Section 3 Urogenital Cancers

*Current Trends in Cancer Management*

[101] Wei AC, Greig PD, Grant D, et al. Survival after hepatic resection for colorectal metastases: A 10-year experience. Annals of Surgical Oncology. 2006;**13**(5):668-676

[102] Abdalla EK, Vauthey JN, Ellis LM, et al. Recurrence and outcomes

radiofrequency ablation, and combined

following hepatic resection,

2004;**239**(6):818-825

resection/ablation for colorectal liver metastases. Annals of Surgery.

**80**

**83**

**Chapter 5**

**Abstract**

**1. Introduction**

cancer development is needed.

Candidates

Exploring New Molecular Targets

in Advanced Ovarian Cancer: The

and Antitumor Benzothiazole

*Andrea I. Loaiza Perez and Tracey D. Bradshaw*

**Keywords:** ovarian cancer, AhR, benzothiazoles

Ligands as Potential Therapeutic

Antitumor benzothiazoles, including 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203; NSC 703786), non-fluorinated parent compound DF 203 (NSC 674495), and Phortress (NSC 710305), the lysyl amide prodrug of 5F 203, are experimental anticancer agents with activity in ovarian and breast cancer models *in vitro* and *in vivo*. These compounds require (and induce their own) metabolism by cytochrome P450 (CYP) enzymes (e.g., CYP1A1) for antitumor action. The aryl hydrocarbon receptor (AhR) is the main transcriptional regulator of CYP1A1, and we have previously demonstrated that DF 203 and 5F 203 are potent AhR ligands and trigger activation of AhR signaling in sensitive breast and ovarian cancer cells, causing nuclear translocation of AhR. We propose that AhR may represent a new molecular target in the treatment of ovarian tumors, and 5F 203 may exemplify a potential novel treatment. Furthermore, putative biomarkers of sensitivity to this agent have been identified.

Ovarian cancer is one of the most lethal gynecological cancers. The National Cancer Institute (NCI) estimated ~22,240 new cases with ~14,070 deaths from ovarian cancer in the US in 2018 [1]. Globally, in 2012, 239,000 women were diagnosed with ovarian carcinoma and 152,000 deaths from this disease were recorded [2]. Unfortunately, the majority of cases are only diagnosed at advanced stages (stage III or IV), a consequence of the cancer's asymptomatic nature, and overall survival lies between 5 and 25% [3, 4]. Hence, the inability to detect this disease during early stages has led to poor prognosis. Despite improvements in medicine and patient care, screening for detection of early-stage ovarian cancer is presently lacking. Thus, a better understanding of the molecular events that underlie ovarian

Aryl Hydrocarbon Receptor (AhR)

#### **Chapter 5**

Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor (AhR) and Antitumor Benzothiazole Ligands as Potential Therapeutic Candidates

*Andrea I. Loaiza Perez and Tracey D. Bradshaw*

### **Abstract**

Antitumor benzothiazoles, including 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203; NSC 703786), non-fluorinated parent compound DF 203 (NSC 674495), and Phortress (NSC 710305), the lysyl amide prodrug of 5F 203, are experimental anticancer agents with activity in ovarian and breast cancer models *in vitro* and *in vivo*. These compounds require (and induce their own) metabolism by cytochrome P450 (CYP) enzymes (e.g., CYP1A1) for antitumor action. The aryl hydrocarbon receptor (AhR) is the main transcriptional regulator of CYP1A1, and we have previously demonstrated that DF 203 and 5F 203 are potent AhR ligands and trigger activation of AhR signaling in sensitive breast and ovarian cancer cells, causing nuclear translocation of AhR. We propose that AhR may represent a new molecular target in the treatment of ovarian tumors, and 5F 203 may exemplify a potential novel treatment. Furthermore, putative biomarkers of sensitivity to this agent have been identified.

**Keywords:** ovarian cancer, AhR, benzothiazoles

### **1. Introduction**

Ovarian cancer is one of the most lethal gynecological cancers. The National Cancer Institute (NCI) estimated ~22,240 new cases with ~14,070 deaths from ovarian cancer in the US in 2018 [1]. Globally, in 2012, 239,000 women were diagnosed with ovarian carcinoma and 152,000 deaths from this disease were recorded [2]. Unfortunately, the majority of cases are only diagnosed at advanced stages (stage III or IV), a consequence of the cancer's asymptomatic nature, and overall survival lies between 5 and 25% [3, 4]. Hence, the inability to detect this disease during early stages has led to poor prognosis. Despite improvements in medicine and patient care, screening for detection of early-stage ovarian cancer is presently lacking. Thus, a better understanding of the molecular events that underlie ovarian cancer development is needed.

The current strategy for treatment of ovarian cancer is surgery followed by radiotherapy and chemotherapy [3, 4]. Although ~70% of ovarian cancer patients respond initially to a combination of platinum and taxane-based chemotherapy, drug-resistance emerges and current treatments are of limited efficacy in preventing tumor recurrence and progression [3, 4]. Thus, despite surgery, radiotherapy, and chemotherapy, most patients present with recurrent disease within 12–18 months, which spreads rapidly within the peritoneal cavity. In platinumresistant disease, survival rarely exceeds 5 months [1, 5]. Thus, new anti-neoplastic agents are urgently needed to improve the prognoses for ovarian cancer patients. Recently, evidence has emerged revealing the importance of genomic aberrations in the progression of ovarian cancer [6–8]. Through the use of high-throughput technologies (i.e., array comparative genomic hybridization (aCGH), microarray, and SNP arrays), specific genomic regions have been identified to be either amplified or silenced in tumor progression [6, 7].

Molecularly targeted cancer therapies recently introduced into the clinic include drugs designed to interfere with specific proteins ("molecular targets") that are involved in the growth, progression, and spread of cancer. Used in the treatment of ovarian cancer are bevacizumab (a VEGF inhibitor) [9], olaparib [10], and niraparib (PARP inhibitors) [11].

The objective of our work has been for many years to validate the aryl hydrocarbon receptor as a molecular target of antitumor drugs, for the treatment of different cancers including ovarian cancer.

#### **1.1 The aryl hydrocarbon receptor as a putative molecular target for cancer therapeutics**

#### *1.1.1 AhR structure*

The aryl hydrocarbon receptor (AhR) was initially defined as a receptor for environmental toxins such as dioxin. It has been described as a "pioneer member" of the basic-helix/loop/helix PER-ARNT-SIM (bHLH/PAS) family of "sensors" of foreign and endogenous signals [12].

As intimated, other members include AhR nuclear translocator (ARNT); *drosophila* proteins, single-minded (SIM) and period (PER); and hypoxia inducible factor 1*α* (HIF 1*α*) [13–16]. AhR is a ligand-activated transcription factor. The most commonly known ligands of AhR are polycyclic and polyhalogenated hydrocarbons (benzopyrene, 3-methyl-colanthrene), xenobiotics (phenobarbital), and other pesticides such as tetrachlorodibenzo-p-dioxin (TCDD).

#### *1.1.2 AhR activation and signaling*

AhR is localized within the cell cytosol constitutively where it is part of an inactivated complex composed of two heat-shock proteins: heat-shock protein 90 (Hsp90), the aryl hydrocarbon receptor interacting protein (AIP), and a 23-kDa protein (p23) (**Figure 1**). Hsp90 acts as a chaperone maintaining AhR in a favorable ligand-binding configuration while preventing its nuclear translocation. Hydrophobic ligands of AhR enter the cell by diffusion and bind to the receptor associated to Hsp90. Ligand binding to the receptor triggers a conformational change in AhR to a form that exhibits stronger affinity for DNA. This event leads to dissociation of the cytoplasmic complex and AhR nuclear translocation. In the nucleus, AhR interacts with ARNT forming a heterodimer that binds to specific DNA sequences—the xenobiotic response elements (XREs) in the promoter regions of genes within the AhR gene battery. Binding leads to transcriptional activation of

**85**

investigation.

**Figure 1.**

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

genes activated by AhR including those encoding phase I and II metabolic enzymes such as cytochrome P450 (CYP) 1A1, CYP1A2, and CYP1B1. AhR activation was first described as a cellular response to promote elimination of ambient contaminants and xenobiotics [17–19]. In humans, AhR is localized in liver, lungs, kidneys, placenta, lymphocytes, ovary, and breast. AhR/ARNT complex activation is tissue-specific and depends on co-regulators and repressors present in different cell types [18].

Functional AhR has been reported in rat and mouse uteri. AhR knockout mice exhibited no histopathological changes in uterine tissues; however, dioxin inhibited estrogen-induced responses including estrogen receptor (ER) binding in rat uteri. In addition, *in utero* and lactational exposure to dioxin results in decreased uterine weights in female offspring during estrus and diestrus. The female reproductive tract expresses mRNA for the transcription factors AhR and ARNT, and changes in their expression at select target sites in specific pathological conditions such as endometriosis and uterine leiomyomas suggest a potential role for these factors in

The role of the AhR and AhR agonists has not been extensively investigated in endometrial and ovarian cancer cell lines; however, there is evidence that AhR-ERα cross talk and growth inhibitory pathways are operative [21–23], requiring further

Intriguingly, immune suppression in ovarian cancer has been described, with a particular focus on the potential involvement of the c-KIT/PI3K/AKT, wnt/β-catenin, IL-6/STAT3, and AhR signaling pathways in regulation of indoleamine 2,3-dioxygen-

AhR has important roles in homeostasis and evidence is accumulating to support

its contribution to disease pathogenesis—including cancer. AhR expression has been detected in multiple tumor types and its function probed by RNA interference, over-expression, and inhibition studies. AhR knockdown led to decreased cancer cell proliferation, migration, and invasion *in vitro*, and *in vivo*, constitutive over-expression resulted in enhanced stomach and liver cancers, suggesting a pro-oncogenic role. In contrast, loss of AhR in transgenic mice that spontaneously

*DOI: http://dx.doi.org/10.5772/intechopen.81194*

**1.2 The importance of AhR in ovarian cancer**

*The aryl hydrocarbon receptor signaling pathway.*

the pathogenesis of these conditions [20].

ase expression in tumor-associated macrophages [24].

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.81194*

**Figure 1.** *The aryl hydrocarbon receptor signaling pathway.*

*Current Trends in Cancer Management*

fied or silenced in tumor progression [6, 7].

rib (PARP inhibitors) [11].

**therapeutics**

*1.1.1 AhR structure*

cancers including ovarian cancer.

foreign and endogenous signals [12].

*1.1.2 AhR activation and signaling*

pesticides such as tetrachlorodibenzo-p-dioxin (TCDD).

The current strategy for treatment of ovarian cancer is surgery followed by radiotherapy and chemotherapy [3, 4]. Although ~70% of ovarian cancer patients respond initially to a combination of platinum and taxane-based chemotherapy, drug-resistance emerges and current treatments are of limited efficacy in preventing tumor recurrence and progression [3, 4]. Thus, despite surgery, radiotherapy, and chemotherapy, most patients present with recurrent disease within 12–18 months, which spreads rapidly within the peritoneal cavity. In platinumresistant disease, survival rarely exceeds 5 months [1, 5]. Thus, new anti-neoplastic agents are urgently needed to improve the prognoses for ovarian cancer patients. Recently, evidence has emerged revealing the importance of genomic aberrations in the progression of ovarian cancer [6–8]. Through the use of high-throughput technologies (i.e., array comparative genomic hybridization (aCGH), microarray, and SNP arrays), specific genomic regions have been identified to be either ampli-

Molecularly targeted cancer therapies recently introduced into the clinic include drugs designed to interfere with specific proteins ("molecular targets") that are involved in the growth, progression, and spread of cancer. Used in the treatment of ovarian cancer are bevacizumab (a VEGF inhibitor) [9], olaparib [10], and nirapa-

The objective of our work has been for many years to validate the aryl hydrocarbon receptor as a molecular target of antitumor drugs, for the treatment of different

**1.1 The aryl hydrocarbon receptor as a putative molecular target for cancer** 

The aryl hydrocarbon receptor (AhR) was initially defined as a receptor for environmental toxins such as dioxin. It has been described as a "pioneer member" of the basic-helix/loop/helix PER-ARNT-SIM (bHLH/PAS) family of "sensors" of

As intimated, other members include AhR nuclear translocator (ARNT); *drosophila* proteins, single-minded (SIM) and period (PER); and hypoxia inducible factor 1*α* (HIF 1*α*) [13–16]. AhR is a ligand-activated transcription factor. The most commonly known ligands of AhR are polycyclic and polyhalogenated hydrocarbons (benzopyrene, 3-methyl-colanthrene), xenobiotics (phenobarbital), and other

AhR is localized within the cell cytosol constitutively where it is part of an inactivated complex composed of two heat-shock proteins: heat-shock protein 90 (Hsp90), the aryl hydrocarbon receptor interacting protein (AIP), and a 23-kDa protein (p23) (**Figure 1**). Hsp90 acts as a chaperone maintaining AhR in a favorable ligand-binding configuration while preventing its nuclear translocation. Hydrophobic ligands of AhR enter the cell by diffusion and bind to the receptor associated to Hsp90. Ligand binding to the receptor triggers a conformational change in AhR to a form that exhibits stronger affinity for DNA. This event leads to dissociation of the cytoplasmic complex and AhR nuclear translocation. In the nucleus, AhR interacts with ARNT forming a heterodimer that binds to specific DNA sequences—the xenobiotic response elements (XREs) in the promoter regions of genes within the AhR gene battery. Binding leads to transcriptional activation of

**84**

genes activated by AhR including those encoding phase I and II metabolic enzymes such as cytochrome P450 (CYP) 1A1, CYP1A2, and CYP1B1. AhR activation was first described as a cellular response to promote elimination of ambient contaminants and xenobiotics [17–19]. In humans, AhR is localized in liver, lungs, kidneys, placenta, lymphocytes, ovary, and breast. AhR/ARNT complex activation is tissue-specific and depends on co-regulators and repressors present in different cell types [18].

#### **1.2 The importance of AhR in ovarian cancer**

Functional AhR has been reported in rat and mouse uteri. AhR knockout mice exhibited no histopathological changes in uterine tissues; however, dioxin inhibited estrogen-induced responses including estrogen receptor (ER) binding in rat uteri. In addition, *in utero* and lactational exposure to dioxin results in decreased uterine weights in female offspring during estrus and diestrus. The female reproductive tract expresses mRNA for the transcription factors AhR and ARNT, and changes in their expression at select target sites in specific pathological conditions such as endometriosis and uterine leiomyomas suggest a potential role for these factors in the pathogenesis of these conditions [20].

The role of the AhR and AhR agonists has not been extensively investigated in endometrial and ovarian cancer cell lines; however, there is evidence that AhR-ERα cross talk and growth inhibitory pathways are operative [21–23], requiring further investigation.

Intriguingly, immune suppression in ovarian cancer has been described, with a particular focus on the potential involvement of the c-KIT/PI3K/AKT, wnt/β-catenin, IL-6/STAT3, and AhR signaling pathways in regulation of indoleamine 2,3-dioxygenase expression in tumor-associated macrophages [24].

AhR has important roles in homeostasis and evidence is accumulating to support its contribution to disease pathogenesis—including cancer. AhR expression has been detected in multiple tumor types and its function probed by RNA interference, over-expression, and inhibition studies. AhR knockdown led to decreased cancer cell proliferation, migration, and invasion *in vitro*, and *in vivo*, constitutive over-expression resulted in enhanced stomach and liver cancers, suggesting a pro-oncogenic role. In contrast, loss of AhR in transgenic mice that spontaneously

develop colorectal carcinoma (CRC) and carcinogen-induced tumors led to increased carcinogenesis suggesting a tumor-suppressive role for AhR [25].

Specific to this review, AhR has been found to be widely expressed in many histotypes of ovarian cancer tissue; in the ovarian cancer tissue microarray, the AhR immunoreactivity was present in disgerminoma (DISG), adenocarcinoma (ADEN), teratoma malignant change (TMC), yolk sac tumor (YST), mucinous adenocarcinoma (Mu-ADEN), serous adenocarcinoma (low grade (L-Se-ADEN) and high grade (H-Se-ADEN), but not in normal tissue (NORM). The semi-quantification analysis revealed that the value in NORM was similar to that in DISG and ADEN, but was much lower than that in TMC, YST, Mu-ADEN, and L- and H-Se-ADEN. No difference was detected between the grades, stages, and tumor node metastasis classifications in each histotype of ovarian cancer tissues studied. Moreover, the endogenous AhR ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) inhibited proliferation of OVCAR-3 cells and migration of SKOV-3 cells *in vitro* and suppressed growth of OVCAR-3 cell xenografts in mice [26].

#### **1.3 Benzothiazoles and aminoflavone: AhR-targeted anticancer therapies**

The benzothiazole (Bz) class of experimental antitumor agent includes 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203; NSC 703786; **Figure 2**); nonfluorinated parent compound DF 203 (NSC 674495); and Phortress (NSC 710305), the lysyl amide prodrug of 5F 203. These experimental antitumor agents elicit potent and selective antitumor activity *in vitro* against certain cell lines originating from breast (irrespective of ER status) and ovarian carcinomas. Empirical screening in the NCI cell line anticancer drug screen revealed that the Bzs [27–32] and also aminoflavone (AF) [33] were noteworthy for their distinct (selective and potent) patterns of growth inhibitory activity. "Sensitive" cell lines caused total carcinoma cell growth inhibition (TGI) between 0.1 and 1 μM, while "resistant" cell lines are refractory to Bz and AF (TGI concentrations *<*10 μM). Among the consistently sensitive cell lines to both compound classes were the ER (+) breast cancer cell lines MCF-7 and T47D, certain ovarian (e.g., IGROV-1) and renal (TK10) cancer cell lines [34]. Intriguingly, IGROV-1 cells developed in the laboratory possessing acquired resistance to cisplatin were equi-sensitive to antitumor Bzs as IGROV-1 parental cells. Detailed mechanistic studies for both Bzs and AF identified a mode of action distinct from current clinical chemotherapeutic agents. In "sensitive" cells, Bzs and AF activate AhR signaling, as might be anticipated from their planar structures [35].

AhR signal transduction induces expression of CYP1A1 and (in certain cell lines) CYP1B1. Prior work has demonstrated that CYP1A1 can metabolize (actively biotransform) Bzs and AF to produce DNA-damaging metabolites [30, 33] (**Figure 2**). For example, DNA adducts, single- and double-strand breaks have been detected exclusively in sensitive cells exposed to 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), and in Bz-sensitive tumor xenografts extracted from Phortress-treated mice [36–39]. Subsequently, it was irrefutably demonstrated that CYP1A1-bioactivation of 5F 203 resulted in generation of an electrophilic species (nitrenium ion) able to form glutathione conjugates and dGuo adducts [40].

#### **1.4 5F 203 activity in ovarian cancer**

Antitumor Bzs, including 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203; NSC 703786; **Figure 2**), non-fluorinated parent compound DF 203 (NSC 674495), and Phortress (NSC 710305), the lysyl amide prodrug of 5F 203, are experimental anticancer agents with activity in ovarian and breast cancer models *in vitro* and *in vivo* (**Figure 3A** and **B**) [36, 41].

**87**

**Figure 2.**

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

As introduced, these compounds require metabolism by cytochrome P450 (CYP) enzymes (e.g., CYP1A1) for antitumor action. The aryl hydrocarbon receptor (AhR) is the main transcriptional regulator of CYP1A1 and we have previously demonstrated that DF 203 and 5F 203 induce activation of AhR signaling in sensitive breast cancer cells, causing nuclear translocation of AhR [27–29]. Also, IGROV-1 human ovarian cancer cells, sensitive to 5F 203 treatment, show induction of CYP1A1 expression by 5F 203 and Phortress (**Figure 3B**), whereas SKOV-3 cells, resistant to these agents, express neither constitutive nor inducible CYP1A1 [42]. Fine needle aspirates obtained from IGROV-1 human xenografts, treated *ex vivo* with 5F 203, resulted in induction of CYP1A1 expression. This was not observed in 5F 203-resistant tumors. It was proposed that induction of *cyp1a1* mRNA in response to 5F 203 treatment *ex vivo* might provide a possible biomarker for determination of drug-sensitive ovarian tumors in patients [42]. Compounds that activate AhR signaling and induce CYP expression frequently generate reactive oxygen species (ROS) in susceptible cells. Experimental and clinical evidence has emerged linking oxidative stress to pathologies including carcinogenesis [43]. However, oxidative stress is not always detrimental, and may, if induced in a selective manner, be of therapeutic benefit. Many chemotherapeutic agents induce oxidative stress integral to their antitumor mechanism [44]. High-grade ovarian tumors generally present high ROS levels and respond better to treatment with antitumor agents that induce further ROS, such as paclitaxel [45]. In addition, c-JUN amino terminal kinase (JNK), ERK, and P38MAPK

*5F 203, liberated from Phortress binds cytosolic AhR, triggering nuclear translocation and binding to the XRE within the promoter region of AhR-battery genes including CYP 1A1. This enzyme-catalyzed biotransformation of 5F 203 produces nitrenium species that generate lethal DNA adducts at nucleophilic sites precipitating DNA double-strand breaks and apoptosis, exclusively in sensitive cells. In inherently resistant cells,* 

sustained activation have key roles in cellular stress-induced apoptosis [46].

**sequences in 5F 203-sensitive ovarian cancer cells**

**1.5 5F 203 induces AhR translocation and activation of CYP 1A1-related promoter** 

It has been established that 5F 203 is a potent AhR ligand [35] able to induce nuclear translocation of AhR with subsequent binding to XRE sequences resulting

*DOI: http://dx.doi.org/10.5772/intechopen.81194*

*there is no net cellular uptake of lipophilic 5F 203.*

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.81194*

#### **Figure 2.**

*Current Trends in Cancer Management*

develop colorectal carcinoma (CRC) and carcinogen-induced tumors led to increased carcinogenesis suggesting a tumor-suppressive role for AhR [25]. Specific to this review, AhR has been found to be widely expressed in many histotypes of ovarian cancer tissue; in the ovarian cancer tissue microarray, the AhR immunoreactivity was present in disgerminoma (DISG), adenocarcinoma (ADEN), teratoma malignant change (TMC), yolk sac tumor (YST), mucinous adenocarcinoma (Mu-ADEN), serous adenocarcinoma (low grade (L-Se-ADEN) and high grade (H-Se-ADEN), but not in normal tissue (NORM). The semi-quantification analysis revealed that the value in NORM was similar to that in DISG and ADEN, but was much lower than that in TMC, YST, Mu-ADEN, and L- and H-Se-ADEN. No difference was detected between the grades, stages, and tumor node metastasis classifications in each histotype of ovarian cancer tissues studied. Moreover, the endogenous AhR ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) inhibited proliferation of OVCAR-3 cells and migration of SKOV-3 cells

*in vitro* and suppressed growth of OVCAR-3 cell xenografts in mice [26].

**1.3 Benzothiazoles and aminoflavone: AhR-targeted anticancer therapies**

3-methylphenyl)-5-fluorobenzothiazole (5F 203; NSC 703786; **Figure 2**); non-

anticipated from their planar structures [35].

**1.4 5F 203 activity in ovarian cancer**

*vitro* and *in vivo* (**Figure 3A** and **B**) [36, 41].

The benzothiazole (Bz) class of experimental antitumor agent includes 2-(4-amino-

fluorinated parent compound DF 203 (NSC 674495); and Phortress (NSC 710305), the lysyl amide prodrug of 5F 203. These experimental antitumor agents elicit potent and selective antitumor activity *in vitro* against certain cell lines originating from breast (irrespective of ER status) and ovarian carcinomas. Empirical screening in the NCI cell line anticancer drug screen revealed that the Bzs [27–32] and also aminoflavone (AF) [33] were noteworthy for their distinct (selective and potent) patterns of growth inhibitory activity. "Sensitive" cell lines caused total carcinoma cell growth inhibition (TGI) between 0.1 and 1 μM, while "resistant" cell lines are refractory to Bz and AF (TGI concentrations *<*10 μM). Among the consistently sensitive cell lines to both compound classes were the ER (+) breast cancer cell lines MCF-7 and T47D, certain ovarian (e.g., IGROV-1) and renal (TK10) cancer cell lines [34]. Intriguingly, IGROV-1 cells developed in the laboratory possessing acquired resistance to cisplatin were equi-sensitive to antitumor Bzs as IGROV-1 parental cells. Detailed mechanistic studies for both Bzs and AF identified a mode of action distinct from current clinical chemotherapeutic agents. In "sensitive" cells, Bzs and AF activate AhR signaling, as might be

AhR signal transduction induces expression of CYP1A1 and (in certain cell lines) CYP1B1. Prior work has demonstrated that CYP1A1 can metabolize (actively biotransform) Bzs and AF to produce DNA-damaging metabolites [30, 33] (**Figure 2**). For example, DNA adducts, single- and double-strand breaks have been detected exclusively in sensitive cells exposed to 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), and in Bz-sensitive tumor xenografts extracted from Phortress-treated mice [36–39]. Subsequently, it was irrefutably demonstrated that CYP1A1-bioactivation of 5F 203 resulted in generation of an electrophilic species (nitrenium ion) able to form glutathione conjugates and dGuo adducts [40].

Antitumor Bzs, including 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203; NSC 703786; **Figure 2**), non-fluorinated parent compound DF 203 (NSC 674495), and Phortress (NSC 710305), the lysyl amide prodrug of 5F 203, are experimental anticancer agents with activity in ovarian and breast cancer models *in* 

**86**

*5F 203, liberated from Phortress binds cytosolic AhR, triggering nuclear translocation and binding to the XRE within the promoter region of AhR-battery genes including CYP 1A1. This enzyme-catalyzed biotransformation of 5F 203 produces nitrenium species that generate lethal DNA adducts at nucleophilic sites precipitating DNA double-strand breaks and apoptosis, exclusively in sensitive cells. In inherently resistant cells, there is no net cellular uptake of lipophilic 5F 203.*

As introduced, these compounds require metabolism by cytochrome P450 (CYP) enzymes (e.g., CYP1A1) for antitumor action. The aryl hydrocarbon receptor (AhR) is the main transcriptional regulator of CYP1A1 and we have previously demonstrated that DF 203 and 5F 203 induce activation of AhR signaling in sensitive breast cancer cells, causing nuclear translocation of AhR [27–29]. Also, IGROV-1 human ovarian cancer cells, sensitive to 5F 203 treatment, show induction of CYP1A1 expression by 5F 203 and Phortress (**Figure 3B**), whereas SKOV-3 cells, resistant to these agents, express neither constitutive nor inducible CYP1A1 [42]. Fine needle aspirates obtained from IGROV-1 human xenografts, treated *ex vivo* with 5F 203, resulted in induction of CYP1A1 expression. This was not observed in 5F 203-resistant tumors. It was proposed that induction of *cyp1a1* mRNA in response to 5F 203 treatment *ex vivo* might provide a possible biomarker for determination of drug-sensitive ovarian tumors in patients [42]. Compounds that activate AhR signaling and induce CYP expression frequently generate reactive oxygen species (ROS) in susceptible cells.

Experimental and clinical evidence has emerged linking oxidative stress to pathologies including carcinogenesis [43]. However, oxidative stress is not always detrimental, and may, if induced in a selective manner, be of therapeutic benefit. Many chemotherapeutic agents induce oxidative stress integral to their antitumor mechanism [44]. High-grade ovarian tumors generally present high ROS levels and respond better to treatment with antitumor agents that induce further ROS, such as paclitaxel [45]. In addition, c-JUN amino terminal kinase (JNK), ERK, and P38MAPK sustained activation have key roles in cellular stress-induced apoptosis [46].

#### **1.5 5F 203 induces AhR translocation and activation of CYP 1A1-related promoter sequences in 5F 203-sensitive ovarian cancer cells**

It has been established that 5F 203 is a potent AhR ligand [35] able to induce nuclear translocation of AhR with subsequent binding to XRE sequences resulting

#### **Figure 3.**

*A. In vitro growth inhibitory activity of 5F 203 against ovarian carcinoma cell lines. Representative data generated at the NCI are shown. Cells were exposed to test agent for 48 h before growth was assessed by sulforhodamine blue (SRB) assay. B. In vivo Phortress efficacy against IGROV-1 ovarian tumor xenografts. IGROV-1 ovarian xenografts were transplanted s.c. into flanks of NCR-Nu female nude mice. Animals were treated i.v. with Phortress in saline according to the indicated schedules. Control animals received vehicle alone. Tumor volumes were measured using calipers. Western blot showing induction of CYP1A1 protein in IGROV-1 xenograft tumors retrieved from mice treated with Phortress (20 mg/kg; 24 h). Tumor lysates were prepared and proteins separated by PAGE: Lane 1, +ve control, 5 μg CYP1A1 microsomes; lanes 2 and 3, untreated and vehicle control-treated samples; lanes 4 and 5, 24 h 20 mg/kg Phortress [36].*

in transcriptional activation of target genes including CYP1A1 and CYP1B1 in breast cancer cells sensitive to this agent [28]. As 5F 203 causes IGROV-1 cytotoxicity and consistent with the hypothesis that 5F 203 is an AhR agonist ligand [47], we wished to determine whether 5F 203 activates AhR signaling in IGROV-1 cells with resulting AhR translocation from cytoplasm to nucleus.

The effect of 5F 203 (1 μM) on subcellular distribution of immunoreactive AhR protein was studied by Western blot. We demonstrated (**Figure 4A**), in IGROV-1 cells treated with DMSO only, the cytoplasmic fraction contained relatively high levels of AhR protein compared with the nuclear fraction. In contrast, after treatment of cells with AhR agonists, 1 μM 5F 203 or 10 nM TCDD (positive control), between 1 and 6 h, immunoreactive AhR protein could be detected in the nuclear fraction, indicating AhR translocation [48].

To identify whether 5F 203 caused AhR nuclear translocation in 5F 203-insensitive ovarian carcinoma cells, the effect of 5F 203 (1 μM) on subcellular distribution

**89**

**Figure 4.**

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

of immunoreactive AhR protein was assessed by Western blot in SKOV-3 cells. In DMSO-treated cells, AhR protein levels were high in cytoplasmic and nuclear fractions. After treatment with 1 μM 5F 203 (0.5–6 h), AhR protein in SKOV-3 cytoplasm remained unchanged. AhR was already present in SKOV-3 nuclei and further

After treatment with TCDD, although cytoplasmic AhR was lost, there was no evidence of AhR gain in nuclear fractions (**Figure 4A**). These results were con-

It was then logical to investigate putative activation of CYP1A1-related promoter

CYP1A1 and CYP1B1 promoters are regulated by AhR, which forms a heterodimer with the AhR nuclear transporter (ARNT). Binding of the complete dimer to XRE promoter regions mediates transcription of AhR-responsive genes, including CYP1A1. IGROV-1 and SKOV-3 were transfected with XRE-luciferase reporter construct (pTX.Dir), as a control, the same reporter construct without XRE elements (pT81) was used. Cells were then treated with 0.1% DMSO, 0.1–1 μM 5F 203 or TCDD 10 nM. In IGROV-1 cells transfected with pTX.Dir, 5.5-fold induction of luciferase activity was observed when cells were treated with 1 μM 5F 203 (**Figure 4B**).

*5F 203 induces AhR nuclear translocation and activation of CYP1A1-related promoter sequences in sensitive ovarian cancer cell lines. A: AhR Subcellular localization. IGROV-1 and SKOV-3 cells were incubated with 5F 203 (1 μM) for indicated times, DMSO (0.1%) for 6 h or TCDD (10 nM) for 1 h as a positive control. Nuclear and cytosolic fractions were isolated using a commercial kit and analyzed for AhR content by Western blot. The figure shows representative Western blots. All Western blots were performed three times for each cell line and revealed the same pattern of AhR subcellular distribution. B: Activation of CYP1A1-related promoter sequences. Cells were transfected with a plasmid containing XRE (AhR consensus sequences) upstream of the luciferase reporter gene and a second plasmid containing R. reniformis luciferase gene as an internal control. After 24 h, cells were incubated with 5F 203 for 18 h or pre-treated for 1 h with 1 μM α-NF followed by 18 h of 5F 203 plus 1 μM α-NF. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System* 

*(Promega). Values represent the average of three independent experiments.*

*DOI: http://dx.doi.org/10.5772/intechopen.81194*

firmed by immunofluorescence of AhR.

sequences in 5F 203-sensitive ovarian cancer cells.

translocation was negligible.

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.81194*

of immunoreactive AhR protein was assessed by Western blot in SKOV-3 cells. In DMSO-treated cells, AhR protein levels were high in cytoplasmic and nuclear fractions. After treatment with 1 μM 5F 203 (0.5–6 h), AhR protein in SKOV-3 cytoplasm remained unchanged. AhR was already present in SKOV-3 nuclei and further translocation was negligible.

After treatment with TCDD, although cytoplasmic AhR was lost, there was no evidence of AhR gain in nuclear fractions (**Figure 4A**). These results were confirmed by immunofluorescence of AhR.

It was then logical to investigate putative activation of CYP1A1-related promoter sequences in 5F 203-sensitive ovarian cancer cells.

CYP1A1 and CYP1B1 promoters are regulated by AhR, which forms a heterodimer with the AhR nuclear transporter (ARNT). Binding of the complete dimer to XRE promoter regions mediates transcription of AhR-responsive genes, including CYP1A1. IGROV-1 and SKOV-3 were transfected with XRE-luciferase reporter construct (pTX.Dir), as a control, the same reporter construct without XRE elements (pT81) was used. Cells were then treated with 0.1% DMSO, 0.1–1 μM 5F 203 or TCDD 10 nM. In IGROV-1 cells transfected with pTX.Dir, 5.5-fold induction of luciferase activity was observed when cells were treated with 1 μM 5F 203 (**Figure 4B**).

#### **Figure 4.**

*Current Trends in Cancer Management*

in transcriptional activation of target genes including CYP1A1 and CYP1B1 in breast cancer cells sensitive to this agent [28]. As 5F 203 causes IGROV-1 cytotoxicity and consistent with the hypothesis that 5F 203 is an AhR agonist ligand [47], we wished to determine whether 5F 203 activates AhR signaling in IGROV-1 cells with

*A. In vitro growth inhibitory activity of 5F 203 against ovarian carcinoma cell lines. Representative data generated at the NCI are shown. Cells were exposed to test agent for 48 h before growth was assessed by sulforhodamine blue (SRB) assay. B. In vivo Phortress efficacy against IGROV-1 ovarian tumor xenografts. IGROV-1 ovarian xenografts were transplanted s.c. into flanks of NCR-Nu female nude mice. Animals were treated i.v. with Phortress in saline according to the indicated schedules. Control animals received vehicle alone. Tumor volumes were measured using calipers. Western blot showing induction of CYP1A1 protein in IGROV-1 xenograft tumors retrieved from mice treated with Phortress (20 mg/kg; 24 h). Tumor lysates were prepared and proteins separated by PAGE: Lane 1, +ve control, 5 μg CYP1A1 microsomes; lanes 2 and 3, untreated and* 

The effect of 5F 203 (1 μM) on subcellular distribution of immunoreactive AhR protein was studied by Western blot. We demonstrated (**Figure 4A**), in IGROV-1 cells treated with DMSO only, the cytoplasmic fraction contained relatively high levels of AhR protein compared with the nuclear fraction. In contrast, after treatment of cells with AhR agonists, 1 μM 5F 203 or 10 nM TCDD (positive control), between 1 and 6 h, immunoreactive AhR protein could be detected in the nuclear

To identify whether 5F 203 caused AhR nuclear translocation in 5F 203-insensitive ovarian carcinoma cells, the effect of 5F 203 (1 μM) on subcellular distribution

resulting AhR translocation from cytoplasm to nucleus.

*vehicle control-treated samples; lanes 4 and 5, 24 h 20 mg/kg Phortress [36].*

fraction, indicating AhR translocation [48].

**88**

**Figure 3.**

*5F 203 induces AhR nuclear translocation and activation of CYP1A1-related promoter sequences in sensitive ovarian cancer cell lines. A: AhR Subcellular localization. IGROV-1 and SKOV-3 cells were incubated with 5F 203 (1 μM) for indicated times, DMSO (0.1%) for 6 h or TCDD (10 nM) for 1 h as a positive control. Nuclear and cytosolic fractions were isolated using a commercial kit and analyzed for AhR content by Western blot. The figure shows representative Western blots. All Western blots were performed three times for each cell line and revealed the same pattern of AhR subcellular distribution. B: Activation of CYP1A1-related promoter sequences. Cells were transfected with a plasmid containing XRE (AhR consensus sequences) upstream of the luciferase reporter gene and a second plasmid containing R. reniformis luciferase gene as an internal control. After 24 h, cells were incubated with 5F 203 for 18 h or pre-treated for 1 h with 1 μM α-NF followed by 18 h of 5F 203 plus 1 μM α-NF. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega). Values represent the average of three independent experiments.*

No induction in luciferase activity was observed when SKOV-3 cells transfected with pTX.Dir were treated with 5F 203 (1 μM). Cells transfected with pT81 and treated with 1 μM 5F 203 showed negligible luciferase activity induction (1.1-fold).

These findings clearly demonstrated that 5F 203 induces activation of promoter sequences known to respond to AhR-mediated signals. This is consistent with interaction of protein complexes induced by treatment with 5F 203 through the XRE CYP1A1 promoter sequence.

In IGROV-1 cells, pre-treatment (1 h) with AhR antagonist α-NF (1 μM) followed by co-treatment (18 h) with 5F 203 plus 1 μM α-NF reduced induction (2.65-fold) of luciferase activity. These results support the requirement of AhR in increased XRE-luciferase activity [48].

#### **1.6 5F 203-induced ROS and γH2AX foci formation in sensitive cells is mediated by AhR**

Previous studies demonstrated CYP1A1 induction by 5F 203, and CYP catalyzed 5F 203 biotransformation to DNA reactive species [40]. CYP activity contributed to increased intracellular ROS levels [49]. Oxidative stress is involved in various biological processes including proliferation and apoptosis. Therefore, we compared the effect of 5F 203 on ROS production in Bz-sensitive IGROV-1 cells and Bz-insensitive SKOV-3 cells. To determine whether 5F 203 altered intracellular ROS levels, cells were treated with 5F 203 for 6 h and exposed to 2,7-DCF before ROS levels were evaluated using flow cytometry. 5F 203 increased ROS levels in IGROV-1 cells only. This effect, detectable following 1 h of 1 μM 5F 203 treatment, was partially blocked by pre-incubation of cells with α-NF (**Figure 5A**). Also, pre-treatment with ROS inhibitors N-acetyl cysteine (NAC) and Trolox partially decreased the effect of 5F 203 in IGROV-1 cells (**Figure 5B**). In contrast, 5F 203 strongly inhibited ROS production in SKOV-3 cells, despite detection of neither phenotypic changes nor AhR translocation [48].

It is reported that ROS may cause activation of nuclear factor kappa B (NF-κB) [50]. As activation of this pathway induces NF-κB nuclear translocation, we performed immunostaining of NF-κB using a specific antibody in IGROV-1 and SKOV-3 cells before and after treatment with 5F 203. 5F 203 induced NF-κB translocation in IGROV-1 cells only, and this effect was prevented by pre-treatment of cells with 1 μM α-NF. We then investigated whether 5F 203 caused DNA damage. To determine DNA double-strand breaks (DSB), **γ**H2AX foci were measured in IGROV-1 and SKOV-3 cells following exposure to 5F 203 (1 μM, 2–4 h). DNA DSB formation precipitates serine 139 phosphorylation of histone H2AX, producing **γ**H2AX at DSB sites [48]. **γ**H2AX foci appeared within nuclei of IGROV-1 cells only following treatment with 5F 203 (1 μM; 2–4 h; **Figure 5C**). Foci formation was partially blocked by pre-treatment of cells with α-NF, confirming that activation of AhR is needed for 5F 203-induced DNA damage. In contrast, neither vehicle-treated cells nor SKOV-3 cells challenged with 5F 203 displayed **γ**H2AX foci within their nuclei at any time point examined. These data are consistent with neutral COMET assays performed to examine DNA damage (double-strand breaks) wrought as a consequence of dose-dependent DNA adduct generation which has been detected following treatment of IGROV-1 cells *in vitro* with 5F 203 or Phortress, and in IGROV-1 xenografts retrieved from tumorbearing mice exposed to Phortress [36].

#### **1.7 5F 203 modulates expression and phosphorylation of stress MAPKs**

Mitogen-activated protein kinases (MAPKs) can be modulated by many factors including cell lesions induced by DNA damage [51]. We therefore investigated the effect of 5F 203 on activation of these protein kinases in IGROV-1 cells. As depicted

**91**

**Figure 5.**

203-induced cell death [48].

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

in **Figure 6A**, 5F 203 induced phosphorylation of JNK and P38, the stronger effect was attained after 6 h of treatment in both cases. Also, treatment with 5F 203 increased P38 α levels. Pre-treatment of cells with α-NF decreased phosphorylation of these kinases and P38 α expression, which confirmed that 5F 203 affected expression and activation of these proteins through AhR activation. Exposure to 5F

*AN 0.95 objective, images were processed and analyzed with Nikon C1-EZ package, version 2.20.*

*5F 203 increases ROS levels. Exponentially growing IGROV-1 and SKOV-3 cells were treated with 5F 203 (1 μM) or vehicle control (0.1% DMSO) continuously for 1, 2, 4, or 6 h and ROS levels were measured by flow cytometry after incubation with 2,7-DCF. Data represent the mean of at least two independent experiments where n = 2 per experiment; bars, SEM. \*P < 0.05 when compared to untreated cells, \*\*P < 0.05 when compared to cells treated without AhR inhibitor. B: Trolox and NAC inhibition of 5F 203-mediated ROS induction. IGROV-1 cells were exposed to 0.1% DMSO (control), 5F 203 (1 μM) for 1 or 2 h, or pre-treated with Trolox (250 μM) or NAC (100 μM) for 1 h followed by 5F 203 (1 μM) inhibitor for 1 or 2 h. ROS levels were measured by fluorometry after incubation with 2,7-DCF. Data represent the mean of at least two independent experiments where n = 2 per experiment; bars, SEM. \*P < 0.05 or \*\*P < 0.01 when compared to cells treated without ROS inhibitor. C: Induces γH2AX foci formation in sensitive IGROV-1 cells. A: Measurement of ROS levels. γH2AX foci following 2–4 h of treatment of cells with 5F 203 (1 μM); IGROV-1 cell nuclei were stained with DAPI. Stained cells were visualized on a fluorescence Nikon C1 confocal microscope using a 60X PlanApo* 

The ability of 5F 203 to induce apoptosis was evaluated. Exposure of IGROV-1 cells to 5F 203 (1 μM; 24 h) induced apoptotic body formation (**Figure 6B**). In contrast, SKOV-3 cells, resistant to 5F 203, did not show such features (**Figure 6B**). Also, pre-treatment of cells with α-NF partially blocked the pro-apoptotic effect of 5F 203 in IGROV-1 cells. These data confirmed that AhR is involved in 5F

Trolox (a vitamin E derivative) and NAC are potent ROS scavengers often used as antioxidant agents. We pre-treated IGROV-1 cells with Trolox or NAC in order to investigate the effect of ROS depletion on 5F 203-induced growth inhibition. Cells were exposed to 5F 203 (1 μM, 24 h) after pre-treatment with Trolox (250 μM) or NAC (100 μM) for 1 h. As observed in **Figure 6B,** both inhibitors partially reduced 5F 203-induced apoptotic body formation. These data support the involvement of ROS-generation in 5F 203-induced apoptosis in sensitive IGROV-1 cells [48].

203 (1 h) also increased phosphorylation of ERK in IGROV-1 cells.

*DOI: http://dx.doi.org/10.5772/intechopen.81194*

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.81194*

#### **Figure 5.**

*Current Trends in Cancer Management*

XRE CYP1A1 promoter sequence.

**by AhR**

increased XRE-luciferase activity [48].

bearing mice exposed to Phortress [36].

No induction in luciferase activity was observed when SKOV-3 cells transfected with pTX.Dir were treated with 5F 203 (1 μM). Cells transfected with pT81 and treated with 1 μM 5F 203 showed negligible luciferase activity induction (1.1-fold).

These findings clearly demonstrated that 5F 203 induces activation of promoter

sequences known to respond to AhR-mediated signals. This is consistent with interaction of protein complexes induced by treatment with 5F 203 through the

In IGROV-1 cells, pre-treatment (1 h) with AhR antagonist α-NF (1 μM) followed by co-treatment (18 h) with 5F 203 plus 1 μM α-NF reduced induction (2.65-fold) of luciferase activity. These results support the requirement of AhR in

**1.6 5F 203-induced ROS and γH2AX foci formation in sensitive cells is mediated** 

Previous studies demonstrated CYP1A1 induction by 5F 203, and CYP catalyzed 5F 203 biotransformation to DNA reactive species [40]. CYP activity contributed to increased intracellular ROS levels [49]. Oxidative stress is involved in various biological processes including proliferation and apoptosis. Therefore, we compared the effect of 5F 203 on ROS production in Bz-sensitive IGROV-1 cells and Bz-insensitive SKOV-3 cells. To determine whether 5F 203 altered intracellular ROS levels, cells were treated with 5F 203 for 6 h and exposed to 2,7-DCF before ROS levels were evaluated using flow cytometry. 5F 203 increased ROS levels in IGROV-1 cells only. This effect, detectable following 1 h of 1 μM 5F 203 treatment, was partially blocked by pre-incubation of cells with α-NF (**Figure 5A**). Also, pre-treatment with ROS inhibitors N-acetyl cysteine (NAC) and Trolox partially decreased the effect of 5F 203 in IGROV-1 cells (**Figure 5B**). In contrast, 5F 203 strongly inhibited ROS production in SKOV-3 cells,

despite detection of neither phenotypic changes nor AhR translocation [48].

**1.7 5F 203 modulates expression and phosphorylation of stress MAPKs**

Mitogen-activated protein kinases (MAPKs) can be modulated by many factors including cell lesions induced by DNA damage [51]. We therefore investigated the effect of 5F 203 on activation of these protein kinases in IGROV-1 cells. As depicted

It is reported that ROS may cause activation of nuclear factor kappa B (NF-κB) [50]. As activation of this pathway induces NF-κB nuclear translocation, we performed immunostaining of NF-κB using a specific antibody in IGROV-1 and SKOV-3 cells before and after treatment with 5F 203. 5F 203 induced NF-κB translocation in IGROV-1 cells only, and this effect was prevented by pre-treatment of cells with 1 μM α-NF. We then investigated whether 5F 203 caused DNA damage. To determine DNA double-strand breaks (DSB), **γ**H2AX foci were measured in IGROV-1 and SKOV-3 cells following exposure to 5F 203 (1 μM, 2–4 h). DNA DSB formation precipitates serine 139 phosphorylation of histone H2AX, producing **γ**H2AX at DSB sites [48]. **γ**H2AX foci appeared within nuclei of IGROV-1 cells only following treatment with 5F 203 (1 μM; 2–4 h; **Figure 5C**). Foci formation was partially blocked by pre-treatment of cells with α-NF, confirming that activation of AhR is needed for 5F 203-induced DNA damage. In contrast, neither vehicle-treated cells nor SKOV-3 cells challenged with 5F 203 displayed **γ**H2AX foci within their nuclei at any time point examined. These data are consistent with neutral COMET assays performed to examine DNA damage (double-strand breaks) wrought as a consequence of dose-dependent DNA adduct generation which has been detected following treatment of IGROV-1 cells *in vitro* with 5F 203 or Phortress, and in IGROV-1 xenografts retrieved from tumor-

**90**

*5F 203 increases ROS levels. Exponentially growing IGROV-1 and SKOV-3 cells were treated with 5F 203 (1 μM) or vehicle control (0.1% DMSO) continuously for 1, 2, 4, or 6 h and ROS levels were measured by flow cytometry after incubation with 2,7-DCF. Data represent the mean of at least two independent experiments where n = 2 per experiment; bars, SEM. \*P < 0.05 when compared to untreated cells, \*\*P < 0.05 when compared to cells treated without AhR inhibitor. B: Trolox and NAC inhibition of 5F 203-mediated ROS induction. IGROV-1 cells were exposed to 0.1% DMSO (control), 5F 203 (1 μM) for 1 or 2 h, or pre-treated with Trolox (250 μM) or NAC (100 μM) for 1 h followed by 5F 203 (1 μM) inhibitor for 1 or 2 h. ROS levels were measured by fluorometry after incubation with 2,7-DCF. Data represent the mean of at least two independent experiments where n = 2 per experiment; bars, SEM. \*P < 0.05 or \*\*P < 0.01 when compared to cells treated without ROS inhibitor. C: Induces γH2AX foci formation in sensitive IGROV-1 cells. A: Measurement of ROS levels. γH2AX foci following 2–4 h of treatment of cells with 5F 203 (1 μM); IGROV-1 cell nuclei were stained with DAPI. Stained cells were visualized on a fluorescence Nikon C1 confocal microscope using a 60X PlanApo AN 0.95 objective, images were processed and analyzed with Nikon C1-EZ package, version 2.20.*

in **Figure 6A**, 5F 203 induced phosphorylation of JNK and P38, the stronger effect was attained after 6 h of treatment in both cases. Also, treatment with 5F 203 increased P38 α levels. Pre-treatment of cells with α-NF decreased phosphorylation of these kinases and P38 α expression, which confirmed that 5F 203 affected expression and activation of these proteins through AhR activation. Exposure to 5F 203 (1 h) also increased phosphorylation of ERK in IGROV-1 cells.

The ability of 5F 203 to induce apoptosis was evaluated. Exposure of IGROV-1 cells to 5F 203 (1 μM; 24 h) induced apoptotic body formation (**Figure 6B**). In contrast, SKOV-3 cells, resistant to 5F 203, did not show such features (**Figure 6B**). Also, pre-treatment of cells with α-NF partially blocked the pro-apoptotic effect of 5F 203 in IGROV-1 cells. These data confirmed that AhR is involved in 5F 203-induced cell death [48].

Trolox (a vitamin E derivative) and NAC are potent ROS scavengers often used as antioxidant agents. We pre-treated IGROV-1 cells with Trolox or NAC in order to investigate the effect of ROS depletion on 5F 203-induced growth inhibition. Cells were exposed to 5F 203 (1 μM, 24 h) after pre-treatment with Trolox (250 μM) or NAC (100 μM) for 1 h. As observed in **Figure 6B,** both inhibitors partially reduced 5F 203-induced apoptotic body formation. These data support the involvement of ROS-generation in 5F 203-induced apoptosis in sensitive IGROV-1 cells [48].

#### **Figure 6.**

*5F 203 induces MAPK activation and apoptosis in sensitive IGROV-1 cells. A: MAPK expression and activation. IGROV-1 and SKOV-3 cells were incubated with 5F203 (1 μM) for indicated times or DMSO (0.1%) for 6 h. Whole cell extracts were obtained and subjected to SDS-PAGE and Western blotting with pERK, ERK, pJNK, JNK, pp38, and p38 α antibodies. The figure shows representative Western blots. All Western blots were performed three times for each cell line and revealed the same pattern of protein phosphorylation and expression. B: Evaluation of cell apoptosis. Cells were incubated with 1 μM 5F 203 or DMSO (0.1%) for 24 h or pre-treated with α-NF (1 μM) Trolox (250 μM) or NAC (100 μM) for 1 h followed by 5F 203 (1 μM) for 24 h. Then, non-adherent cells were obtained by cytocentrifugation in the culture medium. Once fixed, cells were stained with DAPI and observed under a fluorescence microscope. Condensed and fragmented nuclei were considered apoptotic. Representative fields are shown.*

#### **1.8 5F 203 alters cell cycle distribution and evokes apoptosis in sensitive ovarian cancer cells**

As results indicated that 5F 203 induced DNA damage (e.g., **Figure 6B**), perturbations in cell cycle were explored. IGROV-1 and SKOV-3 cells were exposed to 1 μM 5F 203 or 0.1% DMSO for 24 and 48 h and prepared for cell cycle analyses. As illustrated (**Figure 7A**), 5F 203 caused an increase in G1 phase IGROV-1 events (44% control; 50% at 24 h and 59% at 48 h), coinciding with decreased G2/M phase (20% control to 13% at 24 h and 9% at 48 h). Accumulation of sub-G1 events was also detected from 5% (control) to 12 and 20% at 24 and 48 h, respectively. When cells were pre-incubated with α-NF, sub-G1 events diminished, indicating that AhR

**93**

**Figure 7.**

*phosphorylated form of P53.*

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

activation is necessary for 5F 203-induced apoptosis. In contrast, SKOV-3 cell cycle was not perturbed following treatment with 5F 203.The data demonstrate that 5F 203-induced DNA damage may lead to accumulation of cells in G1 phase concomitant with growth inhibition. As IGROV-1 cells are p53 wild type, their response to 5F 203 is consistent with operation of a G1 checkpoint arrest to cell cycle progression after DNA damage. As G1 phase arrest was observed in 5F 203-sensitive cells, cyclin D1 levels were examined. Exposure of IGROV-1 cells to 5F 203 reduced cyclin D1 protein levels by 50 and 75% after 24 and 48 h, respectively. In contrast, only a 35% decrease in cyclin D1 levels was observed in SKOV-3 cells after 48 h treatment with

*5F 203 induces AhR-dependent cell cycle arrest in G1 phase, a decrease in cyclin D1 and caspase‐3 mediated apoptosis in sensitive ovarian cancer cells. A: Exponentially growing IGROV‐1 cells were exposed to either 0.1% DMSO (control) or 5F 203 (1 µM) for 24 and 48 h, (upper panel) or pre‐incubated for 1 h with ‐α NF followed by 24 or 48 h 5F 203 +α ‐NF (1 µM) (lower panel). Exponentially growing SKOV‐3 cells were exposed to either 0.1% DMSO (control) or 5F203 (1 µM) for 24 or 48 h. Then both cell lines were harvested, washed in PBS, and fixed in 70% ethanol. DNA was stained by incubating cells in PBS containing propidium iodide and fluorescence measured and analyzed as described in Materials and Methods section. The experiment was repeated three times (significant difference between treatments with p <0.01). Data of one representative experiment are shown in the figure. B: Effect of 5F 203 on cyclin D1 expression. IGROV‐1 cells were exposed to either 0.1% DMSO (control) or 5F 203 (1 µM) for 3, 6, 24, or 48 h. Proteins in total lysates were resolved by SDS-PAGE and Western blot performed with anti‐cyclin D1 Ab. Anti‐actin Ab was used as a loading control. C: Effect of 5F 203 on p53 signaling pathway, caspase‐3 activation, and PARP cleavage in ovarian cancer cells. Cells were incubated with 1 µM 5F 203 during indicated times or TCDD (T) 10 nM for 1 h. Cells treated with 10 µM Etoposide (Et) were used as a positive control for apoptosis. Whole cell extracts were obtained and subjected to SDS-PAGE and Western blotting with pp53 and P21, caspase‐3 and PARP antibodies. PP53,* 

*DOI: http://dx.doi.org/10.5772/intechopen.81194*

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.81194*

#### **Figure 7.**

*Current Trends in Cancer Management*

**92**

**cancer cells**

*considered apoptotic. Representative fields are shown.*

**Figure 6.**

**1.8 5F 203 alters cell cycle distribution and evokes apoptosis in sensitive ovarian** 

*activation. IGROV-1 and SKOV-3 cells were incubated with 5F203 (1 μM) for indicated times or DMSO (0.1%) for 6 h. Whole cell extracts were obtained and subjected to SDS-PAGE and Western blotting with pERK, ERK, pJNK, JNK, pp38, and p38 α antibodies. The figure shows representative Western blots. All Western blots were performed three times for each cell line and revealed the same pattern of protein phosphorylation and expression. B: Evaluation of cell apoptosis. Cells were incubated with 1 μM 5F 203 or DMSO (0.1%) for 24 h or pre-treated with α-NF (1 μM) Trolox (250 μM) or NAC (100 μM) for 1 h followed by 5F 203 (1 μM) for 24 h. Then, non-adherent cells were obtained by cytocentrifugation in the culture medium. Once fixed, cells were stained with DAPI and observed under a fluorescence microscope. Condensed and fragmented nuclei were* 

*5F 203 induces MAPK activation and apoptosis in sensitive IGROV-1 cells. A: MAPK expression and* 

As results indicated that 5F 203 induced DNA damage (e.g., **Figure 6B**), perturbations in cell cycle were explored. IGROV-1 and SKOV-3 cells were exposed to 1 μM 5F 203 or 0.1% DMSO for 24 and 48 h and prepared for cell cycle analyses. As illustrated (**Figure 7A**), 5F 203 caused an increase in G1 phase IGROV-1 events (44% control; 50% at 24 h and 59% at 48 h), coinciding with decreased G2/M phase (20% control to 13% at 24 h and 9% at 48 h). Accumulation of sub-G1 events was also detected from 5% (control) to 12 and 20% at 24 and 48 h, respectively. When cells were pre-incubated with α-NF, sub-G1 events diminished, indicating that AhR

*5F 203 induces AhR-dependent cell cycle arrest in G1 phase, a decrease in cyclin D1 and caspase‐3 mediated apoptosis in sensitive ovarian cancer cells. A: Exponentially growing IGROV‐1 cells were exposed to either 0.1% DMSO (control) or 5F 203 (1 µM) for 24 and 48 h, (upper panel) or pre‐incubated for 1 h with ‐α NF followed by 24 or 48 h 5F 203 +α ‐NF (1 µM) (lower panel). Exponentially growing SKOV‐3 cells were exposed to either 0.1% DMSO (control) or 5F203 (1 µM) for 24 or 48 h. Then both cell lines were harvested, washed in PBS, and fixed in 70% ethanol. DNA was stained by incubating cells in PBS containing propidium iodide and fluorescence measured and analyzed as described in Materials and Methods section. The experiment was repeated three times (significant difference between treatments with p <0.01). Data of one representative experiment are shown in the figure. B: Effect of 5F 203 on cyclin D1 expression. IGROV‐1 cells were exposed to either 0.1% DMSO (control) or 5F 203 (1 µM) for 3, 6, 24, or 48 h. Proteins in total lysates were resolved by SDS-PAGE and Western blot performed with anti‐cyclin D1 Ab. Anti‐actin Ab was used as a loading control. C: Effect of 5F 203 on p53 signaling pathway, caspase‐3 activation, and PARP cleavage in ovarian cancer cells. Cells were incubated with 1 µM 5F 203 during indicated times or TCDD (T) 10 nM for 1 h. Cells treated with 10 µM Etoposide (Et) were used as a positive control for apoptosis. Whole cell extracts were obtained and subjected to SDS-PAGE and Western blotting with pp53 and P21, caspase‐3 and PARP antibodies. PP53, phosphorylated form of P53.*

activation is necessary for 5F 203-induced apoptosis. In contrast, SKOV-3 cell cycle was not perturbed following treatment with 5F 203.The data demonstrate that 5F 203-induced DNA damage may lead to accumulation of cells in G1 phase concomitant with growth inhibition. As IGROV-1 cells are p53 wild type, their response to 5F 203 is consistent with operation of a G1 checkpoint arrest to cell cycle progression after DNA damage. As G1 phase arrest was observed in 5F 203-sensitive cells, cyclin D1 levels were examined. Exposure of IGROV-1 cells to 5F 203 reduced cyclin D1 protein levels by 50 and 75% after 24 and 48 h, respectively. In contrast, only a 35% decrease in cyclin D1 levels was observed in SKOV-3 cells after 48 h treatment with

5F 203 (**Figure 7B**). In order to study, whether 5F 203 treatment caused caspase 3 activation, PARP cleavage, and p53 phosphorylation in IGROV-1 cells as a result of its pro-apoptotic action, we carried out Western blot experiments upon separated proteins of whole cell lysates following treatment of cells with 1 μM 5F 203 between 3 and 48 h. We observed caspase-3 activation, PARP cleavage, and 2.7- and 4-fold increase in p53 phosphorylation between 24 and 48 h, respectively. A similar pattern of increased p21 protein levels was observed after treatment of IGROV-1 cells

#### **Figure 8.**

*5F 203 activity in ascites-derived ovarian cancer cell strains isolated from patients. A: 5F 203 cytotoxicity assay. Cells derived from three patients were incubated with 5F 203 for 5 days. Cellular viability was evaluated by MTS assay. Values represent the average of two independent experiments using cells from one patient with n = 4, \*P < 0.05 compared with untreated cells. B: Cells derived from patient A (sensitive to 5F 203) were incubated with 5F 203 for 5 days or pre-treated for 1 h with α-NF (1 μM) and then treated with 5F 203 plus α-NF (1 μM) for 5 days. Cellular viability was evaluated by MTS assay. The values represent the average of two independent experiments using cells from one patient with n = 4, \*\*P < 0.01 compared with cells incubated without AhR inhibitor. C: 5F 203 induces translocation of AhR to the nucleus in sensitive ascites-derived ovarian cancer cells. The staining of cells from one representative patient is shown. Cells were grown on coverslips and treated with DMSO for 1 h, 5F 203 for 30 min, or α-NF (1 μM) followed by 1 h of 5F 203, α-NF (1 μM), or 10 nM TCDD for 1 h. After fixation, cells were double-stained for AhR (green) and propidium iodide (red). Cells treated with 10 nM TCDD were incubated only with secondary antibody to determine nonspecific background. Stained cells were visualized on a fluorescence microscope using a 40X PlanApo AN 0.95 objective, and images were processed and analyzed with Nikon C1-EZ package, version 2.20.*

**95**

**2. Discussion**

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

with 5F 203 (**Figure 7C**). In contrast, caspase activation and PARP cleavage were not detected in SKOV-3 cells treated with 5F 203; these cells showed decreased levels

Thus, clear distinction can be seen—in terms of AhR signal transduction activation, CYP1A1 induction, DNA damage, ROS generation and apoptosis—between Bz-sensitive and Bz-insensitive ovarian cancer models *in vitro* and *in vivo*. It is important to evaluate whether such distinction can be translated to the clinic to

**1.9 5F 203-induced cytotoxicity in cells isolated from ovarian cancer ascites is** 

It has been proposed that high-grade advanced stage papillary serous ovarian adenocarcinoma ascites fluid is enriched for "metastatic," or "tumor-initiating" cells, and that these cells may represent a therapy-resistant population. Thus, ascites is considered a good model for disease study [52]. Cancer cells derived from ascites fluid produced by ovarian tumors from three patients were authenticated by pathologists. All tumors were high-grade (G3), serous, papillary histological type. Cells were treated *ex vivo* with 5F 203 for 5 days and cytotoxicity measured using MTS assays. Two cell strains were sensitive to 5F 203 and one was resistant (**Figure 8A**). In patient A, 1 and 10 μM 5F 203 decreased cell viability by 40 and 50%, respectively (compared to control considered 100%). This decrease in cell viability diminished to 20 and 30%, respectively, when cells were pre-treated with 1 μM α-NF followed by incubation with 5F 203/α-NF. We observed similar results in cells derived from patient B (data not shown). Results indicate that AhR mediates the effect of 5F 203 in these papillary tumors sensitive to 5F 203 (**Figure 8B**). AhR localization and nuclear translocation were then investigated. As demonstrated in **Figure 4A**, in ovarian cancer cells treated with vehicle (DMSO), high levels of cytosolic AhR protein were detected with some nuclear AhR staining present. However, after treatment for just 1 h with 1 μM 5F 203 or 10 nM TCDD, immunofluorescent AhR protein levels increased in the nucleus and decreased in the cytosol. In contrast, constitutive nuclear AhR localization was detected in cells of patient C, resistant to 5F 203. CYP1A1 mRNA levels were measured by real-time PCR in cells from patients A, B (sensitive to 5F 203), and C (resistant to 5F 203) following exposure to 5F 203 (1 μM; 24 h). In cells from patients A and B, we observed induction of *cyp1a1* expression (**Figure 8A**), which was partially reduced by α-NF. In contrast, reduction of (constitutive) *cyp1a1* expression was observed after treatment of patient C cells with 5F 203. ROS levels were also evaluated after treatment of patient ascites cells with 5F 203 and increased levels were detected only in cells sensitive to 5F 203, ROS were not

induced in the 5F 203-insensitive ascites cells of patient C (**Figure 8B**) [48].

medicine" are models applicable to antitumor Bzs.

activated anticancer agents for the treatment of ovarian cancer.

These promising data represent only a small clinical sample, but nevertheless support the hypothesis that in a clinical setting, "patient selection" and "precision

In this chapter, we propose that AhR represents a novel molecular target for ovarian cancer treatment and that the Bz class signifies AhR-targeted, CYP-

5F 203 activates AhR signaling in cultured and patient ovarian carcinoma cells sensitive to this agent, demonstrating that 5F 203 cytotoxicity is AhR dependent. In sensitive IGROV-1 cells, 5F 203, a known AhR ligand [35], triggers

*DOI: http://dx.doi.org/10.5772/intechopen.81194*

**mediated by AhR**

of pp53 after treatment with 5F 203 (**Figure 7C**) [48].

enable identification of sensitive ovarian cancer phenotypes.

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.81194*

with 5F 203 (**Figure 7C**). In contrast, caspase activation and PARP cleavage were not detected in SKOV-3 cells treated with 5F 203; these cells showed decreased levels of pp53 after treatment with 5F 203 (**Figure 7C**) [48].

Thus, clear distinction can be seen—in terms of AhR signal transduction activation, CYP1A1 induction, DNA damage, ROS generation and apoptosis—between Bz-sensitive and Bz-insensitive ovarian cancer models *in vitro* and *in vivo*. It is important to evaluate whether such distinction can be translated to the clinic to enable identification of sensitive ovarian cancer phenotypes.

#### **1.9 5F 203-induced cytotoxicity in cells isolated from ovarian cancer ascites is mediated by AhR**

It has been proposed that high-grade advanced stage papillary serous ovarian adenocarcinoma ascites fluid is enriched for "metastatic," or "tumor-initiating" cells, and that these cells may represent a therapy-resistant population. Thus, ascites is considered a good model for disease study [52]. Cancer cells derived from ascites fluid produced by ovarian tumors from three patients were authenticated by pathologists. All tumors were high-grade (G3), serous, papillary histological type. Cells were treated *ex vivo* with 5F 203 for 5 days and cytotoxicity measured using MTS assays. Two cell strains were sensitive to 5F 203 and one was resistant (**Figure 8A**). In patient A, 1 and 10 μM 5F 203 decreased cell viability by 40 and 50%, respectively (compared to control considered 100%). This decrease in cell viability diminished to 20 and 30%, respectively, when cells were pre-treated with 1 μM α-NF followed by incubation with 5F 203/α-NF. We observed similar results in cells derived from patient B (data not shown). Results indicate that AhR mediates the effect of 5F 203 in these papillary tumors sensitive to 5F 203 (**Figure 8B**). AhR localization and nuclear translocation were then investigated. As demonstrated in **Figure 4A**, in ovarian cancer cells treated with vehicle (DMSO), high levels of cytosolic AhR protein were detected with some nuclear AhR staining present. However, after treatment for just 1 h with 1 μM 5F 203 or 10 nM TCDD, immunofluorescent AhR protein levels increased in the nucleus and decreased in the cytosol. In contrast, constitutive nuclear AhR localization was detected in cells of patient C, resistant to 5F 203. CYP1A1 mRNA levels were measured by real-time PCR in cells from patients A, B (sensitive to 5F 203), and C (resistant to 5F 203) following exposure to 5F 203 (1 μM; 24 h). In cells from patients A and B, we observed induction of *cyp1a1* expression (**Figure 8A**), which was partially reduced by α-NF. In contrast, reduction of (constitutive) *cyp1a1* expression was observed after treatment of patient C cells with 5F 203. ROS levels were also evaluated after treatment of patient ascites cells with 5F 203 and increased levels were detected only in cells sensitive to 5F 203, ROS were not induced in the 5F 203-insensitive ascites cells of patient C (**Figure 8B**) [48].

These promising data represent only a small clinical sample, but nevertheless support the hypothesis that in a clinical setting, "patient selection" and "precision medicine" are models applicable to antitumor Bzs.

#### **2. Discussion**

In this chapter, we propose that AhR represents a novel molecular target for ovarian cancer treatment and that the Bz class signifies AhR-targeted, CYPactivated anticancer agents for the treatment of ovarian cancer.

5F 203 activates AhR signaling in cultured and patient ovarian carcinoma cells sensitive to this agent, demonstrating that 5F 203 cytotoxicity is AhR dependent. In sensitive IGROV-1 cells, 5F 203, a known AhR ligand [35], triggers

*Current Trends in Cancer Management*

5F 203 (**Figure 7B**). In order to study, whether 5F 203 treatment caused caspase 3 activation, PARP cleavage, and p53 phosphorylation in IGROV-1 cells as a result of its pro-apoptotic action, we carried out Western blot experiments upon separated proteins of whole cell lysates following treatment of cells with 1 μM 5F 203 between 3 and 48 h. We observed caspase-3 activation, PARP cleavage, and 2.7- and 4-fold increase in p53 phosphorylation between 24 and 48 h, respectively. A similar pattern of increased p21 protein levels was observed after treatment of IGROV-1 cells

*5F 203 activity in ascites-derived ovarian cancer cell strains isolated from patients. A: 5F 203 cytotoxicity assay. Cells derived from three patients were incubated with 5F 203 for 5 days. Cellular viability was evaluated by MTS assay. Values represent the average of two independent experiments using cells from one patient with n = 4, \*P < 0.05 compared with untreated cells. B: Cells derived from patient A (sensitive to 5F 203) were incubated with 5F 203 for 5 days or pre-treated for 1 h with α-NF (1 μM) and then treated with 5F 203 plus α-NF (1 μM) for 5 days. Cellular viability was evaluated by MTS assay. The values represent the average of two independent experiments using cells from one patient with n = 4, \*\*P < 0.01 compared with cells incubated without AhR inhibitor. C: 5F 203 induces translocation of AhR to the nucleus in sensitive ascites-derived ovarian cancer cells. The staining of cells from one representative patient is shown. Cells were grown on coverslips and treated with DMSO for 1 h, 5F 203 for 30 min, or α-NF (1 μM) followed by 1 h of 5F 203, α-NF (1 μM), or 10 nM TCDD for 1 h. After fixation, cells were double-stained for AhR (green) and propidium iodide (red). Cells treated with 10 nM TCDD were incubated only with secondary antibody to determine nonspecific background. Stained cells were visualized on a fluorescence microscope using a 40X PlanApo AN 0.95* 

*objective, and images were processed and analyzed with Nikon C1-EZ package, version 2.20.*

**94**

**Figure 8.**

AhR translocation from cytosol to nucleus, activating CYP1A1-related promoter sequences driving transcription of AhR-responsive genes as reported by XRE-luciferase.

It was recognized several years ago that certain ovarian cancer cell lines were exquisitely sensitive to antitumor Bzs [32]. 5F 203 potency and selectivity against ovarian cell lines within the NCI panel have been demonstrated [32, 53]: IGROV-1, OVCAR4 and OVCAR5 displayed GI50 values <100 nM in contrast, whereas GI50 values >100 μM were observed in OVCAR8 and SKOV-3 cell lines. Subsequently, induction of CYP1A1 by 5F 203 in sensitive cancer cells only inferred significant correlation between sensitivity and CYP1A1 induction [48]. *In vivo*, significant antitumor efficacy of 5F 203 prodrug Phortress was demonstrated against IGROV-1 ovarian (as well as breast) tumor xenografts. Moreover, CYP1A1 expression was detected in IGROV-1 (and sensitive breast) tumors of mice receiving Phortress treatment. No CYP1A1 protein was detected in insensitive breast tumor tissue following treatment of mice with Phortress [36]. Phortress was well tolerated, it possessed excellent solubility, bioavailability, and pharmacokinetic properties (liberating efficacious, sustained 5F 203 plasma concentrations), and Phase 1 clinical trials were initiated. Clinical evaluation revealed long-term stable disease in lung, colorectal, and kidney cancer patients; however, neither ovarian nor breast cancer patients were recruited to trial and short patent life precluded continuation of development.

AhR ligands such as 5F 203 induce their own CYP-catalyzed bioactivation; therefore, potential drug-drug or indeed drug-xenobiotic interactions were considered prior to commencement of the clinical trial. For example, many oral contraceptives are steroid based and any drug inducing CYP1A1 activity will lead to rapid metabolism and reduced contraceptive efficacy [54]. In the Phortress trial protocol, it was cautioned not to drink grapefruit juice, as this is able to inhibit CYP1A1 potentially reducing Phortress efficacy [55]. Red wine consumption was also discouraged as resveratrol is a competitive antagonist of AhR ligands; it promotes AhR nuclear translocation and binding to DNA, but transactivation of AhR-inducible genes such as *cyp1a1* is inhibited [56].

Differential sensitivity to 5F 203 may be a consequence of differential regulation by AhR of CYP1A1 expression in different cell types. In resistant cells, we observed constitutive nuclear localization of AhR. In resistant cells, AhR may be associated with co-repressors [57]; lack of AhR degradation (by ubiquitination) or recycling may lead to inappropriate AhR function [58]. Also, different AhR nuclear localization sequences [57] or polymorphisms may cause inappropriate receptor function [59]. Additionally, mutations in the CYP1A1 promoter in insensitive cells may lead to decreased CYP1A1 activation [60, 61]. Considering the clinical potential of 5F 203, its mechanism of action was further investigated. 5F 203 induced ROS formation in sensitive cells (**Figure 5**). In IGROV-1 cells, 5F 203 evoked DNA damage detected as H2AX foci (2–4 h), increased pp53 levels and P21 expression, decreased cyclin D1 expression, caused G1 cell cycle arrest and apoptosis. In contrast, SKOV-3 cells showed decreased levels of pp53 after treatment with 5F 203; the reason for this effect is unclear, but it may contribute to cellular resistance to 5F 203 (**Figure 7**). 5F 203-induced growth inhibition and apoptosis in IGROV-1 cells may in part be a consequence of elevated ROS (**Figure 5A**) and caspase-3 activation (**Figure 7C**) resulting in oxidative DNA damage and cell death [48]. Oxidative stress may activate caspases and is implicated in a number of cellular processes including apoptosis. Many chemotherapeutic agents are known to induce cytotoxicity by ROS-mediated mechanisms, for example, doxorubicin [62] and AhR ligand aminoflavone [63]. It was demonstrated that ROS might trigger apoptosis signaling mediated by p53 in IGROV-1 cells [64–66]. IGROV-1 cells possess wild type p53 and show sensitivity

**97**

or i.p. [34].

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

to 5F 203. However, 5F 203 activity is independent of p53 exemplified by (i) potent activity of 5F 203 in MDA-MB-468 p53 mutant cells [32] and (ii) IGROV-1 variant populations demonstrating acquired resistance to cisplatin retaining sensitivity to 5F 203 (Bradshaw et al. unpublished results). Cisplatin resistance is associated with p53 mutations in IGROV-1 cells [64]. In IGROV-1 cells, p53 may be attempting (failed) repair rather than mediating apoptosis. Our results show that antioxidant agents such as NAC and Trolox decrease ROS formation and protect IGROV-1 cells from apoptosis induced by 5F 203 (**Figure 5B**). Previous work has demonstrated that stress signaling pathways are also activated by cisplatin and retinoids in IGROV-1 and cisplatin-resistant IGROV-1 cells; furthermore activation of JNK and P38 by these agents is stronger in cisplatin-resistant IGROV-1 cells. Reflecting the integral to antitumor activity, 5F 203 induces ROS in sensitive IGROV-1 cells and that IGROV-1 cells resistant to cisplatin retain sensitivity to 5F 203, we propose that 5F 203 could be a putative treatment for ovarian tumors. MAPK p38 α acts as an oxidative stress sensor; ROS-induced activation of p38 promotes apoptosis and prevents further oncogenic/carcinogenic ROS formation [44]. Also, micro-RNAs (miRNA) expression can be altered by different stress conditions, and they are well-known stress response regulators. It has been described that two members of the miR-200 family, miR-141 and miR-200a, inhibit p38 α and have an essential role in the redox response. In animal models, accumulation of miRNAs mimics p38 α deficiency and promotes malignancy. Human ovarian adenocarcinomas demonstrating high oxidative stress show high expression of miR-200a and low basal levels of p38 α. Chemotherapy drugs that induce ROS also induce p38 α in these tumors, leading to apoptosis. It was proposed that in ovarian tumors, high levels of miR-200 s and low levels of p38 α could be predictive markers of good clinical response to chemotherapy [45]. Our results are consistent with these observations, IGROV-1 cells have low levels of basal p38 α and treatment with 5F 203 induced p38 α expression and pp38 (**Figure 6A**), which may lead to apoptosis. In contrast, SKOV-3 cells show high basal levels of p38 α and treatment

with 5F 203 did not modulate p38 α expression or activation (**Figure 6A**). We hypothesize that this may contribute to the lack of apoptosis induction in SKOV-3 cells. Finally, compatible with results from ovarian cancer cell lines, we identified putative surrogate markers of sensitivity to 5F 203 in a small sample of patient tumors. Clear distinction was demonstrated between ovarian cancer patient tumor cells that were sensitive to 5F 203 and those that were inherently 5F 203-resistant. Only in ascites-isolated patient tumor cells sensitive to 5F 203 were (i) cytosolic AhR translocation to cell nuclei, (ii) CYP1A1 mRNA induction, and (iii) increased ROS levels observed (**Figures 8** and **9**) in response to *ex vivo* treatment. Such pharmacodynamic endpoints are readily obtained from bioassays that could be adopted clinically to detect candidate 5F 203-responsive patients. In this way, unresponsive patients would be spared unnecessary treatment. Sensitivity to 5F 203 and AhR activation should be examined in a larger sample of ovarian carcinoma patient tumors of different histological types in future studies (**Figure 10**). However, the fact that cells isolated from patients with high-grade ovarian tumors were sensitive to 5F 203 shows that this agent may offer alternative treatment for patients with advanced disease. Intraperitoneal (i.p.) chemotherapy is currently used in treatment of ovarian tumors, and both 5F 203 and its prodrug Phortress have demonstrated antitumor efficacy administered either intravenously

Nanoformulations of 5F 203 are currently under evaluation to maximize tumortargeting and sustained, controlled release. Future studies will include investigation of miRNA profiles in 5F 203-sensitive ovarian cancer cells compared with those of insensitive ones. In addition, the activity of 5F 203 against ovarian cancer stem

cell-like/initiating populations remains to be evaluated.

*DOI: http://dx.doi.org/10.5772/intechopen.81194*

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.81194*

to 5F 203. However, 5F 203 activity is independent of p53 exemplified by (i) potent activity of 5F 203 in MDA-MB-468 p53 mutant cells [32] and (ii) IGROV-1 variant populations demonstrating acquired resistance to cisplatin retaining sensitivity to 5F 203 (Bradshaw et al. unpublished results). Cisplatin resistance is associated with p53 mutations in IGROV-1 cells [64]. In IGROV-1 cells, p53 may be attempting (failed) repair rather than mediating apoptosis. Our results show that antioxidant agents such as NAC and Trolox decrease ROS formation and protect IGROV-1 cells from apoptosis induced by 5F 203 (**Figure 5B**). Previous work has demonstrated that stress signaling pathways are also activated by cisplatin and retinoids in IGROV-1 and cisplatin-resistant IGROV-1 cells; furthermore activation of JNK and P38 by these agents is stronger in cisplatin-resistant IGROV-1 cells. Reflecting the integral to antitumor activity, 5F 203 induces ROS in sensitive IGROV-1 cells and that IGROV-1 cells resistant to cisplatin retain sensitivity to 5F 203, we propose that 5F 203 could be a putative treatment for ovarian tumors. MAPK p38 α acts as an oxidative stress sensor; ROS-induced activation of p38 promotes apoptosis and prevents further oncogenic/carcinogenic ROS formation [44]. Also, micro-RNAs (miRNA) expression can be altered by different stress conditions, and they are well-known stress response regulators. It has been described that two members of the miR-200 family, miR-141 and miR-200a, inhibit p38 α and have an essential role in the redox response. In animal models, accumulation of miRNAs mimics p38 α deficiency and promotes malignancy. Human ovarian adenocarcinomas demonstrating high oxidative stress show high expression of miR-200a and low basal levels of p38 α. Chemotherapy drugs that induce ROS also induce p38 α in these tumors, leading to apoptosis. It was proposed that in ovarian tumors, high levels of miR-200 s and low levels of p38 α could be predictive markers of good clinical response to chemotherapy [45]. Our results are consistent with these observations, IGROV-1 cells have low levels of basal p38 α and treatment with 5F 203 induced p38 α expression and pp38 (**Figure 6A**), which may lead to apoptosis. In contrast, SKOV-3 cells show high basal levels of p38 α and treatment with 5F 203 did not modulate p38 α expression or activation (**Figure 6A**). We hypothesize that this may contribute to the lack of apoptosis induction in SKOV-3 cells. Finally, compatible with results from ovarian cancer cell lines, we identified putative surrogate markers of sensitivity to 5F 203 in a small sample of patient tumors. Clear distinction was demonstrated between ovarian cancer patient tumor cells that were sensitive to 5F 203 and those that were inherently 5F 203-resistant. Only in ascites-isolated patient tumor cells sensitive to 5F 203 were (i) cytosolic AhR translocation to cell nuclei, (ii) CYP1A1 mRNA induction, and (iii) increased ROS levels observed (**Figures 8** and **9**) in response to *ex vivo* treatment. Such pharmacodynamic endpoints are readily obtained from bioassays that could be adopted clinically to detect candidate 5F 203-responsive patients. In this way, unresponsive patients would be spared unnecessary treatment. Sensitivity to 5F 203 and AhR activation should be examined in a larger sample of ovarian carcinoma patient tumors of different histological types in future studies (**Figure 10**). However, the fact that cells isolated from patients with high-grade ovarian tumors were sensitive to 5F 203 shows that this agent may offer alternative treatment for patients with advanced disease. Intraperitoneal (i.p.) chemotherapy is currently used in treatment of ovarian tumors, and both 5F 203 and its prodrug Phortress have demonstrated antitumor efficacy administered either intravenously or i.p. [34].

Nanoformulations of 5F 203 are currently under evaluation to maximize tumortargeting and sustained, controlled release. Future studies will include investigation of miRNA profiles in 5F 203-sensitive ovarian cancer cells compared with those of insensitive ones. In addition, the activity of 5F 203 against ovarian cancer stem cell-like/initiating populations remains to be evaluated.

*Current Trends in Cancer Management*

XRE-luciferase.

of development.

genes such as *cyp1a1* is inhibited [56].

AhR translocation from cytosol to nucleus, activating CYP1A1-related promoter sequences driving transcription of AhR-responsive genes as reported by

It was recognized several years ago that certain ovarian cancer cell lines were exquisitely sensitive to antitumor Bzs [32]. 5F 203 potency and selectivity against ovarian cell lines within the NCI panel have been demonstrated [32, 53]: IGROV-1, OVCAR4 and OVCAR5 displayed GI50 values <100 nM in contrast, whereas GI50 values >100 μM were observed in OVCAR8 and SKOV-3 cell lines. Subsequently, induction of CYP1A1 by 5F 203 in sensitive cancer cells only inferred significant correlation between sensitivity and CYP1A1 induction [48]. *In vivo*, significant antitumor efficacy of 5F 203 prodrug Phortress was demonstrated against IGROV-1 ovarian (as well as breast) tumor xenografts. Moreover, CYP1A1 expression was detected in IGROV-1 (and sensitive breast) tumors of mice receiving Phortress treatment. No CYP1A1 protein was detected in insensitive breast tumor tissue following treatment of mice with Phortress [36]. Phortress was well tolerated, it possessed excellent solubility, bioavailability, and pharmacokinetic properties (liberating efficacious, sustained 5F 203 plasma concentrations), and Phase 1 clinical trials were initiated. Clinical evaluation revealed long-term stable disease in lung, colorectal, and kidney cancer patients; however, neither ovarian nor breast cancer patients were recruited to trial and short patent life precluded continuation

AhR ligands such as 5F 203 induce their own CYP-catalyzed bioactivation; therefore, potential drug-drug or indeed drug-xenobiotic interactions were considered prior to commencement of the clinical trial. For example, many oral contraceptives are steroid based and any drug inducing CYP1A1 activity will lead to rapid metabolism and reduced contraceptive efficacy [54]. In the Phortress trial protocol, it was cautioned not to drink grapefruit juice, as this is able to inhibit CYP1A1 potentially reducing Phortress efficacy [55]. Red wine consumption was also discouraged as resveratrol is a competitive antagonist of AhR ligands; it promotes AhR nuclear translocation and binding to DNA, but transactivation of AhR-inducible

Differential sensitivity to 5F 203 may be a consequence of differential regulation by AhR of CYP1A1 expression in different cell types. In resistant cells, we observed constitutive nuclear localization of AhR. In resistant cells, AhR may be associated with co-repressors [57]; lack of AhR degradation (by ubiquitination) or recycling may lead to inappropriate AhR function [58]. Also, different AhR nuclear localization sequences [57] or polymorphisms may cause inappropriate receptor function [59]. Additionally, mutations in the CYP1A1 promoter in insensitive cells may lead to decreased CYP1A1 activation [60, 61]. Considering the clinical potential of 5F 203, its mechanism of action was further investigated. 5F 203 induced ROS formation in sensitive cells (**Figure 5**). In IGROV-1 cells, 5F 203 evoked DNA damage detected as H2AX foci (2–4 h), increased pp53 levels and P21 expression, decreased cyclin D1 expression, caused G1 cell cycle arrest and apoptosis. In contrast, SKOV-3 cells showed decreased levels of pp53 after treatment with 5F 203; the reason for this effect is unclear, but it may contribute to cellular resistance to 5F 203 (**Figure 7**). 5F 203-induced growth inhibition and apoptosis in IGROV-1 cells may in part be a consequence of elevated ROS (**Figure 5A**) and caspase-3 activation (**Figure 7C**) resulting in oxidative DNA damage and cell death [48]. Oxidative stress may activate caspases and is implicated in a number of cellular processes including apoptosis. Many chemotherapeutic agents are known to induce cytotoxicity by ROS-mediated mechanisms, for example, doxorubicin [62] and AhR ligand aminoflavone [63]. It was demonstrated that ROS might trigger apoptosis signaling mediated by p53 in IGROV-1 cells [64–66]. IGROV-1 cells possess wild type p53 and show sensitivity

**96**

#### **Figure 9.**

*5F 203 induces CYP1A1 over-expression and increases ROS levels in sensitive ascites-derived ovarian cancer cells isolated from patients. A: Induction of CYP1A1 gene expression. Cells derived from three patients were exposed to 0.1% DMSO (control), 5F 203 (1 μM) for 24 h, or pre-treated with α-NF (1 μM) for 1 h followed by 5F 203 (1 μM) α-NF (1 μM) for 24 h. RNA was isolated and real-time PCR was performed to measure CYP1A1 expression. A: Each bar represents mean ± SD of triplicate measurements in drug treated, compared to untreated cells, using GAPDH expression as an endogenous control. \*P < 0.01 or \*P < 0.05 when compared to untreated cells \*\*P < 0.01 or \*\*P < 0.05 when compared to cells treated without AhR inhibitor. B: Measurement of ROS levels. Cells derived from three patients were treated with 5F 203 (1 μM) or vehicle control (0.1% DMSO) continuously for 1 or 2 h and ROS levels were measured by fluorometry after incubation with 2,7- DCF. Incubation with H2O2 (100 μM) was used as positive control. Data represent the mean of at least two independent experiments where n = 4 per experiment; bars, SEM. \*P < 0.05, \*\*P < 0.01 when compared to untreated cells.*

In summary, we have demonstrated AhR-dependent cytotoxicity of 5F 203 in ovarian carcinoma cells, we conclude that AhR may represent a new molecular target in the treatment of ovarian cancer and that 5F 203 may offer a potential novel treatment for newly diagnosed and cisplatin-resistant disease.

Tumor cells will be isolated from ascites fluid and cultured *ex vivo*. Following exposure of carcinoma cells *ex vivo* to escalating Bz concentrations (i) Bz sensitivity; (ii) AhR localization and nuclear translocation; (iii) CYP 1A1 expression and inducibility (by Bz); and (iv) ROS generation will be determined. If tumor cells are identified that show dose-dependent growth inhibition, AhR translocation,

**99**

therapy.

**Figure 10.**

**Acknowledgements**

assistance with figures.

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

CYP 1A1 induction, and ROS generation following exposure to Bz, the patient from whom cells were isolated may be identified as a suitable candidate to receive Bz

*Proposed procedure for identification of patients whose tumors may be responsive to Bz treatment.*

We are very grateful to Dr. Eduardo Sandes from Área de Investigaciones, Instituto de Oncología "Ángel H. Roffo," Universidad de Buenos Aires, for his

*DOI: http://dx.doi.org/10.5772/intechopen.81194*

*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.81194*

**Figure 10.**

*Current Trends in Cancer Management*

**98**

**Figure 9.**

*untreated cells.*

In summary, we have demonstrated AhR-dependent cytotoxicity of 5F 203 in ovarian carcinoma cells, we conclude that AhR may represent a new molecular target in the treatment of ovarian cancer and that 5F 203 may offer a potential novel

*5F 203 induces CYP1A1 over-expression and increases ROS levels in sensitive ascites-derived ovarian cancer cells isolated from patients. A: Induction of CYP1A1 gene expression. Cells derived from three patients were exposed to 0.1% DMSO (control), 5F 203 (1 μM) for 24 h, or pre-treated with α-NF (1 μM) for 1 h followed by 5F 203 (1 μM) α-NF (1 μM) for 24 h. RNA was isolated and real-time PCR was performed to measure CYP1A1 expression. A: Each bar represents mean ± SD of triplicate measurements in drug treated, compared to untreated cells, using GAPDH expression as an endogenous control. \*P < 0.01 or \*P < 0.05 when compared to untreated cells \*\*P < 0.01 or \*\*P < 0.05 when compared to cells treated without AhR inhibitor. B: Measurement of ROS levels. Cells derived from three patients were treated with 5F 203 (1 μM) or vehicle control (0.1% DMSO) continuously for 1 or 2 h and ROS levels were measured by fluorometry after incubation with 2,7- DCF. Incubation with H2O2 (100 μM) was used as positive control. Data represent the mean of at least two independent experiments where n = 4 per experiment; bars, SEM. \*P < 0.05, \*\*P < 0.01 when compared to* 

Tumor cells will be isolated from ascites fluid and cultured *ex vivo*. Following exposure of carcinoma cells *ex vivo* to escalating Bz concentrations (i) Bz sensitivity; (ii) AhR localization and nuclear translocation; (iii) CYP 1A1 expression and inducibility (by Bz); and (iv) ROS generation will be determined. If tumor cells are identified that show dose-dependent growth inhibition, AhR translocation,

treatment for newly diagnosed and cisplatin-resistant disease.

*Proposed procedure for identification of patients whose tumors may be responsive to Bz treatment.*

CYP 1A1 induction, and ROS generation following exposure to Bz, the patient from whom cells were isolated may be identified as a suitable candidate to receive Bz therapy.

### **Acknowledgements**

We are very grateful to Dr. Eduardo Sandes from Área de Investigaciones, Instituto de Oncología "Ángel H. Roffo," Universidad de Buenos Aires, for his assistance with figures.

*Current Trends in Cancer Management*

#### **Author details**

Andrea I. Loaiza Perez1,2\* and Tracey D. Bradshaw3

1 National Scientific Council (CONICET), Ciudad de Buenos Aires, Argentina

2 Área de Investigaciones, Instituto de Oncología "Ángel H. Roffo", Universidad de Buenos Aires, Ciudad de Buenos Aires, Argentina

3 Centre for Biomolecular Science, University of Nottingham, Nottingham, UK

\*Address all correspondence to: loaizaa2012@gmail.com

© 2018 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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*Exploring New Molecular Targets in Advanced Ovarian Cancer: The Aryl Hydrocarbon Receptor…*

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*DOI: http://dx.doi.org/10.5772/intechopen.81194*

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**Author details**

provided the original work is properly cited.

Andrea I. Loaiza Perez1,2\* and Tracey D. Bradshaw3

Buenos Aires, Ciudad de Buenos Aires, Argentina

\*Address all correspondence to: loaizaa2012@gmail.com

© 2018 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,

1 National Scientific Council (CONICET), Ciudad de Buenos Aires, Argentina

2 Área de Investigaciones, Instituto de Oncología "Ángel H. Roffo", Universidad de

3 Centre for Biomolecular Science, University of Nottingham, Nottingham, UK

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[39] Leong C-O, Suggitt M, Swaine MJ, Bibby MC, Stevens MFG, Bradshaw TD. In vitro, in vivo and in silico analyses of the antitumor activity of 2-(4-amino-3-methylphenyl)-5 fluorobenzothiazoles. Molecular Cancer

FP. Bioactivation of fluorinated 2-arylbenzothiazole antitumor molecules by human cytochrome P450s 1A1 and 2W1 and deactivation by cytochrome P450 2S1. Chemical Research in Toxicology.

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Molecular Cancer Therapeutics.

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2003;**2**:1265-1272

2011;**711**:167-173

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2003;**88**:470-477

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[34] Bradshaw TD, Stevens MFG, Calvert H, Plummer R. Clinical trials: Poster presentations. Abstract B59: Preliminary clinical experiences of phortress: Putative role for c-MET inhibition in antitumor activity. In: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; Nov 15-19, 2009; Boston, MA. Molecular Cancer Therapeutics. 2009;**8**(12 Supplement).

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1998;**78**:421-442

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2-[4-amino-3-methylphenyl]-5 fluoro-benzothiazole: A potential surrogate marker for patient sensitivity. Molecular Cancer Therapeutics. 2003;**2**(12):1265-1272

*Current Trends in Cancer Management*

[18] Whitlock JP Jr. Induction of cytochrome P4501A1. Annual Review of Pharmacology and Toxicology.

Design. 2002;**8**(27):2475-2490

10.1093/molehr/8.1.75

2002;**61**(6):1393-1403

1999;**39**:103-125

[17] Rowlands JC, Gustafsson JA. Aryl hydrocarbon receptor-mediated signal transduction. Critical Reviews in Toxicology. 1997;**27**(2):109-134

[25] Safe S, Lee S-O, Jin U-H. Role of the aryl hydrocarbon receptor in carcinogenesis and potential as a drug target. Toxicological Sciences. 2013;**135**(1):1-16. DOI: 10.1093/toxsci/ kft128. Advance Access Publication June

[26] Wang K, Li Y, Jiang YZ, Dai CF, Patankar MS, Song JS, et al. An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells. Cancer Letters. 2013;**340**(1):63-71

[27] Chua MS, Kashiyama E,

Bradshaw T, et al. Role of CYP1A1 in modulation of antitumor properties of the novel agent 2-(4-amino-3 methylphenyl)benzothiazole (DF 203, NSC 674495) in human breast cancer cells. Cancer Research. 2000;**60**(18):

[28] Loaiza-Perez AI, Trapani V, Hose C, et al. Aryl hydrocarbon receptor mediates sensitivity of MCF-7 breast cancer cells to antitumor agent 2-(4-Amino-3-methylphenyl)

benzothiazole. Molecular Pharmacology. 2002;**61**(1):13-19

[29] Monks A, Harris E, Hose C, et al. Genotoxic profiling of MCF-7 breast cancer cell line elucidates gene expression modifications underlying toxicity of the anticancer drug 2-(4-amino-3-methylphenyl)- 5-fluorobenzothiazole. Molecular Pharmacology. 2003;**63**(3):766-772

[30] Trapani V, Patel V, Leong CO, et al. DNA damage and cell cycle arrest induced by 2-(4-amino-3 methylphenyl)-5- fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells. British Journal of Cancer.

[31] Hose CD, Hollingshead M, Sausville EA, Monks A. Induction of CYP1A1 in tumor cells by the antitumor agent

2003;**88**(4):599-605

14, 2013

5196-5203

[19] Bradshaw TD, Trapani V, Vasselin DA, Westwell AD. The aryl hydrocarbon receptor in anticancer drug discovery: Friend or foe? Current Pharmaceutical

[20] Khorram O, Garthwaite M, Golos T. Uterine and ovarian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA expression in benign and malignant gynaecological conditions. MHR: Basic Science of Reproductive Medicine. 2002;**8**(1):75-80. DOI:

[21] Castro-Rivera E, Wormke M, Safe S. Estrogen and aryl hydrocarbon responsiveness of ECC-1 endometrial cancer cells. Molecular and Cellular Endocrinology. 1999;**150**(1-2):11-21

[22] Rogers JM, Denison MS. Analysis of the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in human ovarian carcinoma BG-1 cells. Molecular Pharmacology.

[23] Rowlands C, Krishnan V, Wang X, Santostefano M, Safe S, Miller WR, et al. Characterization of the aryl hydrocarbon receptor and aryl hydrocarbon responsiveness in human ovarian carcinoma cell lines. Cancer

[24] Cannon MJ, Debopam G, Gujja S. Signaling circuits and regulation of immune suppression by ovarian tumorassociated macrophages. Vaccines (Basel). 2015;**3**(2):448-466. DOI:

Research. 1993;**53**:1802-1807

10.3390/vaccines3020448

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[33] Loaiza-Perez AI, Kenney S, Boswell J, et al. Aryl hydrocarbon receptor activation of an antitumor aminoflavone: Basis of selective toxicity for MCF-7 breast tumor cells. Molecular Cancer Therapeutics. 2004;**3**(6):715-725

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[35] Bazzi R, Bradshaw TD, Rowlands C, Stevens MFG, Bell D. 2-(4-Amino-3 methylphenyl) -5-fluorobenzothiazole is a ligand and shows species-specific partial agonism of the aryl hydrocarbon receptor. Toxicology and Applied Pharmacology. 2009;**237**:102-110

[36] Bradshaw TD, Bibby MJ, Double JA, Fitchner I, Cooper PA, Alley MC, et al. Preclinical evaluation of amino acid prodrugs of novel antitumor 2-(4-amino-3-methylphenyl) benzothiazoles. Molecular Cancer Therapeutics. 2002;**1**:239-246

[37] Leong C-O, Gaskell M, Martin EA, Heydon RT, Farmer PB, Bibby MC, et al. Antitumour 2-(4-aminophenyl)

benzothiazoles generate DNA adducts in sensitive tumour cells in vitro and in vivo. British Journal of Cancer. 2003;**88**:470-477

[38] Bradshaw TD, Chua M-S, Browne HL, Trapani V, Sausville EA, Stevens MFG. In vitro evaluation of amino acid prodrugs of novel antitumour 2-(4-amino-3-methylphenyl) benzothiazoles. British Journal of Cancer. 2002;**86**:1348-1354

[39] Leong C-O, Suggitt M, Swaine MJ, Bibby MC, Stevens MFG, Bradshaw TD. In vitro, in vivo and in silico analyses of the antitumor activity of 2-(4-amino-3-methylphenyl)-5 fluorobenzothiazoles. Molecular Cancer Therapeutics. 2004;**3**:1565-1575

[40] Wang K, Guengerich FP. Bioactivation of fluorinated 2-arylbenzothiazole antitumor molecules by human cytochrome P450s 1A1 and 2W1 and deactivation by cytochrome P450 2S1. Chemical Research in Toxicology. 2012;**25**:1740-1751

[41] Fichtner I, Monks A, Hose C, Stevens MFG, Bradshaw TD. The experimental antitumor agent phortress and doxorubicin are equiactive against human-derived breast carcinoma xenograft models. Breast Cancer Research and Treatment. 2004;**87**:97-107

[42] Hose CD, Hollingshead M, Sausville EA, Monks A. Induction of CYP1A1 in tumor cells by the antitumor agent 2-[4-amino-3-methylphenyl]- 5-fluorobenzothiazole: A potential surrogate marker for patient sensitivity. Molecular Cancer Therapeutics. 2003;**2**:1265-1272

[43] Ziech D, Franco R, Pappa A, Panayiotidis MI. Reactive oxygen species (ROS)-induced genetic and epigenetic alterations in human carcinogenesis. Mutation Research. 2011;**711**:167-173

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jcb.24589

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[55] Santes-Palacios R, Romo-Mancillas A, Camacho-Carranza R, Espinosa-Aguirre JJ. Inhibition of human and rat CYP1A1 enzyme by grapefruit juice compounds. Toxicology Letters. Sep 6, 2016;**258**:267-275. DOI: 10.1016/j. toxlet.2016.07.023. Epub 2016 Jul 18

[56] Casper RF, Quesne M, Rogers IM, Shirota T, Jolivet A, Milgrom E, Savouret JF. Resveratrol has antagonist activity on the aryl hydrocarbon receptor: implications for prevention of dioxin toxicity. Molecular Pharmacology. Oct 1999;**56**(4):784-790. https://www.ncbi.

nlm.nih.gov/pubmed/10496962

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2000;**275**:9390-9395

pone.0029079

2001;**44**:1446-1455

org/10.1002/tre.107

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[46] Zuco V, Zanchi C, Cassinelli G, Lanzi C, Supino R, Pisano C, et al. Induction of apoptosis and stress response in ovarian carcinoma cell lines treated with ST 1926, an atypical retinoid. Cell Death and Differentiation.

[47] Bradshaw TD, Stone EL, Trapani V, Leong C-O, Matthews CS, te Poele R, et al. Mechanisms of acquired resistance

benzothiazole in breast cancer cell lines. Breast Cancer Research and Treatment.

[48] Callero M, Luzzani G, De Dios D,

to 2-(4-amino-3-methylphenyl)

Bradshaw T, Loaiza Perez A. Biomarkers of sensitivity to potent and selective antitumor 2-(4-amino-3 methylphenyl)-5-fluorobenzothiazole

(5F203) in ovarian cancer. Journal of Cellular Biochemistry. 2013;**114**(10):2392-2404. DOI: 10.1002/

[49] Vibhuti A, Arif E, Mishra A, Deepak D, Singh B, Rahman I, et al. CYP1A1, CYP1A2, and CYBA gene polymorphisms associated with oxidative stress in COPD. Clinica Chimica Acta. 2010;**411**:474-480

[50] Morgan MJ, Liu Z. Crosstalk of reactive oxygen species and NF-kB signaling. Cell Research. 2011;**21**:103-115

[51] Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM. Initiation

of DNA fragmentation during apoptosis induces phosphorylation

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[59] Ikuta T, Eguchi H, Tachibana T, Yoneda Y, Kawajiri K. Nuclear localization and export signals of the human aryl hydrocarbon receptor. The Journal of Biological Chemistry. 1998;**273**:2895-2904

[60] Rowlands CJ, Staskal DF, Gollapudi B, Budinsky R. The human AHR: Identification of single nucleotide polymorphisms from six ethnic populations. Pharmacogenetics and Genomics. 2010;**20**:283-290

[61] Zheng Q, Sha X, Liu J, Heath E, Lorusso P, Li J. Association of human cytochrome P450 1A1 (CYP1A1) and sulfotransferase 1A1 (SULT1A1) polymorphisms with differential metabolism and cytotoxicity of aminoflavone. Molecular Cancer Therapeutics. 2010;**9**:2803-2813

[62] Lin X, Li Q, Wang YJ, Ju YW, Chi ZQ, Wang MW, et al. Morphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor kappaB transcriptional activation in neuroblastoma SH-SY5Y cells. The Biochemical Journal. 2007;**406**:215-221

[63] McLean L, Davis W Jr, Sowers E, Brantley EJ. 5F 203-Induced Apoptosis Involves Oxidative Stress and Caspase Activation in Sensitive Breast Cancer Cells. Washington DC, USA: American Association for Cancer Research; 2006

[64] Righetti SC, Perego P, Corna E, Pierotti MA, Zunino F. Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: Spontaneously mutant selection. Cell Growth & Differentiation. 1999;**10**:473-478

[65] Assinelli G, Supino R, Perego P, Polizzi D, Lanzi C, Pratesi G, et al.

A role for loss of p53 function in sensitivity of ovarian carcinoma cells to taxanes. International Journal of Cancer. 2001;**92**:738-747

[66] Perego P, Giarola M, Righetti SC, Supino R, Caserini C, Delia D, et al. Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems. Cancer Research. 1996;**56**:556-562

**107**

**Chapter 6**

*Hulya Yazici*

**1. Introduction**

frequently [2].

**Abstract**

Functions of miRNAs in the

Development, Diagnosis, and

Treatment of Ovarian Carcinoma

miRNAs (miRNA) are small RNA molecules that are not to expressed to proteins. Their size is 20–22 nucleotides in length and they are highly conserved molecules among the species. miRNAs are synthesized in the nucleus as a primary miRNA. Primary miRNA is transferred to cytoplasm by Xpo5 protein (exportin-5) and then is processed by Dicer enzyme to a 22-nucleotide-sized long mature miRNA. miRNAs are differentially expressed in different diseases and are released into plasma by normal and tumor tissues during the cell metabolism. Ovarian carcinoma is the deadliest cancer among women. When the disease was diagnosed, the disease usually progressed. Currently, there is no biological marker to detect ovarian carcinoma at an early stage. Furthermore, there is a need for markers that are sensitive to chemotherapy changes and early detection of the disease. Because of this, miRNAs can be detected in plasma and can be used as highly significant biological markers and therapeutic targets for ovarian carcinoma. When the literature of the last 5 years is searched, there are many studies about miRNA and ovarian carcinoma. In this chapter, studies examining the relationship between ovarian carcinoma and miRNA from different angles are summarized under different sections.

**Keywords:** miRNAs, ovarian carcinoma, diagnosis and treatment

Ovarian cancer (OC) is the sixth common cancer in women in the United States. According to GLOBOCAN data published in 2018, 295,414 new cases of ovarian cancer have been reported in the world. A total of 184,799 of these cases have been reported to have died due to ovarian carcinoma. The patients who died because of ovarian carcinoma had constituted 62.5% of the cases diagnosed in 2018 [1]. Ovarian carcinoma is diagnosed in women at peri- and postmenopausal status

Genetics, syndromes (breast and ovarian carcinoma syndrome and Lynch syndrome), family history, personal history of cancer or endometriosis, increasing age, reproductive history and infertility, hormone replacement therapy, and obesity are factors that may increase the risk of ovarian carcinoma. However, oral contraceptive usage, having pregnancy before age 26, breastfeeding, removal of the ovaries and fallopian tubes, hysterectomy, and tubal ligation are factors that may reduce the risk of ovarian carcinoma. Ovarian carcinoma comprises a heterogeneous group of

#### **Chapter 6**

## Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma

*Hulya Yazici*

#### **Abstract**

miRNAs (miRNA) are small RNA molecules that are not to expressed to proteins. Their size is 20–22 nucleotides in length and they are highly conserved molecules among the species. miRNAs are synthesized in the nucleus as a primary miRNA. Primary miRNA is transferred to cytoplasm by Xpo5 protein (exportin-5) and then is processed by Dicer enzyme to a 22-nucleotide-sized long mature miRNA. miRNAs are differentially expressed in different diseases and are released into plasma by normal and tumor tissues during the cell metabolism. Ovarian carcinoma is the deadliest cancer among women. When the disease was diagnosed, the disease usually progressed. Currently, there is no biological marker to detect ovarian carcinoma at an early stage. Furthermore, there is a need for markers that are sensitive to chemotherapy changes and early detection of the disease. Because of this, miRNAs can be detected in plasma and can be used as highly significant biological markers and therapeutic targets for ovarian carcinoma. When the literature of the last 5 years is searched, there are many studies about miRNA and ovarian carcinoma. In this chapter, studies examining the relationship between ovarian carcinoma and miRNA from different angles are summarized under different sections.

**Keywords:** miRNAs, ovarian carcinoma, diagnosis and treatment

#### **1. Introduction**

Ovarian cancer (OC) is the sixth common cancer in women in the United States. According to GLOBOCAN data published in 2018, 295,414 new cases of ovarian cancer have been reported in the world. A total of 184,799 of these cases have been reported to have died due to ovarian carcinoma. The patients who died because of ovarian carcinoma had constituted 62.5% of the cases diagnosed in 2018 [1]. Ovarian carcinoma is diagnosed in women at peri- and postmenopausal status frequently [2].

Genetics, syndromes (breast and ovarian carcinoma syndrome and Lynch syndrome), family history, personal history of cancer or endometriosis, increasing age, reproductive history and infertility, hormone replacement therapy, and obesity are factors that may increase the risk of ovarian carcinoma. However, oral contraceptive usage, having pregnancy before age 26, breastfeeding, removal of the ovaries and fallopian tubes, hysterectomy, and tubal ligation are factors that may reduce the risk of ovarian carcinoma. Ovarian carcinoma comprises a heterogeneous group of

tumors with different histologic subtypes which have particular genetic structures and different response to treatment. The most common histological subtype is epithelial ovarian carcinomas accounting for about 90% of cases and can be classified as serous, endometrioid, and clear-cell and mucinous carcinomas [3, 4].

However, a majority of women are diagnosed in an advanced stage because of the asymptomatic issue in the early stage and due to the lack of an adequate early detection screening method [5]. Ovarian cancer still remains as one of the leading causes of cancer-related deaths, and the treatments could be improved using predictive biomarkers to measure a response to ovarian cancer therapy. Currently, there is no available proven single biomarker in the clinical use for detecting ovarian carcinoma in the early stage with adequate sensitivity and specificity. To solve this problem, researchers have aimed at the identification and validation of novel biomarkers for the early detection of ovarian carcinoma using new technologies. Diagnostic markers for population screening would be a simple blood testing with 95% specificity and sensitivity.

Similar to regulatory RNAs, microRNAs (miRNAs) are frequently deregulated in carcinogenesis. In ovarian tumorigenesis, numerous miRNAs were found altered, and some of these genes might represent ideal targets for diagnosis, prognosis, and treatment [6].

This chapter will focus on the recent advancements in miRNAs in the diagnosis, prognosis, and the treatments resistant to ovarian carcinoma.

#### **2. Methods**

PubMed search was performed using the keywords "ovarian carcinoma and miRNA" to prepare a comprehensive literature review. The results were filtered by published manuscripts in the last 5 years. A total of 193 associated papers, and review articles were found in the initial search. Additional searches were performed using the keywords genetic markers in ovarian cancer, multidrug resistance in ovarian cancer, and prognostic markers to supplement the information. 180 papers were selected for inclusion in the manuscript following the careful review of the abstracts. This chapter was written using the data of 3 meta-analyses, 20 reviews, and 67 original papers published in the last 5 years.

#### **3. Biogenesis and functions of miRNAs**

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules which regulate gene expression at the posttranscriptional level [7, 8]. Thousands of miRNA sequences have been identified in a wide range of organisms after the discovery of small noncoding RNAs [9, 10] and have currently been shown to be highly conserved among a wide range of species [11]. The miRNA database contains 38,589 entries representing hairpin precursor miRNAs, expressing 48,885 mature miRNA for 271 species (http://microrna.sanger.ac.uk). Each miRNA directly or indirectly regulates approximately 100 mRNA transcripts; however, a single gene coding protein could be regulated by more than one miRNA.

MicroRNAs are transcribed by RNA polymerase II or III, generating primary transcripts as short RNA hairpin structures (pre-miRNA) which are subsequently processed by cytoplasmic RNase III-type enzymes, Drosha and Dicer. The processed, mature miRNA incorporates into the RNA-induced silencing protein complex (RISC) to regulate the function of genes through degradation of mRNA and inhibition of translation [12, 13]. RISC usually binds to the 3′-UTR region of

**109**

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma*

target mRNAs to repress translation. The degree of translation depends on the degree of complementarity between miRNA and target mRNA. miRNAs usually bind to specific sequences with partial complementarity on target RNA transcripts, called microRNA response elements (MREs), which result in translational repres-

**3.1 Important miRNAs as diagnostic and prognostic biomarkers and** 

MicroRNAs were shown to have a crucial function in oncogenesis by regulating cell proliferation, cell differentiation, and apoptosis as oncogenes or tumor suppressors. The deregulation of miRNAs was suggested to be involved in a mechanism for cancer development. Also, miRNAs have been used as potential diagnostic or

Ovarian carcinoma is the most lethal cancer among gynecological malignancies. Therefore, there is still a need for good diagnostic markers to detect the disease at an early stage and good prognostic markers to follow the effects of therapeutic agents during the chemotherapy of patients with ovarian carcinoma. Li et al. investigated the miR-193b expression level in the tissues of patients diagnosed with ovarian carcinoma and ovarian carcinoma cell lines. They found the aberrant expression level of miR-193b in the tissue of ovarian carcinoma patients. miR-193b showed decreased expression in tumor tissues of patients with ovarian carcinoma and was correlated with FIGO stage, histologic grade, ascites, lymph node metastasis, tumor size, and also poor survival. Therefore, they suggested that the level of miR193b expression could be a potential biomarker for ovarian carcinoma patients [16]. Fukagawa et al. investigated the expression level of miR-135a-3p in the serum of ovarian carcinoma patients compared to ovarian cysts and normal ovarian tissue and also analyzed the expression level of miR-135a-3p in ovarian carcinoma cell lines such as SKOV3, ES2, and xenograft under cisplatin and paclitaxel. According to their result, they suggested that the miR-135a-3p expression could be used as a noninvasive biomarker in serum of patients with ovarian carcinoma in the diagnosis and follow-up of the disease [17]. Zuberi et al. evaluated the impact of the miR-125b expression level in patients with ovarian carcinoma and found that the expression level of miR-125b was statistically significant and that it was increased in serum specimens of patients with ovarian carcinoma compared to the levels in serum specimens of the healthy controls. They also demonstrated that the upregulation of miR-125b was associated with FIGO stage and lymph node involvement and distant metastasis and was correlated with hypermethylation of some tumor suppressor genes such as p16, p14, BRCA1, DAPK1, PTEN, and RASSF1A. Their results suggested that the expression level of mi-125b might be an early diagnostic biomarker to predict distant metastasis and lymph node status [18]. Zhang et al. investigated miR-613 expression in tissue of patients with ovarian carcinoma compared to matched normal adjacent tissue of patients. They found that low expression of miR-613 was associated with the FIGO stage, tumor grade, lymph node involvement, short progression-free survival (PFS), and overall survival (OS) in ovarian carcinoma patients. Their results indicated that miR-613 might be a good prognostic biomarker in patients with retinoblastoma [19]. Yanaihara et al. searched five miRNAs such as miR-132, miR-9, miR-126, miR-34a, and miR-21 in 12 highgrade serous ovarian carcinoma and 15 clear-cell ovarian carcinoma patients. They found that five miRNAs showed statistically higher expression in patients with clear-cell ovarian carcinoma. They also investigated further biological significance of miR-9 expression especially and demonstrated that miR-9 might have distinguished histologic subtypes of ovarian carcinoma and might be used a therapeutic

*DOI: http://dx.doi.org/10.5772/intechopen.85100*

therapeutic targets in cancer treatment.

**therapeutic targets in ovarian carcinoma**

sion in humans [14, 15].

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85100*

target mRNAs to repress translation. The degree of translation depends on the degree of complementarity between miRNA and target mRNA. miRNAs usually bind to specific sequences with partial complementarity on target RNA transcripts, called microRNA response elements (MREs), which result in translational repression in humans [14, 15].

MicroRNAs were shown to have a crucial function in oncogenesis by regulating cell proliferation, cell differentiation, and apoptosis as oncogenes or tumor suppressors. The deregulation of miRNAs was suggested to be involved in a mechanism for cancer development. Also, miRNAs have been used as potential diagnostic or therapeutic targets in cancer treatment.

#### **3.1 Important miRNAs as diagnostic and prognostic biomarkers and therapeutic targets in ovarian carcinoma**

Ovarian carcinoma is the most lethal cancer among gynecological malignancies. Therefore, there is still a need for good diagnostic markers to detect the disease at an early stage and good prognostic markers to follow the effects of therapeutic agents during the chemotherapy of patients with ovarian carcinoma. Li et al. investigated the miR-193b expression level in the tissues of patients diagnosed with ovarian carcinoma and ovarian carcinoma cell lines. They found the aberrant expression level of miR-193b in the tissue of ovarian carcinoma patients. miR-193b showed decreased expression in tumor tissues of patients with ovarian carcinoma and was correlated with FIGO stage, histologic grade, ascites, lymph node metastasis, tumor size, and also poor survival. Therefore, they suggested that the level of miR193b expression could be a potential biomarker for ovarian carcinoma patients [16]. Fukagawa et al. investigated the expression level of miR-135a-3p in the serum of ovarian carcinoma patients compared to ovarian cysts and normal ovarian tissue and also analyzed the expression level of miR-135a-3p in ovarian carcinoma cell lines such as SKOV3, ES2, and xenograft under cisplatin and paclitaxel. According to their result, they suggested that the miR-135a-3p expression could be used as a noninvasive biomarker in serum of patients with ovarian carcinoma in the diagnosis and follow-up of the disease [17]. Zuberi et al. evaluated the impact of the miR-125b expression level in patients with ovarian carcinoma and found that the expression level of miR-125b was statistically significant and that it was increased in serum specimens of patients with ovarian carcinoma compared to the levels in serum specimens of the healthy controls. They also demonstrated that the upregulation of miR-125b was associated with FIGO stage and lymph node involvement and distant metastasis and was correlated with hypermethylation of some tumor suppressor genes such as p16, p14, BRCA1, DAPK1, PTEN, and RASSF1A. Their results suggested that the expression level of mi-125b might be an early diagnostic biomarker to predict distant metastasis and lymph node status [18]. Zhang et al. investigated miR-613 expression in tissue of patients with ovarian carcinoma compared to matched normal adjacent tissue of patients. They found that low expression of miR-613 was associated with the FIGO stage, tumor grade, lymph node involvement, short progression-free survival (PFS), and overall survival (OS) in ovarian carcinoma patients. Their results indicated that miR-613 might be a good prognostic biomarker in patients with retinoblastoma [19]. Yanaihara et al. searched five miRNAs such as miR-132, miR-9, miR-126, miR-34a, and miR-21 in 12 highgrade serous ovarian carcinoma and 15 clear-cell ovarian carcinoma patients. They found that five miRNAs showed statistically higher expression in patients with clear-cell ovarian carcinoma. They also investigated further biological significance of miR-9 expression especially and demonstrated that miR-9 might have distinguished histologic subtypes of ovarian carcinoma and might be used a therapeutic

*Current Trends in Cancer Management*

95% specificity and sensitivity.

treatment [6].

**2. Methods**

tumors with different histologic subtypes which have particular genetic structures and different response to treatment. The most common histological subtype is epithelial ovarian carcinomas accounting for about 90% of cases and can be classified

However, a majority of women are diagnosed in an advanced stage because of the asymptomatic issue in the early stage and due to the lack of an adequate early detection screening method [5]. Ovarian cancer still remains as one of the leading causes of cancer-related deaths, and the treatments could be improved using predictive biomarkers to measure a response to ovarian cancer therapy. Currently, there is no available proven single biomarker in the clinical use for detecting ovarian carcinoma in the early stage with adequate sensitivity and specificity. To solve this problem, researchers have aimed at the identification and validation of novel biomarkers for the early detection of ovarian carcinoma using new technologies. Diagnostic markers for population screening would be a simple blood testing with

Similar to regulatory RNAs, microRNAs (miRNAs) are frequently deregulated in carcinogenesis. In ovarian tumorigenesis, numerous miRNAs were found altered, and some of these genes might represent ideal targets for diagnosis, prognosis, and

This chapter will focus on the recent advancements in miRNAs in the diagnosis,

PubMed search was performed using the keywords "ovarian carcinoma and miRNA" to prepare a comprehensive literature review. The results were filtered by published manuscripts in the last 5 years. A total of 193 associated papers, and review articles were found in the initial search. Additional searches were performed using the keywords genetic markers in ovarian cancer, multidrug resistance in ovarian cancer, and prognostic markers to supplement the information. 180 papers were selected for inclusion in the manuscript following the careful review of the abstracts. This chapter was written using the data of 3 meta-analyses, 20 reviews,

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules which regulate gene expression at the posttranscriptional level [7, 8]. Thousands of miRNA sequences have been identified in a wide range of organisms after the discovery of small noncoding RNAs [9, 10] and have currently been shown to be highly conserved among a wide range of species [11]. The miRNA database contains 38,589 entries representing hairpin precursor miRNAs, expressing 48,885 mature miRNA for 271 species (http://microrna.sanger.ac.uk). Each miRNA directly or indirectly regulates approximately 100 mRNA transcripts; however, a single gene

MicroRNAs are transcribed by RNA polymerase II or III, generating primary transcripts as short RNA hairpin structures (pre-miRNA) which are subsequently processed by cytoplasmic RNase III-type enzymes, Drosha and Dicer. The processed, mature miRNA incorporates into the RNA-induced silencing protein complex (RISC) to regulate the function of genes through degradation of mRNA and inhibition of translation [12, 13]. RISC usually binds to the 3′-UTR region of

prognosis, and the treatments resistant to ovarian carcinoma.

and 67 original papers published in the last 5 years.

coding protein could be regulated by more than one miRNA.

**3. Biogenesis and functions of miRNAs**

as serous, endometrioid, and clear-cell and mucinous carcinomas [3, 4].

**108**

target for treatment of ovarian carcinoma [20]. Yang et al. showed that miR-506 was associated with poor prognosis in ovarian carcinoma (OC) patients [21]. Sun et al. demonstrated in their study that miR-506 expression was correlated with early FIGO stage and good and longer survival [22]. The results of both studies suggested that miR-506 might be used as a prognostic biomarker. Yuan et al. showed that miR-494 had an antitumor effect in the tissue of OC patients and that miR-494 suppressed the cell proliferation and cell migration in epithelial ovarian carcinoma through the c-myc gene [23].

Agostini et al. studied 155 tissues of ovarian carcinoma including 30 sex cordstromal tumors, 22 borderline tumors, and 103 ovarian carcinomas and investigated the HMGA2 gene and its two miRNA targets in the study. They found that let-7a and miR-30c were highly decreased in all tumors in the study. Their results showed that Let-7a and miR30c were deregulated in OC patients, and the cause of deregulation in let-7a and miR30c might be due to the genomic imbalances, and the genomic imbalances resulted with the upregulation of HMGA2 gene. They suggested further research to better understand the associations between genetic imbalance and miRNA expression and prognostic and diagnostic importance in ovarian carcinoma [24]. Ma et al. investigated the expression level of miR-486-5p and its target, OLFM4 gene. Their results suggested that the decreased expression level of OLFM4 was associated with higher-grade FIGO tumors and poor differentiation. OLFM4 is downregulated by miR-486-5p which contributed to the tumorigenesis of ovarian carcinoma, and the opposite might be possible. OLFM4 and miR-485-5p might be the therapeutic targets for ovarian carcinoma [25]. Teng et al. published an article explaining an association between DNMT3A/3B and miR-29b. The results showed that the downregulation of miR29b was controlled by high levels of DNMT3A/3B expression. The results were suggested that was a cross talk and feedback between DNMT3A/3B and miR-29b and that the expression of miR-29b negatively controlled DNMTs, especially DNMT3A/3B. The findings showed that miR-29b and inhibitors of DNMT3A/B might be therapeutic target for patients with ovarian carcinoma [26]. According to articles published by different authors, miR-199 was downregulated in epithelial ovarian carcinoma and targeted to c-Met, HIF1-alpha, HIF2-beta, and IKK-beta proteins. Therefore, c-Met and/or miR-199 might be a target for patients with metastatic ovarian carcinoma [27–29]. Although many studies have been conducted so far, there are many candidate molecules that can be used in the diagnosis and prognosis of ovarian cancer; however, most of them should be validated with larger patient groups.

#### **3.2 miRNAs responsible for drug resistance in treatment of ovarian carcinoma**

The response of a patient with ovarian cancer to chemotherapy is actually the most important factor determining the survival of the patient. The primary treatment for ovarian cancer is surgery. After surgery, the first-line treatment is platinum-based combination [cisplatin or carboplatin and/or taxane (paclitaxel)] [30]. The majority of patients, almost 70%, receive this treatment and show complete remission. Patients were extremely sensitive to chemotherapy when they first received the treatment, and this situation changed during the next relapse period. The pharmacokinetics and pharmacodynamics of platinum-based therapies are known to be influenced by germ line genetic factors [31].

It is stated that many events associated with cisplatin resistance may be effective. It has been thought that these mechanisms can be generated by genetic and epigenetic alterations and miRNAs [32]. It is known that women with BRCA1 and BRCA2 gene mutations had better response to chemotherapy and had longer survival. It was reported that the mechanism underlying situation was associated with miR-9 in

**111**

as standard in the clinic.

**sites in ovarian carcinoma**

*3.3.1 3*′*UTR miRNA binding site of the KRAS gene*

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma*

animal models. miR-9 downregulates the BRCA1 gene, causing the DNA repair mechanism not to function, and thus increases the sensitivity to chemotherapy. In a study by Sun et al., It was shown that miR-9 overexpression in 58 tumor tissue samples was associated with BRCA1 gene mutation. Accordingly, these patients have been reported to be extremely sensitive to chemotherapy, especially platinumbased drugs and PARP inhibitors [33]. Furthermore, a similar relationship between BRCA1-miR-9 is present between the miR-93 and the PTEN gene. High miR-93 and low PTEN expressions were investigated in ovarian carcinoma cell lines which were OVCAR3 and SKOV3 with and without platinum resistance. In this study by Fu et al., the relationship between PTEN-miR-93 was also shown in tumor tissues of 10 ovarian cancer patients [34]. Let-7 expression has been reported to play a role in the response to chemotherapy. In particular, when paclitaxel was added to platinum regimen, it was shown that chemotherapy is beneficial in patients with low expression of let-7 [35]. It has been reported that high-level expression of miR622 is responsible for the development of resistance to platinum drugs and PARP inhibitors in patients with high-grade serous ovarian cancer with BRCA1 mutation. It is emphasized that this effect of miR622 can be affected by correcting the disorders in the homologous recombination mechanism [36]. The blockade of PD-L1, PD-1, and CTLA-4, which are immune system inhibitor receptors, has been extremely successful in some advanced cancers. High expression of miR-424 (322), especially in tumors, prolongs progression-free survival in ovarian cancer patients. miR-424 (322) blocks PD-L1 and CD80 expressions. The expression of miR424 (322) with PD-L1 immune checkpoint inhibitors is corrected, i.e., it is converted to normal. In in vivo and in vitro conditions, the restoration of miR424 (322), i.e., normalization, eliminates the resistance to chemotherapy by activation of the T cell immune response via blocking PD-L1. There is a synergetic development of chemotherapy and immunotherapy. PD-L1 and chemoresistance are controlled via miRNAs [37]. In the literature, there are a number of studies showing the relationship between miRNAs and chemosensitivity and chemoresistance. Some miRNAs have a highly significant role in the use of combined therapies such as chemotherapy and immunotherapy. There is no doubt that the success of cancer treatments will increase as the relationship between miRNA molecules and cancer treatments is determined. However, there is no doubt that more studies should be done to use these molecules

**3.3 Important polymorphisms and mutations of miRNA processing and binding** 

In 2008, Ratner et al. identified a germ line SNP in 3′UTR of the KRAS oncogene (rs61764370). The functional KRAS-variant was disrupted by the binding of let-7 to 3′UTR region of KRAS gene [38]. In 2010, Ratner et al. reported that a single nucleotide polymorphism (SNP), rs61764370, located in the 3′UTR of the *KRAS* oncogene was associated with the risk of unselected epithelial ovarian cancer [39]. They also showed that the variant was associated with hereditary ovarian cancer patients carrying *BRCA1* mutations and ovarian cancer patients with family history not carrying *BRCA1* or *BRCA2* [39]. This SNP was thought to be a strong candidate for cancer risk. These observations suggested that miRNAs can function as tumor suppressors or oncogenes [40]. An assay has subsequently been marketed to determine genotype at rs61764370 as a commercial test to determine the risk in women with a family history of ovarian cancer (http://www.miradx.com). However, in June 2011, Pharoah et al. showed in an extensive study with 8.669 unselected cases

*DOI: http://dx.doi.org/10.5772/intechopen.85100*

#### *Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85100*

animal models. miR-9 downregulates the BRCA1 gene, causing the DNA repair mechanism not to function, and thus increases the sensitivity to chemotherapy. In a study by Sun et al., It was shown that miR-9 overexpression in 58 tumor tissue samples was associated with BRCA1 gene mutation. Accordingly, these patients have been reported to be extremely sensitive to chemotherapy, especially platinumbased drugs and PARP inhibitors [33]. Furthermore, a similar relationship between BRCA1-miR-9 is present between the miR-93 and the PTEN gene. High miR-93 and low PTEN expressions were investigated in ovarian carcinoma cell lines which were OVCAR3 and SKOV3 with and without platinum resistance. In this study by Fu et al., the relationship between PTEN-miR-93 was also shown in tumor tissues of 10 ovarian cancer patients [34]. Let-7 expression has been reported to play a role in the response to chemotherapy. In particular, when paclitaxel was added to platinum regimen, it was shown that chemotherapy is beneficial in patients with low expression of let-7 [35]. It has been reported that high-level expression of miR622 is responsible for the development of resistance to platinum drugs and PARP inhibitors in patients with high-grade serous ovarian cancer with BRCA1 mutation. It is emphasized that this effect of miR622 can be affected by correcting the disorders in the homologous recombination mechanism [36]. The blockade of PD-L1, PD-1, and CTLA-4, which are immune system inhibitor receptors, has been extremely successful in some advanced cancers. High expression of miR-424 (322), especially in tumors, prolongs progression-free survival in ovarian cancer patients. miR-424 (322) blocks PD-L1 and CD80 expressions. The expression of miR424 (322) with PD-L1 immune checkpoint inhibitors is corrected, i.e., it is converted to normal. In in vivo and in vitro conditions, the restoration of miR424 (322), i.e., normalization, eliminates the resistance to chemotherapy by activation of the T cell immune response via blocking PD-L1. There is a synergetic development of chemotherapy and immunotherapy. PD-L1 and chemoresistance are controlled via miRNAs [37]. In the literature, there are a number of studies showing the relationship between miRNAs and chemosensitivity and chemoresistance. Some miRNAs have a highly significant role in the use of combined therapies such as chemotherapy and immunotherapy. There is no doubt that the success of cancer treatments will increase as the relationship between miRNA molecules and cancer treatments is determined. However, there is no doubt that more studies should be done to use these molecules as standard in the clinic.

#### **3.3 Important polymorphisms and mutations of miRNA processing and binding sites in ovarian carcinoma**

#### *3.3.1 3*′*UTR miRNA binding site of the KRAS gene*

In 2008, Ratner et al. identified a germ line SNP in 3′UTR of the KRAS oncogene (rs61764370). The functional KRAS-variant was disrupted by the binding of let-7 to 3′UTR region of KRAS gene [38]. In 2010, Ratner et al. reported that a single nucleotide polymorphism (SNP), rs61764370, located in the 3′UTR of the *KRAS* oncogene was associated with the risk of unselected epithelial ovarian cancer [39]. They also showed that the variant was associated with hereditary ovarian cancer patients carrying *BRCA1* mutations and ovarian cancer patients with family history not carrying *BRCA1* or *BRCA2* [39]. This SNP was thought to be a strong candidate for cancer risk. These observations suggested that miRNAs can function as tumor suppressors or oncogenes [40]. An assay has subsequently been marketed to determine genotype at rs61764370 as a commercial test to determine the risk in women with a family history of ovarian cancer (http://www.miradx.com). However, in June 2011, Pharoah et al. showed in an extensive study with 8.669 unselected cases

*Current Trends in Cancer Management*

through the c-myc gene [23].

validated with larger patient groups.

target for treatment of ovarian carcinoma [20]. Yang et al. showed that miR-506 was associated with poor prognosis in ovarian carcinoma (OC) patients [21]. Sun et al. demonstrated in their study that miR-506 expression was correlated with early FIGO stage and good and longer survival [22]. The results of both studies suggested that miR-506 might be used as a prognostic biomarker. Yuan et al. showed that miR-494 had an antitumor effect in the tissue of OC patients and that miR-494 suppressed the cell proliferation and cell migration in epithelial ovarian carcinoma

Agostini et al. studied 155 tissues of ovarian carcinoma including 30 sex cordstromal tumors, 22 borderline tumors, and 103 ovarian carcinomas and investigated the HMGA2 gene and its two miRNA targets in the study. They found that let-7a and miR-30c were highly decreased in all tumors in the study. Their results showed that Let-7a and miR30c were deregulated in OC patients, and the cause of deregulation in let-7a and miR30c might be due to the genomic imbalances, and the genomic imbalances resulted with the upregulation of HMGA2 gene. They suggested further research to better understand the associations between genetic imbalance and miRNA expression and prognostic and diagnostic importance in ovarian carcinoma [24]. Ma et al. investigated the expression level of miR-486-5p and its target, OLFM4 gene. Their results suggested that the decreased expression level of OLFM4 was associated with higher-grade FIGO tumors and poor differentiation. OLFM4 is downregulated by miR-486-5p which contributed to the tumorigenesis of ovarian carcinoma, and the opposite might be possible. OLFM4 and miR-485-5p might be the therapeutic targets for ovarian carcinoma [25]. Teng et al. published an article explaining an association between DNMT3A/3B and miR-29b. The results showed that the downregulation of miR29b was controlled by high levels of DNMT3A/3B expression. The results were suggested that was a cross talk and feedback between DNMT3A/3B and miR-29b and that the expression of miR-29b negatively controlled DNMTs, especially DNMT3A/3B. The findings showed that miR-29b and inhibitors of DNMT3A/B might be therapeutic target for patients with ovarian carcinoma [26]. According to articles published by different authors, miR-199 was downregulated in epithelial ovarian carcinoma and targeted to c-Met, HIF1-alpha, HIF2-beta, and IKK-beta proteins. Therefore, c-Met and/or miR-199 might be a target for patients with metastatic ovarian carcinoma [27–29]. Although many studies have been conducted so far, there are many candidate molecules that can be used in the diagnosis and prognosis of ovarian cancer; however, most of them should be

**3.2 miRNAs responsible for drug resistance in treatment of ovarian carcinoma**

The response of a patient with ovarian cancer to chemotherapy is actually the most important factor determining the survival of the patient. The primary treatment for ovarian cancer is surgery. After surgery, the first-line treatment is platinum-based combination [cisplatin or carboplatin and/or taxane (paclitaxel)] [30]. The majority of patients, almost 70%, receive this treatment and show complete remission. Patients were extremely sensitive to chemotherapy when they first received the treatment, and this situation changed during the next relapse period. The pharmacokinetics and pharmacodynamics of platinum-based therapies are

It is stated that many events associated with cisplatin resistance may be effective. It has been thought that these mechanisms can be generated by genetic and epigenetic alterations and miRNAs [32]. It is known that women with BRCA1 and BRCA2 gene mutations had better response to chemotherapy and had longer survival. It was reported that the mechanism underlying situation was associated with miR-9 in

known to be influenced by germ line genetic factors [31].

**110**

of invasive epithelial ovarian cancer and 10.012 controls that the SNP was clinically useless for risk prediction in sporadic or familial ovarian cancer [41].

#### *3.3.2 3*′*UTR miRNA binding site of the BRCA1 and BRCA2*

BRCA1/2 mutations and targeted miRNAs to BRCA genes were demonstrated in many studies in the last decade [42] [43–45]. Moskwa et al. suggested that tumors overexpressing miRNAs such as miR-182 which target BRCA proteins can also be susceptible to PARP inhibition.

They suggested that the high level of miR-182 expression may affect BRCA1 regulation for sporadic breast tumors. The changing level of miR-182 expression in different types of breast tumor cell lines affected the level of protein expression of BRCA1 and changed the sensitivity to PARP1 inhibition, both in breast cancer cell lines and in xenograft models [42]. Bioinformatic tools showed a binding site for miR-146a and miR-146b-5p in the upstream of BRCA1. This information suggested that BRCA1 gene can be downregulated by miR-146a and miR-146b-5p in basallike breast cancer cell lines and triple-negative breast tumors. This downregulation of BRCA1 increased a cell proliferation and a reduced homologous recombination repair rate controlled by BRCA1. Garcia et al. showed that the highest levels of miR-146a and/or miR-146b-5p were found in basal-like epithelial mammary tumor cell lines and breast tumors with triple-negative histology, and also the characteristics of these types of tumors are the closest tumors having carriers of BRCA1 mutations [43–45]. miRNAs are known to regulate tumor suppressor genes and oncogenes. The genetic alterations in the binding sites of miRNAs on DNA sequence of miRNA could affect the expression of tumor suppressor genes and oncogenes. Shen et al. searched the selected 17 miRNAs which have an important role in the development of breast cancer in 42 patients with familial breast carcinoma. miR-30c-1 and miR-17 among 17 miRNAs were only observed in noncarriers of BRCA1/2 mutations. They showed that these two miRNAs, miR-30c-1 and miR-17, resulted in conformational changes in their secondary structures and altered the expression in functional assays. They also showed that miR-17 could bind to the 3′UTR of BRCA1 mRNAs. Their results suggested that functional genetic alterations in miRNA genes can potentially alter the regulation of BRCA1 gene which is important for breast cancer [45]. The same perspective and scenario may be valid for patients with sporadic ovarian carcinoma having an overexpression of BRCA1, and the effect of PARP inhibitors can also be increased by eliminating BRCA expression via miR182 in ovarian carcinoma.

#### **3.4 miRNAs as angiogenetic and metastatic biomarkers in ovarian carcinoma**

The major challenge in treatment of ovarian carcinoma is the lack of good diagnostic and prognostic factors to follow and to diagnose in each stage of the disease. Li et al. established the study using SKOV3 and OVCAR3 ovarian carcinoma cell lines. They demonstrated that miR-125a-5p, miR125b-5p, miR22-3p, miR205-5p, and miR-152 were significantly downregulated in SKOV3 cell lines and also showed the negative correlation between miR-152 and expression level of ERBB3. The findings of the study showed that miR-152 was associated with the regulation of the proliferation and metastasis of ovarian cancer cells via the repression of ERBB3 expression. miR-152 is an important molecule to suppress the proliferation, invasion, and migration of ovarian carcinoma cell lines. Their results suggested that miR-152 may be a potential therapeutic target for ovarian carcinoma [46].

**113**

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma*

The interaction between HOTAIR and both miR-214 and miR-217 was shown in the study of Dong et al. on SKOV3 ovarian carcinoma cell line. Their results demonstrated that HOTAIR, which had an interaction with PIK3R3, regulated the proliferation, migration, and invasion in SKOV3 ovarian cell line via miR-214 and

Li et al. investigated miR-340 expression in five different ovarian carcinoma cell lines such as OVCAR3, CAOV3, HO-8910, ES-2, A2780, and FTE187. They showed that miR-340 was decreased in ovarian carcinoma cell lines and induced apoptosis in cells with downregulation of NF-ƘB1 to inhibit metastasis in ovarian carcinoma cell lines. They emphasized that miR-340-NF-ƘB1 interaction might be a potential

Wang et al. examined the expression of MTA1 and miR-30c in ovarian cancer line, SKOV3, and normal human ovarian surface epithelial cell line, HOSE. They found that miR-30c expression was significantly reduced when MTA1 expression was higher and localized in the cytoplasm of the cells. Their results suggested that MTA1 and miR30c expression were altered in ovarian carcinoma cell line and might be associated in invasion and metastasis of patients with ovarian carcinoma [49].

**3.5 Important miRNAs in exosomal and peripheral circulations in ovarian** 

Numerous studies demonstrated the clinical importance of circulating miRNAs as diagnostic and prognostic biomarkers in all types of cancer. Circulating miRNAs in ovarian cancer were published in many studies using blood plasma, serum,

The first study was published by Taylor et al. demonstrating that the levels of eight exosomal microRNAs extracted from sera which were miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214 were elevated at an advanced-stage ovarian carcinoma [50, 51]. The miRNA signature of exosomes showed that the circulating miRNAs can present the characteristics of the tumor.

Many researchers investigated different miRNAs in the sera of OC patients [51–59].The microRNAs miR182, miR200a, miR200b, and miR200c from miR200 family were investigated by Kan et al. in the sera of OC patients and healthy controls. They found significant differences between patients and controls and suggested that miR200b and miR200c had a power to discriminate serous ovarian cancer from healthy controls and had a potential as a biomarker of sera [53]. Chung et al. showed that the miR-132, miR-26a, miR-let7b, miR-145, and miR-143 were decreased in serum specimens of patients with ovarian carcinoma and healthy individuals [54]. Xu et al. showed significantly higher miR-21 levels in sera of patients with epithelial ovarian cancer than the levels in healthy controls. They also indicated the correlation between the increased miR-21 expression in sera and advanced FIGO stage, high tumor grade, and shortened overall survival. Their findings suggested that serum miR-21 may be a novel diagnostic and prognostic marker and could be used as a therapeutic target in advanced-stage ovarian carcinoma [55]. Hong et al. investigated the serum levels of miR-221 in patients with epithelial ovarian carcinoma and in controls. miR-221 was found to be upregulated in patients with EOC compared with the healthy controls. The expression level

therapeutic target or agent for patients with ovarian carcinoma [48].

*DOI: http://dx.doi.org/10.5772/intechopen.85100*

miR-217 [47].

**carcinoma**

ascites, and urine.

*3.5.1 Exosomal miRNAs*

*3.5.2 miRNAs in sera*

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85100*

The interaction between HOTAIR and both miR-214 and miR-217 was shown in the study of Dong et al. on SKOV3 ovarian carcinoma cell line. Their results demonstrated that HOTAIR, which had an interaction with PIK3R3, regulated the proliferation, migration, and invasion in SKOV3 ovarian cell line via miR-214 and miR-217 [47].

Li et al. investigated miR-340 expression in five different ovarian carcinoma cell lines such as OVCAR3, CAOV3, HO-8910, ES-2, A2780, and FTE187. They showed that miR-340 was decreased in ovarian carcinoma cell lines and induced apoptosis in cells with downregulation of NF-ƘB1 to inhibit metastasis in ovarian carcinoma cell lines. They emphasized that miR-340-NF-ƘB1 interaction might be a potential therapeutic target or agent for patients with ovarian carcinoma [48].

Wang et al. examined the expression of MTA1 and miR-30c in ovarian cancer line, SKOV3, and normal human ovarian surface epithelial cell line, HOSE. They found that miR-30c expression was significantly reduced when MTA1 expression was higher and localized in the cytoplasm of the cells. Their results suggested that MTA1 and miR30c expression were altered in ovarian carcinoma cell line and might be associated in invasion and metastasis of patients with ovarian carcinoma [49].

#### **3.5 Important miRNAs in exosomal and peripheral circulations in ovarian carcinoma**

Numerous studies demonstrated the clinical importance of circulating miRNAs as diagnostic and prognostic biomarkers in all types of cancer. Circulating miRNAs in ovarian cancer were published in many studies using blood plasma, serum, ascites, and urine.

#### *3.5.1 Exosomal miRNAs*

*Current Trends in Cancer Management*

susceptible to PARP inhibition.

expression via miR182 in ovarian carcinoma.

**ovarian carcinoma**

**3.4 miRNAs as angiogenetic and metastatic biomarkers in** 

The major challenge in treatment of ovarian carcinoma is the lack of good diagnostic and prognostic factors to follow and to diagnose in each stage of the disease. Li et al. established the study using SKOV3 and OVCAR3 ovarian carcinoma cell lines. They demonstrated that miR-125a-5p, miR125b-5p, miR22-3p, miR205-5p, and miR-152 were significantly downregulated in SKOV3 cell lines and also showed the negative correlation between miR-152 and expression level of ERBB3. The findings of the study showed that miR-152 was associated with the regulation of the proliferation and metastasis of ovarian cancer cells via the repression of ERBB3 expression. miR-152 is an important molecule to suppress the proliferation, invasion, and migration of ovarian carcinoma cell lines. Their results suggested that miR-152 may be a potential therapeutic target for ovarian

of invasive epithelial ovarian cancer and 10.012 controls that the SNP was clinically

BRCA1/2 mutations and targeted miRNAs to BRCA genes were demonstrated in many studies in the last decade [42] [43–45]. Moskwa et al. suggested that tumors overexpressing miRNAs such as miR-182 which target BRCA proteins can also be

They suggested that the high level of miR-182 expression may affect BRCA1 regulation for sporadic breast tumors. The changing level of miR-182 expression in different types of breast tumor cell lines affected the level of protein expression of BRCA1 and changed the sensitivity to PARP1 inhibition, both in breast cancer cell lines and in xenograft models [42]. Bioinformatic tools showed a binding site for miR-146a and miR-146b-5p in the upstream of BRCA1. This information suggested that BRCA1 gene can be downregulated by miR-146a and miR-146b-5p in basallike breast cancer cell lines and triple-negative breast tumors. This downregulation of BRCA1 increased a cell proliferation and a reduced homologous recombination repair rate controlled by BRCA1. Garcia et al. showed that the highest levels of miR-146a and/or miR-146b-5p were found in basal-like epithelial mammary tumor cell lines and breast tumors with triple-negative histology, and also the characteristics of these types of tumors are the closest tumors having carriers of BRCA1 mutations [43–45]. miRNAs are known to regulate tumor suppressor genes and oncogenes. The genetic alterations in the binding sites of miRNAs on DNA sequence of miRNA could affect the expression of tumor suppressor genes and oncogenes. Shen et al. searched the selected 17 miRNAs which have an important role in the development of breast cancer in 42 patients with familial breast carcinoma. miR-30c-1 and miR-17 among 17 miRNAs were only observed in noncarriers of BRCA1/2 mutations. They showed that these two miRNAs, miR-30c-1 and miR-17, resulted in conformational changes in their secondary structures and altered the expression in functional assays. They also showed that miR-17 could bind to the 3′UTR of BRCA1 mRNAs. Their results suggested that functional genetic alterations in miRNA genes can potentially alter the regulation of BRCA1 gene which is important for breast cancer [45]. The same perspective and scenario may be valid for patients with sporadic ovarian carcinoma having an overexpression of BRCA1, and the effect of PARP inhibitors can also be increased by eliminating BRCA

useless for risk prediction in sporadic or familial ovarian cancer [41].

*3.3.2 3*′*UTR miRNA binding site of the BRCA1 and BRCA2*

**112**

carcinoma [46].

The first study was published by Taylor et al. demonstrating that the levels of eight exosomal microRNAs extracted from sera which were miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214 were elevated at an advanced-stage ovarian carcinoma [50, 51]. The miRNA signature of exosomes showed that the circulating miRNAs can present the characteristics of the tumor.

#### *3.5.2 miRNAs in sera*

Many researchers investigated different miRNAs in the sera of OC patients [51–59].The microRNAs miR182, miR200a, miR200b, and miR200c from miR200 family were investigated by Kan et al. in the sera of OC patients and healthy controls. They found significant differences between patients and controls and suggested that miR200b and miR200c had a power to discriminate serous ovarian cancer from healthy controls and had a potential as a biomarker of sera [53]. Chung et al. showed that the miR-132, miR-26a, miR-let7b, miR-145, and miR-143 were decreased in serum specimens of patients with ovarian carcinoma and healthy individuals [54]. Xu et al. showed significantly higher miR-21 levels in sera of patients with epithelial ovarian cancer than the levels in healthy controls. They also indicated the correlation between the increased miR-21 expression in sera and advanced FIGO stage, high tumor grade, and shortened overall survival. Their findings suggested that serum miR-21 may be a novel diagnostic and prognostic marker and could be used as a therapeutic target in advanced-stage ovarian carcinoma [55]. Hong et al. investigated the serum levels of miR-221 in patients with epithelial ovarian carcinoma and in controls. miR-221 was found to be upregulated in patients with EOC compared with the healthy controls. The expression level

of serum miR-221 was significantly associated with the International Federation of Gynecology and Obstetrics (FIGO) stage and tumor grade. In addition, higher serum miR-221 expression was shown to be an independent prognostic factor for epithelial ovarian carcinoma [56].

#### *3.5.3 miRNAs in plasma*

Some other scientists used plasma in investigating the circulating biomarkers for ovarian carcinoma [60–63]. Zheng et al. showed higher plasma miR-205 and lower let-7f expression in patients with ovarian carcinoma than in healthy controls. The joint use of both MiR-205 and let-7f provided higher diagnostic accuracy for epithelial ovarian carcinoma, especially in patients with stage I disease. They also demonstrated that the combination of these two miRNAs and carbohydrate antigen-125 (CA-125) further improved the accuracy of detection of epithelial carcinoma in plasma samples and that the elevated miR-483-5p expression was found in patients with ovarian carcinoma with stages III and IV compared with stages I and II. Moreover, they demonstrated that lower levels of let-7f was predictive for poor prognosis in patients with epithelial ovarian carcinoma. Their findings suggested that plasma miR-205 and let-7f can be biomarkers for ovarian cancer detection and prognosis [60]. Shapira et al. assessed the expression levels of 754 miRNAs in presurgical plasma samples of 42 women with serous epithelial ovarian cancer and 36 plasma samples collected from women who had a benign pelvic mass at surgery. There were six miRNAs, miR-106b, miR-126, miR-150, miR-17, miR-20a, and miR-92a which were distinguished as benign in histology before surgery. They showed that 10 miRNAs in plasma can distinguish healthy controls from women with ovarian cancer and a benign neoplasm before surgery. In the comparison of healthy controls with patient's plasma samples, they found that five miRNAs, miR-1274a, miR-30b, miR-30c, miR-625-3p, and miR720, were differentially expressed and also that the level of miR-139-5p, miR-142-3p, miR-484, miR-486, and miR-660 were higher in healthy controls when analyzed against patients having benign mass in their body. They demonstrated that MiR-720 and miR-20a were higher in women who died 2 years after their diagnosis, and women who survived 44 years after diagnosis had higher levels of miR-223, miR-126-3p, and miR-1290 in their plasma before surgery [62].

#### *3.5.4 miRNAs in ascites*

Vaksman et al. investigated the effusion supernatants in 86 patients with ovarian carcinoma. In the study, they demonstrated that there were significant associations between clinicopathologic parameters and the levels of miR-21, miR-23a, miR-23b, miR-29a, miR-99a, miR-125b, miR-200c, miR-320a, and miR-484 and also between miRNAs 21, 23b, and 29a and poor survival. It was shown that the higher expression of miR-21 in metastatic ovarian carcinoma constituted chemoresistance in ovarian carcinoma, and the higher expression of miR-23a and miR-29a was associated with significantly shorter PFS [64].

#### *3.5.5 miRNAs in urine*

Researchers detected miRNAs on the urine of patients with ovarian carcinoma in the studies in the literature [65, 66]. Zavesky et al. investigated the expression of miRNAs in the urine of patients with ovarian carcinoma and endometrial carcinoma. They compared the expression levels of 18 miRNAs in OC patients before and after surgery. The expression levels of miR-92a, miR100, miR106b, and miR-200b were found significantly different between patients with ovarian carcinoma

**115**

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma*

and healthy controls. The expression levels of miR100 and miR106b were lower; however, the expression levels of miR-92a and miR-200b were higher in patients with ovarian carcinoma compared with the levels in healthy controls [65]. Zhou et al. examined the urine specimen obtained from 39 OC patients, from 26 patients with benign gynecologic disease, and from 30 healthy controls. They found that miR30a-5p was upregulated in OC patients compared with the healthy controls, and they also showed that the level of miR30a-5p can be used to follow excess tissues of

Some miRNAs participated in the control of autophagy by regulating ATGs proteins [67]. Yang et al. showed that the higher expression of mir-30d regulated autophagy through inhibiting LC3B-I conversion to LC3B-II enzymes and formation of autophagosome. Their results suggested that mir-30d disrupts the process of autophagy targeting multiple genes in the autophagy pathway. The data suggested that miR-30d might participate to oncogenesis and be used in the cancer therapy strategy [68]. Dai et al. investigated the expression levels of miR29b that targeted to genes of myeloid cell leukemia sequence 1 (MCL1), mitogen-activated protein kinase 10 (MAPK10), and autophagy-related protein 9A (ATG9A) and suggested that lower level of miR29b was an independent poor prognostic marker in ovarian carcinoma [69]. He et al. examined the downregulation of ATG14 through EGR1 miR-152 in cisplatin resistance ovarian carcinoma cell lines of A2780, CP70, SKOV3, and DDP. They determined that miR-152 expression level was extremely low in the cisplatin-resistant cell lines. Therefore, they suggested that the overexpression of miR-152 might be a useful therapeutic strategy to overcome cisplatin resistance by

Invasion into surrounding tissue is an important step of metastasis. Therefore, understanding the molecular mechanism of invasion may help to understand the metastasis process and identify novel biomarkers and therapeutic agents to treat and to protect patients against metastasis. Zhang et al. showed that there was a higher expression of miR-630 in 30 patients with ovarian carcinoma [71]. They also showed the effects of higher miR-360 expression in SKOV3 cell line. The results of the cell line study indicated that miR-630 targeted the KLF6 gene (Krüppel-like factor 6). KLF6 gene is responsible for cancer cell proliferation and migration. They demonstrated that miR-630 supported the epithelial cancer proliferation and invasion via targeting KLF6 gene, and overexpression of miR-630 stimulated growth of ovarian carcinoma tumor in vivo. Therefore, miR-630 was suggested to be a possible therapeutic target in ovarian carcinoma [71]. Sun et al. determined that the expression of miR-548c decreased in ovarian and endometrium carcinoma. Their results suggested that miR548c affected the expression of Twist. Higher expression level of Twist was shown in ovarian and endometrium carcinoma. Therefore, they emphasized that miR-548c might be used for therapeutic purposes to impress the expression level of TWIST in overexpressing tumors such as ovarian and endometrium carcinoma [72]. Wei et al. examined miR-205 expression level in 30 patients with ovarian carcinoma and in 12 normal ovarian tissues, and they found miR-205 overexpression in ovarian carcinoma tissue of patients. Also, the behavior of miR-205 was investigated in ovarian carcinoma cell lines of OVCAR5, OVCA8, and SKOV3. They determined that miR205 targeted to TCF21 gene (transcription factor 21) which significantly decreased in tumor tissue of OC patients [73]. They concluded that miR-205 was

*DOI: http://dx.doi.org/10.5772/intechopen.85100*

ovarian carcinoma after surgery [66].

**3.6 Important miRNAs for autophagy in ovarian carcinoma**

inhibiting ATG14 expression in ovarian carcinoma [70].

**3.7 Important miRNAs for invasion in ovarian carcinoma**

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85100*

and healthy controls. The expression levels of miR100 and miR106b were lower; however, the expression levels of miR-92a and miR-200b were higher in patients with ovarian carcinoma compared with the levels in healthy controls [65]. Zhou et al. examined the urine specimen obtained from 39 OC patients, from 26 patients with benign gynecologic disease, and from 30 healthy controls. They found that miR30a-5p was upregulated in OC patients compared with the healthy controls, and they also showed that the level of miR30a-5p can be used to follow excess tissues of ovarian carcinoma after surgery [66].

#### **3.6 Important miRNAs for autophagy in ovarian carcinoma**

Some miRNAs participated in the control of autophagy by regulating ATGs proteins [67]. Yang et al. showed that the higher expression of mir-30d regulated autophagy through inhibiting LC3B-I conversion to LC3B-II enzymes and formation of autophagosome. Their results suggested that mir-30d disrupts the process of autophagy targeting multiple genes in the autophagy pathway. The data suggested that miR-30d might participate to oncogenesis and be used in the cancer therapy strategy [68]. Dai et al. investigated the expression levels of miR29b that targeted to genes of myeloid cell leukemia sequence 1 (MCL1), mitogen-activated protein kinase 10 (MAPK10), and autophagy-related protein 9A (ATG9A) and suggested that lower level of miR29b was an independent poor prognostic marker in ovarian carcinoma [69]. He et al. examined the downregulation of ATG14 through EGR1 miR-152 in cisplatin resistance ovarian carcinoma cell lines of A2780, CP70, SKOV3, and DDP. They determined that miR-152 expression level was extremely low in the cisplatin-resistant cell lines. Therefore, they suggested that the overexpression of miR-152 might be a useful therapeutic strategy to overcome cisplatin resistance by inhibiting ATG14 expression in ovarian carcinoma [70].

#### **3.7 Important miRNAs for invasion in ovarian carcinoma**

Invasion into surrounding tissue is an important step of metastasis. Therefore, understanding the molecular mechanism of invasion may help to understand the metastasis process and identify novel biomarkers and therapeutic agents to treat and to protect patients against metastasis. Zhang et al. showed that there was a higher expression of miR-630 in 30 patients with ovarian carcinoma [71]. They also showed the effects of higher miR-360 expression in SKOV3 cell line. The results of the cell line study indicated that miR-630 targeted the KLF6 gene (Krüppel-like factor 6). KLF6 gene is responsible for cancer cell proliferation and migration. They demonstrated that miR-630 supported the epithelial cancer proliferation and invasion via targeting KLF6 gene, and overexpression of miR-630 stimulated growth of ovarian carcinoma tumor in vivo. Therefore, miR-630 was suggested to be a possible therapeutic target in ovarian carcinoma [71]. Sun et al. determined that the expression of miR-548c decreased in ovarian and endometrium carcinoma. Their results suggested that miR548c affected the expression of Twist. Higher expression level of Twist was shown in ovarian and endometrium carcinoma. Therefore, they emphasized that miR-548c might be used for therapeutic purposes to impress the expression level of TWIST in overexpressing tumors such as ovarian and endometrium carcinoma [72]. Wei et al. examined miR-205 expression level in 30 patients with ovarian carcinoma and in 12 normal ovarian tissues, and they found miR-205 overexpression in ovarian carcinoma tissue of patients. Also, the behavior of miR-205 was investigated in ovarian carcinoma cell lines of OVCAR5, OVCA8, and SKOV3. They determined that miR205 targeted to TCF21 gene (transcription factor 21) which significantly decreased in tumor tissue of OC patients [73]. They concluded that miR-205 was

*Current Trends in Cancer Management*

epithelial ovarian carcinoma [56].

*3.5.3 miRNAs in plasma*

*3.5.4 miRNAs in ascites*

significantly shorter PFS [64].

*3.5.5 miRNAs in urine*

of serum miR-221 was significantly associated with the International Federation of Gynecology and Obstetrics (FIGO) stage and tumor grade. In addition, higher serum miR-221 expression was shown to be an independent prognostic factor for

Some other scientists used plasma in investigating the circulating biomarkers for ovarian carcinoma [60–63]. Zheng et al. showed higher plasma miR-205 and lower let-7f expression in patients with ovarian carcinoma than in healthy controls. The joint use of both MiR-205 and let-7f provided higher diagnostic accuracy for epithelial ovarian carcinoma, especially in patients with stage I disease. They also demonstrated that the combination of these two miRNAs and carbohydrate antigen-125 (CA-125) further improved the accuracy of detection of epithelial carcinoma in plasma samples and that the elevated miR-483-5p expression was found in patients with ovarian carcinoma with stages III and IV compared with stages I and II. Moreover, they demonstrated that lower levels of let-7f was predictive for poor prognosis in patients with epithelial ovarian carcinoma. Their findings suggested that plasma miR-205 and let-7f can be biomarkers for ovarian cancer detection and prognosis [60]. Shapira et al. assessed the expression levels of 754 miRNAs in presurgical plasma samples of 42 women with serous epithelial ovarian cancer and 36 plasma samples collected from women who had a benign pelvic mass at surgery. There were six miRNAs, miR-106b, miR-126, miR-150, miR-17, miR-20a, and miR-92a which were distinguished as benign in histology before surgery. They showed that 10 miRNAs in plasma can distinguish healthy controls from women with ovarian cancer and a benign neoplasm before surgery. In the comparison of healthy controls with patient's plasma samples, they found that five miRNAs, miR-1274a, miR-30b, miR-30c, miR-625-3p, and miR720, were differentially expressed and also that the level of miR-139-5p, miR-142-3p, miR-484, miR-486, and miR-660 were higher in healthy controls when analyzed against patients having benign mass in their body. They demonstrated that MiR-720 and miR-20a were higher in women who died 2 years after their diagnosis, and women who survived 44 years after diagnosis had higher levels of miR-223,

miR-126-3p, and miR-1290 in their plasma before surgery [62].

Vaksman et al. investigated the effusion supernatants in 86 patients with ovarian carcinoma. In the study, they demonstrated that there were significant associations between clinicopathologic parameters and the levels of miR-21, miR-23a, miR-23b, miR-29a, miR-99a, miR-125b, miR-200c, miR-320a, and miR-484 and also between miRNAs 21, 23b, and 29a and poor survival. It was shown that the higher expression of miR-21 in metastatic ovarian carcinoma constituted chemoresistance in ovarian carcinoma, and the higher expression of miR-23a and miR-29a was associated with

Researchers detected miRNAs on the urine of patients with ovarian carcinoma in the studies in the literature [65, 66]. Zavesky et al. investigated the expression of miRNAs in the urine of patients with ovarian carcinoma and endometrial carcinoma. They compared the expression levels of 18 miRNAs in OC patients before and after surgery. The expression levels of miR-92a, miR100, miR106b, and miR-200b were found significantly different between patients with ovarian carcinoma

**114**

associated with the invasive behavior of ovarian tumor cells by targeting and with the decrease of TCF21 expression. miR-205 and TCF21 were suggested to be used for anticancer purposes [73]. Human telomerase reverse transcriptase (hTERT) is another important molecule in ovarian carcinoma. Bai et al. investigated expression levels of miR-532 and miR-3064 and found that they were downregulated in 60 tumor tissues of ovarian cancer patients, and there was an association between the decreased miR-532 and miR-3064 and poor survival of patients with ovarian carcinoma. Bai et al. also demonstrated that miR-532 and miR-3064 targeted to hTERT gene and inhibited the proliferation, epithelial-mesenchymal transitions (EMT), and invasion of ovarian carcinoma cells. Their results showed that miR-3064 controlled the expression level of hTERT, and the role of miR-532 was limited in ovarian carcinoma. They suggested that both miR-532 and miR-3064 might be a good therapeutic agent for treatment of ovarian carcinoma [74].

#### **3.8 An impact of miRNAs on epithelial-mesenchymal transitions (EMT) in ovarian carcinoma**

#### *3.8.1 miR-125a*

The epithelial-to-mesenchymal transition (EMT) and its reversion, mesenchymal-to-epithelial transition (MET), are important mechanisms in carcinoma progression and tumor metastasis. The important regulators of this process are growth factors, transcription factors, and adhesion molecules in that the activity of microRNA (miRNA) is suggested to contribute to EMT, MET, and metastatic progression. In ovarian cancer cells, EMT induces by overexpression of EGFR which leads to transcriptional repression of the miR-125a. MiR125a is suggested to be a negative regulator of EMT. Therefore, the repression of miR-125a was suggested to be a potential novel therapeutic approach for invasive behavior of ovarian cancer [75].

#### *3.8.2 miR-125b*

miR-125b expression was lower in epithelial ovarian carcinoma. The expression of miR125b in ovarian carcinoma blocked the tumor invasion. The expression of miR125b was associated with EMT and also with the expression of SET gene. Functional studies showed that SET gene was a target for miR-125b. The downregulated SET gene may be observed during tumor migration [76].

#### *3.8.3 miR-200 family*

Various studies on miR-200 family showed that the miR-200 family was associated with the inhibition of cancer metastasis via epithelial-to-mesenchymal transition. mRNAs of SMAD and ZEB gene families are the key targets for the inhibition of cancer cell metastasis stimulated by miR-200 via EMT. ZEB2 has specific sequences on its' 3′ UTR region for miR-200a, miR-141, miR-200b, miR-200c, 429, miR-200a, and miR-141. ZEB1 and ZEB2 are transcriptional repressor of E-Cadherin [77]. Wang et al. determined that the higher expressions of miR-429 and miR-200 families in mesenchymal-like ovarian carcinoma cell lines elevated the MET and the sensitivity to cisplatin [78]. In addition, TET3 was a gene downregulated during the epithelial-mesenchymal transition (EMT) induced with TGF-β1 in ovarian carcinoma cell lines. miR-30d was associated as a downstream target of TET3 gene. miR-30d could not bind to the promoter of TET3 gene, and TGF-β1-associated EMT was stimulated owing to the demethylation on binding site of miR-30d [79].

**117**

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma*

Fu et al. found higher miR-222-3p expression level in tumors of OC patients. They determined that the overexpression of miR-222-3p was associated with good survival in patients with epithelial ovarian carcinoma. As a further research, they also investigated biological function of miR-222-3p in cell lines and in mouse models. The data of the in vitro experiments determined that miR-222-3p suppressed the cell proliferation and migration in ovarian cancer cell lines and downregulated AKT activation by decreased phosphorylation of AKT protein. They showed that GNAI2 is a target for miR-222-3p and also induced PI3K/AKT pathway. They suggested that miR222-3p/GNAI2/AKT interactions might be used as a therapeutic target in ovarian carcinoma [80]. Zhou et al. showed that miR-595 is a significant biomarker to show poor prognosis in patients in ovarian carcinoma. They investigated miR-595 in tumors in epithelial ovarian carcinoma, and the lower expression of miR-595 was found associated with advanced FIGO stage and distant metastasis and short overall survival [81]. Shi et al. published a meta-analysis about miR-200 and miR-30. They showed that the expression levels of miR-200 family had significant association with overall survival (OS) and insignificant association with progression-free survival (PFS) in general evaluation. They also evaluated their results in subgroup analysis and found that an increased expression level of miR-200a, miR-200c, and miR-141 was associated with better PFS for patients with ovarian carcinoma. A higher expression level of miR-30 was associated with good overall survival and progression-free survival [82]. Therefore, they suggested that both miR200 family and miR-30 might be good prognostic biomarkers in patients with ovarian carcinoma. Kleeman et al. examined the prognostic and apoptotic potentials of miR-147b, miR-1912, and miR-3073a in ovarian carcinoma cell lines which have different genetic backgrounds such as SKOV3 (TP53 null), OVCAAR3 (TP53R248Q ), TOV21G, TOV112D (TP53R175H), A2780, and A2780cis (TP53K351N) with/without adding the chemotherapeutic agent of carboplatin. They showed that the expression level of miR-147b and miR-1912 was higher after carboplatin treatment in ovarian cancer cell lines, while the expression level of miR-147b and miR-1912 was lower in untreated ovarian cancer cell line with carboplatin. They underlined that these two-miRNA were leaded pro apoptotic signals and decreased the expression level of Bcl2 and affected to median survival of ovarian carcinoma cell lines [83]. Yoshioka et al. published that the expression level of WNT7A gene was higher in 300 FFPE tissue of ovarian specimens including ovarian tumor, benign/borderline, and normal ovarian tissue. They used ovarian carcinoma cell lines and mouse models to characterize the role of WNT7A gene in ovarian tumor development and progression. They suggested that the re-expression of WNT7A gene could play an important role in malignant transformation of ovarian tissue and progression of ovarian carcinoma [84]. After the article was published by Yoshioka et al., MacLean et al. demonstrated that miR-15b expression targeted WNT7A gene and found an inverse association between WNT7A and miR-15b. Higher expression level of WNT7A gene and lower expression level of miR-15b were associated with poor survival in patients with ovarian carcinoma. Their data showed that WNT7A was controlled by miR-15b expression reduced by promoter methylation through the DNMT1 gene, a responsible methylation in the genome of ovarian carcinoma [85]. Sun et al. published meta-analysis on miR-9 and its prognostic importance in ovarian carcinoma. Their results revealed that the decreased expression level of miR-9 was found to be associated with poor overall survival (OS) and PFS in patients with ovarian carcinoma [86]. Wang et al. demonstrated that higher level expression of miR-532-5p was associated with the survival of patients with ovarian carcinoma in TCGA data and

*DOI: http://dx.doi.org/10.5772/intechopen.85100*

**3.9 Important miRNAs on survival in ovarian carcinoma**

also ovarian carcinoma cell lines, such as SKOV3 and OVCAR3 [87].

#### **3.9 Important miRNAs on survival in ovarian carcinoma**

*Current Trends in Cancer Management*

**ovarian carcinoma**

*3.8.1 miR-125a*

*3.8.2 miR-125b*

*3.8.3 miR-200 family*

associated with the invasive behavior of ovarian tumor cells by targeting and with the decrease of TCF21 expression. miR-205 and TCF21 were suggested to be used for anticancer purposes [73]. Human telomerase reverse transcriptase (hTERT) is another important molecule in ovarian carcinoma. Bai et al. investigated expression levels of miR-532 and miR-3064 and found that they were downregulated in 60 tumor tissues of ovarian cancer patients, and there was an association between the decreased miR-532 and miR-3064 and poor survival of patients with ovarian carcinoma. Bai et al. also demonstrated that miR-532 and miR-3064 targeted to hTERT gene and inhibited the proliferation, epithelial-mesenchymal transitions (EMT), and invasion of ovarian carcinoma cells. Their results showed that miR-3064 controlled the expression level of hTERT, and the role of miR-532 was limited in ovarian carcinoma. They suggested that both miR-532 and miR-3064 might be a

good therapeutic agent for treatment of ovarian carcinoma [74].

**3.8 An impact of miRNAs on epithelial-mesenchymal transitions (EMT) in** 

The epithelial-to-mesenchymal transition (EMT) and its reversion, mesenchymal-to-epithelial transition (MET), are important mechanisms in carcinoma progression and tumor metastasis. The important regulators of this process are growth factors, transcription factors, and adhesion molecules in that the activity of microRNA (miRNA) is suggested to contribute to EMT, MET, and metastatic progression. In ovarian cancer cells, EMT induces by overexpression of EGFR which leads to transcriptional repression of the miR-125a. MiR125a is suggested to be a negative regulator of EMT. Therefore, the repression of miR-125a was suggested to be a potential novel therapeutic approach for invasive behavior of ovarian cancer [75].

miR-125b expression was lower in epithelial ovarian carcinoma. The expression of miR125b in ovarian carcinoma blocked the tumor invasion. The expression of miR125b was associated with EMT and also with the expression of SET gene. Functional studies showed that SET gene was a target for miR-125b. The downregu-

Various studies on miR-200 family showed that the miR-200 family was associated with the inhibition of cancer metastasis via epithelial-to-mesenchymal transition. mRNAs of SMAD and ZEB gene families are the key targets for the inhibition of cancer cell metastasis stimulated by miR-200 via EMT. ZEB2 has specific sequences on its' 3′ UTR region for miR-200a, miR-141, miR-200b, miR-200c, 429, miR-200a, and miR-141. ZEB1 and ZEB2 are transcriptional repressor of E-Cadherin [77]. Wang et al. determined that the higher expressions of miR-429 and miR-200 families in mesenchymal-like ovarian carcinoma cell lines elevated the MET and the sensitivity to cisplatin [78]. In addition, TET3 was a gene downregulated during the epithelial-mesenchymal transition (EMT) induced with TGF-β1 in ovarian carcinoma cell lines. miR-30d was associated as a downstream target of TET3 gene. miR-30d could not bind to the promoter of TET3 gene, and TGF-β1-associated EMT was stimulated owing to the demethylation on binding site

lated SET gene may be observed during tumor migration [76].

**116**

of miR-30d [79].

Fu et al. found higher miR-222-3p expression level in tumors of OC patients. They determined that the overexpression of miR-222-3p was associated with good survival in patients with epithelial ovarian carcinoma. As a further research, they also investigated biological function of miR-222-3p in cell lines and in mouse models. The data of the in vitro experiments determined that miR-222-3p suppressed the cell proliferation and migration in ovarian cancer cell lines and downregulated AKT activation by decreased phosphorylation of AKT protein. They showed that GNAI2 is a target for miR-222-3p and also induced PI3K/AKT pathway. They suggested that miR222-3p/GNAI2/AKT interactions might be used as a therapeutic target in ovarian carcinoma [80]. Zhou et al. showed that miR-595 is a significant biomarker to show poor prognosis in patients in ovarian carcinoma. They investigated miR-595 in tumors in epithelial ovarian carcinoma, and the lower expression of miR-595 was found associated with advanced FIGO stage and distant metastasis and short overall survival [81]. Shi et al. published a meta-analysis about miR-200 and miR-30. They showed that the expression levels of miR-200 family had significant association with overall survival (OS) and insignificant association with progression-free survival (PFS) in general evaluation. They also evaluated their results in subgroup analysis and found that an increased expression level of miR-200a, miR-200c, and miR-141 was associated with better PFS for patients with ovarian carcinoma. A higher expression level of miR-30 was associated with good overall survival and progression-free survival [82]. Therefore, they suggested that both miR200 family and miR-30 might be good prognostic biomarkers in patients with ovarian carcinoma. Kleeman et al. examined the prognostic and apoptotic potentials of miR-147b, miR-1912, and miR-3073a in ovarian carcinoma cell lines which have different genetic backgrounds such as SKOV3 (TP53 null), OVCAAR3 (TP53R248Q ), TOV21G, TOV112D (TP53R175H), A2780, and A2780cis (TP53K351N) with/without adding the chemotherapeutic agent of carboplatin. They showed that the expression level of miR-147b and miR-1912 was higher after carboplatin treatment in ovarian cancer cell lines, while the expression level of miR-147b and miR-1912 was lower in untreated ovarian cancer cell line with carboplatin. They underlined that these two-miRNA were leaded pro apoptotic signals and decreased the expression level of Bcl2 and affected to median survival of ovarian carcinoma cell lines [83]. Yoshioka et al. published that the expression level of WNT7A gene was higher in 300 FFPE tissue of ovarian specimens including ovarian tumor, benign/borderline, and normal ovarian tissue. They used ovarian carcinoma cell lines and mouse models to characterize the role of WNT7A gene in ovarian tumor development and progression. They suggested that the re-expression of WNT7A gene could play an important role in malignant transformation of ovarian tissue and progression of ovarian carcinoma [84]. After the article was published by Yoshioka et al., MacLean et al. demonstrated that miR-15b expression targeted WNT7A gene and found an inverse association between WNT7A and miR-15b. Higher expression level of WNT7A gene and lower expression level of miR-15b were associated with poor survival in patients with ovarian carcinoma. Their data showed that WNT7A was controlled by miR-15b expression reduced by promoter methylation through the DNMT1 gene, a responsible methylation in the genome of ovarian carcinoma [85]. Sun et al. published meta-analysis on miR-9 and its prognostic importance in ovarian carcinoma. Their results revealed that the decreased expression level of miR-9 was found to be associated with poor overall survival (OS) and PFS in patients with ovarian carcinoma [86]. Wang et al. demonstrated that higher level expression of miR-532-5p was associated with the survival of patients with ovarian carcinoma in TCGA data and also ovarian carcinoma cell lines, such as SKOV3 and OVCAR3 [87].

#### **4. Conclusion remarks**

Over the last 5 years, a large number of studies have been carried out to reveal the relationship between ovarian carcinoma and miRNAs. All of these studies about miRNA are promising for early detection of ovarian cancer, monitoring treatment, determining chemotherapy resistance, and discovering new therapeutic agents. With more extensive studies in the future and the use of effective miRNA molecules found in the clinic, ovarian cancer will be more manageable and early detectable. There is a need for a large number of well-selected patient groups and validated studies for miRNAs to be involved and used in the routine clinic practice. However, when all studies are completed, it is undoubted that miRNAs will contribute to cancer diagnosis, prognosis, and development of new chemotherapeutic drugs and beneficial to individualized medicine. It will be understood that the effects of these small molecules are actually greater than it is thought in the future.

#### **Author details**

Hulya Yazici Oncology Institute, Department of Basic Oncology, Cancer Genetics Division, Istanbul University, Istanbul, Turkey

\*Address all correspondence to: hulyayazici67@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.

**119**

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma*

[11] Wheeler BM et al. The deep evolution of metazoan microRNAs.

[12] Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function.

[14] Shah MY, Calin GA. MicroRNAs as therapeutic targets in human cancers. Wiley Interdisciplinary Reviews: RNA.

[15] Bhartiya D, Scaria V. Genomic variations in non-coding RNAs: Structure, function and regulation. Genomics. 2016;**107**(2-3):59-68

[16] Li H et al. Tissue miR-193b as a novel biomarker for patients with ovarian cancer. Medical Science Monitor. 2015;**21**:3929-3934

[17] Fukagawa S et al. MicroRNA-135a-3p as a promising biomarker and nucleic acid therapeutic agent for ovarian cancer. Cancer Science.

[18] Zuberi M et al. Utility of serum miR-125b as a diagnostic and prognostic

[19] Zhang X, Zhang H. Diminished miR-613 expression as a novel prognostic biomarker for human ovarian cancer. European Review for Medical and Pharmacological Sciences.

[20] Yanaihara N et al. MicroRNA gene expression signature driven

indicator and its alliance with a panel of tumor suppressor genes in epithelial ovarian cancer. PLoS One.

2017;**108**(5):886-896

2016;**11**(4):e0153902

2016;**20**(5):837-841

Evolution & Development.

Cell. 2004;**116**(2):281-297

2009;**10**(2):126-139

2014;**5**(4):537-548

[13] Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nature Reviews. Molecular Cell Biology.

2009;**11**(1):50-68

*DOI: http://dx.doi.org/10.5772/intechopen.85100*

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[2] Jones MB. Borderline ovarian

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[4] Seidman JD et al. The histologic type and stage distribution of ovarian carcinomas of surface epithelial origin. International Journal of Gynecological

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*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85100*

#### **References**

*Current Trends in Cancer Management*

**4. Conclusion remarks**

**118**

**Author details**

Hulya Yazici

provided the original work is properly cited.

Istanbul University, Istanbul, Turkey

\*Address all correspondence to: hulyayazici67@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,

Oncology Institute, Department of Basic Oncology, Cancer Genetics Division,

Over the last 5 years, a large number of studies have been carried out to reveal the relationship between ovarian carcinoma and miRNAs. All of these studies about miRNA are promising for early detection of ovarian cancer, monitoring treatment, determining chemotherapy resistance, and discovering new therapeutic agents. With more extensive studies in the future and the use of effective miRNA molecules found in the clinic, ovarian cancer will be more manageable and early detectable. There is a need for a large number of well-selected patient groups and validated studies for miRNAs to be involved and used in the routine clinic practice. However, when all studies are completed, it is undoubted that miRNAs will contribute to cancer diagnosis, prognosis, and development of new chemotherapeutic drugs and beneficial to individualized medicine. It will be understood that the effects of these

small molecules are actually greater than it is thought in the future.

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[3] Cho KR, Shih Ie M. Ovarian cancer. Annual Review of Pathology. 2009;**4**:287-313

[4] Seidman JD et al. The histologic type and stage distribution of ovarian carcinomas of surface epithelial origin. International Journal of Gynecological Pathology. 2004;**23**(1):41-44

[5] Jemal A et al. Cancer statistics, 2009. CA: A Cancer Journal for Clinicians. 2009;**59**(4):225-249

[6] Bartels CL, Tsongalis GJ. MicroRNAs: Novel biomarkers for human cancer. Clinical Chemistry. 2009;**55**(4):623-631

[7] Cullen BR. Derivation and function of small interfering RNAs and microRNAs. Virus Research. 2004;**102**(1):3-9

[8] Liu X, Fortin K, Mourelatos Z. MicroRNAs: Biogenesis and molecular functions. Brain Pathology. 2008;**18**(1):113-121

[9] Lee RC, Feinbaum RL, Ambros V. The *C. elegans* heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;**75**(5):843-854

[10] Reinhart BJ et al. The 21-nucleotide let-7 RNA regulates developmental timing in *Caenorhabditis elegans*. Nature. 2000;**403**(6772):901-906

[11] Wheeler BM et al. The deep evolution of metazoan microRNAs. Evolution & Development. 2009;**11**(1):50-68

[12] Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;**116**(2):281-297

[13] Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nature Reviews. Molecular Cell Biology. 2009;**10**(2):126-139

[14] Shah MY, Calin GA. MicroRNAs as therapeutic targets in human cancers. Wiley Interdisciplinary Reviews: RNA. 2014;**5**(4):537-548

[15] Bhartiya D, Scaria V. Genomic variations in non-coding RNAs: Structure, function and regulation. Genomics. 2016;**107**(2-3):59-68

[16] Li H et al. Tissue miR-193b as a novel biomarker for patients with ovarian cancer. Medical Science Monitor. 2015;**21**:3929-3934

[17] Fukagawa S et al. MicroRNA-135a-3p as a promising biomarker and nucleic acid therapeutic agent for ovarian cancer. Cancer Science. 2017;**108**(5):886-896

[18] Zuberi M et al. Utility of serum miR-125b as a diagnostic and prognostic indicator and its alliance with a panel of tumor suppressor genes in epithelial ovarian cancer. PLoS One. 2016;**11**(4):e0153902

[19] Zhang X, Zhang H. Diminished miR-613 expression as a novel prognostic biomarker for human ovarian cancer. European Review for Medical and Pharmacological Sciences. 2016;**20**(5):837-841

[20] Yanaihara N et al. MicroRNA gene expression signature driven

by miR-9 overexpression in ovarian clear cell carcinoma. PLoS One. 2016;**11**(9):e0162584

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[26] Teng Y et al. A double-negative feedback interaction between MicroRNA-29b and DNMT3A/3B contributes to ovarian cancer progression. Cellular Physiology and Biochemistry. 2016;**39**(6):2341-2352

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[29] Chen R et al. Regulation of IKKbeta by miR-199a affects NF-kappaB activity in ovarian cancer cells. Oncogene. 2008;**27**(34):4712-4723

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[32] Cao J et al. DNA methylationmediated repression of miR-886-3p predicts poor outcome of human small cell lung cancer. Cancer Research. 2013;**73**(11):3326-3335

[33] Sun C et al. miR-9 regulation of BRCA1 and ovarian cancer sensitivity to cisplatin and PARP inhibition. Journal of the National Cancer Institute. 2013;**105**(22):1750-1758

[34] Fu X et al. Involvement of microRNA-93, a new regulator of PTEN/ Akt signaling pathway, in regulation of chemotherapeutic drug cisplatin chemosensitivity in ovarian cancer cells. FEBS Letters. 2012;**586**(9):1279-1286

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[39] Ratner E et al. A KRAS-variant in ovarian cancer acts as a genetic marker of cancer risk. Cancer Research.

[40] Esquela-Kerscher A, Slack FJ. Oncomirs—MicroRNAs with a role in cancer. Nature Reviews. Cancer.

[41] Pharoah PD et al. The role of KRAS rs61764370 in invasive epithelial ovarian cancer: Implications for clinical testing. Clinical Cancer Research.

[42] Moskwa P et al. miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors. Molecular Cell.

[43] Garcia AI et al. Down-regulation of BRCA1 expression by miR-146a and miR-146b-5p in triple negative sporadic breast cancers. EMBO Molecular Medicine. 2011;**3**(5):279-290

[44] Shen J et al. A functional polymorphism in the miR-146a gene and age of familial breast/ovarian cancer diagnosis. Carcinogenesis.

[45] Shen J, Ambrosone CB, Zhao H. Novel genetic variants in microRNA genes and familial breast cancer. International Journal of Cancer.

2008;**29**(10):1963-1966

2009;**124**(5):1178-1182

2018;**41**(3):1529-1535

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2010;**70**(16):6509-6515

2006;**6**(4):259-269

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2011;**41**(2):210-220

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[38] Chin LJ et al. A SNP in a let-7 microRNA complementary site in the KRAS 3′ untranslated region increases non-small cell lung cancer risk. Cancer Research. 2008;**68**(20):8535-8540

*Current Trends in Cancer Management*

by miR-9 overexpression in ovarian clear cell carcinoma. PLoS One.

[29] Chen R et al. Regulation of IKKbeta by miR-199a affects NF-kappaB activity in ovarian cancer cells. Oncogene.

[30] Pignata S et al. Chemotherapy in epithelial ovarian cancer. Cancer

[31] Permuth-Wey J et al. STAT3 polymorphisms may predict an unfavorable response to first-line platinum-based therapy for women with advanced serous epithelial ovarian cancer. International Journal of Cancer.

[32] Cao J et al. DNA methylationmediated repression of miR-886-3p predicts poor outcome of human small cell lung cancer. Cancer Research.

[33] Sun C et al. miR-9 regulation of BRCA1 and ovarian cancer sensitivity to cisplatin and PARP inhibition. Journal of the National Cancer Institute.

2008;**27**(34):4712-4723

Letters. 2011;**303**(2):73-83

2016;**138**(3):612-619

2013;**73**(11):3326-3335

2013;**105**(22):1750-1758

[34] Fu X et al. Involvement of

microRNA-93, a new regulator of PTEN/ Akt signaling pathway, in regulation of chemotherapeutic drug cisplatin chemosensitivity in ovarian cancer cells. FEBS Letters. 2012;**586**(9):1279-1286

[35] Iorio MV, Croce CM. Commentary on microRNA fingerprint in human epithelial ovarian cancer. Cancer Research. 2016;**76**(21):6143-6145

[36] Choi YE et al. Platinum and PARP inhibitor resistance due to overexpression of MicroRNA-622 in BRCA1-mutant ovarian cancer. Cell Reports. 2016;**14**(3):429-439

[37] Xu S et al. miR-424(322) reverses chemoresistance via T-cell immune response activation by blocking the PD-L1 immune checkpoint. Nature Communications. 2016;**7**:11406

[21] Yang D et al. Integrated analyses identify a master microRNA regulatory network for the mesenchymal subtype in serous ovarian cancer. Cancer Cell.

[22] Sun Y et al. MiR-506 inhibits multiple targets in the epithelial-tomesenchymal transition network and is associated with good prognosis in epithelial ovarian cancer. The Journal of

Pathology. 2015;**235**(1):25-36

Monitor. 2016;**22**:617-624

[23] Yuan J, Wang K, Xi M. MiR-494 inhibits epithelial ovarian cancer growth by targeting c-Myc. Medical Science

[24] Agostini A et al. Expressions of miR-30c and let-7a are inversely correlated with HMGA2 expression in squamous cell carcinoma of the vulva. Oncotarget. 2016;**7**(51):85058-85062

[25] Ma H et al. Estrogen receptormediated miR-486-5p regulation of OLFM4 expression in ovarian cancer. Oncotarget. 2016;**7**(9):10594-10605

[26] Teng Y et al. A double-negative feedback interaction between MicroRNA-29b and DNMT3A/3B contributes to ovarian cancer

progression. Cellular Physiology and Biochemistry. 2016;**39**(6):2341-2352

[27] Kinose Y et al. The hypoxiarelated microRNA miR-199a-3p displays tumor suppressor functions in ovarian carcinoma. Oncotarget.

[28] Joshi HP et al. Dynamin 2 along with microRNA-199a reciprocally regulate hypoxia-inducible factors and ovarian cancer metastasis. Proceedings of the National Academy of Sciences of the United States of America.

2015;**6**(13):11342-11356

2014;**111**(14):5331-5336

2016;**11**(9):e0162584

2013;**23**(2):186-199

**120**

[39] Ratner E et al. A KRAS-variant in ovarian cancer acts as a genetic marker of cancer risk. Cancer Research. 2010;**70**(16):6509-6515

[40] Esquela-Kerscher A, Slack FJ. Oncomirs—MicroRNAs with a role in cancer. Nature Reviews. Cancer. 2006;**6**(4):259-269

[41] Pharoah PD et al. The role of KRAS rs61764370 in invasive epithelial ovarian cancer: Implications for clinical testing. Clinical Cancer Research. 2011;**17**(11):3742-3750

[42] Moskwa P et al. miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors. Molecular Cell. 2011;**41**(2):210-220

[43] Garcia AI et al. Down-regulation of BRCA1 expression by miR-146a and miR-146b-5p in triple negative sporadic breast cancers. EMBO Molecular Medicine. 2011;**3**(5):279-290

[44] Shen J et al. A functional polymorphism in the miR-146a gene and age of familial breast/ovarian cancer diagnosis. Carcinogenesis. 2008;**29**(10):1963-1966

[45] Shen J, Ambrosone CB, Zhao H. Novel genetic variants in microRNA genes and familial breast cancer. International Journal of Cancer. 2009;**124**(5):1178-1182

[46] Li LW et al. miR-152 is involved in the proliferation and metastasis of ovarian cancer through repression of ERBB3. International Journal of Molecular Medicine. 2018;**41**(3):1529-1535

[47] Dong L, Hui L. HOTAIR promotes proliferation, migration, and invasion of ovarian cancer SKOV3 cells through regulating PIK3R3. Medical Science Monitor. 2016;**22**:325-331

[48] Li P, Sun Y, Liu Q. MicroRNA-340 induces apoptosis and inhibits metastasis of ovarian cancer cells by inactivation of NF-x03BA;B1. Cellular Physiology and Biochemistry. 2016;**38**(5):1915-1927

[49] Wang X et al. MicroRNA-30c inhibits metastasis of ovarian cancer by targeting metastasis-associated gene 1. Journal of Cancer Research and Therapeutics. 2017;**13**(4):676-682

[50] Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecologic Oncology. 2008;**110**(1):13-21

[51] Zuberi M et al. Expression of serum miR-200a, miR-200b, and miR-200c as candidate biomarkers in epithelial ovarian cancer and their association with clinicopathological features. Clinical & Translational Oncology. 2015;**17**(10):779-787

[52] Resnick KE et al. The detection of differentially expressed microRNAs from the serum of ovarian cancer patients using a novel real-time PCR platform. Gynecologic Oncology. 2009;**112**(1):55-59

[53] Kan CW et al. Elevated levels of circulating microRNA-200 family members correlate with serous epithelial ovarian cancer. BMC Cancer. 2012;**12**:627

[54] Chung YW et al. Detection of microRNA as novel biomarkers of epithelial ovarian cancer from the serum of ovarian cancer patients. International Journal of Gynecological Cancer. 2013;**23**(4):673-679

[55] Xu YZ et al. Identification of serum microRNA-21 as a biomarker for early detection and prognosis in human epithelial ovarian cancer. Asian Pacific Journal of Cancer Prevention. 2013;**14**(2):1057-1060

[56] Hong F et al. Prognostic significance of serum microRNA-221 expression in human epithelial ovarian cancer. The Journal of International Medical Research. 2013;**41**(1):64-71

[57] Meng X et al. Diagnostic and prognostic potential of serum miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429 in ovarian cancer patients. British Journal of Cancer. 2015;**113**(9):1358-1366

[58] Gao YC, Wu J. MicroRNA-200c and microRNA-141 as potential diagnostic and prognostic biomarkers for ovarian cancer. Tumour Biology. 2015;**36**(6):4843-4850

[59] Liang H et al. Serum microRNA-145 as a novel biomarker in human ovarian cancer. Tumour Biology. 2015;**36**(7):5305-5313

[60] Zheng H et al. Plasma miRNAs as diagnostic and prognostic biomarkers for ovarian cancer. PLoS One. 2013;**8**(11):e77853

[61] Suryawanshi S et al. Plasma microRNAs as novel biomarkers for endometriosis and endometriosisassociated ovarian cancer. Clinical Cancer Research. 2013;**19**(5):1213-1224

[62] Shapira I et al. Circulating biomarkers for detection of ovarian cancer and predicting cancer outcomes. British Journal of Cancer. 2014;**110**(4):976-983

[63] Langhe R et al. A novel serum microRNA panel to discriminate benign from malignant ovarian disease. Cancer Letters. 2015;**356**(2 Pt B):628-636

[64] Vaksman O et al. Exosome-derived miRNAs and ovarian carcinoma progression. Carcinogenesis. 2014;**35**(9):2113-2120

[65] Zavesky L et al. Evaluation of cell-free urine microRNAs expression for the use in diagnosis of ovarian and endometrial cancers. A pilot study. Pathology Oncology Research. 2015;**21**(4):1027-1035

[66] Zhou J et al. Urinary microRNA-30a-5p is a potential biomarker for ovarian serous adenocarcinoma. Oncology Reports. 2015;**33**(6):2915-2923

[67] Titone R et al. Epigenetic control of autophagy by microRNAs in ovarian cancer. BioMed Research International. 2014;**2014**:343542

[68] Yang X et al. mir-30d Regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells. Biochemical and Biophysical Research Communications. 2013;**431**(3):617-622

[69] Dai F, Zhang Y, Chen Y. Involvement of miR-29b signaling in the sensitivity to chemotherapy in patients with ovarian carcinoma. Human Pathology. 2014;**45**(6):1285-1293

[70] He J et al. Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatininduced apoptosis by inhibiting cyto-protective autophagy. Autophagy. 2015;**11**(2):373-384

[71] Zhang S et al. MiR-630 promotes epithelial ovarian cancer proliferation and invasion via targeting KLF6. European Review for Medical and Pharmacological Sciences. 2017;**21**(20):4542-4547

[72] Sun X et al. MiR-548c impairs migration and invasion of endometrial and ovarian cancer cells via downregulation of Twist. Journal

**123**

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma*

ovarian cancer cell growth and associates with good overall survival. Oncotarget. 2016;**7**(49):80633-80654

[81] Zhou QH et al. Mir-595 is a significant indicator of poor patient prognosis in epithelial ovarian cancer. European Review for Medical and Pharmacological Sciences.

[82] Shi M et al. MicroRNA-200 and microRNA-30 family as prognostic molecular signatures in ovarian cancer: A meta-analysis. Medicine (Baltimore).

[83] Kleemann M et al. Investigation on tissue specific effects of proapoptotic micro RNAs revealed miR-147b as a potential biomarker in ovarian cancer prognosis. Oncotarget.

[84] Yoshioka S et al. WNT7A regulates tumor growth and progression in ovarian cancer through the WNT/ beta-catenin pathway. Molecular Cancer

2017;**21**(19):4278-4282

2018;**97**(32):e11505

2017;**8**(12):18773-18791

Research. 2012;**10**(3):469-482

[85] MacLean JA 2nd et al. WNT7A regulation by miR-15b in ovarian cancer.

[86] Sun H et al. Prognostic value of microRNA-9 in cancers: A systematic review and meta-analysis. Oncotarget.

[87] Wang F et al. High expression of miR-532-5p, a tumor suppressor, leads to better prognosis in ovarian cancer both in vivo and in vitro. Molecular Cancer Therapeutics.

PLoS One. 2016;**11**(5):e0156109

2016;**7**(41):67020-67032

2016;**15**(5):1123-1131

*DOI: http://dx.doi.org/10.5772/intechopen.85100*

[73] Wei J et al. MicroRNA-205 promotes cell invasion by repressing TCF21 in human ovarian cancer. Journal of Ovarian Research. 2017;**10**(1):33

of Experimental & Clinical Cancer

[74] Bai L et al. MicroRNA-532 and microRNA-3064 inhibit cell proliferation and invasion by acting as direct regulators of human telomerase reverse transcriptase in ovarian cancer.

PLoS One. 2017;**12**(3):e0173912

[75] Cowden Dahl KD et al. The epidermal growth factor receptor responsive miR-125a represses mesenchymal morphology in ovarian cancer cells. Neoplasia.

[76] Ying X et al. MicroRNA-125b suppresses ovarian cancer progression via suppression of the epithelialmesenchymal transition pathway by targeting the SET protein. Cellular Physiology and Biochemistry.

[77] Park SM et al. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes & Development. 2008;**22**(7):894-907

[78] Wang L et al. Ectopic overexpression of miR-429 induces mesenchymal-to-epithelial transition (MET) and increased drug sensitivity in metastasizing ovarian cancer cells. Gynecologic Oncology.

[79] Ye Z et al. TET3 inhibits TGFbeta1-induced epithelial-mesenchymal transition by demethylating miR-30d precursor gene in ovarian cancer cells. Journal of Experimental & Clinical Cancer Research. 2016;**35**:72

[80] Fu X et al. MicroRNA-222-3p/ GNAI2/AKT axis inhibits epithelial

2009;**11**(11):1208-1215

2016;**39**(2):501-510

2014;**134**(1):96-103

Research. 2016;**35**:10

*Functions of miRNAs in the Development, Diagnosis, and Treatment of Ovarian Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.85100*

of Experimental & Clinical Cancer Research. 2016;**35**:10

*Current Trends in Cancer Management*

[55] Xu YZ et al. Identification of serum microRNA-21 as a biomarker for early detection and prognosis in human epithelial ovarian cancer. Asian Pacific Journal of Cancer Prevention.

[56] Hong F et al. Prognostic significance of serum microRNA-221 expression in human epithelial ovarian cancer. The Journal of International Medical

[64] Vaksman O et al. Exosome-derived miRNAs and ovarian carcinoma progression. Carcinogenesis.

[65] Zavesky L et al. Evaluation of cell-free urine microRNAs expression for the use in diagnosis of ovarian and endometrial cancers. A pilot study. Pathology Oncology Research.

[66] Zhou J et al. Urinary microRNA-30a-5p is a potential biomarker for ovarian serous adenocarcinoma.

Oncology Reports. 2015;**33**(6):2915-2923

[67] Titone R et al. Epigenetic control of autophagy by microRNAs in ovarian cancer. BioMed Research International.

[68] Yang X et al. mir-30d Regulates multiple genes in the autophagy

pathway and impairs autophagy process in human cancer cells. Biochemical and Biophysical Research Communications.

[69] Dai F, Zhang Y, Chen Y. Involvement of miR-29b signaling in the sensitivity to chemotherapy in patients with ovarian carcinoma. Human Pathology.

[70] He J et al. Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatininduced apoptosis by inhibiting cyto-protective autophagy. Autophagy.

[71] Zhang S et al. MiR-630 promotes epithelial ovarian cancer proliferation and invasion via targeting KLF6. European Review for Medical and Pharmacological Sciences.

[72] Sun X et al. MiR-548c impairs migration and invasion of endometrial

and ovarian cancer cells via downregulation of Twist. Journal

2014;**35**(9):2113-2120

2015;**21**(4):1027-1035

2014;**2014**:343542

2013;**431**(3):617-622

2014;**45**(6):1285-1293

2015;**11**(2):373-384

2017;**21**(20):4542-4547

2013;**14**(2):1057-1060

Research. 2013;**41**(1):64-71

2015;**113**(9):1358-1366

2015;**36**(6):4843-4850

2015;**36**(7):5305-5313

2013;**8**(11):e77853

[57] Meng X et al. Diagnostic and prognostic potential of serum miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429 in ovarian cancer patients. British Journal of Cancer.

[58] Gao YC, Wu J. MicroRNA-200c and microRNA-141 as potential diagnostic and prognostic biomarkers for ovarian cancer. Tumour Biology.

[59] Liang H et al. Serum microRNA-145

[60] Zheng H et al. Plasma miRNAs as diagnostic and prognostic biomarkers

as a novel biomarker in human ovarian cancer. Tumour Biology.

for ovarian cancer. PLoS One.

[61] Suryawanshi S et al. Plasma microRNAs as novel biomarkers for endometriosis and endometriosisassociated ovarian cancer. Clinical Cancer Research. 2013;**19**(5):1213-1224

[62] Shapira I et al. Circulating biomarkers for detection of ovarian cancer and predicting cancer outcomes. British Journal of Cancer.

[63] Langhe R et al. A novel serum microRNA panel to discriminate benign from malignant ovarian disease. Cancer Letters. 2015;**356**(2 Pt B):628-636

2014;**110**(4):976-983

**122**

[73] Wei J et al. MicroRNA-205 promotes cell invasion by repressing TCF21 in human ovarian cancer. Journal of Ovarian Research. 2017;**10**(1):33

[74] Bai L et al. MicroRNA-532 and microRNA-3064 inhibit cell proliferation and invasion by acting as direct regulators of human telomerase reverse transcriptase in ovarian cancer. PLoS One. 2017;**12**(3):e0173912

[75] Cowden Dahl KD et al. The epidermal growth factor receptor responsive miR-125a represses mesenchymal morphology in ovarian cancer cells. Neoplasia. 2009;**11**(11):1208-1215

[76] Ying X et al. MicroRNA-125b suppresses ovarian cancer progression via suppression of the epithelialmesenchymal transition pathway by targeting the SET protein. Cellular Physiology and Biochemistry. 2016;**39**(2):501-510

[77] Park SM et al. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes & Development. 2008;**22**(7):894-907

[78] Wang L et al. Ectopic overexpression of miR-429 induces mesenchymal-to-epithelial transition (MET) and increased drug sensitivity in metastasizing ovarian cancer cells. Gynecologic Oncology. 2014;**134**(1):96-103

[79] Ye Z et al. TET3 inhibits TGFbeta1-induced epithelial-mesenchymal transition by demethylating miR-30d precursor gene in ovarian cancer cells. Journal of Experimental & Clinical Cancer Research. 2016;**35**:72

[80] Fu X et al. MicroRNA-222-3p/ GNAI2/AKT axis inhibits epithelial ovarian cancer cell growth and associates with good overall survival. Oncotarget. 2016;**7**(49):80633-80654

[81] Zhou QH et al. Mir-595 is a significant indicator of poor patient prognosis in epithelial ovarian cancer. European Review for Medical and Pharmacological Sciences. 2017;**21**(19):4278-4282

[82] Shi M et al. MicroRNA-200 and microRNA-30 family as prognostic molecular signatures in ovarian cancer: A meta-analysis. Medicine (Baltimore). 2018;**97**(32):e11505

[83] Kleemann M et al. Investigation on tissue specific effects of proapoptotic micro RNAs revealed miR-147b as a potential biomarker in ovarian cancer prognosis. Oncotarget. 2017;**8**(12):18773-18791

[84] Yoshioka S et al. WNT7A regulates tumor growth and progression in ovarian cancer through the WNT/ beta-catenin pathway. Molecular Cancer Research. 2012;**10**(3):469-482

[85] MacLean JA 2nd et al. WNT7A regulation by miR-15b in ovarian cancer. PLoS One. 2016;**11**(5):e0156109

[86] Sun H et al. Prognostic value of microRNA-9 in cancers: A systematic review and meta-analysis. Oncotarget. 2016;**7**(41):67020-67032

[87] Wang F et al. High expression of miR-532-5p, a tumor suppressor, leads to better prognosis in ovarian cancer both in vivo and in vitro. Molecular Cancer Therapeutics. 2016;**15**(5):1123-1131

**125**

Section 4

Paraneoplastic Syndromes

Section 4

## Paraneoplastic Syndromes

**127**

**Chapter 7**

**Abstract**

mentioned.

**1. Introduction**

aggressive and lethal disease.

Paraneoplastic Pemphigus Is a

Paraneoplastic pemphigus is a multiorganic autoimmune disease, usually triggered by neoplasias, mainly of lymphoproliferative origin such as chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, Castleman disease, and thymoma. This disorder is characterized by the presence of autoantibodies that react against proteins, such as desmoplakins, desmocollins, and others existing in cell junctions. The prognosis is reserved, and the mortality rate of the disease is very high, thus proving to be an additional challenge in the therapeutic management of onco-hematological diseases. The objective of this chapter is to solve the main clinical aspects of paraneoplastic pemphigus in lymphoproliferative hematological diseases, anatomopathological and immunofluorescence characteristics, as well as associations with the main differential diagnoses and therapeutic management. We will also describe the main differential diagnoses of paraneoplastic pemphigus, such as various types of pemphigus including induced drug, bullous pemphigoid, drug eruption, lichen planus, graft versus host disease, erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis. In addition, the prognosis and quality of life will be

**Keywords:** paraneoplastic pemphigus, neoplasms disease, autoimmune disease

Paraneoplastic pemphigus (PNP) was first described in 1990 by Anhalt et al. as a rare autoimmune disease that causes ulcerated lesions and vesicular eruptions in the mucocutaneous regions [1]. In 2001, the researcher Nguyen et al. introduced the term multiorganic autoimmune paraneoplastic syndrome, since it is a systemic disease that can affect the kidneys, bladder, and smooth and striated muscles [2]. PNP is a disease triggered mainly by B-cell lymphomas and malignant hematological diseases [3]. Other neoplasms also demonstrate the onset of this disease, as well as carcinoma of the stomach, lung, and colon [3]. The patients with PNP present high mortality rates, being around 90% of the cases, besides presenting an extremely complex and difficult diagnosis, since it resembles several other diseases [4, 5]. The treatment and management of this disease are often ineffective, as it is an extremely

In this chapter, we will address the epidemiological aspects, the main triggers, pathophysiology, main manifestations, diagnosis, differential diagnoses, treatments

used, prognosis, and the quality of life of patients affected by PNP.

Life-Threatening Disease

*Richard Lucas Konichi-Dias*

#### **Chapter 7**

## Paraneoplastic Pemphigus Is a Life-Threatening Disease

*Richard Lucas Konichi-Dias*

#### **Abstract**

Paraneoplastic pemphigus is a multiorganic autoimmune disease, usually triggered by neoplasias, mainly of lymphoproliferative origin such as chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, Castleman disease, and thymoma. This disorder is characterized by the presence of autoantibodies that react against proteins, such as desmoplakins, desmocollins, and others existing in cell junctions. The prognosis is reserved, and the mortality rate of the disease is very high, thus proving to be an additional challenge in the therapeutic management of onco-hematological diseases. The objective of this chapter is to solve the main clinical aspects of paraneoplastic pemphigus in lymphoproliferative hematological diseases, anatomopathological and immunofluorescence characteristics, as well as associations with the main differential diagnoses and therapeutic management. We will also describe the main differential diagnoses of paraneoplastic pemphigus, such as various types of pemphigus including induced drug, bullous pemphigoid, drug eruption, lichen planus, graft versus host disease, erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis. In addition, the prognosis and quality of life will be mentioned.

**Keywords:** paraneoplastic pemphigus, neoplasms disease, autoimmune disease

#### **1. Introduction**

Paraneoplastic pemphigus (PNP) was first described in 1990 by Anhalt et al. as a rare autoimmune disease that causes ulcerated lesions and vesicular eruptions in the mucocutaneous regions [1]. In 2001, the researcher Nguyen et al. introduced the term multiorganic autoimmune paraneoplastic syndrome, since it is a systemic disease that can affect the kidneys, bladder, and smooth and striated muscles [2]. PNP is a disease triggered mainly by B-cell lymphomas and malignant hematological diseases [3]. Other neoplasms also demonstrate the onset of this disease, as well as carcinoma of the stomach, lung, and colon [3]. The patients with PNP present high mortality rates, being around 90% of the cases, besides presenting an extremely complex and difficult diagnosis, since it resembles several other diseases [4, 5]. The treatment and management of this disease are often ineffective, as it is an extremely aggressive and lethal disease.

In this chapter, we will address the epidemiological aspects, the main triggers, pathophysiology, main manifestations, diagnosis, differential diagnoses, treatments used, prognosis, and the quality of life of patients affected by PNP.

#### **2. Epidemiology**

Because PNP is an extremely rare disease, there is still no data on the incidence of this disease in the world population [3]. To date, about 500 cases have been reported in the literature, with PNP representing 3–5% of all cases of pemphigus in the population [6–8]. The vast majority of affected patients demonstrate lymphoproliferative disorders (LPD) [9]. Although this disease can affect children and adolescents, the most common age group is between 45 and 70 years of age and is not correlated with place of origin, race, and sex [7, 10–14].

#### **3. Association with malignancy and genetic background**

PNP can be triggered by several types of neoplasias; however, about 84% of all patients present neoplasias or hematological disorders [3, 7, 15]. Non-Hodgkin's lymphoma is the most common disorder with 38.6% of cases, followed by chronic lymphocytic leukemia and Castleman disease with 18.4% each (**Table 1**). Among the non-hematological neoplasms, sarcomas present approximately 8.6% of the cases, such as leiomyosarcoma, malignant nerve sheath tumor, poorly differentiated sarcoma, reticular cell sarcoma, dendritic cell sarcoma, liposarcoma, and inflammatory myofibroblastoma [15–17]. Other less common diseases described in the literature that provide PNP are malignant thymoma, squamous cell carcinoma of the esophagus, colon carcinoma, CD8+ T-cell lymphoma, retroperitoneal Kaposi's sarcoma, and lymphoepithelioma-like carcinoma [18–23]. Although the PNP is triggered by several neoplasias, the manifestations of this disease may precede the hematological disorders and other malignancies, thus requiring the frequent and continuous follow-up of these patients [15]. In addition, there are reports of the occurrence of PNP without a detecting the cause [24, 25].

It is known that the major histocompatibility complex (MHC) has important relationships in increasing the susceptibility of autoimmune diseases. Although there are few papers that analyze the relationship between PNP and genetics, some studies in the Caucasian and Chinese population showed the relationships of the HLA class II alleles DRB1\*03 and HLA-Cw\*14 in the PNP's trigger [26, 27]. HLA-Cw\* 14 proved to be a more specific allele type of PNP. Its importance has been associated with PNP, regardless of whether it is a Castleman disease or other tumors, in addition to Castleman disease. [26]. However, to date, these studies are preliminary studies that suggest the association between genetic factors and PNP. To better understand this relationship, it is important to conduct studies with larger numbers of patients and that are affected by different tumors, as well as the realization of this association in different populations.


**129**

*Paraneoplastic Pemphigus Is a Life-Threatening Disease DOI: http://dx.doi.org/10.5772/intechopen.84956*

demonstrate the immunological complexity of the disease.

PNP even being a disease not yet known at the present time, it is known that both autoantibodies, as cell-mediated immunity, are involved [28]. Certainly, it deduces that the immune system is paramount in the pathophysiology of

PNP triggers immune changes with the production of autoantibodies capable of acting on various proteins in the body. The major target proteins of the autoantibodies are desmoglein 1 (DSG-1) and desmoglein 3 (DSG-3); desmocollins 1, 2, and 3; desmoplakins 1 and 2; BP230; BP130; and envoplakin, in addition to several other epitopes affected by autoantigens found in the individual [29]. These characteristics

Proteins of the plakin family, such as desmoplakins 1 and 2, envoplakin, periplakin, plectin and BP230, demonstrate the major targets of autoantibodies [30]. In contrast, the proteins of the cadherin family are the second most affected, with proteins such as DSG-1 and DSG-3 and desmocollin [31]. It is known that the presence of autoantibodies to some proteins are not related to the clinical practice of the patients, although there is a study that has mentioned DSG-3 relation with genital

Other autoantibodies such as alpha-2 macroglobulin-like 1 (A2ML1), a broad-range protease inhibitor, have been shown to be important in some patients. This protein has been shown to increase in the oral mucosa, intestine, esophagus, and muscles. However, its true function in the epithelium is

PNP studies with tumor resection demonstrate that tumors have the capacity to secrete autoantibodies capable of affecting the proteins of the epidermal region [35]. While knowing that most PNPs are involved in neoplastic and LPD diseases, triggering by solid tumors is still poorly understood and demonstrates other mechanisms involved in the production of autoantibodies to plakin

The involvement of the humoral immunity of PNP presents the desmoplakins 1 and 2, envoplakin, periplakin, BP230, A2ML1, and DSG-1 and DSG-3 as the main proteins of concern [1]. However, 16% of all affected do not demonstrate the presence of these autoantibodies, and this makes, in some cases, the accomplishment of the early diagnosis difficult. A study conducted in patients with PNP and who developed muscle weakness demonstrated autoantibodies against neuromuscular junction proteins and muscle tissue. These muscle-associated proteins were autoantibodies to anti-acetylcholinesterase receptors and anti-titin and anti-ryanodine

Cellular immunity has evidenced important roles in the immunophenotyping of PNP. Pathological analyses have demonstrated inflammatory infiltrates with the presence of CD8+ T cells, CD68+ monocytes, and non-major histocompatibility complex-restricted CD56+ in the subepidermal region [2, 37]. Besides that, in the places of affection, the increase in tumor necrosis factor, as well as interferon gamma, was evidenced [38]. These findings show the importance of cellular immunity in the pathogenesis of the disease, since they present abundantly in the sites of

**4. Pathogenesis**

**4.1 Autoantibodies**

involvement [32].

unknown [33, 34].

proteins.

receptor [36].

**4.2 Cellular immunity**

PNP involvement.

this disease.

#### **Table 1.**

*Paraneoplastic pemphigus associated with neoplasms.*

### **4. Pathogenesis**

*Current Trends in Cancer Management*

detecting the cause [24, 25].

realization of this association in different populations.

*Paraneoplastic pemphigus associated with neoplasms.*

Because PNP is an extremely rare disease, there is still no data on the incidence

PNP can be triggered by several types of neoplasias; however, about 84% of all patients present neoplasias or hematological disorders [3, 7, 15]. Non-Hodgkin's lymphoma is the most common disorder with 38.6% of cases, followed by chronic lymphocytic leukemia and Castleman disease with 18.4% each (**Table 1**). Among the non-hematological neoplasms, sarcomas present approximately 8.6% of the cases, such as leiomyosarcoma, malignant nerve sheath tumor, poorly differentiated sarcoma, reticular cell sarcoma, dendritic cell sarcoma, liposarcoma, and inflammatory myofibroblastoma [15–17]. Other less common diseases described in the literature that provide PNP are malignant thymoma, squamous cell carcinoma of the esophagus, colon carcinoma, CD8+ T-cell lymphoma, retroperitoneal Kaposi's sarcoma, and lymphoepithelioma-like carcinoma [18–23]. Although the PNP is triggered by several neoplasias, the manifestations of this disease may precede the hematological disorders and other malignancies, thus requiring the frequent and continuous follow-up of these patients [15]. In addition, there are reports of the occurrence of PNP without a

It is known that the major histocompatibility complex (MHC) has important relationships in increasing the susceptibility of autoimmune diseases. Although there are few papers that analyze the relationship between PNP and genetics, some studies in the Caucasian and Chinese population showed the relationships of the HLA class II alleles DRB1\*03 and HLA-Cw\*14 in the PNP's trigger [26, 27]. HLA-Cw\* 14 proved to be a more specific allele type of PNP. Its importance has been associated with PNP, regardless of whether it is a Castleman disease or other tumors, in addition to Castleman disease. [26]. However, to date, these studies are preliminary studies that suggest the association between genetic factors and PNP. To better understand this relationship, it is important to conduct studies with larger numbers of patients and that are affected by different tumors, as well as the

**Neoplasms Frequencies (%)** Non-Hodgkin's lymphoma 38.6 Chronic lymphocytic leukemia 18.4 Castleman disease 18.4 Sarcoma 8.6 Others 16

of this disease in the world population [3]. To date, about 500 cases have been reported in the literature, with PNP representing 3–5% of all cases of pemphigus in the population [6–8]. The vast majority of affected patients demonstrate lymphoproliferative disorders (LPD) [9]. Although this disease can affect children and adolescents, the most common age group is between 45 and 70 years of age and is

not correlated with place of origin, race, and sex [7, 10–14].

**3. Association with malignancy and genetic background**

**2. Epidemiology**

**128**

**Table 1.**

PNP even being a disease not yet known at the present time, it is known that both autoantibodies, as cell-mediated immunity, are involved [28]. Certainly, it deduces that the immune system is paramount in the pathophysiology of this disease.

#### **4.1 Autoantibodies**

PNP triggers immune changes with the production of autoantibodies capable of acting on various proteins in the body. The major target proteins of the autoantibodies are desmoglein 1 (DSG-1) and desmoglein 3 (DSG-3); desmocollins 1, 2, and 3; desmoplakins 1 and 2; BP230; BP130; and envoplakin, in addition to several other epitopes affected by autoantigens found in the individual [29]. These characteristics demonstrate the immunological complexity of the disease.

Proteins of the plakin family, such as desmoplakins 1 and 2, envoplakin, periplakin, plectin and BP230, demonstrate the major targets of autoantibodies [30]. In contrast, the proteins of the cadherin family are the second most affected, with proteins such as DSG-1 and DSG-3 and desmocollin [31]. It is known that the presence of autoantibodies to some proteins are not related to the clinical practice of the patients, although there is a study that has mentioned DSG-3 relation with genital involvement [32].

Other autoantibodies such as alpha-2 macroglobulin-like 1 (A2ML1), a broad-range protease inhibitor, have been shown to be important in some patients. This protein has been shown to increase in the oral mucosa, intestine, esophagus, and muscles. However, its true function in the epithelium is unknown [33, 34].

PNP studies with tumor resection demonstrate that tumors have the capacity to secrete autoantibodies capable of affecting the proteins of the epidermal region [35]. While knowing that most PNPs are involved in neoplastic and LPD diseases, triggering by solid tumors is still poorly understood and demonstrates other mechanisms involved in the production of autoantibodies to plakin proteins.

The involvement of the humoral immunity of PNP presents the desmoplakins 1 and 2, envoplakin, periplakin, BP230, A2ML1, and DSG-1 and DSG-3 as the main proteins of concern [1]. However, 16% of all affected do not demonstrate the presence of these autoantibodies, and this makes, in some cases, the accomplishment of the early diagnosis difficult. A study conducted in patients with PNP and who developed muscle weakness demonstrated autoantibodies against neuromuscular junction proteins and muscle tissue. These muscle-associated proteins were autoantibodies to anti-acetylcholinesterase receptors and anti-titin and anti-ryanodine receptor [36].

#### **4.2 Cellular immunity**

Cellular immunity has evidenced important roles in the immunophenotyping of PNP. Pathological analyses have demonstrated inflammatory infiltrates with the presence of CD8+ T cells, CD68+ monocytes, and non-major histocompatibility complex-restricted CD56+ in the subepidermal region [2, 37]. Besides that, in the places of affection, the increase in tumor necrosis factor, as well as interferon gamma, was evidenced [38]. These findings show the importance of cellular immunity in the pathogenesis of the disease, since they present abundantly in the sites of PNP involvement.

#### **5. Clinical features**

PNP presents several symptoms and clinical evolutions. The first symptoms as well as the progression of the disease are very varied from one patient to another. However, there are more frequent clinical features of these individuals.

#### **5.1 Oral lesions**

The oral mucosa is often affected in patients with PNP [3, 39, 40]. Oral symptoms may be the first symptoms in these patients, even before skin lesions [41]. The most common symptoms are oral and labial erosions with bleeding that may be associated with blisters, macules, papules, vesicles, and erythema (**Figure 1**). In addition, these patients may present a positive Nikolsky sign [41].

PNP lesions may be similar to oral manifestations of other diseases. Pemphigus vulgaris is a disease that initially triggers blisters and ulcers in the oral mucosa (especially on the cheeks) and may even reach the body. Erythema multiforme also affects the region of the oral mucosa with the appearance of erythema, edema, and some superficial erosions with formation of pseudomembrane. Lichen planus causes erythematous lesions where Wickham striae are present and may in rare cases develop erosions. In most cases of oral lichen planus, these are asymptomatic manifestations with few complications. Even though these diseases show some similarity to PNP, they are less aggressive, lethal, painful, and incapacitating, with less ability to spread to all mucosal and other body sites when compared to PNP [28, 42, 43].

#### **5.2 Secondary mucosal lesions**

Lesions can also affect regions such as the oropharynx, esophagus, stomach, duodenum, large intestine, conjunctiva, and anogenital region [2, 3, 7, 39, 41, 44, 45]. The involvement of the oropharynx and esophagus commonly triggers painful sensations and dysphagia [4]. The anogenital lesions demonstrate red-violet erythema in the glans or its surroundings (**Figure 2**). In some cases, lichen planus presents a possible differential diagnosis. However, unlike red-violet lesions, lichen planus forms linear white streaks that may arise in the glans, scrotum, and vulva, in addition to the presence of dyspareunia and pruritus [43]. In these patients, both necrosis and loss of epidermis are absent, unlike patients with PNP who present this clinical [43].

**131**

**5.3 Skin lesions**

**Figure 3.**

**Figure 2.**

*Red-violet lesion in the genital organ.*

*Extensive erosions and blisters in the dorsal region.*

*Paraneoplastic Pemphigus Is a Life-Threatening Disease DOI: http://dx.doi.org/10.5772/intechopen.84956*

About 70% of the patients present conjunctival lesions such as bilateral bulbar conjunctival hyperemia, diffuse papillary tarsal conjunctival reactions, conjunctival epithelium desquamation, forniceal shortening, painful ocular irritation, poor vision, conjunctival and corneal erosions, and pseudomembranous conjunctivitis [2, 46, 47].

Skin lesions usually appear soon after the onset of mucosal involvement [48]. The most affected sites are the dorsal region (**Figure 3**), head, and neck (**Figure 4**), in addition to the nearby extremities [4, 39, 49]. Patients with PNP started the study in very different ways, with the first signs being erythema, bullous and vesicular lesions, papules, skin scaling with Nikolsky sign, exfoliative erythema, and ulcers with hematic crust. Often, the first clinical sign on the skin is erythema that may progress with bullous and ulcerated lesions [24, 50]. Unlike adults, PNP in the skin of children appears in the form of lichenoid lesions, rather than bullous lesions.

Similar to PNP, bullous pemphigoid (BP) provides blistering with erythematous base or normal skin. However, BP lesions occur more frequently in the lower abdomen and lower limbs, and in most individuals, mucosal lesions are not affected [51]. In addition, pruritus is present in the vast majority of these patients, unlike PNP, which show painful

and disseminated lesions mainly in the upper body and mucosal regions [28, 51].

**Figure 1.** *Severe erosive mucositis with hematic crusting on the lips and oral mucosa.*

*Paraneoplastic Pemphigus Is a Life-Threatening Disease DOI: http://dx.doi.org/10.5772/intechopen.84956*

*Current Trends in Cancer Management*

**5.2 Secondary mucosal lesions**

PNP presents several symptoms and clinical evolutions. The first symptoms as well as the progression of the disease are very varied from one patient to another.

The oral mucosa is often affected in patients with PNP [3, 39, 40]. Oral symptoms may be the first symptoms in these patients, even before skin lesions [41]. The most common symptoms are oral and labial erosions with bleeding that may be associated with blisters, macules, papules, vesicles, and erythema (**Figure 1**). In

PNP lesions may be similar to oral manifestations of other diseases. Pemphigus vulgaris is a disease that initially triggers blisters and ulcers in the oral mucosa (especially on the cheeks) and may even reach the body. Erythema multiforme also affects the region of the oral mucosa with the appearance of erythema, edema, and some superficial erosions with formation of pseudomembrane. Lichen planus causes erythematous lesions where Wickham striae are present and may in rare cases develop erosions. In most cases of oral lichen planus, these are asymptomatic manifestations with few complications. Even though these diseases show some similarity to PNP, they are less aggressive, lethal, painful, and incapacitating, with less ability to spread

Lesions can also affect regions such as the oropharynx, esophagus, stomach, duodenum, large intestine, conjunctiva, and anogenital region [2, 3, 7, 39, 41, 44, 45]. The involvement of the oropharynx and esophagus commonly triggers painful sensations and dysphagia [4]. The anogenital lesions demonstrate red-violet erythema in the glans or its surroundings (**Figure 2**). In some cases, lichen planus presents a possible differential diagnosis. However, unlike red-violet lesions, lichen planus forms linear white streaks that may arise in the glans, scrotum, and vulva, in addition to the presence of dyspareunia and pruritus [43]. In these patients, both necrosis and loss of epidermis are absent, unlike patients with PNP who present this

However, there are more frequent clinical features of these individuals.

addition, these patients may present a positive Nikolsky sign [41].

to all mucosal and other body sites when compared to PNP [28, 42, 43].

*Severe erosive mucositis with hematic crusting on the lips and oral mucosa.*

**5. Clinical features**

**5.1 Oral lesions**

**130**

**Figure 1.**

clinical [43].

**Figure 2.** *Red-violet lesion in the genital organ.*

#### **Figure 3.** *Extensive erosions and blisters in the dorsal region.*

About 70% of the patients present conjunctival lesions such as bilateral bulbar conjunctival hyperemia, diffuse papillary tarsal conjunctival reactions, conjunctival epithelium desquamation, forniceal shortening, painful ocular irritation, poor vision, conjunctival and corneal erosions, and pseudomembranous conjunctivitis [2, 46, 47].

#### **5.3 Skin lesions**

Skin lesions usually appear soon after the onset of mucosal involvement [48]. The most affected sites are the dorsal region (**Figure 3**), head, and neck (**Figure 4**), in addition to the nearby extremities [4, 39, 49]. Patients with PNP started the study in very different ways, with the first signs being erythema, bullous and vesicular lesions, papules, skin scaling with Nikolsky sign, exfoliative erythema, and ulcers with hematic crust. Often, the first clinical sign on the skin is erythema that may progress with bullous and ulcerated lesions [24, 50]. Unlike adults, PNP in the skin of children appears in the form of lichenoid lesions, rather than bullous lesions.

Similar to PNP, bullous pemphigoid (BP) provides blistering with erythematous base or normal skin. However, BP lesions occur more frequently in the lower abdomen and lower limbs, and in most individuals, mucosal lesions are not affected [51]. In addition, pruritus is present in the vast majority of these patients, unlike PNP, which show painful and disseminated lesions mainly in the upper body and mucosal regions [28, 51].

#### **Figure 4.** *Confluent erosions with hematic crusts in the head and neck region.*

Already erythema multiforme shows prodromal symptoms such as fever and myalgia before the appearance of lesions on the mucosal and skin. Their skin lesions change in feature according to the course of the disease and resemble insect bites or hives that result in the well-known targetoid lesions that are common in this disease. Although cases of necrosis and blisters occur in the center of the lesions, this disease shows less aggression and fewer blisters and ulcers with hematic crusts than the patients affected by PNP [42].

Lichen planus affects flexor surfaces of the wrists, forearm, and legs. These lesions have round reticular white lines such as Wickham striae. They may arise in places that suffer trauma (Koebner's phenomenon), in addition to making the site pigmented after inflation, thus demonstrating clinical differences in cutaneous erosions seen in the course of PNP progression [43].

The graft versus host disease causes rash and maculopapular rash that present itching and can spread to the entire body, less in the scalp. In very severe cases, there may be some sites with necrosis at the base of epidermal rete pegs [52]. Generally, these severe cases are differentiated from the PNP both by the patient's clinical history and by skin biopsy that demonstrate distinct histopathological characteristics.

#### **5.4 Pulmonary manifestations**

Approximately 92.8% of the cases described in the literature show pulmonary involvement [3]. The pulmonary clinical signs of PNP are dyspnea, obstructive pulmonary disease, and bronchiolitis obliterans. The resolution of pulmonary problems is of extreme importance, since it is the main cause of death in individuals with PNP [53]. The patients with the greatest pulmonary involvement are Chinese children and patients with Castleman disease [53]. Studies show that 71% of the patients had bronchiolitis obliterans organizing pneumonia, and they give worse prognosis even if treatment of the neoplasia occurs [12, 54].

#### **6. Histopathological examination**

The pathological analyses demonstrate many varied aspects, since they show them peculiar characteristics according to the evaluated lesions [55]. When

**133**

**Figure 5.**

dermal junctions in band [28, 55].

**7. Immunological studies**

*Paraneoplastic Pemphigus Is a Life-Threatening Disease DOI: http://dx.doi.org/10.5772/intechopen.84956*

analyzing the biopsy of blisters, we found acantholysis with inflammatory infiltrates (**Figure 5**) [55]. However, when it presents inflammatory maculopapular lesions, the most common findings are lichenoid interface dermatitis [55]. In the presence of lesions with the presence of blisters and maculopapular lesions, mixed characteristics of each type of lesion may occur in the pathology. The findings with dyskeratosis and suprabasal acantholysis are one of the most important characteristics that lead to the definitive diagnosis of PNP [6]. Dyskeratosis is an abnormal formation of epidermal keratinization, whereas acantholysis is the loss of adhesion between skin cells [28]. These findings may help in the diagnosis even when there is no possibility of performing direct immunofluorescence (DIF) or when they are negative [39, 55]. DIF is a laboratory technique capable of detecting the deposition of autoantibodies and immune cells in the sites affected by the disease. The use of DIF demonstrates an extremely important technique for the diagnosis of PNP, since it can analyze both specific autoantibodies and cytotoxic cells of the human immune system, such as CD8+ T cells that act by attacking several layers with keratin and demonstrating intracellular staining of cementum and/or marking of epidermal

*Histopathological examination of the biopsy specimen showing keratinocyte apoptosis and* 

*acantholysis (hematoxylin and eosin, original magnification × 100).*

The use of DIF demonstrates great importance in the diagnosis of PNP even though approximately 50% of the cases show negative [3]. This technique shows a staining in IgG deposition intracellular chicken wire pattern (linear formation of autoantibodies deposition) along the dermoepidermal junction in both the linear form, as granulate [15]. The presence of IgG deposition in the dermoepidermal region

is very characteristic of the PNP; however, only 25% presents this pattern [56].

alerting to PNP investigation, since it shows high specificity [56].

The use of indirect immunofluorescence (IIF) shows involvement of the epidermis by the deposition of IgG in the intercellular regions. Other techniques used as cytoplasmic fluorescence (intracellular staining) demonstrate a prominent basal staining. IIF marking is extremely strong in the layers of the epithelium, and this,

*Paraneoplastic Pemphigus Is a Life-Threatening Disease DOI: http://dx.doi.org/10.5772/intechopen.84956*

#### **Figure 5.**

*Current Trends in Cancer Management*

than the patients affected by PNP [42].

**Figure 4.**

**5.4 Pulmonary manifestations**

**6. Histopathological examination**

erosions seen in the course of PNP progression [43].

*Confluent erosions with hematic crusts in the head and neck region.*

prognosis even if treatment of the neoplasia occurs [12, 54].

Already erythema multiforme shows prodromal symptoms such as fever and myalgia before the appearance of lesions on the mucosal and skin. Their skin lesions change in feature according to the course of the disease and resemble insect bites or hives that result in the well-known targetoid lesions that are common in this disease. Although cases of necrosis and blisters occur in the center of the lesions, this disease shows less aggression and fewer blisters and ulcers with hematic crusts

Lichen planus affects flexor surfaces of the wrists, forearm, and legs. These lesions have round reticular white lines such as Wickham striae. They may arise in places that suffer trauma (Koebner's phenomenon), in addition to making the site pigmented after inflation, thus demonstrating clinical differences in cutaneous

The graft versus host disease causes rash and maculopapular rash that present itching and can spread to the entire body, less in the scalp. In very severe cases, there may be some sites with necrosis at the base of epidermal rete pegs [52]. Generally, these severe cases are differentiated from the PNP both by the patient's clinical history and by skin biopsy that demonstrate distinct histopathological characteristics.

Approximately 92.8% of the cases described in the literature show pulmonary involvement [3]. The pulmonary clinical signs of PNP are dyspnea, obstructive pulmonary disease, and bronchiolitis obliterans. The resolution of pulmonary problems is of extreme importance, since it is the main cause of death in individuals with PNP [53]. The patients with the greatest pulmonary involvement are Chinese children and patients with Castleman disease [53]. Studies show that 71% of the patients had bronchiolitis obliterans organizing pneumonia, and they give worse

The pathological analyses demonstrate many varied aspects, since they show

them peculiar characteristics according to the evaluated lesions [55]. When

**132**

*Histopathological examination of the biopsy specimen showing keratinocyte apoptosis and acantholysis (hematoxylin and eosin, original magnification × 100).*

analyzing the biopsy of blisters, we found acantholysis with inflammatory infiltrates (**Figure 5**) [55]. However, when it presents inflammatory maculopapular lesions, the most common findings are lichenoid interface dermatitis [55]. In the presence of lesions with the presence of blisters and maculopapular lesions, mixed characteristics of each type of lesion may occur in the pathology. The findings with dyskeratosis and suprabasal acantholysis are one of the most important characteristics that lead to the definitive diagnosis of PNP [6]. Dyskeratosis is an abnormal formation of epidermal keratinization, whereas acantholysis is the loss of adhesion between skin cells [28]. These findings may help in the diagnosis even when there is no possibility of performing direct immunofluorescence (DIF) or when they are negative [39, 55]. DIF is a laboratory technique capable of detecting the deposition of autoantibodies and immune cells in the sites affected by the disease. The use of DIF demonstrates an extremely important technique for the diagnosis of PNP, since it can analyze both specific autoantibodies and cytotoxic cells of the human immune system, such as CD8+ T cells that act by attacking several layers with keratin and demonstrating intracellular staining of cementum and/or marking of epidermal dermal junctions in band [28, 55].

#### **7. Immunological studies**

The use of DIF demonstrates great importance in the diagnosis of PNP even though approximately 50% of the cases show negative [3]. This technique shows a staining in IgG deposition intracellular chicken wire pattern (linear formation of autoantibodies deposition) along the dermoepidermal junction in both the linear form, as granulate [15]. The presence of IgG deposition in the dermoepidermal region is very characteristic of the PNP; however, only 25% presents this pattern [56].

The use of indirect immunofluorescence (IIF) shows involvement of the epidermis by the deposition of IgG in the intercellular regions. Other techniques used as cytoplasmic fluorescence (intracellular staining) demonstrate a prominent basal staining. IIF marking is extremely strong in the layers of the epithelium, and this, alerting to PNP investigation, since it shows high specificity [56].

Other serological methods may also be used, such as immunoprecipitation, immunoblot and anti-EP enzyme-linked immunosorbent assay (ELISA) [57–59]. Studies evidenced 95 and 100% sensitivity in radioactive and nonradioactive immunoprecipitation techniques, respectively, and this demonstrates that immunoprecipitation is the most serologically sensitive test for PNP diagnosis [57, 60, 61]. Currently the immunoprecipitation is considered gold standard in the diagnosis of PNP, that is, the main criterion to diagnose [62, 63].

#### **8. Diagnosis**

The criteria for diagnosis according to Anhalt et al. in 1990 are based on five criteria, such as clinical characteristics, histopathological analysis, direct and indirect immunofluorescence, and immunoprecipitation [1]. These criteria have been modified and adapted. In 1993, researchers included to perform the diagnosis the presence of three main criteria or two major and two minor [63]. Already in 2002, Mimouni et al. reviewed the Anhalt criteria and considered four minimum criteria of high confidence in diagnosis (**Table 2**) [12]. DIF is a nonessential criterion because of its low sensitivity. As for IIF on rat bladder epithelia and monkey esophagus, they were considered useful for tracking and detecting PNP [57, 64]. Negative IIF cannot exclude PNP, and other techniques such as immunoblotting and immunoprecipitation should be used to confirm or rule out a diagnosis.


#### **Table 2.**

*Minimum criteria for diagnosis.*

#### **9. Differential diagnosis**

The diagnosis of PNP can be complex and difficult to perform because there are several similar diseases (**Table 3**). PNP and pemphigus vulgaris (PV) are very similar clinically, but some details differentiate them. PNP develops with inflammatory papules or macules that progress to blisters, while PV presents bullous lesions with a reddish background. Molecularly, the PNP presents some antibodies specific for this disease, such as the presence of anti-A2ML1, anti-envoplakin, and anti-periplakin, and demonstrates patterns of IgG deposition on cell surfaces with accumulation in the basement membrane zone [57, 64–66]. Even though bullous autoimmune diseases resemble each other, PNP differentiates it by the presence of antibody that stains the mouse bladder. In bullous pemphigoid (PB), BP230 and BP180 can be found, as well as in PNP. However, the use of DIF differentiates them by the IgG deposition patterns found in the PNP. The involvement by morbilliform-like erythema, toxic epidermal necrolysis, and Stevens-Johnson syndrome can also be confused with PNP. However, the detection of antibodies, pathological analysis of the lesions, and the patient's clinic can differentiate these diseases [1, 10, 39, 57, 64–66].

Despite some cases that both clinically and histologically resemble each other, it is important to perform other techniques to rule out differential diagnoses. The use of otorhinolaryngological examination is very important to differentiate the diseases

**135**

**10. Treatment**

*Differential diagnosis.*

Pemphigus vulgaris

Bullous pemphigoid

Erythema multiforme

**Table 3.**

Toxic epidermal necrolysis

Stevens-Johnson syndrome

*Paraneoplastic Pemphigus Is a Life-Threatening Disease DOI: http://dx.doi.org/10.5772/intechopen.84956*

**Disease Causers Pathophysiology**

hypersensitivity by infection,

Drug reaction that affects more than 30% of the body

Drug reaction that affects less than 10% of the body

viruses and drugs

Lichen planus Autoimmune reaction Autoantigens anti-keratinocyte and

Autoimmune reaction Autoantigens anti-desmoglein 1,3

Autoimmune reaction Autoantigens anti-BP180 and anti-BP230

antinuclear

leukotrienes

tumor necrosis factor-α

and increased granulysin

and increased granulysin

Infiltration of cytotoxic T cell and increased

Infiltration of cytotoxic T cell, natural killer,

Infiltration of cytotoxic T cell, natural killer,

eosinophils, and increased histamine and

that affect the mucous membranes. Well-done physical examination of the oral cavity, histopathological analysis characteristics, cutaneous involvement, and the presence

Drug eruption Drug reaction Perivascular infiltration by lymphocytes,

Effective treatment for PNP is still a major puzzle because of its rarity. Although several drugs are used in the literature, PNP has shown great resistance when compared to other forms of pemphigus [50, 68]. When there is suspicion or evidence of PNP, the performance of the six steps described on 2011 by Frew et al. may provide better management of individuals (**Table4**) [69]. Stabilization of patients, according to the first step, is the most important step, since it is the major cause of death in patients [69]. Currently, the first-line treatment for PNP is still high doses of corticosteroids [70]. This treatment improves the cutaneous lesions, but the mucosal involvement is little altered. The use of other drugs also shows little efficacy in the lesions of the

Several studies have shown that the combination of drugs has been effective and safe. These associations were prednisolone used with other therapies, such as mycophenolate mofetil, cyclosporine A, azathioprine, plasmapheresis, and intravenous immunoglobulin [72–77]. Even though treatment is more effective, mucosal

The use of monoclonal antibody has been effective in the treatment of PNP in some case reports described in the literature. Administration of rituximab, an anti-CD20, has shown good PNP therapy due to B-cell lymphoma [78, 79]. This therapy

infusions for 4 weeks of corticosteroid and administration of other immunosup-

The use of alemtuzumab, a humanized monoclonal antibody that binds to CD52, has been reported. Reported in the treatment of PNP remission in patients whose presence of chronic lymphoid leukemia [80]. Alemtuzumab has been used in a patient with resistance to other drugs such as corticosteroids, intravenous immunoglobulin,

weekly for 4 weeks followed by eight weekly

mucosa, this resistance being the characteristic of the disease [69, 71].

involvement is still resistant to such combined therapies [71].

is based on an infusion of 375 mg/m2

pressive drugs such as cyclosporine A [69].

of IIF strongly suggest for the diagnosis of PNP [40, 44, 67].


#### **Table 3.**

*Current Trends in Cancer Management*

**8. Diagnosis**

**9. Differential diagnosis**

*Minimum criteria for diagnosis.*

**Table 2.**

can differentiate these diseases [1, 10, 39, 57, 64–66].

PNP, that is, the main criterion to diagnose [62, 63].

Other serological methods may also be used, such as immunoprecipitation, immunoblot and anti-EP enzyme-linked immunosorbent assay (ELISA) [57–59]. Studies evidenced 95 and 100% sensitivity in radioactive and nonradioactive immunoprecipitation techniques, respectively, and this demonstrates that immunoprecipitation is the most serologically sensitive test for PNP diagnosis [57, 60, 61]. Currently the immunoprecipitation is considered gold standard in the diagnosis of

The criteria for diagnosis according to Anhalt et al. in 1990 are based on five criteria, such as clinical characteristics, histopathological analysis, direct and indirect immunofluorescence, and immunoprecipitation [1]. These criteria have been modified and adapted. In 1993, researchers included to perform the diagnosis the presence of three main criteria or two major and two minor [63]. Already in 2002, Mimouni et al. reviewed the Anhalt criteria and considered four minimum criteria of high confidence in diagnosis (**Table 2**) [12]. DIF is a nonessential criterion because of its low sensitivity. As for IIF on rat bladder epithelia and monkey esophagus, they were considered useful for tracking and detecting PNP [57, 64]. Negative IIF cannot exclude PNP, and other techniques such as immunoblotting and

The diagnosis of PNP can be complex and difficult to perform because there are several similar diseases (**Table 3**). PNP and pemphigus vulgaris (PV) are very similar clinically, but some details differentiate them. PNP develops with inflammatory papules or macules that progress to blisters, while PV presents bullous lesions with a reddish background. Molecularly, the PNP presents some antibodies specific for this disease, such as the presence of anti-A2ML1, anti-envoplakin, and anti-periplakin, and demonstrates patterns of IgG deposition on cell surfaces with accumulation in the basement membrane zone [57, 64–66]. Even though bullous autoimmune diseases resemble each other, PNP differentiates it by the presence of antibody that stains the mouse bladder. In bullous pemphigoid (PB), BP230 and BP180 can be found, as well as in PNP. However, the use of DIF differentiates them by the IgG deposition patterns found in the PNP. The involvement by morbilliform-like erythema, toxic epidermal necrolysis, and Stevens-Johnson syndrome can also be confused with PNP. However, the detection of antibodies, pathological analysis of the lesions, and the patient's clinic

Despite some cases that both clinically and histologically resemble each other, it is important to perform other techniques to rule out differential diagnoses. The use of otorhinolaryngological examination is very important to differentiate the diseases

immunoprecipitation should be used to confirm or rule out a diagnosis.

2. Histologic features of acantholysis or lichenoid or interface dermatitis

4. The presence of an underlying neoplasm, especially lymphoproliferative tumors

3. Demonstration of antiplakin autoantibodies

1. Clinical features of severe and protracted mucosal involvement and polymorphic cutaneous eruptions

**134**

*Differential diagnosis.*

that affect the mucous membranes. Well-done physical examination of the oral cavity, histopathological analysis characteristics, cutaneous involvement, and the presence of IIF strongly suggest for the diagnosis of PNP [40, 44, 67].

#### **10. Treatment**

Effective treatment for PNP is still a major puzzle because of its rarity. Although several drugs are used in the literature, PNP has shown great resistance when compared to other forms of pemphigus [50, 68]. When there is suspicion or evidence of PNP, the performance of the six steps described on 2011 by Frew et al. may provide better management of individuals (**Table4**) [69]. Stabilization of patients, according to the first step, is the most important step, since it is the major cause of death in patients [69].

Currently, the first-line treatment for PNP is still high doses of corticosteroids [70]. This treatment improves the cutaneous lesions, but the mucosal involvement is little altered. The use of other drugs also shows little efficacy in the lesions of the mucosa, this resistance being the characteristic of the disease [69, 71].

Several studies have shown that the combination of drugs has been effective and safe. These associations were prednisolone used with other therapies, such as mycophenolate mofetil, cyclosporine A, azathioprine, plasmapheresis, and intravenous immunoglobulin [72–77]. Even though treatment is more effective, mucosal involvement is still resistant to such combined therapies [71].

The use of monoclonal antibody has been effective in the treatment of PNP in some case reports described in the literature. Administration of rituximab, an anti-CD20, has shown good PNP therapy due to B-cell lymphoma [78, 79]. This therapy is based on an infusion of 375 mg/m2 weekly for 4 weeks followed by eight weekly infusions for 4 weeks of corticosteroid and administration of other immunosuppressive drugs such as cyclosporine A [69].

The use of alemtuzumab, a humanized monoclonal antibody that binds to CD52, has been reported. Reported in the treatment of PNP remission in patients whose presence of chronic lymphoid leukemia [80]. Alemtuzumab has been used in a patient with resistance to other drugs such as corticosteroids, intravenous immunoglobulin,


#### **Table 4.**

*Management of the patient with suspected PNP.*

and cyclosporine A. In this patient, intravenous 30 mg was infused three times a week for 3 months. Even though there was improvement in both skin and mucosal lesions, the patient continued maintenance treatment with 500 mg of mycophenolate mofetil and 5 mg of prednisone [80]. Although there are several treatment alternatives, new therapies that reduce the resistance of PNP to drugs are still fundamental. Daclizumab, a monoclonal antibody against T-cell interleukin-2, has been shown to be a promising therapy [81].

It is known that in order to avoid large amounts of autoantibodies released into the bloodstream during tumor excision surgery, it is necessary to block blood flow and prevent compression of neoplastic tissue. In addition, the use of intravenous immunoglobulin before and during operations has demonstrated a significant reduction in mortality caused by bronchiolitis obliterans. Even after complete tumor resolution, immunoglobulin administration is required until 2 years to provide remission of autoimmunity triggered by PNP [82, 83].

In addition to the treatment of neoplasia and PNP, other ducts must be performed. When there is loss of skin integrity or immunosuppression, antimicrobial therapy is recommended early to prevent sepsis. Medications for pain control are also useful, since patients have pain in regions with ulceration and erosions [50].

Although there are several treatments stipulated in the literature, there are still no known drugs that reduce the mortality of patients, since the PNP proves highly resistant to more aggressive therapies. However, it is known that management, diagnosis, and early treatment are indispensable methods for a better response of the patients in the prescribed procedures.

#### **11. Prognosis**

The prognosis of PNP is extremely poor. Mortality can reach 90% of the cases in the first year, 41% of mortality in the second year, and 38% of death in the third year with the disease [84]. Commonly, death is triggered by systemic complications such as bronchiolitis obliterans, sepsis, and bleeding in the gastrointestinal tract [6, 50]. It is known that regardless of the cure or control of the neoplasia, the PNP progresses, demonstrating itself autonomous to the triggering factor [6, 10, 11, 13, 50]. Patients who exhibit morbilliform erythema and necrosis of skin biopsy keratinocytes demonstrate a worse overall survival [84]. In some cases, the removal of Castleman disease and benign thymoma has shown better results than other underlying diseases [84, 85].

Even with a high mortality rate, the prognosis depends very much on the proper management of the patient, such as monitoring of vital signs, control of oral and skin lesions, treatment of the triggering disease, and prevention of sepsis and bronchitis obliterans. For this, it is essential to follow the patient closely and treat the disease aggressively [50].

**137**

*Paraneoplastic Pemphigus Is a Life-Threatening Disease DOI: http://dx.doi.org/10.5772/intechopen.84956*

at key points by multidisciplinary teams.

immunologists, psychologists, nurses, and social workers.

of Health Sciences Barretos, São Paulo, Brazil.

A2ML1 alpha-2 macroglobulin-like 1

DIF direct immunofluorescence

IIF indirect immunofluorescence LPD lymphoproliferative disorders PNP paraneoplastic pemphigus

The author has declared no conflicts of interest.

Studies have mentioned severe losses in the quality of life of patients with pemphigus. The main criteria that impair the quality of life were the greater severity of the disease, anxiety, and depression. However, there was no clear measurement of gender, age, type of pemphigus, duration of disease, skin involvement, disease activity, itching, burning sensation in the skin, or treatment in use [86]. There is still a great need in the standardization and validation of PNP-specific questionnaires, as this proves to be extremely important in order to know and enable actions

PNP demonstrates a great challenge for physicians, since it presents several clinical aspects and varied degrees of bodily involvement. Early diagnosis, management of the patient, treatment of the underlying neoplasia, and aggressive treatment for PNP are of paramount importance for the best prognosis of the patient, since it is an extremely lethal disease. For this, more studies are needed to better understand the disease and cooperation between multidisciplinary teams involving dermatologists, oncologists, hematologists, otorhinolaryngologists, surgeons, ophthalmologists,

The author would like to acknowledge the help of Dr. Paulo Prata and the School

**12. Quality of life**

**13. Conclusions**

**Acknowledgements**

**Conflict of interest**

**Appendices and nomenclature**

BP bullous pemphigoid

DSG desmoglein

### **12. Quality of life**

*Current Trends in Cancer Management*

1. Stabilization of vital parameters

3. Diagnosis of PNP

5. Treatment of PNP

2. Assessment of any underlying malignancy

4. Removal and therapy for the triggering tumor

*Management of the patient with suspected PNP.*

therapy [81].

**Table 4.**

erosions [50].

**11. Prognosis**

underlying diseases [84, 85].

the disease aggressively [50].

and cyclosporine A. In this patient, intravenous 30 mg was infused three times a week for 3 months. Even though there was improvement in both skin and mucosal lesions, the patient continued maintenance treatment with 500 mg of mycophenolate mofetil and 5 mg of prednisone [80]. Although there are several treatment alternatives, new therapies that reduce the resistance of PNP to drugs are still fundamental. Daclizumab, a monoclonal antibody against T-cell interleukin-2, has been shown to be a promising

It is known that in order to avoid large amounts of autoantibodies released into the bloodstream during tumor excision surgery, it is necessary to block blood flow and prevent compression of neoplastic tissue. In addition, the use of intravenous immunoglobulin before and during operations has demonstrated a significant reduction in mortality caused by bronchiolitis obliterans. Even after complete tumor resolution, immunoglobulin administration is required until 2 years to

In addition to the treatment of neoplasia and PNP, other ducts must be performed. When there is loss of skin integrity or immunosuppression, antimicrobial therapy is recommended early to prevent sepsis. Medications for pain control are also useful, since patients have pain in regions with ulceration and

Although there are several treatments stipulated in the literature, there are still no known drugs that reduce the mortality of patients, since the PNP proves highly resistant to more aggressive therapies. However, it is known that management, diagnosis, and early treatment are indispensable methods for a better response of

The prognosis of PNP is extremely poor. Mortality can reach 90% of the cases in the first year, 41% of mortality in the second year, and 38% of death in the third year with the disease [84]. Commonly, death is triggered by systemic complications such as bronchiolitis obliterans, sepsis, and bleeding in the gastrointestinal tract [6, 50]. It is known that regardless of the cure or control of the neoplasia, the PNP progresses, demonstrating itself autonomous to the triggering factor [6, 10, 11, 13, 50]. Patients who exhibit morbilliform erythema and necrosis of skin biopsy keratinocytes demonstrate a worse overall survival [84]. In some cases, the removal of Castleman disease and benign thymoma has shown better results than other

Even with a high mortality rate, the prognosis depends very much on the proper management of the patient, such as monitoring of vital signs, control of oral and skin lesions, treatment of the triggering disease, and prevention of sepsis and bronchitis obliterans. For this, it is essential to follow the patient closely and treat

provide remission of autoimmunity triggered by PNP [82, 83].

the patients in the prescribed procedures.

**136**

Studies have mentioned severe losses in the quality of life of patients with pemphigus. The main criteria that impair the quality of life were the greater severity of the disease, anxiety, and depression. However, there was no clear measurement of gender, age, type of pemphigus, duration of disease, skin involvement, disease activity, itching, burning sensation in the skin, or treatment in use [86]. There is still a great need in the standardization and validation of PNP-specific questionnaires, as this proves to be extremely important in order to know and enable actions at key points by multidisciplinary teams.

### **13. Conclusions**

PNP demonstrates a great challenge for physicians, since it presents several clinical aspects and varied degrees of bodily involvement. Early diagnosis, management of the patient, treatment of the underlying neoplasia, and aggressive treatment for PNP are of paramount importance for the best prognosis of the patient, since it is an extremely lethal disease. For this, more studies are needed to better understand the disease and cooperation between multidisciplinary teams involving dermatologists, oncologists, hematologists, otorhinolaryngologists, surgeons, ophthalmologists, immunologists, psychologists, nurses, and social workers.

### **Acknowledgements**

The author would like to acknowledge the help of Dr. Paulo Prata and the School of Health Sciences Barretos, São Paulo, Brazil.

### **Conflict of interest**

The author has declared no conflicts of interest.

#### **Appendices and nomenclature**


*Current Trends in Cancer Management*

#### **Author details**

Richard Lucas Konichi-Dias School of Health Sciences, Dr. Paulo Prata-FACISB, Barretos, Sao Paulo, Brazil

\*Address all correspondence to: richardkonichi95@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.

**139**

*Paraneoplastic Pemphigus Is a Life-Threatening Disease DOI: http://dx.doi.org/10.5772/intechopen.84956*

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paraneoplastic autoimmune multiorgan syndrome. International Journal of Dermatology. 2009;**48**:162-169

[12] Mimouni D, Anhalt GJ, Lazarova Z, et al. Paraneoplastic pemphigus in children and adolescents. British Journal of Dermatology. 2002;**147**:725-732

[13] Lane JE, Woody C, Davis LS, et al. Paraneoplastic autoimmune multiorgan syndrome (paraneoplastic pemphigus) in a child: Case report and review of the literature. Pediatrics.

[14] Geller S, Gat A, Harel A, et al. Childhood pemphigus foliaceus with exclusive immunoglobulin G autoantibodies to desmocollins.

[15] Amber KT, Valdebran M, Grando SA. Paraneoplastic autoimmune multiorgan

syndrome (PAMS): Beyond the single phenotype of paraneoplastic pemphigus. Autoimmunity Reviews.

[16] Kaplan I, Hodak E, Ackerman L, et al. Neoplasms associated with paraneoplastic pemphigus: A review with emphasis on non-hematologic malignancy and oral mucosal manifestations. Oral Oncology.

2004;**114**:e513-e516

Pediatric Dermatology.

2016;**33**:e10-e13

2018;**17**:1002-1010

2004;**40**:553-562

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**Author details**

Richard Lucas Konichi-Dias

provided the original work is properly cited.

School of Health Sciences, Dr. Paulo Prata-FACISB, Barretos, Sao Paulo, Brazil

© 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,

\*Address all correspondence to: richardkonichi95@gmail.com

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**144**

### *Edited by Liliana Streba, Dan Ionut Gheonea and Michael Schenker*

The field of cancer diagnosis, prognosis, and treatment is constantly advancing. From novel biomarkers to cutting-edge imaging solutions, changing chemotherapy protocols and novel immune-targeting agents, medical teams develop and test new ways to manage this ever-growing threat to the modern age.

Imaging has been a reliable method for initial diagnosis and later surveillance of premalignant and cancerous lesions of the digestive tract.

This book project aims to characterize the main diagnostic procedures and novel medical and surgical treatments, as well as provide an updated view on current guidelines, premalignant lesions management, and minimally invasive curative techniques.

Published in London, UK © 2019 IntechOpen © Ugreen / iStock

Current Trends in Cancer Management

Current Trends in

Cancer Management

*Edited by Liliana Streba,* 

*Dan Ionut Gheonea and Michael Schenker*