**2. Pleiotropic effects of metformin against cancer**

The early days of laboratory research on metformin's anti-cancer mechanisms focused mainly on its metabolic effects on cell proliferation, which naturally follows from the initial use of metformin as a treatment for T2DM as a metabolic disorder. Eventually, it became gradually apparent that unlike modern day targeted therapies, metformin's anti-neoplastic bioactivity is broad ranged and pleiotropic, encompassing not only its established metabolic effects, but also involving antiangiogenic, anti-inflammatory, epigenetic, apoptotic and autophagic, and immunologic actions as well as effects on the microbiome and on cancer stem cells (CSCs) that all synergistically contribute to overall cancer prevention and control. Furthermore, within each category of its bioactivity, it further exerts multiple molecular actions, and it has thus become increasingly apparent that metformin could be properly conceived of as a multi-faceted multi-tasking molecule with direct and indirect actions against cancer. In summary, the anticancer effects of metformin is based on 1) its main action on cellular metabolism via the maintenance of plasma glucose and insulin levels, 2) targeted action against cancer cells with pleiotropic inhibitory effects on multiple pathways involved in cancer cell survival and metastasis, and 3) indirect anti-angiogenic anti-inflammatory as well as immunomodulatory effects and also its actions on the microbiome and CSCs. The complex pleiotropic nature of metformin effects on cancer is illustrated in **Figure 2**.

#### **2.1 Metformin metabolic effects**

To understand the metabolic impact of metformin on cancer, we must first recognize the intimate relationship between glucose energy metabolism and cellular proliferation as well as a unique propensity of cancer cells to utilize glucose anaerobically even in the presence of oxygen in contrast to non-cancer cells which utilize oxidative phosphorylation to generate energy. This phenomenon was first noted by Otto Warburg almost a hundred years ago, and subsequently termed the "Warburg effect" [7]. This altered energy metabolism of cancer cells may underline their proliferation, invasiveness, and chemoresistance and this altered metabolic pattern in cancer is regulated by oncogenic and tumor suppressor signals such as hypoxia inducible factor 1 (HIF-1), myelocytomatosis oncogene cellular homolog

#### **Figure 2.**

*Representation of some of the pleiotropic direct and indirect anticancer effects of metformin as illustrated by molecular and cellular pathways. Metformin effects key energy and metabolic processes such as the mitochondrial respiration (complex I), TCA cycle, fatty acid* β*-oxidation, gluconeogenesis, and glycolysis. Metformin affects the cell cycle, cell growth, immune response, autophagy, and apoptosis, angiogenesis and cancer stem cells. Abbreviations: 4EBP1, 4E-binding protein 1; ACC, acetyl-CoA carboxylase; AKT, AKT serine/threonine kinase 1; AMPK, AMP-activated protein kinase; BCL2, apoptosis regulator, BCL2; CCND1, cyclin D1; CSC, cancer stem cell; DDIT4, DNA damage inducible transcript 4; EMT, epithelial-to-mesenchymal transition; FOXO3, forkhead box O3; GPDH, glycerol-3-phosphate dehydrogenase; IPMK, inositol polyphosphate multikinase; LKB1, liver kinase B1; miRNA, micro RNA; mTORC1, target of rapamycin complex 1; SREBF1, sterol regulatory element binding transcription factor 1; STAT3, signal transducer and activator of transcription 3; TCA, tricarboxylic acid; TGFB1, transforming growth factor beta 1; VEGF, vascular endothelial growth factor. Phosphorylated molecules are indicated by a prefix p. source: [6], Licensed under CC BY 4.0.*

(Myc), p53, and the phosphoinositide 3 kinase (PI3K)/AKT8 virus oncogene cellular homolog (Akt)/mammalian target of rapamycin (mTOR) pathways.

Metformin's main pharmacologic action is the reducing elevated plasma glucose is largely due to the improvement in hepatic insulin resistance leading to a reduction in hepatic glucose output from gluconeogenesis, increases glucose uptake in muscle, decreased absorption of sugar from the intestines, and improved insulin sensitivity, mainly via activation of a cellular energy sensor known as AMP-activated protein kinase (AMPK). The major downstream target of AMPK is mTOR, which is very important in cellular growth processes and cancer dynamics, and mTOR is inhibited by AMPK [8]. Since glucose metabolism is at the center of the metabolic derangement that is a hallmark of cancer cells, and metformin chiefly targets glucose metabolism, it follows that the altered metabolic pathway may be a target by metformin for cancer prevention or therapy.

It is through its main effects above on metabolism and cellular energetics that metformin can attenuate cancer cell proliferation (See **Figure 3**). Furthermore, these metabolic effects in turn impact the immune system, epigenetics, inflammation, cellular apoptotic and autophagic pathways as well as the microbiome and CSCs which all play a role in cancer development.

#### **2.2 Metformin immuno-modulatory effects**

The immune system participates broadly in the prevention and control of cancer and interacts with biological pathways of metabolism and inflammation, *Repurposing of Metformin as a Multifaceted and Multitasking Preventative and Treatment… DOI: http://dx.doi.org/10.5772/intechopen.96101*

#### **Figure 3.**

*The effect of metformin in suppressing cancer cell growth via metabolic pathways. Metformin inhibits complex I of the electron transport chain, which leads to increased AMP/ATP ratio and activation of AMPK by LKB1. Activated AMPK subsequently inhibits mTOR and its downstream targets by the following two pathways: 1. AMPK stabilizes TSC1/2, which inhibits Rheb, an activator of mTOR; 2. AMPK inhibits mTOR binding protein raptor. Metformin directly inhibits mTOR by up-regulating REDD1 and suppressing rags. AMPK, AMP-activated protein kinase; Rheb, Ras homolog enriched in brain; LKB1, liver kinase B1; REDD1, regulated in development and DNA damage response 1; TSC, tuberous sclerosis complex; rags, rag GTPases; mTOR, mammalian target of rapamycin; 4EBP1, eukaryotic initiation factor 4E binding protein 1; S6K, S6 kinase. Source: [9], Licensed under CC BY 3.0.*

and metformin again acts in a multifaceted fashion to bolster immunity against cancer with effects on almost every aspect of the immune system, especially with reference to cancer immunty (**Figure 4**). One of metformin's actions is the enhancement of CD8+ T lymphocytes and rescues them from exhaustion. CD8+ T cells which is one of the key components in cellular immunity against tumors, as these cells can expand and transform into effector cytotoxic T lymphocytes (CTL) which targets cancer. This phenomenon of the rescue of exhausted CD8+ T lymphocytes has been confirmed *in vitro* in leukemia, melanoma, renal cell carcinoma, non–small-cell lung carcinoma (NSCLC), gastrointestinal carcinoma, and breast cancer. Also, metformin-induced activation of AMPK as one of its main metabolic actions mentioned above promotes immune check-point programmed death ligand 1 (PD-L1) degradation, which allows CTL-mediated tumor cell death [11]. Additionally, metformin can also enhance local as well as systemic cytokine responses to tumors [12]. Furthermore, metformin also has indirect effects on the immune system via its influence on the microbiome and its anti-inflammatory effects, which has been reviewed exhaustively and is briefly summarized below.

#### **Figure 4.**

*Metformin effects related to anticancer immunity. Metformin indirectly increases T-cell activity by negatively regulating (a) chronic inflammation, (B) hypoxia, and (C) PD-L1 levels that inhibit T-cell activity. Metformin directly relieves T-cell exhaustion by means of metabolic reprogramming of TIL and promotes memory T-cell differentiation (D). Metformin shifts the profile of gut microbiota more favorably to T-cell immunity (TAM) tumor-associated macrophages (E); (M*φ*) macrophages; (MDSC) myeloid-derived suppressor cells; (T) T-cell; (DAMPs) damage-associated molecular patterns; (APC) antigen presenting; (SCFA) short-chain fatty acid. Source: [10], CC BY-NC 3.0.*

#### **2.3 Metformin effects on the microbiome**

Whereas science has become increasingly aware of the central role the gut microbiome plays in health and diseases including cancer, particularly via its effects on the immune system [13], metformin's beneficial role on host metabolism has also been found to be in part related to the microflora in the gut. The microbiome modulates our immune system and inflammatory response and both of these are key factors in determining cancer development and are associated with inflammatory immune response [14] highlights the crosstalk between metformin effects on metabolism, immunity, inflammation and the microbiome, which in turn can modulate cancer biodyamanics, and part of the mechanisms involved in this complex interplay is illustrated in **Figure 5** below.

#### **2.4 Metformin anti-inflammatory effects**

Inflammation effects on cancer promotion is well known. In 1863, Rudolf Virchow first proposed the role of inflammation in cancer based on the observation of leukocytes in cancerous tissue. Subsequently, accumulated evidence has identified inflammation both as a cause and result of malignancy [16], with numerous studies in past decades implicating chronic inflammation in the promotion of malignancy [17] (**Figure 6**). Not surprisingly then, given the T2DM's known association with chronic low-grade subclinical inflammation which is part and parcel of its the insulin resistance that is its hallmark [19], and metformin's effects on the immune and metabolic systems, that metformin must also modulate the inflammatory response. This connection has been well demonstrated by animal experiments where metformin treated rodents reveal dampened pro-inflammatory pathways nuclear factor k B (NF-k) and Jun N-terminal kinase (JNK) and increased anti-inflammatory cytokine IL-10 [20].

*Repurposing of Metformin as a Multifaceted and Multitasking Preventative and Treatment… DOI: http://dx.doi.org/10.5772/intechopen.96101*

#### **Figure 5.**

*Crosstalk between metformin action and gut microbiota. GLP1: Glucagon-like peptide-1; GLP2: Glucagon-like peptide-2; LPS: Lipopolysaccharide; SCFA: Short-chain fatty acid. Source: [15], Licensed under CC BY-NC 4.0.*

#### **Figure 6.**

*Inflammatory cytokines released by immune cells within the tumor microenvironment has a direct effect on pre-malignant and cancer cells by increasing their proliferation and resistance to cell death and stresses thus directly promoting tumor growth and progression. Additionally, inflammatory signals can suppress antitumor immunity via action of regulatory T-cells, myeloid cells and enhance other cancer promoting cells (such as fibroblasts, myeloid cells and endothelium of new blood vessels); altogether, these inflammation driven changes also significantly contribute to tumor growths and progression. TME: Tumor microenvironment, Treg: Regulatory T cells. Source: [18], Licensed under CC BY 3.0.*

#### **2.5 Metformin epigenetic effects**

Epigenetics is the genomic mechanism that reversibly modulates gene expression independent of DNA sequences. Epigenetic processes which allow for the gene modulatory effect involve DNA methylation, histone modification,

#### **Figure 7.**

*Schematic of histone modifications via metabolic effects of metformin. Glycolysis determines the NAD+ /NADH ratio, which affects the activity of histone deacetylases to reduce histone acetylation. Source: [9], Licensed under CC BY 3.0.*

the readout of these modifications, chromatin remodeling and the effects of noncoding RNA all of which affects cellular activities such as growth and differentiation. Thus, epigenetics can in one sense be conceived of as a master switch of cancer biological processes. Recently, there has been growing interest in epigenetic targeting as a promising therapeutic option for cancer [21]. And since cellular metabolism is tightly linked to epigenetic modifications, it is again not surprising that metformin as a modulator of cellular metabolism may also possess significant epigenetic effects mainly via histone modification (**Figure 7**), which in turn is another avenue whereby metformin may exert its anti-cancer effects [9].

#### **2.6 Metformin apoptotic and autophagic effects**

Both apoptosis or programmed cell death and autophagy are important catabolic and tumor-suppressive pathways that control cell survival and cell death and are thus increasingly important therapeutic targets in cancer [22]. While apoptosis involves cellular suicide and cell death pathways, autophagy involves recycling and degradation of cellular waste which if maladapted and excessive can also lead to cell death and there is significant cross-talk between these two pathways [23]. In cancer biology, autophagy is cancer suppressive as it facilitates the degradation of oncogenic molecules thus pre-empting the development of cancers, while apoptosis leads to cellular suicide and limits the survival of cancer cells. As a result, defective or inadequate autophagy or apoptosis can both lead to cancer. The complexity of the crosstalk between the apoptosis and autophagy is illustrated in **Figure 8**.

In the case of these pathways, metformin has been shown to promote apoptosis in a variety of cancers via various biological pathways [24] while also promoting autophagy [25] as two other dimensions of its anti-cancer bioactivity.

*Repurposing of Metformin as a Multifaceted and Multitasking Preventative and Treatment… DOI: http://dx.doi.org/10.5772/intechopen.96101*

#### **Figure 8.**

*Complex crosstalk between autophagy and apoptosis pathways. Various proteins involved at the different points of crosstalk are shown and labeled. Lines denote interactions or processes, with solid lines corresponding to intrapathway processes and dashed lines corresponding to inter-pathway connections. Red lines denote inhibitory interactions, while lines with arrows indicate facilitating interactions. Source: [24], Licensed under CC BY 3.0.*

#### **2.7 Metformin effects on cancer stem cells**

CSCs were only identified in the 1990s, and they have been hypothesized to persist in tumors as a distinct cell population capable of self-renewal and maybe responsible for cancer relapse and metastasis by giving rise to new tumors. These CSCs are also believed to be resistant to traditional chemotherapy and radiation. A complex regulatory network consisting of microRNAs and Wnt/β-catenin, Notch, and Hedgehog signaling pathways control the properties of CSCs. Therefore, the development of specific therapies targeting and its regulatory pathways is another avenue for improved cancer treatments to prevent relapse and metastases, and improve survival [26]. In this regard, metformin has been reported to target CSCs perhaps via blunting of the Warburg effect and consequently down-regulates their growth. In animal studies, it has been found that metformin exposure was associated with a ~ 2-fold reduction in ovarian CSCs and increased in chemotherapy response and translational studies completed as part of a multi-center phase 2 clinical trial was able to demonstrate a 2.4-fold CSC reduction as well as improved survival in ovarian cancer patients [27].

#### **2.8 Metformin's antiangiogenic effects**

Angiogenesis is the process where a tumor can induce its own blood supply via neovascularization to enhance its own nutrient source as well as increase its propensity to metastasize*.* It follows that antiangiogenesis which involves the suppression of vascular supply to tumors may be an effective method of cancer control as initially proposed by Folkman [28]. In this regard, preclinical studies with metformin have reported that it indirectly modulates tumor angiogenesis most likely via metabolic pathways affecting proangiogenic signals. As an example, metformin is known to decrease HIF-1α stability in cancer cells, reducing the expression of HIF-1 targeted genes and thus resulting in smaller tumor vessel size, reduced microvessel density and slower tumor growth [29]. Another murine experiment analyzing angiogenesis in a matrigel plug model found that metformin treatment lead to a decrease in angiogenesis [30].
