered (more or less effective in transporting metformin into the cell) by polymorphism or genetic error [52]. Polymorphisms have also been associated with a decrease in metformin efficacy in diabetic patients [53–55]. In BC cells, we have demonstrated that OCT1 expression is associated with the anticancer activity *in vivo* [44]. Once inside the cell, metformin may directly interact with the metabolic sensor AMPK to induce activation, restoring homeostasis and blocking cellular replication and motility under low energy (stress) conditions. The AMPK 'switch' is also influenced by the intracellular AMP:ATP ratio, which in turn is influenced by fatty acid oxidation and

*Metformin*

facilitate cancer growth and survival [5]. More specifically, it may increase intracellular reactive oxygen species by disruption of the mitochondrial electron transport chain to reduce the mitochondrial membrane potential in BC or act directly to inhibit the mitochondrial respiratory-chain complex 1 (MRCC1) [6–8].

Chronic energy excess and physical inactivity lead to systemic alterations of carbohydrate and fatty acid metabolism characterized by systemic hyperglycemia, hyperinsulinemia with insulin resistance followed by hypoinsulinemia, an increase in inflammatory cytokines and adipokines, alterations of steroid and growth hormones, and downregulation of immune surveillance and tissue oxygenation [3, 9, 10]. These changes are frequent but variable in patients with obesity and type 2 diabetes and can be modified by drugs, exercise, body weight, socioeconomic factors, access to healthcare, genetic risk, and other factors. Patients with these disorders are at an increased risk of cardiovascular disease, cancer, and other diseases associated with significant morbidity and mortality. In the U.S., there are ~13.8 million type 2 diabetics, 5 million undiagnosed diabetics, and 41 million persons with prediabetes/metabolic syndrome [11–13]. Obesity

is a frequent comorbidity, often proceeding diabetes by years or decades.

equate vascular supply to meet the needs of increased cell replication.

and growth factors that contribute to systemic energy stress.

**2. Metabolic dysregulation, breast cancer, and metformin**

Abundant epidemiologic and clinical data have shown that obesity and type 2 diabetes increase the risk and severity of cardiovascular disease and human cancer. Each of these chronic metabolic disorders as a single variable significantly increases the risk of breast cancer (BC) [10, 14]. In combination, the risk is increased by 20–50%, depending on the severity of disease and other variables. It is highest in women with abdominal (central) obesity in the postmenopausal setting, in women of all ethnic backgrounds [15–17]. Obesity also promotes BC in premenopausal women of color, especially African Americans and Latinos [18–23]. In patients with obesity and diabetes, BC also presents at a higher disease stage and is more resistant to treatment, resulting in a shorter disease-free interval and a significantly higher

Steroid receptor-positive BC (luminal A) and basal (triple negative) BC cells are the most responsive to extracellular glucose at or above 7 mM of glucose to promote cell replication, tumor growth, and motility. In contrast, steroid receptor-positive BC cells that also express high HER2 (luminal B) and steroid receptor-negative, HER2 positive (the HER2 subtype) are less responsive to hyperglycemia, even at levels associated with untreated type 2 diabetes (10 mM glucose or higher) [26].

Energy-sensing systems are integral to maintaining homeostasis in normal and transformed cells. Energy deprivation is frequent in cancer cells due to an inad-

In energy-stressed cells, AMPK is allosterically modified by binding to AMP and ADP, rendering them targetable by AMPK kinases. AMPK activation induces signaling, upregulates energy production, and inhibits energy programming for cell growth and motility. In cancerous cells, this shift often fails to occur even with stress. As a result, cancer cells typically prioritize replication and motility to favor cancer growth and metastasis. Drugs that activate AMPK, most notably metformin, reengage the AMPK failsafe to inhibit proliferation and motility. Thus, metformin provides a unique and generally less-toxic approach to combat the emergence or growth of cancers through inhibition of cell replication. This is particularly important for patients with obesity and type 2 diabetes, who lack homeostasis and experience wide swings in systemic glucose, insulin, and other energy precursors

**182**

mortality rate [24, 25].

glucose metabolism. Thus, metformin can indirectly affect AMPK, through reduction of gluconeogenesis and thus changing of the AMP:ATP ratio. These mechanisms are represented in **Figure 1**. These processes are modulated by P53 status. It is mutated in many BCs, particularly tumors that are high grade, late-stage or nonluminal in subtype. In BCs that are P53 competent, AMPK activation (from metformin or other triggers) upregulates P53 tumor suppressor activity as a downstream target. This induces activation of cell cycle checkpoint proteins, to inhibit cell proliferation [56]. In P53 incompetent cells, AMPK activation from metformin may be less effective through P53 mechanisms. Given the numerous other actions of metformin, as well as the molecular subtype specificity of the drug, we postulated that P53 status alone would not have a major impact on anticancer effects of metformin. We have demonstrated that this is the case in preclinical studies of numerous BC cell lines [57]. In other cells, metformin may induce cell cycle arrest and death through activation of apoptotic pathways and downregulation of p53 [58, 59] or PARP cleavage, especially in triple negative BC [60, 61].

Activation of mTOR-dependent protein synthesis and cell growth (downstream of the PI3K/Akt signaling axis), along with AMPK, provides a robust signaling platform for BC cell growth, proliferation, and chemotherapy resistance. In addition to activating AMPK, metformin inhibits mTOR and downstream signaling components of this critical pathway. Mutation of the PI3K catalytic subunit (PIK3CA) occurs in 20-35% of BCs [62, 63]. Mutation or loss of the tumor suppressor gene PTEN has also been demonstrated in 40% of BC [64, 65]. Metformin can also inhibit gluconeogenesis and mTOR signaling independent of AMPK and the tuberous sclerosis 2 (TSC2) gene in some experimental systems (in hepatic cells that lack AMPK or its kinase, LKB1). In this model system, metformin induces

### **Figure 1.**

*Metformin AMPK-dependent mechanism of action on breast cancer. Metformin activates AMPK directly through insulin-like growth factor (IGF-I) or insulin receptor, which in turn can activate PI3K/Akt/mTOR or RAS/Raf/MEK/ERK to increase cell growth, survival, angiogenesis, migration, and invasion. Metformin indirectly activates AMPK, which activates mTORC2, CREB, and gluconeogenesis. Lastly, glucose can enter BC cell through GLUT-1, and metformin can directly downregulate GLUT-1 receptor.*

**185**

patients [74].

**in breast cancer**

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

downregulation of hepatic gluconeogenesis through non-AMPK–associated

Signaling systems and thus metformin sensitivity by dose or mechanism vary by the molecular subtype of BC as well as unique genomic changes in each patient's BC. For example, we have shown that metformin-induced partial S phase arrest increased P-AMPK and reduced P-EGFR, P-MAPK, P-Src, cyclin D1, and cyclin E, with the induction of PARP cleavage and apoptosis only in triple negative BCs [44]. In this tumor subtype, metformin specifically targets Stat3 and is not dependent on mTOR signaling [44]. In non-triple negative BCs (luminal and HER2), metformin induces partial cell cycle arrest at the G1 checkpoint, reduces cyclin D1 and E2F1 expression, and inhibits AMPK, MAPK, Akt, and mTOR activity [44, 57, 68]. Metformin-associated AMPK activation may also inactivate the insulin receptor substrate 1 (IRS1), which in turn regulates IGF-IR and PI3K/Akt signaling pathways to block the progrowth effects of hyperinsulinemia and insulin-like growth factors

Metformin is unique in the breadth and complexity of AMPK-dependent direct and indirect targets that inhibit cancer. Several new mechanisms fall into the rapidly expanding field of immuno-oncology. Metformin-induced activation of AMPK activates the programmed death ligand-1 (PDL-1) at S195, reducing stability and membrane localization and thus increasing PDL-1 degradation [70]. Metformin also promotes cytotoxic T cell lymphocyte activity in tumor tissue and enhances tumor-associated immune surveillance [6, 70, 71]. Additionally, metformin upregulates pro-inflammatory cytokines (tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), IL-1β, the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), the hypoxia-inducible factor 1-alpha (HIF-1α), and the vascular

AMPK-dependent mechanisms of action have been validated using clinical trial–derived BC samples as well as preclinical model systems, reviewed in detail elsewhere [43]. Some of these were especially important to spur the expansion of metformin use in BC patients. The timing, dose, and duration of metformin treatment in BC patients with or without other chemotherapy are actively under investigation. Neoadjuvant metformin, in particular, has shown benefit with a higher rate of complete pathological response, as compared to similar BC

**3.1 Metformin targets cell cycle proteins in AMPK-dependent manner** 

AMPK plays an integral role in the regulation of cell cycle and cell division. The ability of metformin to activate AMPK thus has a significant inhibitory effect on cell-cycle associated proteins. This mechanism is represented in **Figure 2**. Expression profiling of BC derived from metformin-treated patients as compared to controls has shown consistent downregulation of many gene encoding proteins involved in mitosis, including kinesins, tubulins, histones, Aurora, as well as Polo-like kinases and ribosomal proteins (critical for protein and macromolecular biosynthesis, respectively) [75]. Given the targeted effects of metformin, it is not surprising that its actions are synergistic with drugs like paclitaxel that induce defects in mitotic spindle assembly, chromosome segregation, and cell division. In combination, metformin and paclitaxel dramatically increase the number of cells arrested in G2-M and apoptosis, as compared to either agent alone [76]. Metformin may also induce GO/G1 arrest due to activation of AMPK, downregulation of cyclin D1, and enhanced binding of CDK2 by p27Kip1 and p21cip1 [60, 61], especially in non-triple negative cells. Some have shown that metformin sensitivity to GO/G1

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

typically associated with type 2 diabetes [66, 67, 69].

endothelial growth factor (VEGF), reviewed in [72, 73]).

mechanisms [66, 67].

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*

downregulation of hepatic gluconeogenesis through non-AMPK–associated mechanisms [66, 67].

Signaling systems and thus metformin sensitivity by dose or mechanism vary by the molecular subtype of BC as well as unique genomic changes in each patient's BC. For example, we have shown that metformin-induced partial S phase arrest increased P-AMPK and reduced P-EGFR, P-MAPK, P-Src, cyclin D1, and cyclin E, with the induction of PARP cleavage and apoptosis only in triple negative BCs [44]. In this tumor subtype, metformin specifically targets Stat3 and is not dependent on mTOR signaling [44]. In non-triple negative BCs (luminal and HER2), metformin induces partial cell cycle arrest at the G1 checkpoint, reduces cyclin D1 and E2F1 expression, and inhibits AMPK, MAPK, Akt, and mTOR activity [44, 57, 68]. Metformin-associated AMPK activation may also inactivate the insulin receptor substrate 1 (IRS1), which in turn regulates IGF-IR and PI3K/Akt signaling pathways to block the progrowth effects of hyperinsulinemia and insulin-like growth factors typically associated with type 2 diabetes [66, 67, 69].

Metformin is unique in the breadth and complexity of AMPK-dependent direct and indirect targets that inhibit cancer. Several new mechanisms fall into the rapidly expanding field of immuno-oncology. Metformin-induced activation of AMPK activates the programmed death ligand-1 (PDL-1) at S195, reducing stability and membrane localization and thus increasing PDL-1 degradation [70]. Metformin also promotes cytotoxic T cell lymphocyte activity in tumor tissue and enhances tumor-associated immune surveillance [6, 70, 71]. Additionally, metformin upregulates pro-inflammatory cytokines (tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), IL-1β, the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), the hypoxia-inducible factor 1-alpha (HIF-1α), and the vascular endothelial growth factor (VEGF), reviewed in [72, 73]).

AMPK-dependent mechanisms of action have been validated using clinical trial–derived BC samples as well as preclinical model systems, reviewed in detail elsewhere [43]. Some of these were especially important to spur the expansion of metformin use in BC patients. The timing, dose, and duration of metformin treatment in BC patients with or without other chemotherapy are actively under investigation. Neoadjuvant metformin, in particular, has shown benefit with a higher rate of complete pathological response, as compared to similar BC patients [74].

### **3.1 Metformin targets cell cycle proteins in AMPK-dependent manner in breast cancer**

AMPK plays an integral role in the regulation of cell cycle and cell division. The ability of metformin to activate AMPK thus has a significant inhibitory effect on cell-cycle associated proteins. This mechanism is represented in **Figure 2**. Expression profiling of BC derived from metformin-treated patients as compared to controls has shown consistent downregulation of many gene encoding proteins involved in mitosis, including kinesins, tubulins, histones, Aurora, as well as Polo-like kinases and ribosomal proteins (critical for protein and macromolecular biosynthesis, respectively) [75]. Given the targeted effects of metformin, it is not surprising that its actions are synergistic with drugs like paclitaxel that induce defects in mitotic spindle assembly, chromosome segregation, and cell division. In combination, metformin and paclitaxel dramatically increase the number of cells arrested in G2-M and apoptosis, as compared to either agent alone [76]. Metformin may also induce GO/G1 arrest due to activation of AMPK, downregulation of cyclin D1, and enhanced binding of CDK2 by p27Kip1 and p21cip1 [60, 61], especially in non-triple negative cells. Some have shown that metformin sensitivity to GO/G1

*Metformin*

in triple negative BC [60, 61].

glucose metabolism. Thus, metformin can indirectly affect AMPK, through reduction of gluconeogenesis and thus changing of the AMP:ATP ratio. These mechanisms are represented in **Figure 1**. These processes are modulated by P53 status. It is mutated in many BCs, particularly tumors that are high grade, late-stage or nonluminal in subtype. In BCs that are P53 competent, AMPK activation (from metformin or other triggers) upregulates P53 tumor suppressor activity as a downstream target. This induces activation of cell cycle checkpoint proteins, to inhibit cell proliferation [56]. In P53 incompetent cells, AMPK activation from metformin may be less effective through P53 mechanisms. Given the numerous other actions of metformin, as well as the molecular subtype specificity of the drug, we postulated that P53 status alone would not have a major impact on anticancer effects of metformin. We have demonstrated that this is the case in preclinical studies of numerous BC cell lines [57]. In other cells, metformin may induce cell cycle arrest and death through activation of apoptotic pathways and downregulation of p53 [58, 59] or PARP cleavage, especially

Activation of mTOR-dependent protein synthesis and cell growth (downstream

of the PI3K/Akt signaling axis), along with AMPK, provides a robust signaling platform for BC cell growth, proliferation, and chemotherapy resistance. In addition to activating AMPK, metformin inhibits mTOR and downstream signaling components of this critical pathway. Mutation of the PI3K catalytic subunit (PIK3CA) occurs in 20-35% of BCs [62, 63]. Mutation or loss of the tumor suppressor gene PTEN has also been demonstrated in 40% of BC [64, 65]. Metformin can also inhibit gluconeogenesis and mTOR signaling independent of AMPK and the tuberous sclerosis 2 (TSC2) gene in some experimental systems (in hepatic cells that lack AMPK or its kinase, LKB1). In this model system, metformin induces

*Metformin AMPK-dependent mechanism of action on breast cancer. Metformin activates AMPK directly through insulin-like growth factor (IGF-I) or insulin receptor, which in turn can activate PI3K/Akt/mTOR or RAS/Raf/MEK/ERK to increase cell growth, survival, angiogenesis, migration, and invasion. Metformin indirectly activates AMPK, which activates mTORC2, CREB, and gluconeogenesis. Lastly, glucose can enter BC* 

*cell through GLUT-1, and metformin can directly downregulate GLUT-1 receptor.*

**184**

**Figure 1.**

### **Figure 2.**

*AMPK-dependent action on cell cycle and alternate mechanisms. Metformin activates AMPK directly through insulin-like growth factor (IGF-I) or insulin receptor, which in turn can activate PI3K/Akt/mTOR or RAS/Raf/MEK/ERK signaling pathway. Metformin can also inhibit downstream signaling intermediates to attenuate autophagy, mRNA translation, cell growth, ribosome biogenesis, protein synthesis, and cell cycle growth. Metformin can also activate AMPK, which blocks P53 and induces cell cycle arrest. Lastly, metformin can block complex I of mitochondrial biogenesis to increase intracellular O2, which can block HIF-1 and VEGF production.*

arrest is linked to overexpression of p27Kip1 p21cip1 [60, 61]. We have demonstrated that metformin induces cycle arrest at the G1 checkpoint in luminal A, B and HER2 BC [75] associated with a reduction of cyclin D1 and E2F1 expression, with no changes in p27Kip1 or p21waf1. While these authors describe how metformin can increase CDK chemical inhibitors to control BC growth [57, 61], others have utilized cell cycle-dependent kinases (CDK) inhibitors with metformin and report that this combination should be used with caution [77].

In addition to downregulating cell replication under stress, metformin upregulates the cellular DNA-damage response, resulting in a decline in the mutational burden for those cancer cells that survive. Mechanisms underlying this effect include selective activation of the ataxia telangiectasia mutated (ATM) gene as well as ATM targets, such as protein kinase CHK2 gene and attenuation of reactive oxygen species ROS that result in DNA damage [78]. Algire et al. have postulated that downregulation of ROS production and thus somatic mutation are likely contributing mechanisms for the reduction in cancer risk associated with metformin use [8].

In summary, AMPK plays a central regulatory role in human cells, including BC where it regulates energy metabolism, cell growth and motility, response to insulin and growth factors, and estrogen production. Metformin induces AMPK activation in a robust manner, to affect numerous target pathways and intermediate molecules. The activity of AMPK and thus metformin can be modified by interacting factors including hormones, growth factors, and energy sensors. Selective targeting of AMPK-dependent pathways has shown less efficacy than metformin alone against BC [79], consistent with the findings that not all mechanisms of metformin action are AMPK dependent.

**187**

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

Upregulation of bioavailable glucose, insulin, and other growth factors increase the risk and promote BC aggression [16, 23, 27, 80, 81]. In addition to shifts in host metabolism, glycolytic reprogramming occurs in breast epithelial cells during malignant transformation. This process is accentuated by systemic dysregulation of carbohydrate and lipid metabolism, as bioavailable sugars and fat typically increase in these patients. Glycolytic reprogramming includes dependence on aerobic respiration, providing less-efficient energy (ATP) production per molecule of glucose from and incomplete oxidative phosphorylation. Cancer cell reprogramming includes activation of numerous signaling intermediaries, including phosphatidylinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), phosphatase and tensin homolog (PTEN), and AMPK [82–84]. Changes in other factors including c-MYC, hypoxia-inducible factor 1-alpha (HIF1α), epidermal growth factor receptor (EGFR), tumor protein 53 (P53), and the Met receptor may also facilitate cancer cell dependence on aerobic glycolysis [16, 85–87]. We have focused on the effects of extracellular glucose and other carbohydrates, combined with or without metformin using BC cell lines and animal models of obesity, metabolic syndrome, and mammary tumorigenesis, summarized in **Figure 3** and detailed elsewhere [26, 47, 49, 52, 57, 68, 88–93]. Importantly, most *in vitro* studies of metformin use commercially purchased media containing ~17 mM glucose (incompatible with human life, above concentrations achieved in diabetes). This is significantly higher than serum derived from normal persons (~5 mM), metabolic syndrome patients (~7 mM), or uncontrolled diabetes (~10 mM) [26]. We have shown that all molecular subtypes of BC cells grown with high glucose media require significantly more metformin to achieve the same anticancer efficacy (i.e., much higher EC50 of metformin) [26]. Normalization of glucose concentration in the culture media significantly reduced the EC50 of metformin for all BC cell types to induce BC growth inhibition or death. This hyperglycemic override of metformin action by dose makes biologic sense, given the ability of glucose to enter cells and promote many of the same pathways we have shown that are critical to metformin action. Similar issues may arise in animal models, particularly if the animals are overfed or obese. In both mouse and rat model systems, we have achieved plasma metformin concentrations equivalent to the normal range in humans, by providing it in the drinking water. We have also shown that metformin accumulates in the cytoplasm, markedly higher than serum levels in

**4. AMP-independent mechanisms of action on metformin**

mammary tumor cells with functional and sufficient OCT1 protein [26].

Luminal A and some subsets of triple negative BC cell lines show the greatest increase in proliferation when cultured in media with supraphysiologic glucose or insulin. In contrast, luminal B and HER2 BC cells were significantly less responsive to glucose or insulin, even at the highest concentrations examined. This responsivity pattern was similar to the cellular response to metformin by molecular BC subtype, with triple negative being the most responsive. From a molecular standpoint, triple negative BC cell responsivity to high glucose and metformin by dose was unique (efficacy at lower EC50s). Triple negative BC cells are especially dependent on glucose/glucosamine (metabolized through glycolysis) and lipids for energy and building block production, cell division, phenotypic aggression, and motility [94]. When grown with media containing supraphysiologic glucose, they upregulate specific genes, including EGFR, P-EGFR, IGF1R, P-IGF1R, IRS2, cyclin D1, and cyclin E expression, and inhibit AMPK/P-AMPK and p38 in a dose-dependent manner [26]. With the addition of metformin, there is a downregulation of these

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

**4.1 Metformin action on glucose and metabolism**

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*

### **4. AMP-independent mechanisms of action on metformin**

### **4.1 Metformin action on glucose and metabolism**

*Metformin*

**Figure 2.**

*production.*

arrest is linked to overexpression of p27Kip1 p21cip1 [60, 61]. We have demonstrated that metformin induces cycle arrest at the G1 checkpoint in luminal A, B and HER2 BC [75] associated with a reduction of cyclin D1 and E2F1 expression, with no changes in p27Kip1 or p21waf1. While these authors describe how metformin can increase CDK chemical inhibitors to control BC growth [57, 61], others have utilized cell cycle-dependent kinases (CDK) inhibitors with metformin and report that this

*AMPK-dependent action on cell cycle and alternate mechanisms. Metformin activates AMPK directly through insulin-like growth factor (IGF-I) or insulin receptor, which in turn can activate PI3K/Akt/mTOR or RAS/Raf/MEK/ERK signaling pathway. Metformin can also inhibit downstream signaling intermediates to attenuate autophagy, mRNA translation, cell growth, ribosome biogenesis, protein synthesis, and cell cycle growth. Metformin can also activate AMPK, which blocks P53 and induces cell cycle arrest. Lastly, metformin can block complex I of mitochondrial biogenesis to increase intracellular O2, which can block HIF-1 and VEGF* 

In addition to downregulating cell replication under stress, metformin upregulates the cellular DNA-damage response, resulting in a decline in the mutational burden for those cancer cells that survive. Mechanisms underlying this effect include selective activation of the ataxia telangiectasia mutated (ATM) gene as well as ATM targets, such as protein kinase CHK2 gene and attenuation of reactive oxygen species ROS that result in DNA damage [78]. Algire et al. have postulated that downregulation of ROS production and thus somatic mutation are likely contributing mechanisms for the reduction in cancer risk associated with metformin use [8]. In summary, AMPK plays a central regulatory role in human cells, including BC where it regulates energy metabolism, cell growth and motility, response to insulin and growth factors, and estrogen production. Metformin induces AMPK activation in a robust manner, to affect numerous target pathways and intermediate molecules. The activity of AMPK and thus metformin can be modified by interacting factors including hormones, growth factors, and energy sensors. Selective targeting of AMPK-dependent pathways has shown less efficacy than metformin alone against BC [79], consistent with the findings that not all mechanisms of metformin action

combination should be used with caution [77].

**186**

are AMPK dependent.

Upregulation of bioavailable glucose, insulin, and other growth factors increase the risk and promote BC aggression [16, 23, 27, 80, 81]. In addition to shifts in host metabolism, glycolytic reprogramming occurs in breast epithelial cells during malignant transformation. This process is accentuated by systemic dysregulation of carbohydrate and lipid metabolism, as bioavailable sugars and fat typically increase in these patients. Glycolytic reprogramming includes dependence on aerobic respiration, providing less-efficient energy (ATP) production per molecule of glucose from and incomplete oxidative phosphorylation. Cancer cell reprogramming includes activation of numerous signaling intermediaries, including phosphatidylinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), phosphatase and tensin homolog (PTEN), and AMPK [82–84]. Changes in other factors including c-MYC, hypoxia-inducible factor 1-alpha (HIF1α), epidermal growth factor receptor (EGFR), tumor protein 53 (P53), and the Met receptor may also facilitate cancer cell dependence on aerobic glycolysis [16, 85–87].

We have focused on the effects of extracellular glucose and other carbohydrates, combined with or without metformin using BC cell lines and animal models of obesity, metabolic syndrome, and mammary tumorigenesis, summarized in **Figure 3** and detailed elsewhere [26, 47, 49, 52, 57, 68, 88–93]. Importantly, most *in vitro* studies of metformin use commercially purchased media containing ~17 mM glucose (incompatible with human life, above concentrations achieved in diabetes). This is significantly higher than serum derived from normal persons (~5 mM), metabolic syndrome patients (~7 mM), or uncontrolled diabetes (~10 mM) [26]. We have shown that all molecular subtypes of BC cells grown with high glucose media require significantly more metformin to achieve the same anticancer efficacy (i.e., much higher EC50 of metformin) [26]. Normalization of glucose concentration in the culture media significantly reduced the EC50 of metformin for all BC cell types to induce BC growth inhibition or death. This hyperglycemic override of metformin action by dose makes biologic sense, given the ability of glucose to enter cells and promote many of the same pathways we have shown that are critical to metformin action. Similar issues may arise in animal models, particularly if the animals are overfed or obese. In both mouse and rat model systems, we have achieved plasma metformin concentrations equivalent to the normal range in humans, by providing it in the drinking water. We have also shown that metformin accumulates in the cytoplasm, markedly higher than serum levels in mammary tumor cells with functional and sufficient OCT1 protein [26].

Luminal A and some subsets of triple negative BC cell lines show the greatest increase in proliferation when cultured in media with supraphysiologic glucose or insulin. In contrast, luminal B and HER2 BC cells were significantly less responsive to glucose or insulin, even at the highest concentrations examined. This responsivity pattern was similar to the cellular response to metformin by molecular BC subtype, with triple negative being the most responsive. From a molecular standpoint, triple negative BC cell responsivity to high glucose and metformin by dose was unique (efficacy at lower EC50s). Triple negative BC cells are especially dependent on glucose/glucosamine (metabolized through glycolysis) and lipids for energy and building block production, cell division, phenotypic aggression, and motility [94]. When grown with media containing supraphysiologic glucose, they upregulate specific genes, including EGFR, P-EGFR, IGF1R, P-IGF1R, IRS2, cyclin D1, and cyclin E expression, and inhibit AMPK/P-AMPK and p38 in a dose-dependent manner [26]. With the addition of metformin, there is a downregulation of these

### **Figure 3.**

*Metformin action on glucose and metabolism breast cancer. Metformin enters the BC cell through OCT 1 transporter to attenuate inner membrane fluidity/permeability, the Krebs cycle (TCA), and complex I of the mitochondria. Metformin can also block downstream signaling intermediates involved in the PI3K/Akt/mTOR or RAS/Raf/MEK/ERK signaling pathways, which can control BC cell growth. Lastly, metformin blocks GLUT1 transporter and key enzymes that are involved in carbohydrate synthesis.*

genes and the upregulation of genes associated with cell killing and growth control [49, 94]. Our report showed that glucose promotes phenotypic aggression and reduces metformin efficacy by targeting key enzymes that are required for glucose metabolism in TNBC. Such enzymes include G6PD, Fructose-2-6-BP, PGK, PGM, ENO, PKM2, and LDH-A (shown in **Figure 3** and reviewed in [49]). Further, we reported that metformin attenuated the expression of over 20 critical genes involved in glucose metabolism, glucose transporters, gluconeogenesis, and tricarboxylic acid cycle [49]. Metformin-associated gene expression changes also reduced phenotypic aggressiveness and stem-like progenitor cell pool [26, 49, 90, 92]. Metformin treatment also restricted cell proliferation with S phase arrest, motility (through downregulation of intermediate filament proteins), and increased apoptosis (through activation of both the intrinsic and extrinsic pathways) [26, 47, 57, 88, 89, 92]. Metformin significantly inhibits carbohydrate induced pro-oncogenic metabolic and biologic characteristics of triple negative BC cells [26]. Altogether, metformin's ability to target key glucose transporters, such a GLUT1, along with key genes involved in glucose and carbohydrate metabolism, highlights the role that this agent may play to control highly aggressive malignant BC cells via downregulation of the cellular metabolic machinery.

We have also shown that inhibition of lipid biosynthesis was requisite to the anticancer effects of metformin in triple negative BC cells. It downregulates both

**189**

patients.

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

**4.2 Metformin action on cholesterol, EGFR signaling, and lipid rafts**

The mevalonate pathway, also known as the β-hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase pathway, is critical for cancer cell survival. Inhibition of the pathway by statins or other agents has been shown to have anticancer effects [96, 97]. In contrast, elevated cholesterol has been strongly associated with BC risk, a worse BC-associated outcome and chemotherapeutic resistance. This reflects the pivotal role of lipids including cholesterol in cancer survival and growth, including upregulation of signaling through membrane-bound receptors, facilitation of intracellular signaling pathways, and serving as an anchor for intracytoplasmic filaments to promote motility and invasion and as a precursor for cellular metabolism to generate energy and facilitate replication [98, 99]. We have shown that triple negative BC cells are especially dependent on the upregulation of

Statins are widely prescribed for patients with high cholesterol or lipid abnormalities, most often to reduce the risk of cardiovascular disease. Statins also benefit women to reduce the risk and disease progression of BC. Two population-based studies from Northern Europe are particularly compelling. A Finnish study involving over 30,000 women showed that statin use, pre- or post-BC diagnosis, reduced BC-specific mortality by about 50% [101]. A large Danish study showed a benefit for BC patients as well, with significantly lower recurrence rates in statin users as compared to nonusers. They also reported that lipophilic statins (rather than hydrophilic satins) had the most anti-BC activity [102]. A recent study from MD Anderson Cancer Center suggests that statin use is particularly beneficial for BC patients with triple negative tumors, especially in patients with higher stage disease [95]. Their data are consistent with our preclinical data, showing significant upregulation of lipid metabolism-associated gene triple negative BC as compared to other molecular subtypes. See for further discussion elsewhere [103]. A major issue with statin use is toxicity, which reportedly occurs in up to half of patients. Some statin drugs are also expensive and thus may be unaffordable by many

Metformin, in contrast, is relatively nontoxic and inexpensive. We have demonstrated that metformin has potent effects in lipid and cholesterol biosynthesis in BC cells. More specifically, it inhibits transcriptional activation of HMGCo-A

fatty acid synthase (FASN) and the cholesterol biosynthesis pathway, as detailed below. Other studies have focused on interactions between obesity, weight gain, hormonal status, and BC, and more specifically if metformin could be used to disrupt this process. Using a rat model of mammary tumor development after exposure to a carcinogen, animals were overfed and then segregated into lean and obese. Both subsets were subjected to ovary removal, half were given metformin, and they were followed for the development and progression of mammary tumors [52, 93, 95]. Obese rats experienced marked changes in metabolism, akin to metabolic syndrome. Mammary tumors from these obese rats showed enhanced tumor growth and tumor-associated glucose uptake, 50% higher than nonobese rats in association with upregulation of the progesterone receptor. In contrast, the lean rats preferentially deposited excess nutrients in mammary (nontumor) and peripheral tissues. Metformin abrogated systemic metabolic dysregulation, reduced tumorigenesis, tumor progression, and tumor-associated PR expression in obese rats. Similar changes in body weight and obesity are frequent after female menopause has been observed in BC of postmenopausal females with obesity, providing additional clues for the use and timing of metformin associated with BC risk and

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

treatment for future study.

lipid and cholesterol biosynthesis [100].

### *Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*

fatty acid synthase (FASN) and the cholesterol biosynthesis pathway, as detailed below. Other studies have focused on interactions between obesity, weight gain, hormonal status, and BC, and more specifically if metformin could be used to disrupt this process. Using a rat model of mammary tumor development after exposure to a carcinogen, animals were overfed and then segregated into lean and obese. Both subsets were subjected to ovary removal, half were given metformin, and they were followed for the development and progression of mammary tumors [52, 93, 95]. Obese rats experienced marked changes in metabolism, akin to metabolic syndrome. Mammary tumors from these obese rats showed enhanced tumor growth and tumor-associated glucose uptake, 50% higher than nonobese rats in association with upregulation of the progesterone receptor. In contrast, the lean rats preferentially deposited excess nutrients in mammary (nontumor) and peripheral tissues. Metformin abrogated systemic metabolic dysregulation, reduced tumorigenesis, tumor progression, and tumor-associated PR expression in obese rats. Similar changes in body weight and obesity are frequent after female menopause has been observed in BC of postmenopausal females with obesity, providing additional clues for the use and timing of metformin associated with BC risk and treatment for future study.

### **4.2 Metformin action on cholesterol, EGFR signaling, and lipid rafts**

The mevalonate pathway, also known as the β-hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase pathway, is critical for cancer cell survival. Inhibition of the pathway by statins or other agents has been shown to have anticancer effects [96, 97]. In contrast, elevated cholesterol has been strongly associated with BC risk, a worse BC-associated outcome and chemotherapeutic resistance. This reflects the pivotal role of lipids including cholesterol in cancer survival and growth, including upregulation of signaling through membrane-bound receptors, facilitation of intracellular signaling pathways, and serving as an anchor for intracytoplasmic filaments to promote motility and invasion and as a precursor for cellular metabolism to generate energy and facilitate replication [98, 99]. We have shown that triple negative BC cells are especially dependent on the upregulation of lipid and cholesterol biosynthesis [100].

Statins are widely prescribed for patients with high cholesterol or lipid abnormalities, most often to reduce the risk of cardiovascular disease. Statins also benefit women to reduce the risk and disease progression of BC. Two population-based studies from Northern Europe are particularly compelling. A Finnish study involving over 30,000 women showed that statin use, pre- or post-BC diagnosis, reduced BC-specific mortality by about 50% [101]. A large Danish study showed a benefit for BC patients as well, with significantly lower recurrence rates in statin users as compared to nonusers. They also reported that lipophilic statins (rather than hydrophilic satins) had the most anti-BC activity [102]. A recent study from MD Anderson Cancer Center suggests that statin use is particularly beneficial for BC patients with triple negative tumors, especially in patients with higher stage disease [95]. Their data are consistent with our preclinical data, showing significant upregulation of lipid metabolism-associated gene triple negative BC as compared to other molecular subtypes. See for further discussion elsewhere [103]. A major issue with statin use is toxicity, which reportedly occurs in up to half of patients. Some statin drugs are also expensive and thus may be unaffordable by many patients.

Metformin, in contrast, is relatively nontoxic and inexpensive. We have demonstrated that metformin has potent effects in lipid and cholesterol biosynthesis in BC cells. More specifically, it inhibits transcriptional activation of HMGCo-A

*Metformin*

**Figure 3.**

**188**

of the cellular metabolic machinery.

genes and the upregulation of genes associated with cell killing and growth control [49, 94]. Our report showed that glucose promotes phenotypic aggression and reduces metformin efficacy by targeting key enzymes that are required for glucose metabolism in TNBC. Such enzymes include G6PD, Fructose-2-6-BP, PGK, PGM, ENO, PKM2, and LDH-A (shown in **Figure 3** and reviewed in [49]). Further, we reported that metformin attenuated the expression of over 20 critical genes involved in glucose metabolism, glucose transporters, gluconeogenesis, and tricarboxylic acid cycle [49]. Metformin-associated gene expression changes also reduced phenotypic aggressiveness and stem-like progenitor cell pool [26, 49, 90, 92]. Metformin treatment also restricted cell proliferation with S phase arrest, motility (through downregulation of intermediate filament proteins), and increased apoptosis (through activation of both the intrinsic and extrinsic pathways) [26, 47, 57, 88, 89, 92]. Metformin significantly inhibits carbohydrate induced pro-oncogenic metabolic and biologic characteristics of triple negative BC cells [26]. Altogether, metformin's ability to target key glucose transporters, such a GLUT1, along with key genes involved in glucose and carbohydrate metabolism, highlights the role that this agent may play to control highly aggressive malignant BC cells via downregulation

*Metformin action on glucose and metabolism breast cancer. Metformin enters the BC cell through OCT 1 transporter to attenuate inner membrane fluidity/permeability, the Krebs cycle (TCA), and complex I of the mitochondria. Metformin can also block downstream signaling intermediates involved in the PI3K/Akt/mTOR or RAS/Raf/MEK/ERK signaling pathways, which can control BC cell growth. Lastly, metformin blocks* 

*GLUT1 transporter and key enzymes that are involved in carbohydrate synthesis.*

We have also shown that inhibition of lipid biosynthesis was requisite to the anticancer effects of metformin in triple negative BC cells. It downregulates both (the enzyme targeted by statins), as well as over 20 other genes in the cholesterol biosynthesis pathway. We have also shown that it induces translational activation of downstream signaling, including the genes ACAA2, HMGCS1, HMGCR, MVK, MVD, LSS, and DHCR24 (**Figure 4**). Through broad inhibition of cholesterol biosynthesis in triple negative BC, metformin induces a significant reduction of membrane-associated and intracellular cholesterol and reduces GM1 lipid rafts through decreased synthesis and destabilization (disassociation). GM1 lipid raft stability has a profound effect on some receptors that rely on GM1 lipid rafts (like EGFR) for stability, ligand binding, and thus activation, resulting in downstream signaling. We have shown that metformin inhibits cholesterol biosynthesis and raft production, reducing membranous EGFR and its activation associated with downstream signaling in TNBC [91]. We have also shown that in combination, metformin and the statin-mimetic MβCD were synergistic in attenuating cholesterol biosynthesis and cell proliferation [91]. Others have validated our observation that metformin downregulates genes involved in cholesterol biosynthesis, reporting downregulation of HMGCR, LDLR, and SREBP1 [104]. A particularly exciting corollary of these findings is the potential of metformin to synergize with receptor tyrosine kinase inhibitors (RTKIs) against BC. This is an underexplored area of breast oncology research with tremendous translational potential, given the growing use of RTKIs against BC.

### **Figure 4.**

*Metformin action on cholesterol synthesis and lipid rafts. Metformin blocks epidermal growth factor receptor (EGFR), human epidermal growth factor receptors 2/3 (HER2/HER3), which in turn can block key enzymes involved in cholesterol synthesis pathway. Metformin and statins both can inhibit rate limiting step HMG-CoA Reductase, HMGCR. Metformin can also decrease cellular membrane rigidity, increase fluidity, and decrease cholesterol content to allow for the internalization of EGFR, HER2, or HER3 receptors. Internalization of these receptors is through GM1 lipid rafts, which are degraded and allow for BC cell death.*

**191**

**Figure 5.**

*cell death.*

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

MiRNAs are endogenous, short (21-25) nucleotide sequences that control gene expression during post-transcriptional translation. It has previously been reported that more than half of human genes are regulated by miRNAs [105]. A growing body of evidence has highlighted the role of miRNAs as master regulators of metabolic processes, such as lipid and cholesterol synthesis [92, 105, 106]. Perturbations of these processes are important for tumor development. Modulation of these regulators using synthetic antagomirs to block the activity of specific miRNAs is an important new area of breast research. Metformin exerts some of its anticancer activity through modulation of miRNAs that target genes in metabolic and other pathways (**Figure 5**) [92, 107, 108]. miRNAs have been reported to be potential biomarkers for BC (i.e., *miR-9, miR-10b,* and *miR-17-5p*), whereas others reportedly have prognostic (i.e., *miR-148a* and *miR-335*) or predictive relevance (i.e., *miR-26a,* 

We have shown that metformin increases several members of the miR-193 family. It upregulates miR-193b, which in turn targets and downregulates the FASN 3'UTR. FASN is an important component of *de novo* fatty acid synthesis. Using an miR-193b mimetic, we induced a drastic reduction in fatty acid synthase (FASN) protein expression as well as increased growth inhibition and apoptosis of TNBC [92]. A separate expression profiling study of metformin-treated TNBC cells has shown similar results [106]. These data show that inhibition of FASN and fatty acid

*Metformin action on lipid synthesis and miRNAs metformin blocks EGFR, HER2, and HER3, which in turn can block key enzymes involved in cholesterol synthesis pathway as described in Figure 4. Metformin can also block acetyl-CoA carboxylase (ACC), which in turn can decrease fatty acid synthase (FASN). Metformin can also increase a myriad of miRNAS (shown in green). One of these miRNAs (miR-193b) can target FASN, which can decrease fatty acid synthesis in BC cells. Additionally metformin can block FASN and increase BC* 

biosynthesis contributes to the potency of metformin against BC cells.

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

*miR-30c, miR-187,* and *miR-339-5p*) [109].

**4.3 Metformin action on miRNA and FASN signaling**

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*

### **4.3 Metformin action on miRNA and FASN signaling**

*Metformin*

ing use of RTKIs against BC.

(the enzyme targeted by statins), as well as over 20 other genes in the cholesterol biosynthesis pathway. We have also shown that it induces translational activation of downstream signaling, including the genes ACAA2, HMGCS1, HMGCR, MVK, MVD, LSS, and DHCR24 (**Figure 4**). Through broad inhibition of cholesterol biosynthesis in triple negative BC, metformin induces a significant reduction of membrane-associated and intracellular cholesterol and reduces GM1 lipid rafts through decreased synthesis and destabilization (disassociation). GM1 lipid raft stability has a profound effect on some receptors that rely on GM1 lipid rafts (like EGFR) for stability, ligand binding, and thus activation, resulting in downstream signaling. We have shown that metformin inhibits cholesterol biosynthesis and raft production, reducing membranous EGFR and its activation associated with downstream signaling in TNBC [91]. We have also shown that in combination, metformin and the statin-mimetic MβCD were synergistic in attenuating cholesterol biosynthesis and cell proliferation [91]. Others have validated our observation that metformin downregulates genes involved in cholesterol biosynthesis, reporting downregulation of HMGCR, LDLR, and SREBP1 [104]. A particularly exciting corollary of these findings is the potential of metformin to synergize with receptor tyrosine kinase inhibitors (RTKIs) against BC. This is an underexplored area of breast oncology research with tremendous translational potential, given the grow-

*Metformin action on cholesterol synthesis and lipid rafts. Metformin blocks epidermal growth factor receptor (EGFR), human epidermal growth factor receptors 2/3 (HER2/HER3), which in turn can block key enzymes involved in cholesterol synthesis pathway. Metformin and statins both can inhibit rate limiting step HMG-CoA Reductase, HMGCR. Metformin can also decrease cellular membrane rigidity, increase fluidity, and decrease cholesterol content to allow for the internalization of EGFR, HER2, or HER3 receptors. Internalization of these* 

*receptors is through GM1 lipid rafts, which are degraded and allow for BC cell death.*

**190**

**Figure 4.**

MiRNAs are endogenous, short (21-25) nucleotide sequences that control gene expression during post-transcriptional translation. It has previously been reported that more than half of human genes are regulated by miRNAs [105]. A growing body of evidence has highlighted the role of miRNAs as master regulators of metabolic processes, such as lipid and cholesterol synthesis [92, 105, 106]. Perturbations of these processes are important for tumor development. Modulation of these regulators using synthetic antagomirs to block the activity of specific miRNAs is an important new area of breast research. Metformin exerts some of its anticancer activity through modulation of miRNAs that target genes in metabolic and other pathways (**Figure 5**) [92, 107, 108]. miRNAs have been reported to be potential biomarkers for BC (i.e., *miR-9, miR-10b,* and *miR-17-5p*), whereas others reportedly have prognostic (i.e., *miR-148a* and *miR-335*) or predictive relevance (i.e., *miR-26a, miR-30c, miR-187,* and *miR-339-5p*) [109].

We have shown that metformin increases several members of the miR-193 family. It upregulates miR-193b, which in turn targets and downregulates the FASN 3'UTR. FASN is an important component of *de novo* fatty acid synthesis. Using an miR-193b mimetic, we induced a drastic reduction in fatty acid synthase (FASN) protein expression as well as increased growth inhibition and apoptosis of TNBC [92]. A separate expression profiling study of metformin-treated TNBC cells has shown similar results [106]. These data show that inhibition of FASN and fatty acid biosynthesis contributes to the potency of metformin against BC cells.

### **Figure 5.**

*Metformin action on lipid synthesis and miRNAs metformin blocks EGFR, HER2, and HER3, which in turn can block key enzymes involved in cholesterol synthesis pathway as described in Figure 4. Metformin can also block acetyl-CoA carboxylase (ACC), which in turn can decrease fatty acid synthase (FASN). Metformin can also increase a myriad of miRNAS (shown in green). One of these miRNAs (miR-193b) can target FASN, which can decrease fatty acid synthesis in BC cells. Additionally metformin can block FASN and increase BC cell death.*

### **4.4 Metformin action on PI3K/Akt/mTOR signaling in breast cancer**

The PI3K/Akt/mTOR pathway plays a central role in regulating protein synthesis, cell proliferation, tumorigenesis, angiogenesis, tumor growth, and metastasis [63]. While AMPK-dependent phosphorylation is frequently described in metformin-mediated inhibition of the PI3K/Akt/mTOR signaling pathway, AMPK activation is not mandatory for these effects; see schematic in **Figure 6** [57]. We have shown that metformin inhibits Akt and mTOR and inhibits cellular proliferation and colony formation and causes a partial G1 cell cycle arrest in all ER-positive, HER2 normal or abnormal BC cell lines examined [57]. Metformin-mediated inhibition of the PI3K/Akt/mTOR signaling pathway has also been shown to induce inhibition of cell replication, S phase arrest, and apoptosis, with a reduction in E2F1 and cyclin D1 expression in triple negative BC cell lines [57].

### **4.5 Metformin action in STAT3 signaling**

TNBC shows high activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway, which in turn promotes cell growth, invasion, migration, metastasis, angiogenesis, immune evasion, and drug resistance and inhibits apoptosis [88]. We have shown that metformin specifically targets STAT3 signaling to reduce P-STAT3 at both Ser727 and Tyr705 phosphorylation sites but not STAT3 expression in TNBC, schematically represented in **Figure 6**. In combination with a Stat3 inhibitor, metformin significantly downregulated STAT3

### **Figure 6.**

*Metformin action in breast cancer. Metformin can block receptor tyrosine kinase (RTK), such as EGFR, HER2, and HER3. Metformin further blocks downstream signaling intermediates involved in PI3K/Akt/mTOR or RAS/Raf/MEK/ERK signaling pathway, such as AKT, mTOR, MEK, or ERK, which can decrease cell growth, angiogenesis, and migration/invasion. Further, metformin can further block cytokine and growth factor receptors such as the TGF-RII. Metformin can block IL-6/STAT3 pathway and TGF-signaling pathway, which in turn can decrease cell growth, angiogenesis, migration/invasion, inflammation, and EMT.*

**193**

patients.

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

expression and was synergistic in reducing cell growth and the induction of apoptosis in TNBC [88]. Given that TNBC also shows an upregulation/activation of the PI3K/Akt/mTOR signaling pathways, we then combined metformin with an mTOR inhibitor rapamycin, to determine if it would reduce metformin efficacy. Significant interactions with metformin were not observed; thus, mechanisms underlying its

The JAK/STAT pathway is upregulated by obesity-associated mechanisms that promote BC growth. Others have demonstrated that metformin attenuates Janus kinase (JAK)/STAT3 signaling at Ser515 and Ser518 within the Src homology 2 domain of JAK1 [110]. Metformin has also been shown to preferentially inhibit nuclear translocation of NK-B and phosphorylation of STAT3 in cancer stem cells (CSCs) as compared to non-CSCs [111]. Given the procarcinogenic and prometastatic role that JAK/STAT pathways play in TNBC, the development of therapeutic strategies to attenuate these pathways using metformin may provide benefit with

A subset of TNBC subclassified as mesenchymal-stem like/claudin-low (MSL/ CL) characteristically shows high expression and activation of TGF-β signaling, phenotypic aggression, and a worse outcome. In addition to TGF-β receptor 2 expression, BC in this group shows upregulation of Smad2, Smad3, ID1, and ID3 [90]. They are especially responsive to TGF-β ligand 1 (TGF-β1), resulting in cell proliferation, migration, and invasion. MSL/CL cell lines also demonstrate downregulation of several growth factor receptors in response to metformin, including fibroblast growth factor receptors (FGFR2 and FGFR3), hormone receptors (AR, ESR1, and PGR), and claudin integral membrane proteins of tight junctions (CLDN3, CLDN4, and CLDN7) in the MSL/CL BC subtypes [90]. Metformin directly attenuated TGF-β signaling pathway by downregulating activation of Smad2/Smad3, ID1, and ID3 (**Figure 6**). In combination with TGF-β inhibitors (TβRI-KIs; LY2197s299 or SB431542), metformin synergistically enhanced cell death in MSL/CL BC cells [90]. Overall, these data suggest that targeting TGF-β signaling using metformin with or without a TGF-β inhibitor may provide benefit

The process of epithelial-mesenchymal transition (EMT) is also common in TNBC and has been associated with biologic aggression and stem-like properties. Metformin reportedly inhibits EMT in a metastatic canine model of mammary cancer [112]. Others have shown that metformin reduces EMT through blockade of transcription factors like ZEB1, TWIST1, and SNAIL (Slug) [113–115]. Given that TGF-β pathway activation and EMT promote breast cancer stem cells (BCSC), therapeutic resistance, dormancy, and a poor outcome [113], and that metformin has been shown to block these in TNBC, inhibitors against TGF-βinduced EMT combined with metformin may provide benefit in some TNBC

**4.7 Metformin action on breast cancer and angiogenesis, and the** 

Clinical studies have demonstrated that diabetic patients treated with metformin are less likely to develop cardiovascular disease, independent of glycemic control. It is unclear whether this outcome reflects downregulation of hyperglycemia and systemic inflammatory triggers or vascular damage, or whether metformin has a direct effect on endothelial cells, vascular resistance, elasticity, and damage

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

effects are not dependent on mTOR.

**4.6 Metformin and TGF-β signaling in TNBC**

for patients with MSL/CL BCs.

**microenvironment**

limited toxicity.

### *Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*

expression and was synergistic in reducing cell growth and the induction of apoptosis in TNBC [88]. Given that TNBC also shows an upregulation/activation of the PI3K/Akt/mTOR signaling pathways, we then combined metformin with an mTOR inhibitor rapamycin, to determine if it would reduce metformin efficacy. Significant interactions with metformin were not observed; thus, mechanisms underlying its effects are not dependent on mTOR.

The JAK/STAT pathway is upregulated by obesity-associated mechanisms that promote BC growth. Others have demonstrated that metformin attenuates Janus kinase (JAK)/STAT3 signaling at Ser515 and Ser518 within the Src homology 2 domain of JAK1 [110]. Metformin has also been shown to preferentially inhibit nuclear translocation of NK-B and phosphorylation of STAT3 in cancer stem cells (CSCs) as compared to non-CSCs [111]. Given the procarcinogenic and prometastatic role that JAK/STAT pathways play in TNBC, the development of therapeutic strategies to attenuate these pathways using metformin may provide benefit with limited toxicity.

### **4.6 Metformin and TGF-β signaling in TNBC**

A subset of TNBC subclassified as mesenchymal-stem like/claudin-low (MSL/ CL) characteristically shows high expression and activation of TGF-β signaling, phenotypic aggression, and a worse outcome. In addition to TGF-β receptor 2 expression, BC in this group shows upregulation of Smad2, Smad3, ID1, and ID3 [90]. They are especially responsive to TGF-β ligand 1 (TGF-β1), resulting in cell proliferation, migration, and invasion. MSL/CL cell lines also demonstrate downregulation of several growth factor receptors in response to metformin, including fibroblast growth factor receptors (FGFR2 and FGFR3), hormone receptors (AR, ESR1, and PGR), and claudin integral membrane proteins of tight junctions (CLDN3, CLDN4, and CLDN7) in the MSL/CL BC subtypes [90]. Metformin directly attenuated TGF-β signaling pathway by downregulating activation of Smad2/Smad3, ID1, and ID3 (**Figure 6**). In combination with TGF-β inhibitors (TβRI-KIs; LY2197s299 or SB431542), metformin synergistically enhanced cell death in MSL/CL BC cells [90]. Overall, these data suggest that targeting TGF-β signaling using metformin with or without a TGF-β inhibitor may provide benefit for patients with MSL/CL BCs.

The process of epithelial-mesenchymal transition (EMT) is also common in TNBC and has been associated with biologic aggression and stem-like properties. Metformin reportedly inhibits EMT in a metastatic canine model of mammary cancer [112]. Others have shown that metformin reduces EMT through blockade of transcription factors like ZEB1, TWIST1, and SNAIL (Slug) [113–115]. Given that TGF-β pathway activation and EMT promote breast cancer stem cells (BCSC), therapeutic resistance, dormancy, and a poor outcome [113], and that metformin has been shown to block these in TNBC, inhibitors against TGF-βinduced EMT combined with metformin may provide benefit in some TNBC patients.

### **4.7 Metformin action on breast cancer and angiogenesis, and the microenvironment**

Clinical studies have demonstrated that diabetic patients treated with metformin are less likely to develop cardiovascular disease, independent of glycemic control. It is unclear whether this outcome reflects downregulation of hyperglycemia and systemic inflammatory triggers or vascular damage, or whether metformin has a direct effect on endothelial cells, vascular resistance, elasticity, and damage

*Metformin*

**4.4 Metformin action on PI3K/Akt/mTOR signaling in breast cancer**

and cyclin D1 expression in triple negative BC cell lines [57].

**4.5 Metformin action in STAT3 signaling**

The PI3K/Akt/mTOR pathway plays a central role in regulating protein synthesis, cell proliferation, tumorigenesis, angiogenesis, tumor growth, and metastasis [63]. While AMPK-dependent phosphorylation is frequently described in metformin-mediated inhibition of the PI3K/Akt/mTOR signaling pathway, AMPK activation is not mandatory for these effects; see schematic in **Figure 6** [57]. We have shown that metformin inhibits Akt and mTOR and inhibits cellular proliferation and colony formation and causes a partial G1 cell cycle arrest in all ER-positive, HER2 normal or abnormal BC cell lines examined [57]. Metformin-mediated inhibition of the PI3K/Akt/mTOR signaling pathway has also been shown to induce inhibition of cell replication, S phase arrest, and apoptosis, with a reduction in E2F1

TNBC shows high activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway, which in turn promotes cell growth, invasion, migration, metastasis, angiogenesis, immune evasion, and drug resistance and inhibits apoptosis [88]. We have shown that metformin specifically targets STAT3 signaling to reduce P-STAT3 at both Ser727 and Tyr705 phosphorylation sites but not STAT3 expression in TNBC, schematically represented in **Figure 6**. In combination with a Stat3 inhibitor, metformin significantly downregulated STAT3

*Metformin action in breast cancer. Metformin can block receptor tyrosine kinase (RTK), such as EGFR, HER2, and HER3. Metformin further blocks downstream signaling intermediates involved in PI3K/Akt/mTOR or RAS/Raf/MEK/ERK signaling pathway, such as AKT, mTOR, MEK, or ERK, which can decrease cell growth, angiogenesis, and migration/invasion. Further, metformin can further block cytokine and growth factor receptors such as the TGF-RII. Metformin can block IL-6/STAT3 pathway and TGF-signaling pathway, which* 

*in turn can decrease cell growth, angiogenesis, migration/invasion, inflammation, and EMT.*

**192**

**Figure 6.**

[12, 80, 116]. In the context of breast cancer, it has long been demonstrated that high-stage and grade cancers with a worse prognosis have the capacity to upregulate peri- and intratumoral neo-angiogenesis [117]. The induction of new vessels provides metabolic and oxygen delivery advantages to the cancer cells, facilitating survival and growth. Neo-angiogenesis is also associated with an increased capacity of the BC to metastasize, particularly to distant sites including the visceral organs and brain. We have demonstrated a reduction in vascular density and growth, in association with metformin treatment in preclinical models. Others have shown that metformin is associated with reduced tumor angiogenesis in many different cancer cell types. Metformin and alternate biguanides, such as phenformin, downregulate VEGF-dependent activation of ERK1, inhibiting neo-angiogenesis and reducing microvessel density (MVD) [118]. Wang et al. have shown that metformin also downregulates the expression of two other genes, platelet-derived growth factor B (PDGF-B) and fibroblast growth factor (FGF-2), to reduce angiogenesis [119]. Downregulation of PDGF-B also restricts BC cell proliferation, survival, and migration, [117]. Metformin's effect on the microenvironment and angiogenesis has also been shown to enhance chemo-sensitivity, via a reduction in MVD leakage and cancer cell hypoxia *in vivo* [117]. Thus, metformin's effects go beyond the cancer cell itself and include the peri- and intratumoral microenvironment and neovasculature.

### **Figure 7.**

*Metformin action on breast cancer stem cells. Metformin can block a myriad of signaling pathways involved in BCSCs, including WNT, transforming growth factor (TGF), NOTCH, hypoxia inducible factor (HIF), and STAT3 signaling pathways. These pathways are thought to enrich for BCSC through the enrichment of CD44 positive receptor and aldehyde dehydrogenase (ALDH+) and decrease in CD24 expression. Metformin can be given as a monotherapy or combinatorial therapy with alternate chemotherapeutic agents, which in turn can induce BCSC death with an increase in apoptosis, cell cycle arrest, and DNA damage. Overall, reduction in BCSCs can result in reduction of tumor growth and prevention in therapy-mediated relapse.*

**195**

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are the progenitor cells that give rise to BC as well as heterogeneity within transformed populations. CSCs are maintained as a subpopulation within the neoplasm that perpetuates clonal expansion and may facilitate dormancy, metastasis, chemoresistance, and relapse. Among the molecular BC subtypes, TNBC shows the highest enrichment of CSCs, identified by expression patterns with flow cytometry as CD44+, CD24−/low CSC [120]. BC CSCs are particularly sensitive to metformin, which induces rapid cell death facilitated through a number of pathways involved in cell differentiation, renewal, metastasis, and metabolism (**Figure 7**). It directly targets key CSC gene signatures such as Notch 1, NF*κ*B, Sox2, KLF-4, Oct4, Lin28, MMP-9, and MMP-2 [121]. Metformin attenuates CSCs in resistant BC, through repression of let-7 miRNA [121]. Its ability to attenuate key metabolic genes, such as FASN via upregulation of miR-193b, also contributes to its anti-CSC activity as stem

The capacity of metformin to induce CSC cell death has significant clinical relevance, given their role in therapeutic resistance, dormancy, and disease progression. Metformin reduces cancer recurrence through the preferential killing of differentiated rather than undifferentiated CSCs [122]. In combination with chemotherapy, metformin is especially active against BC CSCs [111]. In studies of trastuzumab-resistant BC cells as well as xenograft models, the combination of trastuzumab and metformin significantly reduced CD44+, CD24−/low CSC subpopulations and reduced tumor volume [111, 123, 124]. In combination with doxorubicin, paclitaxel, or carboplatin, metformin can also eradicate CSCs and reduce the effective dosage required of the highly toxic chemotherapeutic agents,

**5. Clinical evidence with metformin in breast cancer prevention** 

The pleiotropic oncostatic effects of metformin have been explored as an adjuvant therapeutic option for the management of BC [43, 125, 126]. Epidemiological studies have demonstrated associations between metformin use in patients with type 2 diabetes and decreased cancer incidence and cancer-related mortality [10]. Several observational and randomized trials have evaluated a number of biomarker changes after metformin administration, increasing the footage of metformin as an off-label agent for BC. Over 11 ongoing and 13 completed clinical trials have tested the efficacy of metformin as a monotherapy or in combination with chemotherapy and/or radiotherapy for the management of BC (reviewed in [43, 127]). Goodwin *et al*. have shown that after six months of metformin treatment, a reduction in insulin by 22% had improved metabolic indices, such as insulin sensitivity, body weight, and cholesterol levels in nondiabetic patients with early-stage BC [29]. This information suggests that metformin is effective in the nondiabetic population. These data and other clinical trials further provide support in using metformin as an adjuvant agent as it is the only agent that does not promote BC but actually retards tumor growth. In addition, these clinical trials further support the need to screen for metabolic dysfunction and evaluate whether or not metformin should be integrated into the treatment for BC therapy. Further, BC patients receiving 1500 mg/day of metformin showed a significant reduction in insulin levels and insulin resistance [44, 128]. The effect of metformin in response to neoadjuvant chemotherapy has been examined in diabetic BC patients. This study included 2529

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

**4.8 Metformin action on breast cancer stem cells**

cells are heavily dependent on aerobic glycolysis [92].

minimizing patient risk [111, 123].

**and treatment**

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*

### **4.8 Metformin action on breast cancer stem cells**

*Metformin*

neovasculature.

[12, 80, 116]. In the context of breast cancer, it has long been demonstrated that high-stage and grade cancers with a worse prognosis have the capacity to upregulate peri- and intratumoral neo-angiogenesis [117]. The induction of new vessels provides metabolic and oxygen delivery advantages to the cancer cells, facilitating survival and growth. Neo-angiogenesis is also associated with an increased capacity of the BC to metastasize, particularly to distant sites including the visceral organs and brain. We have demonstrated a reduction in vascular density and growth, in association with metformin treatment in preclinical models. Others have shown that metformin is associated with reduced tumor angiogenesis in many different cancer cell types. Metformin and alternate biguanides, such as phenformin, downregulate VEGF-dependent activation of ERK1, inhibiting neo-angiogenesis and reducing microvessel density (MVD) [118]. Wang et al. have shown that metformin also downregulates the expression of two other genes, platelet-derived growth factor B (PDGF-B) and fibroblast growth factor (FGF-2), to reduce angiogenesis [119]. Downregulation of PDGF-B also restricts BC cell proliferation, survival, and migration, [117]. Metformin's effect on the microenvironment and angiogenesis has also been shown to enhance chemo-sensitivity, via a reduction in MVD leakage and cancer cell hypoxia *in vivo* [117]. Thus, metformin's effects go beyond the cancer cell itself and include the peri- and intratumoral microenvironment and

*Metformin action on breast cancer stem cells. Metformin can block a myriad of signaling pathways involved in BCSCs, including WNT, transforming growth factor (TGF), NOTCH, hypoxia inducible factor (HIF), and STAT3 signaling pathways. These pathways are thought to enrich for BCSC through the enrichment of CD44 positive receptor and aldehyde dehydrogenase (ALDH+) and decrease in CD24 expression. Metformin can be given as a monotherapy or combinatorial therapy with alternate chemotherapeutic agents, which in turn can induce BCSC death with an increase in apoptosis, cell cycle arrest, and DNA damage. Overall, reduction in* 

*BCSCs can result in reduction of tumor growth and prevention in therapy-mediated relapse.*

**194**

**Figure 7.**

Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are the progenitor cells that give rise to BC as well as heterogeneity within transformed populations. CSCs are maintained as a subpopulation within the neoplasm that perpetuates clonal expansion and may facilitate dormancy, metastasis, chemoresistance, and relapse. Among the molecular BC subtypes, TNBC shows the highest enrichment of CSCs, identified by expression patterns with flow cytometry as CD44+, CD24−/low CSC [120]. BC CSCs are particularly sensitive to metformin, which induces rapid cell death facilitated through a number of pathways involved in cell differentiation, renewal, metastasis, and metabolism (**Figure 7**). It directly targets key CSC gene signatures such as Notch 1, NF*κ*B, Sox2, KLF-4, Oct4, Lin28, MMP-9, and MMP-2 [121]. Metformin attenuates CSCs in resistant BC, through repression of let-7 miRNA [121]. Its ability to attenuate key metabolic genes, such as FASN via upregulation of miR-193b, also contributes to its anti-CSC activity as stem cells are heavily dependent on aerobic glycolysis [92].

The capacity of metformin to induce CSC cell death has significant clinical relevance, given their role in therapeutic resistance, dormancy, and disease progression. Metformin reduces cancer recurrence through the preferential killing of differentiated rather than undifferentiated CSCs [122]. In combination with chemotherapy, metformin is especially active against BC CSCs [111]. In studies of trastuzumab-resistant BC cells as well as xenograft models, the combination of trastuzumab and metformin significantly reduced CD44+, CD24−/low CSC subpopulations and reduced tumor volume [111, 123, 124]. In combination with doxorubicin, paclitaxel, or carboplatin, metformin can also eradicate CSCs and reduce the effective dosage required of the highly toxic chemotherapeutic agents, minimizing patient risk [111, 123].

### **5. Clinical evidence with metformin in breast cancer prevention and treatment**

The pleiotropic oncostatic effects of metformin have been explored as an adjuvant therapeutic option for the management of BC [43, 125, 126]. Epidemiological studies have demonstrated associations between metformin use in patients with type 2 diabetes and decreased cancer incidence and cancer-related mortality [10]. Several observational and randomized trials have evaluated a number of biomarker changes after metformin administration, increasing the footage of metformin as an off-label agent for BC. Over 11 ongoing and 13 completed clinical trials have tested the efficacy of metformin as a monotherapy or in combination with chemotherapy and/or radiotherapy for the management of BC (reviewed in [43, 127]). Goodwin *et al*. have shown that after six months of metformin treatment, a reduction in insulin by 22% had improved metabolic indices, such as insulin sensitivity, body weight, and cholesterol levels in nondiabetic patients with early-stage BC [29]. This information suggests that metformin is effective in the nondiabetic population. These data and other clinical trials further provide support in using metformin as an adjuvant agent as it is the only agent that does not promote BC but actually retards tumor growth. In addition, these clinical trials further support the need to screen for metabolic dysfunction and evaluate whether or not metformin should be integrated into the treatment for BC therapy. Further, BC patients receiving 1500 mg/day of metformin showed a significant reduction in insulin levels and insulin resistance [44, 128]. The effect of metformin in response to neoadjuvant chemotherapy has been examined in diabetic BC patients. This study included 2529 women with BC and confirmed that metformin could achieve higher pathological complete response with neoadjuvant therapy relative to non-metformin users [129]. Dowling et al. have further examined neoadjuvant metformin in a prospective window of opportunity study [32]. Clinical and biological effects of metformin on nondiabetic BC patients were evaluated. These patients were treated with 500 mg of metformin three times daily for 2 weeks. Significant attenuated expression of the insulin receptor was observed in treated breast tumors and had high expression of OCT1 (organic cation transporter 1) [32]. The effect of metformin in nondiabetic BC patients was previously reviewed [43]. Systemic reviews and meta-analyses, highlighting a summary of studies involving metformin therapy in nondiabetic patients and diabetic patients, were reviewed in [43].

### **5.1 Metformin dose recommended for breast cancer patients**

Pharmacokinetic profiling of mouse tumors provided preclinical analysis of appropriate human doses to provide efficient inhibition of tumor growth [130]. Based on this evidence, metformin-mediated activation of AMPK and antitumor function was dependent on cellular uptake of the drug, which is primarily controlled by membrane transporters OCT1, OCT2, and OCT3 [131]. Based on the high expression of OCT transporters, 850 mg/day of metformin is required to inhibit tumor growth efficiently. If a tumor expresses low levels of OCT transporter, then 2250 mg/day is recommended [132]. Additionally, a dose of metformin of 500–850 mg/day is typically recommended with standard chemotherapy (including anthracyclines, platinum, taxanes, and capecitabine) for first- or second-line therapy (please see https://www.drugbank.ca/drugs/DB00331). The combination of metformin with a chemotherapeutic agent is recommended for a number of cycles until progression is unacceptable or toxicity develops.

### **5.2 Indications and contraindications for metformin use for breast cancer**

Metformin is not approved for clinical use by the FDA and is still considered investigational for the treatment for BC. While metformin is well established as an inexpensive, well-tolerated, and effective for the treatment of diabetes, adjuvant use of metformin for BC remains to be defined. Current clinical trials have not outlined indications and contraindications for metformin use as adjuvant therapy for BC. Generally, metformin hydrochloride tablets are contraindicated in patients with (1) severe renal impairment (eGFR below 30 mL/min/1.73 m2), (2) hypersensitivity to metformin, and (3) acute or chronic metabolic acidosis including diabetic ketoacidosis. Additionally, current clinical trials with metformin have been listed (https://clinicaltrials.gov/ct2/show/NCT01310231 and https://clinicaltrials.gov/ct2/ show/NCT01101438). The NCIC CTG MA.32 Phase III randomized clinical trial has completed enrollment of 3649 nondiabetic women receiving standard surgical, chemotherapeutic, hormonal, biologic, and radiation treatment for T1-3, N0-3, M0 breast cancer. This trial has provided preliminary findings [33] and has not defined clear indications and/or contraindications for metformin use as adjuvant therapy for breast cancer.

### **6. Conclusions**

A preponderance of clinical, epidemiological, and scientific evidence indicates that metabolic dysregulation of carbohydrate and lipid metabolism promote BC pathogenesis and a worse outcome, for women who have the disease [9, 10, 30, 40, 45, 129, 133].

**197**

especially in these patients.

**Acknowledgements**

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

subtypes, fostering the goal of more personalized cancer care.

One of the therapeutic agents commonly used in patients with metabolic syndrome or type 2 diabetes, metformin, has demonstrated significant anti-BC activity. Metformin inhibits gluconeogenesis, reduces circulating levels of glucose, increases insulin sensitivity, and reduces hyperinsulinemia associated with insulin [134]. These factors have been associated with BC prognosis. Several mechanisms of metformin action involve AMPK-dependent and AMPK-independent signaling pathways, and these effects are remarkably broad and potent. Its ability to target metabolic dysregulation of carbohydrate and lipid metabolism as well as cancer stem cells appear to be equally important in its anticancer activity against BC [129, 133–137]. Furthermore, the effects of metformin are unique among molecular subsets of BC. A better understanding of these mechanisms will facilitate targeted applications in patients with specific

A number of clinical trials are underway to evaluate metformin in BC patients [30, 44–46, 50, 136]. Most have been designed to evaluate its efficacy, in combination with various chemo- or radiotherapy agents; see (https://clinicaltrials.gov/ct2/ results?term=+cancer+AND+metformin). Most ongoing or completed clinical trials have evaluated metformin's effect on cellular proliferation or death, pathological response rate, progression-free or overall survival. Some have also sought to compare its efficacy in patients with or without metabolic dysregulation, as a secondary aim. None have specifically been designed to evaluate interactions with CSCs, or in selected molecular subtypes, although correlative studies have provided some data in this regard. The ALTTO trial has shown that metformin improves outcomes for patients with diabetes and either HER2+ or hormone receptor positive BC [30]. The NCIC Clinical Trials Group (NCIC CTG) MA.32 has shown benefit from metformin, as compared to placebo on outcomes in early stage BC [33]. It demonstrated efficacy with improvements in body weight, insulin, glucose, and leptin levels in BC

patients examined, regardless of baseline BMI or fasting insulin levels [33].

In conclusion, metformin is a unique drug with a long track record of human use, which has demonstrated robust efficacy against type 2 diabetes and metabolic dysregulation. Epidemiologic data show independent and significant benefit in preventing cardiovascular disease and cancer in these patients. Metformin is an inexpensive oral agent that is currently available worldwide. It is generally well tolerated and has a low risk:benefit ratio. Epidemiological and clinical data have shown that metformin reduces BC incidence and mortality in women with metabolic dysregulation, obesity, and type 2 diabetes. This subpopulation of woman is at significantly higher risk for BC, particularly in the postmenopausal setting. Preclinical and clinical evidence shows that metformin inhibits BC cell replication and tumor growth, decreases tumor aggression, reduces the stem cell pool, and slows motility/metastasis and can promote cell death through apoptosis, autophagy, or upregulation of immunity. Metformin has unique effects on molecular subsets of BC, with the aggressive triple negative BC showing the most sensitivity and lowest EC50 data. TNBC is particularly sensitive to metformin's downregulation of fatty acid and cholesterol biosynthesis, glucose transport, and carbohydrate metabolism. This cancer subtype is typically the most aggressive and is less responsive to traditional chemotherapy; thus, metformin's potency may provide significant benefit

Grant support provided in part by Susan G Komen for the Cure K100575 to RSW, SME, and ADT; ACS-IRG 16-184-56 RSW from the American Cancer Society; CCL-

C92110 RSW and ADT from the Colorado Cancer League.

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

### *Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*

One of the therapeutic agents commonly used in patients with metabolic syndrome or type 2 diabetes, metformin, has demonstrated significant anti-BC activity. Metformin inhibits gluconeogenesis, reduces circulating levels of glucose, increases insulin sensitivity, and reduces hyperinsulinemia associated with insulin [134]. These factors have been associated with BC prognosis. Several mechanisms of metformin action involve AMPK-dependent and AMPK-independent signaling pathways, and these effects are remarkably broad and potent. Its ability to target metabolic dysregulation of carbohydrate and lipid metabolism as well as cancer stem cells appear to be equally important in its anticancer activity against BC [129, 133–137]. Furthermore, the effects of metformin are unique among molecular subsets of BC. A better understanding of these mechanisms will facilitate targeted applications in patients with specific subtypes, fostering the goal of more personalized cancer care.

A number of clinical trials are underway to evaluate metformin in BC patients [30, 44–46, 50, 136]. Most have been designed to evaluate its efficacy, in combination with various chemo- or radiotherapy agents; see (https://clinicaltrials.gov/ct2/ results?term=+cancer+AND+metformin). Most ongoing or completed clinical trials have evaluated metformin's effect on cellular proliferation or death, pathological response rate, progression-free or overall survival. Some have also sought to compare its efficacy in patients with or without metabolic dysregulation, as a secondary aim. None have specifically been designed to evaluate interactions with CSCs, or in selected molecular subtypes, although correlative studies have provided some data in this regard. The ALTTO trial has shown that metformin improves outcomes for patients with diabetes and either HER2+ or hormone receptor positive BC [30]. The NCIC Clinical Trials Group (NCIC CTG) MA.32 has shown benefit from metformin, as compared to placebo on outcomes in early stage BC [33]. It demonstrated efficacy with improvements in body weight, insulin, glucose, and leptin levels in BC patients examined, regardless of baseline BMI or fasting insulin levels [33].

In conclusion, metformin is a unique drug with a long track record of human use, which has demonstrated robust efficacy against type 2 diabetes and metabolic dysregulation. Epidemiologic data show independent and significant benefit in preventing cardiovascular disease and cancer in these patients. Metformin is an inexpensive oral agent that is currently available worldwide. It is generally well tolerated and has a low risk:benefit ratio. Epidemiological and clinical data have shown that metformin reduces BC incidence and mortality in women with metabolic dysregulation, obesity, and type 2 diabetes. This subpopulation of woman is at significantly higher risk for BC, particularly in the postmenopausal setting. Preclinical and clinical evidence shows that metformin inhibits BC cell replication and tumor growth, decreases tumor aggression, reduces the stem cell pool, and slows motility/metastasis and can promote cell death through apoptosis, autophagy, or upregulation of immunity. Metformin has unique effects on molecular subsets of BC, with the aggressive triple negative BC showing the most sensitivity and lowest EC50 data. TNBC is particularly sensitive to metformin's downregulation of fatty acid and cholesterol biosynthesis, glucose transport, and carbohydrate metabolism. This cancer subtype is typically the most aggressive and is less responsive to traditional chemotherapy; thus, metformin's potency may provide significant benefit especially in these patients.

### **Acknowledgements**

Grant support provided in part by Susan G Komen for the Cure K100575 to RSW, SME, and ADT; ACS-IRG 16-184-56 RSW from the American Cancer Society; CCL-C92110 RSW and ADT from the Colorado Cancer League.

*Metformin*

women with BC and confirmed that metformin could achieve higher pathological complete response with neoadjuvant therapy relative to non-metformin users [129]. Dowling et al. have further examined neoadjuvant metformin in a prospective window of opportunity study [32]. Clinical and biological effects of metformin on nondiabetic BC patients were evaluated. These patients were treated with 500 mg of metformin three times daily for 2 weeks. Significant attenuated expression of the insulin receptor was observed in treated breast tumors and had high expression of OCT1 (organic cation transporter 1) [32]. The effect of metformin in nondiabetic BC patients was previously reviewed [43]. Systemic reviews and meta-analyses, highlighting a summary of studies involving metformin therapy in nondiabetic

Pharmacokinetic profiling of mouse tumors provided preclinical analysis of appropriate human doses to provide efficient inhibition of tumor growth [130]. Based on this evidence, metformin-mediated activation of AMPK and antitumor function was dependent on cellular uptake of the drug, which is primarily controlled by membrane transporters OCT1, OCT2, and OCT3 [131]. Based on the high expression of OCT transporters, 850 mg/day of metformin is required to inhibit tumor growth efficiently. If a tumor expresses low levels of OCT transporter, then 2250 mg/day is recommended [132]. Additionally, a dose of metformin of 500–850 mg/day is typically recommended with standard chemotherapy (including anthracyclines, platinum, taxanes, and capecitabine) for first- or second-line therapy (please see https://www.drugbank.ca/drugs/DB00331). The combination of metformin with a chemotherapeutic agent is recommended for a number of

patients and diabetic patients, were reviewed in [43].

**5.1 Metformin dose recommended for breast cancer patients**

cycles until progression is unacceptable or toxicity develops.

**5.2 Indications and contraindications for metformin use for breast cancer**

Metformin is not approved for clinical use by the FDA and is still considered investigational for the treatment for BC. While metformin is well established as an inexpensive, well-tolerated, and effective for the treatment of diabetes, adjuvant use of metformin for BC remains to be defined. Current clinical trials have not outlined indications and contraindications for metformin use as adjuvant therapy for BC. Generally, metformin hydrochloride tablets are contraindicated in patients with (1) severe renal impairment (eGFR below 30 mL/min/1.73 m2), (2) hypersensitivity to metformin, and (3) acute or chronic metabolic acidosis including diabetic ketoacidosis. Additionally, current clinical trials with metformin have been listed (https://clinicaltrials.gov/ct2/show/NCT01310231 and https://clinicaltrials.gov/ct2/ show/NCT01101438). The NCIC CTG MA.32 Phase III randomized clinical trial has completed enrollment of 3649 nondiabetic women receiving standard surgical, chemotherapeutic, hormonal, biologic, and radiation treatment for T1-3, N0-3, M0 breast cancer. This trial has provided preliminary findings [33] and has not defined clear indications and/or contraindications for metformin use as adjuvant therapy

A preponderance of clinical, epidemiological, and scientific evidence indicates that metabolic dysregulation of carbohydrate and lipid metabolism promote BC pathogenesis and a worse outcome, for women who have the disease [9, 10, 30, 40, 45, 129, 133].

**196**

for breast cancer.

**6. Conclusions**

### **Conflict of interest**

The authors have declared that no conflict of interest exists.

### **Abbreviations**


**199**

**Author details**

Reema S. Wahdan-Alaswad and Ann D. Thor\*

Aurora, Colorado, United States of America

provided the original work is properly cited.

\*Address all correspondence to: ann.thor@cuanschutz.edu

Department of Pathology, University of Colorado Anschutz Medical Campus,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype…*

NF-B Nuclear factor kappa-light-chain-enhancer of activated B-cells

SREBP1 Sterol regulatory element-binding transcription factor 1 STAT3 Signal transducer and activator of transcription 3

*DOI: http://dx.doi.org/10.5772/intechopen.91183*

MVK Mevalonate kinase

P- Phosphorylated P53 Tumor protein 53

RNA Ribonucleic acid ROS Reactive oxygen species SRC Proto-oncogene c-Src

US United States

PGR Progesterone receptor

MSL Mesenchymal stem-like

NCIC CTG NCIC Clinical Trials Group

OCT1 Organic cation transporter 1 OCT2 Organic cation transporter 2 OCT3 Organic cation transporter 2

PARP Poly (ADP-ribose) polymerase

PI3K Phosphatidyl-inositide 3-kinase PTEN Phosphatase and tensin homolog

TGF-β Transforming growth factor beta TNBC Triple negative breast cancer TNF-α Tumor necrosis factor alpha TSC2 Tuberous sclerosis complex 2

VEGF Vascular endothelial growth factor

MVD Mevalonate diphosphate decarboxylase

*Metformin Activity against Breast Cancer: Mechanistic Differences by Molecular Subtype… DOI: http://dx.doi.org/10.5772/intechopen.91183*


### **Author details**

*Metformin*

**Conflict of interest**

**Abbreviations**

The authors have declared that no conflict of interest exists.

ALTTO Adjuvant lapatinib and/or trastuzumab treatment optimization

CLDN Claudin integral membrane proteins of tight junctions

EC Effective concentration/Inhibitory concentration

HER2 Human epidermal growth factor receptor 2 HER3: Human epidermal growth factor receptor 3

ACAA2 Acetyl-coenzyme A acetyltransferase 2

ADP Adenosine di-phosphate

AMP Adenosine monophosphate AMPK AMP-activated protein kinase AMPKK AMP-activated protein kinase kinase

ATP Adenosine triphosphate AR Androgen receptor BC Breast cancer

BCSCs Breast cancer stem cells CDK Cyclin-dependent kinase

DHCR24 24-dehydrocholesterol reductase

HIF-1α Hypoxia-inducible factor 1-alpha

EGFR Epidermal growth factor receptor EMT Epithelial mesenchymal transition

FDA Food and Drug Administration FGFR2 Fibroblast growth factor receptor 2 FGFR3 Fibroblast growth factor receptor 3

HMGCo-A β-Hydroxy β-methylglutaryl-CoA HMGCS1 Hydroxymethylglutaryl-CoA synthase HMGCR 3-Hydroxy-3-Methylglutaryl-CoA Reductase

LDLR Low-density lipoprotein receptor

MAPK Mitogen-activated protein kinases

MTOR Mammalian target of rapamycin

MRCC1 Mitochondrial respiratory-chain complex 1

ID1 Inhibitor of differentiation-1 IGFIR Insulin-like growth factor receptor IGF1 Insulin-like growth factor-1 IRS1 Insulin receptor substrate 1

IL-1 Interleukin 1 beta IL-6 Interleukin-like 6 JAK Janus kinase

LKB1 Liver kinase B1 LSS Lanosterol synthase

MβCD Methyl-β-cyclodextrin

CL Claudin-low

CSC Cancer stem cells

ESR1 Estrogen receptor

FASN Fatty acid synthase

GLUT1 Glucose transporter 1 GM1 GM1 gangliosidosis marker

AKT Protein kinase B

**198**

Reema S. Wahdan-Alaswad and Ann D. Thor\* Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America

\*Address all correspondence to: ann.thor@cuanschutz.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 4

Metformin and Ageing

Section 4
