Epigenetic Modifications in Ovarian Cancer: A Review

Page 17 of 35 Epigenetic Modifications in Ovarian Cancer: A Review A. H. M. Zuberi Ashraf1,2, Syeda H. Afroze3, Grace A. Osuji2, Saba Y. Kayani3, Natalie Colon3, Ahmed F. Pantho3, Thomas J. Kuehl3, Kimberly A Pilkinton4, M. Nasir Uddin3,5 1Department of Science & Mathematics, Texas A&M University-Central Texas, Killeen, TX, U.S.A. 2Baylor Scott & White Health, Temple, TX, U.S.A. 3Orion Institute for Translational Medicine, Temple, TX, U.S.A. 4Department of Clinical Sciences, University of Houston College of Medicine, Houston, TX, U.S.A. 5Department of Medical Physiology, Texas A&M University College of Medicine, Temple, TX, U.S.A.

Ovarian cancer is responsible for approximately 1.3% of all new cancer cases but ranks fifth for cancer mortalities for women in the world 1 . Data from 2009-2013 suggest 12 new cases of ovarian cancer per 100,000 women per year. In addition, an estimated 22,440 new cases and 14,080 deaths from ovarian cancer occurred in 2017 1 . There is significant patient variation in terms of disease condition and tumor types regarding ovarian cancer. Ovarian cancer is most often discovered at advanced stages as a result of limited screening procedures that are available. Ovarian tumor cell types originate from three broad categories of ovarian cells, which include surface epithelial cells, germ cells, and sex cord-stromal cells 2 . Table 1 reviews the different ovarian cancer cell types. Surface epithelial tumors account for approximately 85-90% of all ovarian cancer cases 3 . Epithelial ovarian cancer (EOC) arises from the epithelium of Müllerian structures including the ovarian surface and tubal epithelium. EOCs fall into two main histological subgroups: Type I include the slower growing low-grade serous, mucinous, endometrioid, and clear cell carcinomas 3 . Type II tumors are high-grade malignancies which lead to rapidly progressive disease. High-grade serous ovarian carcinoma is highly aggressive and metastatic and the most common type II tumor 3 .
Women with familial breast cancer type susceptibility gene (BRCA1 and BRCA2) mutations carry up to 40% elevated risk of developing ovarian cancer 4 . BRCA1 and BRCA2 are well-known human tumor-suppressor genes. They are both poorly conserved except in specific domains such as the BRCA1 C-terminal (BRCT) and N-terminal RING domains in BRCA1 and the BRC repeats in BRCA2. BRCA1 is universally expressed in humans, with highest levels of transcription in ovaries, testis, and thymus. BRCA2 is expressed only in actively dividing cells 4 . These genes are essential for maintenance of genomic stability, ensuring error-proof repair of doublestrand breaks and homologous recombination 2,4 . Family history of ovarian, breast, and colorectal cancer in a firstdegree relative could be associated with mutations in other tumor-suppressor genes such as those involved in DNArepair pathways 4 . Gynecological and obstetric history of low parity, late age of first live birth, late menopause, and greater number of ovulatory cycles are also considered risk factors 4 . Many ovarian cancer cases are diagnosed in women aged 55-64 years, suggesting age to be the most common factor paired with other risk factors 1 .
The lack of effective screening methods has made ovarian cancer one of the deadliest gynecological maladies 3,5 . Unlike pap smears for cervical cancer or colonoscopy for colorectal cancer, ovarian cancer does not have any standardized screening procedure 5 . Transvaginal ultrasound and measuring levels of serum cancer antigen 125 (CA125) have been used as screening tools in randomized trials testing for ovarian cancer. CA125 is a known biomarker for tumors that is expressed at very high concentrations in ovarian cancer cells compared other types of cancers. CA125 is mainly specific to the epithelial ovarian tumors and is not necessarily specific enough for other ovarian tumors 5 . Further review and analysis of the data from the ovarian cancer testing trials indicate that the screening methods used did not contribute to diagnosing ovarian cancer at earlier stages or reducing overall ovarian cancer mortality. In addition, screening resulted in unnecessary surgery, psychological stress, and did not improve quality of life 5 . Improved general screening should involve counseling of each individual patient based on personal risk factors 1,4 . When ovarian cancer is diagnosed at an early stage, the 5-year survival rate is 92%, but, unfortunately, only around 15% of cases are found at this stage 1,4 .
This article reviews the role of some epigenetic modifications in ovarian cancer. The pathogenesis of ovarian cancer, types of epigenetic modifications in signaling pathways, DNA hyper/hypomethylation, histone modifications, enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and polycomb repressive complexes, switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex, microRNAs, cancer stem cells (CSCs), and combination and epigenetic therapies are reviewed. In this review article, we compiled updated information from various scientific studies that investigated the molecular mechanisms in the pathogenesis of ovarian cancer. Clear and comprehensive understanding of the various molecular pathways involved in ovarian cancer formation will inform extensive research studies with a goal to establish improved methods for early disease detection and the development of advanced treatment options, resulting in more favorable prognoses for women diagnosed with ovarian cancer.

Dysgerminoma
The counterpart of testicular seminoma. Tumor consists of cells with clear cytoplasm and central nuclei.

Sex cord-stromal cells Granulosa
Tumor of granulosa cells that secrete large amounts of estrogen. Fibroma Composed of fibroblasts and is hormonally inactive. Thecoma Tumor of theca cells. Composed of lipid droplets and is hormonally active. Fibrothecoma Tumor composed of both fibroma and thecoma components.

Sertoli-Leydig
Tumors that resemble granulosa cells but cause defeminization due to secretion of testosterone.  10 . According to recent studies, the possible sources of progenitor cells or neoplasms for ovarian cancer is not just limited to the ovaries 9,11 . Ovarian neoplasms are mostly of epithelial origin and fewer neoplasms mature from cell types such as germ cell, sex-cord stromal, or mixed cell types (  it signals DNA-repair genes such as BRCA1, but if the damage cannot be repaired, TP53 induces an apoptotic signal in the cell, killing it, and thereby preventing tumor formation 4,10 . In cancers such as breast, bone, leukemia, and sarcomas, frequent TP53 mutations were observed. The majority of the TP53 mutations are missense mutations resulting in single amino acid substitutions 10 . The mutation patterns from an abnormal TP53 can be used to infer the possible mutagens and molecular pathogenesis responsible, thus making this gene a unique avenue of exploration for cancer studies 10 . Abnormalities in homologous recombination repair, retinoblastoma protein, cyclin E1 and NOTCH3 pathways are also observed in type II carcinomas. In addition to genetic alterations in BRCA1-mutant type II tumors, the expression of BRCA1 can also be perturbed by promoter methylation (Figure 1) 10 .

Epigenetic Modifications
Epigenetic changes contribute to disease progression and malignant transformation that may result in ovarian cancer cells being resistant to certain drugs. Epigenetic modification is the temporary change of gene expression as a result of several external factors 12 . Epigenetic alterations are one of the main reasons for the emergence of drugresistant cancer cells, which is a major issue affecting disease remission. Epigenetic modifications are potentially reversible, which opens the possibility of therapeutic interventions. These modifications can affect chromatin structure and gene regulation, which are important characteristics of cancer formation. Cellular functions such as cell differentiation, cell-cycle progression, apoptosis, and gene expression can be severely affected by several different epigenetic changes, such as but not limited to DNA methylation, histone modifications, and chromatin remodeling and repressive complexes. Some of the notable epigenetic modifications that are linked to ovarian cancer are discussed below.

NOTCH Signaling Pathway
This pathway is responsible for governing binary cell fate, patterning, proliferation, growth, and programmed cell death 13 . Dysfunctional NOTCH signaling is known to promote oncogenic effects by inhibiting apoptosis while accelerating cell proliferation. Abnormal NOTCH signaling alters the developmental state of a cell, allowing the cell to adopt a continuous proliferative nature making it cancerous 14 . The NOTCH developmental pathway in mammals includes the NOTCH receptors (NOTCH1-4), delta ligands (DLL1, DLL3, and DLL4), and serrate ligands (JAGGED1 and JAGGED2) 13 . Epigenetic changes, such as hypermethylation and hypomethylation of NOTCH genes, have been linked to ovarian cancer. Lack of DLL1 expression caused by promoter hypermethylation is known to down regulate NOTCH1 expression, ultimately leading to gastric cancer 14 . Numerous studies have identified NOTCH3 to be a candidate oncogene. In comparison to normal ovarian tissue, NOTCH3 expression was preeminent in ovarian cancer tissue 15,16 . Cell-cycle regulator cyclin-dependent kinase inhibitor 1 (CDKN1), related to cell proliferation, is a target gene of NOTCH1 and is known to interact with the p53 pathway to manipulate the cell cycle 13,15 . The NOTCH pathway is also involved in reducing histone H3K4 methylation and down-regulating the retinoblastoma tumor-suppressor gene (RBF) gene. Such epigenetic changes have also been linked to other human cancers 13 . Using real-time polymerase chain reaction (RT-PCR), Jung et al. compared the expression of NOTCH3, JAGGED1 and JAGGED2 mRNAs between ovarian carcinomas and benign tumors. Their study detected a 3-fold and 10-fold increase in expression of JAGGED1 and JAGGED2 respectively in serous carcinomas 17 . Further studies regarding the involvement of the NOTCH pathway in ovarian oncogenesis are in progress, and its implication in ovarian cancer has yet to be fully understood 17 .

Forkhead Box M1 (FOXM1)
FOXM1 plays an important role in cell-cycle progression, apoptosis, angiogenesis, and DNA-damage repair [18][19][20] . During the cell cycle, FOXM1 regulates the G 1 /S and G 2 /M phases and maintains the integrity of the spindle fibers during mitosis. In numerous cancer types, such as ovarian, breast, prostate, lung, and gastric, it has been observed that the 12p13 chromosomal band containing the FOXM1 gene is amplified21. Epigenetic modification of FOXM1 is carried out by lymphoid specific helicase (HELLS). The modification is known to change the pattern of methylation of FOXM1 gene resulting in inducing malignant transformation by disrupting epithelial differentiation in normal cells 22 . Using microarrays, a systemic analysis of gene expression demonstrated the up regulation of FOXM1 mRNA in several ovarian tumors 23 . The up regulation of FOXM1 leads to proliferation, migration, and invasion in breast, pancreatic, and gastric cancer. Up-regulation in extracellular matrix-degrading factors and angiogenic factors, such as urokinase-type plasminogen activator (uPA), uPA receptor, matrix metallopeptidase 2 and vascular endothelial growth factor are also observed in these cancer types 21 . It has been hypothesized that FOXM1 is a crucial regeneration factor for epithelial tissues after injury 21 . When overexpressed, FOXM1 is known to upregulate pluripotent gene expressions such as octamer binding transcription factor 4, homeobox protein NANOG, and sex determining region Y-box 2 24 . Some of the FOXM1induced differentially methylated genes include those that are hypermethylated such as: Chromosome 6 open reading frame 136 (C6orf136), mannosyl (alpha-1,3-)glycoprotein beta-1,2-N-Acetylglucosaminyltransferase (MGAT1), NADH:ubiquinone oxidoreductase subunit A10 (NDUFA10), and platelet-activating factor acetylhydrolase 1b catalytic subunit 3 (PAFAH1B3). On the other hand, genes such as signal peptidase complex subunit 1 (SPCS1), filamin A alpha (FLNA), chondroitin polymerizing factor (CHPF), and glycosyltransferase 8 domain containing 1 (GLT8D1) were found to be hypomethylated as a result of FOXM1 22 . FOXM1 is further capable of initiating an oncogenic pathway by predisposing stem progenitor cells to tumorigenesis 25 . According to a study from the Genome Atlas Consortium, FOXM1 elevation may lead to the growth of pathological EOC 26 . FOXM1 hyperactivity is a steady feature of metastasis and proliferation of EOCs 27,28 .

WNT Signaling Pathway
The WNT signaling transduction pathway is essential for embryonic development and tumorigenesis 29 . It is responsible for proper follicular development and ovarian function. Overactivation of this signaling pathway has reportedly led to colon, liver, cervical, and ovarian cancers 30 . Zhu et al. observed the effects of Dickkopf WNT signaling pathway inhibitor 2 (DKK2) on WNT/β-catenin signaling in ovarian cancer cells by evaluating the β-catenin staining in SKOV3 and ES-2 cells 31 . In their study, downregulation of β-catenin expression in DKK2-transfected cells was noted when compared with untreated cells. In addition, luciferase reporter assay in SKOV3 and ES-2 cells showed that T-cell factor (TCF) activity was significantly blocked in cells transfected with DKK2 31 . Therefore, it appears that DKK2 participates in the negative feedback loop that inhibits WNT signaling. Zhu et al. also found that cells transfected with DKK2 had poor expression of MYC proto-oncogene, BHLH transcription factor (c-MYC) and CDKN1. To further investigate this, the expression of focal adhesion kinase (FAK), MMP2 and MMP9 were measured using western blotting. The results showed FAK and MMP2 expression were lowered in DKK2-transfected cells 31 .
WNT signals are known to be transduced in two main ways: β-Catenin-dependent and independent. During β-catenin-dependent transduction, frizzled family receptor proteins provides an anchor for the WNT ligand to stabilize the β-catenin for proper translocation into the nucleus 30 . Cytoplasmic protein, β-catenin, is a component of cell-cell adhesion junctions and a mediator of the WNT signaling pathway. In normal cells, phosphorylation facilitated β-catenin degrading occurs through the ubiquitin pathway 32 . Nuclear β-catenin is overexpressed in ovarian cancer as a result of up-regulation of the β-catenin gene itself, and due to the phosphorylation of glycogen synthase kinase 3β (GSK3β) which is a component of the β-catenin destruction complex 33 . Mutation of this ubiquitin pathway or the β-catenin destructive complex causes nuclear accumulation of β-catenin which stimulates the transcription and translation of approximately 23 genes involved in ovarian cancer proliferation and growth 30,32 .
E-cadherin is activated by the binding of β-catenin to TCF and upregulated in invasive ovarian cancer cells 32 . In addition, TCF is also known to activate genes such as c-MYC and CDKN1, assisting in the initiation and progression of ovarian tumor 30,33 .

Epithelial-mesenchymal Transition (EMT)
The EMT is recognized as a prerequisite for metastasis since it allows cancer cells to become invasive. This transition can be stimulated by several factors such as transforming growth factor beta (TGFβ), bone morphogenic proteins (BMPs), receptor tyrosine kinases, WNT and NOTCH signaling pathways. These signals influence EMTinducing transcription factors such as Snail (SNAI1), Slug (SNAI2), Twist-related protein 1 (TWIST), and zinc finger transcription factor (ZEB1/2) 34 . During the process of EMT, ovarian cancer cells transition from an epithelial to a mesenchymal morphology while apical and basolateral epithelial cell-specific tight junction proteins, such as E-cadherin and cytokeratin, are down-regulated. On the contrary, expression of vimentin gene and mesenchymal molecule N-cadherin are observed 35 . The EMT mechanism relies on the intrinsic ability of most embryonic epithelial cells to exhibit plasticity and alter their cell-cell contact structures, leading to branching morphogenesis or delamination 36 . Down-regulation of E-cadherin during EMT is a key factor for the invasive and metastatic nature of ovarian clear cell carcinoma. According to Takai et al., expression of E-cadherin reduces as the ovarian cancer increases in stage. The reduction of E-cadherin during stage I was 30.4% and rose to 52.7% reduction in stage IV 35 . EMT is exploited to increase cancer cell aggressiveness through acquiring chemoresistance, regenerative properties, and developing the ability to escape host immune cells 36 . Fang et al. found that in order to sustain the EMT characteristics of ovarian cancer cells, the presence of SOS RAS/RAC guanine nucleotide exchange factor 1 (SOS1)/epidermal growth factor receptor kinase substrate 8 (EPS8)/Abl interactor 1 (ABI1) complex is necessary. This complex supports the invasiveness of ovarian cancer cells and loss of any component of this complex reduces mesenchymallike traits and restores epithelial characteristics to ovarian cancer cells 34 .

DNA Methylation
Most of the DNA methylation takes place at CpG dinucleotides, which can be sporadically distributed throughout the genome or located in CpG-rich regions called CpG islands 37,38 . Normal cells exhibit global hypermethylation of repetitive and satellite sequences accompanied by locus-specific hypomethylation at active gene promoters and enhancers, which regulates the expression of antitumor genes 37,38 . In cancer cells, global hypomethylation accompanied by gene-specific hypermethylation can cause aberrant expression of tumorsuppressor genes or oncogenes. DNA-methylation patterns are widely known to affect gene expression in ovarian cancer. Gene promoter regions, gene bodies, and locations several kilobases upstream or downstream of genes all contain CpG islands 38 . When these CpG islands are methylated in error, it can cause silencing of tumor-suppressor genes. Some of the genes silenced by methylation in ovarian cancer include PTEN 39 , BRCA 40 , paternally expressed gene 3 (PEG3) 41 , RASassociated domain family member 1 (RASSF1) 42 , and mutL homolog 1 (MLH1) 43,44 . Further details about these genes are discussed below.
PTEN. PTEN is an antitumor gene, and the mutation of this specific gene is observed in ovarian cancer 45 . PTEN participates in the negative regulation of PTEN/ phosphatidylinositol 3-kinase (P13K)/protein kinase B (AKT) pathway that regulates the cell cycle, cell division and apoptosis. PTEN is also proposed to induce drug resistance via the P13/AKT pathway in ovarian cancer cells 45,46 . Dysfunction of PTEN protein activated P13K/AKT pathway resulting in ovarian carcinoma becoming resistant to cisplatin 45 . PTEN also contributes to the drug resistance of ovarian cancer cells as a result of DNA methylation 46 . Studies suggest that methylated genes are involved in the process of ovarian carcinoma drug resistance, and PTEN could be a noteworthy regulator 45,46 .

BRCA.
Multiple studies reveal that many EOCs harbor somatic mutations in BRCA1 or BRCA2 47,48 , however, it was found that BRCA proteins are also subject to epigenetic modifications 48 . DNA-damage repair, transcription, cellcycle checkpoint regulation, protein ubiquitination and apoptosis are some of the critical functions of BRCA genes 49 . Due to mutated or non-functional BRCA1 and BRCA2, double-stranded DNA breaks are not repaired, thus increasing the possibility of more mutations 50 . Studies performed to elucidate BRCA mutations revealed that these mutations cause the loss of checkpoint proteins in the cell cycle, leading to chromosomal instability 51,52 . BRCA deficiency is being studied using DNA microarraybased gene-expression analysis to understand its ability to manipulate gene expression 53 . The changes in protein expression as a result of BRCA mutations were thoroughly investigated by studying patients with ovarian carcinoma. The study by Gau et al. explored the different ways by which a deficiency of BRCA1 modulated ovarian cancer progression by regulating the expression of cell motility proteins 54 . They found differential expression of proteins involved in actin cytoskeletal/cell adhesion remodeling in BRCA1 deficiency, which also correlated with higher stages of ovarian cancer 54 . Functional BRCA1 is involved in maintaining optimal expression of regulatory proteins for the cell cytoskeleton, cell adhesion and migration in EOC. The amount of expression varies between early-and advance-stage tumors 48 . Chan et al. found a distinguishing feature between BRCA1 and BRCA2 inactivation whereby epigenetic silencing by promoter hypermethylation results in a significant loss of function only for BRCA1 48 .

RASSF1.
Hypermethylation of the tumor-suppressor gene RASSF1A leads to gene inactivation, which is an early event in ovarian cancer development [55][56][57] . Methylation of CpG islands in the promoter of RASSF1A gene results in its epigenetic inactivation 58 . When compared to a control group with only 0-21% promoter methylation of RASSF1A, the frequency of this methylation in patients with cancer was revealed to be 30-58% 56 . RASSF1A regulates multiple functions such as apoptosis, tubulin dynamics and cell-cycle arrest in cancer cells 59 . RASSF1A promoter methylation is a common epigenetic inactivation which is implicated in human cancer and it results in silencing of RASSF1A expression [57][58][59] . Hypermethylation of RASSF1A promoter is correlated with ovarian cancer and can be important biomarker in detecting the carcinogenic process of ovarian cancer 56 .
PEG3. PEG3 is one of several imprinted genes which play a vital role in human oncogenesis 60 . These imprinted genes are very susceptible to loss of function compared to tumor-suppressor genes because such genes can lose their function as a result of inactivation of just one functional allele 61 . PEG3 is crucial in the p53/c-MYC mediated apoptotic pathway, and, therefore, down-regulation of imprinted genes becomes a key factor in ovarian oncogenesis 62 . It is anticipated that re-expression of PEG3 would inhibit survival and proliferation in human ovarian carcinogenesis 60 . There is prediction that PEG3 is highly associated with carcinogenesis if the promoter of PEG3 was methylated accordingly 63 . Therefore, it was concluded that the loss of PEG3 function as a result of promoter methylation may lead to pathogenesis of multiple types of ovarian cancers 63 .

MLH1.
Promoter hypermethylation of MLH1, an epigenetic inactivation of the MMR system resulting in loss of MMR, was recognized as a potential event for ovarian cancer pathogenesis 64,65 . Studies revealed a staggering frequency of MLH1 promoter hypermethylation, ranging from 6% to 12.5% in ovarian cancer 66 . Previous studies also revealed MLH1 deficiency was linked to resistance to cisplatin and carboplatin 67,68 . The mRNA expression of MLH1 appeared to be inversely correlated with its DNA methylation level, revealing the importance of cytosine methylation in controlling MLH1 expression 69 . These findings confirmed that down-regulation of MLH1 is possible by Histone H3K27 methylation and histone deacetylation. Down-regulation of MLH1 is also a possible way ovarian cancer progresses by further drug resistance 67,68 . The methylation rate in certain genes are linked to the degree of clinical pathogenesis of ovarian cancer. It has been demonstrated that methylation in secreted frizzled-related protein (SFRP)1, -2, -4 and -5; SOX1; paired box 1 (PAX)1 and LIM homeobox transcription factor 1 α (LMX1A) were the highest in those with more aggressive forms of ovarian carcinoma and declined progressively in borderline malignancy 70,71 .

Hypomethylation
The process of hypomethylation in cancer has been less studied than hypermethylation. Hypomethylation at repeat elements is a major cause of genomic instability and epigenetic de-repression of oncogenes in ovarian cancer. There are several examples of genes overexpressed by hypomethylation. Some of these genes include methylationcontrolled-DNAJ Gene (MCJ) 71,72 , synuclein-γ 73,74 , taxolresistant-associated gene 3 (TRAG3) 75 , and brother of the regulator of imprinted sites (BORIS) 76 . A class of repetitive sequences called retrotransposons, specifically long interspersed elements, short interspersed elements, and long terminal repeat retrotransposons are hypothesized to be targets for hypomethylation 70,77 . EOC histotypes exhibit specific differences in global hypermethylation lacking any predictive hypomethylation patterns 78 . Hypomethylation is generally correlated with increasing stage of cancer, which is consistent with the paradigm that DNA hypomethylation is a key oncogenic factor. It is suggested that the degree of hypomethylation of repeat sequences increases from benign precursors to malignant tumors 78 .

Gene Hypomethylation and Ovarian Cancer Pathogenesis
The pathogenesis of ovarian cancer involves hundreds of genes. Past research has established that the hypomethylation of several genes is a major cause in cancer initiation.

Solute carrier family 6 member 12 (SLC6A12).
Sung et al. identified up-regulation of the neurotransmitter transporter, SLC6A12, as a contributor to ovarian cancer metastasis 79 . This gene product functions in the uptake of gamma-aminobutyric acid (GABA) and betaine in the liver, kidneys, and brain surface 80 . SLC6A12-transfected human ovarian cancer (SKOV3) cells showed a two-fold upsurge in cell migration when compared to cells transfected with enhanced green fluorescent (EGF) protein cDNA 79 . Additionally, overexpression of SLC6A12 increased the invasive activity of SKOV3 cells. Regulation of SLC6A12 was found to be influenced by hypomethylation of the promoter region at 18 CpG sites. In metastatic tumor cells, DNA methylation of these sites was significantly reduced in comparison to injected ovarian carcinoma cells. However, gene expression was shown to be enhanced when treated with a DNA methyltransferase inhibitor or histone deacetylase inhibitor 79,80 .

Carbonic anhydrase (CA9). Like SLC6A12, CA9
hypomethylation also affects the pathogenicity of ovarian cancer. The expression of membrane-associated glycoprotein CA9 serves as an indication of tumor hypoxia. Sung et al. observed that hypomethylation resulted in overexpression of CA9, increasing the invasive and migratory capacity of metastatic SKOV3 cancer cells 81 .
Transmembrane protein 88 (TMEM88). The genes TMEM88 and Msh homeobox 1 (MSX1) have been found to result in platinum resistance and thus increased pathogenicity via promoter hypomethylation 82 . TMEM88 regulates the canonical WNT signaling pathway, which causes the accumulation of β-catenin in the cytoplasm and its translocation into the nucleus 82

MSX1.
During the process of embryogenesis, MSX1 is responsible for epithelia-mesenchymal interactions 83 . Researchers observed that in cancer, down-regulation of MSX1 was linked to decrease in six out of eight CpG methylations 83,84 . Cell lines that exhibited platinum resistance had lower MSX1 gene expression in comparison to those that were platinum sensitive. Therefore, the researchers concluded that hypomethylation of the intragenic CpG sites was associated with resistance to platinum chemotherapy 84 . MSX1 expression is of specific importance for the treatment of High-Grade Serous Epithelial Ovarian Cancer (HGSOC). Although, ovarian cancer is responsive to platinum-based chemotherapy, it has been noticed that HGSOCs are particularly resistant to such treatment 84 . Studies revealed and identified CpG sites that were hypomethylated in MSX1 gene resulting in its down regulation in HGSOCs which resulted in its unique insensitivity to platinum-based drugs. Understanding the epigenetic modifications in genes such as MSX1 unlocks different avenues to explore other suppressor genes to elucidate their relation to possible chemoresistance of different types of ovarian cancers to chemotherapeutic agents 83,84 .

Preferentially expressed antigen in melanoma (PRAME) and cancer/testis antigen 45 (CT45).
Ovarian cancer pathogenesis can also be affected by the PRAME gene. PRAME is a cancer-testis antigen that is specific for tumors. Due to these characteristics, cancer-testis antigens have opened the possibility of developing antigen-specific cancer vaccines. Oehler et al. observed DNA hypomethylation of the PRAME promoter in both EOC and in the high-grade serous subtype of ovarian cancer 85 . Hypomethylation was correlated with increased PRAME expression, as EOC with high levels of PRAME expression were seen to have a reduction in methylation. Additionally, promoter and the transposable element, LINE1 hypomethylation (a global methylation status surrogate) were associated, suggesting that hypomethylation commonly observed in EOC is connected to PRAME regulation 85,86 . Similarly, the CT45 gene is also related to cancer pathogenesis. This gene is an X-linked CT gene associated with oncogenesis. In endothelial cancer cells, CT45 promoter activity was directly repressed by DNA methylation, compared to the control cells where CT45 was hypermethylated 86 .

Histone Modification
Chromatin is an important component of the physiological template of eukaryotic genetic information and is composed of DNA, histones, and other proteins condensed into protein complexes. It is an organized structure that consist of nucleosome repeats joined by linker DNA, called H1 87 . Nucleosomes are composed of histone octamers each consisting of two core histones: H2A, H2B, H3 and H4 wrapped by 147 base pairs of DNAs 78,88 . The post-translational modifications of the histone tails such as methylation, acetylation, ubiquitination and sumoylation are catalyzed by specific enzymes resulting in changes in gene expression. Histone modifications also affect accessibility to transcription factors, repressors and proteins that regulate DNA repair and replication 88 . Methylation of lysine residues can have different consequences depending on which histone subunit is affected and on its location within the histone tail. Methylation of histone 3 lysine 27 (H3K27me), histone 3 lysine 9 (H3K9me), or histone 4 lysine 20 (H4K20me) are linked to repression of gene expression by causing the compact state of chromatin 87,88 . Di-and trimethylation of histone 3 lysine 4 (H3K4me2 or me3) are strictly regulated by histone acetyltransferases (HATs) and deacetyltransferases (HDACs) [87][88][89] . Acetylation of lysine residues in histone tails lead to a flexible chromatin structure that is more available to transcription factors increasing gene expression; whereas, deacetylation makes it difficult for transcription factors to access genes, causing transcriptional repression 89 .
In ovarian cancer, abnormal HDAC pathways have been implicated in promoting cancer growth and metastasis 88 . HDAC enzymes are classified into four classes: I, II, III and IV, each with multiple members that have diverse expression profiles. Class III HDAC family members are sirtuin proteins (SIRT1-7) which are nicotinamide adenine dinucleotide (NAD + )-dependent enzymes. The other three classes are zinc dependent. Mammalian homologs of the yeast silent information regulator (ir2), sirtuins can act as HDAC and as a deacetylase for p53 which belongs to non-histone proteins 88 . Jang et al. observed that SIRT1 expression was elevated in malignant EOC compared to benign tumors. Their study also revealed that SIRT1 upregulation in a specific subgroup of malignant serous EOC was correlated to its increased survival capability 90 . Loss of GATA factors (GATA4, GATA5, GATA6) are reported in various cancer types, including ovarian cancer. GATA4 expression is reduced and GATA6 is lost from the nucleus in 85% of ovarian tumors. The study concluded that GATA transcription factors can be silenced by altered histone modification of the promoter loci inhibiting the expression of tumor suppressor disabled-2 (DAB2) 91 . Genome-wide studies revealed that genetic alterations affecting expression of histone-modifying genes are present in various human cancer types including, ovarian carcinomas 58 . Table 3 lists histone-modifying genes that can be altered in ovarian carcinoma.

EZH2 and Polycomb Repressive Complexes PRC2 and PRC1
EZH2 can epigenetically silence the expression of its target genes by catalyzing lysine 27 methylation on histone H3 92

SWI/SNF Chromatin Remodeling Complexes and Ovarian Cancer
The SWI/SNF chromatin remodeling complexes are involved in cancer initiation and progression 97 . Mutations in at least one member of the SWI/SNF complex is common in human tumors 98 . SWI/SNF complexes control crucial cellular processes, such as cell cycle, apoptosis, cell differentiation, genomic stability, and DNA repair 97 . Based on the presence of ARID1A/B or ARID2 and polybromo 1 (PBRM1) subunits, the SWI/SNF complex can be further classified into BRG1-associated factor (BAF) or polybromoassociated BAF complex (PBAF) respectively 99 . In ovarian cancer, mutations in several SWI/SNF complexes were found 100,101 . These mutations lead to altered nucleosome positioning, making it difficult for RNA poly 11 or other transcription factors to access DNA 102 . Through extensive genomic studies, three important limitations in tumor suppression mechanisms by BAF complex were found 102,103 . Firstly, the fundamental mechanism gives rise to genetic supremacy 103 . Secondly, in-vitro chromatin remodeling assays failed to recreate this mechanism 103 . Thirdly, this mechanism is only present in a highly specific tumor environment 103 . The involvement of SWI/SNF complex in regulating chromatin organization, RNA transcription, DNA damage repair and cell division make it extremely difficult to elucidate its role in cancer development 100,102 .
Two important subunits of the SWI/SNF complex are further discussed below ARID1A. The ARID1A is a component of the ARID1 subunit of the SWI/SNF complex which can mobilize nucleosomes and subsequently regulate epigenetics of many genes including those that are responsible for ovarian clear-cell carcinoma, an aggressive form of ovarian cancer known to be resistant to typical chemotherapy. Chromosomal translocation of tumor-suppressor ARID1A gene has been linked to breast and lung cancer, whereas leukemia cells with silenced ARID1A resulted in them being resistant to Fas-mediated apoptosis 104 . Abnormal chromosome remodeling complex and the loss of BAF250a expression of protein are some of the known effects of ARID1A inactivation 105 . Transcriptome sequencing has allowed identification of frequent mutation of the ARID1A gene in endometroid and clear-cell ovarian carcinomas. Abnormality in the ARID1A gene is a consistent feature in cancer, such as gene rearrangement in breast cancer and gene deletion in lung cancer. This suggests that ARID1A has tumor-suppressor properties 105 . In one study, 46% of ovarian clear-cell carcinomas and 30% of endometrial carcinomas were found to have ARID1A mutation using exon resequencing 105,106 . ARID1A mutations were mostly observed in atypical endometriosis and primary malignant lesions 107 105 . This suggests that the loss of BAF250a protein is a pathological factor in the genesis of ovarian clear-cell and endometroid cancer 105,107 . Mutation in the ARID1A gene may encode for a protein like BAF250a but nonfunctional. However, mutated ARID1A inactivates the chromatin remodeling pathway, leading to cancer 105,107 .

SWI/SNF-related,
Matrix-associated, Actindependent Regulator of Chromatin, Subfamily A, Member 4 (SMARCA4). SMARCA4 is a chromatin remodeling gene which when mutated becomes inactivated and contributes to the formation of small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) 108 . SCCOHT is primarily seen in young women, but it is a rare and extremely aggressive type of ovarian cancer. The characteristic features of SCCOHT include abnormal layers of small cells with limited cytoplasm, hyperchromatic nuclei, and small nucleoli. It is suggested immature ovarian cells may be the precursor for SCCOHT 108 . Patients with SCCOHT have a long-term survival rate of only 33% and typically do not live over 2 years after diagnosis, regardless of the cancer stage 109 . Ramos et al. investigated the pathogenesis of SCCOHT and found that 69% of the samples contained SWI/ SNF mutations and 82% had a loss of SMARCA4 110 . Jelinic et al. found that Inactivation through bi-allelic SMARCA4 mutation is a major determinant of SCCOHT 108 . SMARCA4 inactivation was caused by frameshift, nonsense, and splicesite mutations 108,109 . A mutation that causes inactivation in one of the subunit genes results in the loss of tumorsuppression ability of the SWI/SNF complex, leading to uncontrolled cell proliferation and resulting in SCCOHT 109 . In a study to assess the loss of SMARCA4 expression in ovarian tumors, it was found that 42 samples out of 46 lacked SMARCA4 expression which is approximately 91%. In addition, the four samples that did not exhibit the loss of SMARCA4 expression lacked a different protein which is also a component in forming the SWI/SNF complex [108][109][110] .

Role of Micro RNAs (miRNAs)
miRNAs are non-protein coding RNA sequences between 15 to 22 nucleotides in length 111 . Since miRNAs are such short sequences, they do not have to be a perfect complement in order to bind their target mRNA. This means multiple miRNAs can bind to one target mRNA and in turn, one miRNA is able to control multiple mRNA targets 112 . Specific miRNAs have been shown to be either down regulated or up regulated depending on the type of cancer. Some of these miRNAs when up-regulated, can function as oncogenes by inactivating tumor-suppressor genes. In contrast, when down-regulated, they can serve as tumorsuppressor genes by negatively affecting oncogenes 112,113 . Numerous studies have been performed to see if specific miRNAs dysregulation patterns can be detected in ovarian cancer. The Cancer Genome Atlas project analyzed 500 high-grade serous ovarian carcinoma and documented the variations in their miRNA levels 113 . Other studies narrowed the pool further by comparing miRNAs in normal ovaries to miRNAs in ovarian cancer. Out of 310 dysregulated miRNAs discovered across multiple studies, about 34 were seen to be consistently dysregulated in ovarian cancer cells. miR-127 and miR-31 were consistently down-regulated, whereas miR-20a and miR-200b/c was up-regulated 113 .
The expression level of a specific miRNA can provide information regarding the aggressiveness of the cancer 111 . Certain miRNAs may indicate the type of tissue from which the cancer originated, allowing treatment options to be narrowed down. The dysregulation of a specific miRNA, such as an increase in miRNA-17 production, has been noted in patients with chemo-resistant colorectal cancer 112 . Elevated miRNA-17 has also been correlated with an increase in the invasion and proliferation of breast cancer 112 . To explore this relationship between miRNAs and cancer, Lui et al. studied the interaction between liver kinase protein (LKB1), which functions by regulating p53 and p21/WAF1 111 . They found that in presence of miR-17, transcription and translation of LKB1, p53, and p21 in ovarian clear-cell carcinoma were reduced. miR-17 is known to affect cell-cycle regulation and stimulate the proliferation and invasion of ovarian clear-cell carcinoma 111 . They also found that the absence of functional miR-17 caused tumor suppression. Therefore, they concluded that miR-17 interferes with LKB1-p53-p21/WAF1, leading to oncogenesis 111 .
It was found that miR-215 was down-regulated in EOC cell lines and associated with lymph node metastasis 112 . NIN/RPN12 binding protein (NOB1) was identified as a target gene of miR-215. Critical oncogenic pathways through activation of the Mitogen-activated protein kinases (MAPK) pathway and p38MAPK phosphorylation were observed as a result of miR-215 up-regulation 111,113 . Table 4 shows a representative list of different miRNAs involved in dysregulation in EOC. Expression patterns of miRNAs can be used as biomarkers for novel ovarian cancer treatment methods 114 . It was seen that patients with up-regulation of miR-182 displayed reduced sensitivity to cisplatin and paclitaxel, common chemotherapy drugs to treat ovarian  cancer 111,112,114 . miR-18 negatively regulates the tumorsuppressor gene programmed cell death 4 (PDCD4), which regulates the initiation and progression of ovarian cancer 114 . In contrast, miR-199a increases the response to the same drugs by interfering with the ATP Binding Cassette Subfamily G Member 2 (ABCG2) gene which is associated with multidrug resistance in ovarian cancerinitiating cells 114 .

miRNA as Biomarkers
miRNAs can target mRNAs for cleavage or translational repression. They are not capable of coding for proteins but instead possess necessary structural, catalytic, and regulatory functions as they are located at fragile sites and genomic regions related to cancer 115 . Approximately 30% of the genes that code for human proteins are susceptible to miRNAs. Aberrant changes in miRNA expressions can be linked to the expression of oncogenes or protooncogenes 116 . miRNAs can be formidable as they can sustain themselves in fixed tissue, blood, and other body fluids which makes them an excellent source of biomarkers. It was seen that miRNA expression profiles were invaluable in detecting and identifying progenitor cells for different cancer types 117 . miRNAs stored in serum also exhibit astounding resistance to enzymatic cleavage by RNase A, therefore being able to maintain their intrinsic stability for a prolonged period 115 .
Discovery of novel biomarkers is one of the major challenges in diagnostics. It is known that certain miRNAs are involved in regulating cell proliferation, differentiation, and death 111,112,116 . The microarray platform has been used to determine 29 differentially expressed miRNAs with ovarian cancer. From the analysis, it was found that miR -141, miR-200a, miR-200b, and miR-200c were upregulated, whereas, miR-125b1, miR-140, miR-145, and miR-199a were down-regulated 118 . Kosaka et al. detected elevated expression of miR -21, miR-92, miR-93, miR-126, and miR-29a from serum 119 . Additionally, the miRNA expression patterns in ovarian tumors also provide useful insights into the histological subtypes of ovarian tumors 116 . There are multiple ways of detecting miRNAs including in-situ hybridization, northern blotting, microarrays, next-generation sequencing, enzymatic luminescence miRNA assay, and nanopore technology 115 . In a previously reported study, a patient with ovarian cancer negative for the diagnostic Cancer Antigen 125 (CA-125) threshold did, in fact, have elevated levels of miR -21, miR-92, miR-93, and miR-126. This shows that using miRNA as a biomarker may be more accurate for ovarian cancer diagnosis, especially in non-epithelial-types 116,117 . Tables 5-7 lists miRNAs that are used as potential biomarkers for diagnosing various types of ovarian cancers.

Ovarian Cancer Stem Cells
Cancer stem cells (CSCs) related to ovarian cancer are considered the emerging culprit in primary tumorigenesis relapse, metastasis and chemoresistance 11 . CSCs are malignant multi-potent progenitors hypothesized to possess mutations making them drug resistant 119 . They are suspected to exhibit chemo-resistant properties, including low mitotic index, hypoxemia resistance, enhanced DNA-repair mechanisms, and up-regulation of ATPdependent membrane transporters that selectively export chemotherapy agents 119,120 . Studies have demonstrated the emergence of CSCs after chemotherapy treatment with cisplatin and paclitaxel 121,122 . High levels of Zinc Finger CCHC Domain-Containing Protein (LIN28), an RNA-binding protein, are expressed in ovarian cancer cells 123     into an invasive malignant phenotype 123,124 . The exosomes resulted in induction of EMT in ovarian cancer cells 123,125 .
Another study found that ovarian cancer stem cell cultures expressed mesenchymal and epithelial markers, including cytoplasmic E-cadherin and cell surface markers CD133 and CD44 126 . A culture of ovarian clear-cell carcinoma cells with epithelial E-cadherin-positive and mesenchymal vimentinpositive biomarkers demonstrated cooperation between these two phenotypes. These coexist at the epithelial edge of the culture, which suggests a cooperative interaction and the possibility that mesenchymal cells influence epithelial cells to transition 127 .

Effects of HDAC inhibitor, DNA Methyltransferase 1 inhibitor and Calpain inhibitor Combination Therapy on Ovarian Cancer Cells
HDAC inhibitors are emerging as an alternative and novel type of anticancer agent. Acetylation of histone stimulates expression of genes that inhibit proliferation while inducing apoptosis of cancer cells 128 . Deacetylation of histone by deacetylase reduces the expressions of tumor suppressors such as p21WAF1, growth arrest and DNA damage (GADD45) and several others [129][130][131] . Hydroxamic acid was reported as potent HDAC inhibitor which interacts with the zinc in deacetylase 132 . It was seen that HDAC was inhibited in neuroblastoma cell line SH-SY5Y by 2-amino-5-(thiophen-2-yl) benzamide series (K560). The 2-aminobenzamide moiety in K560 exerts selective inhibition by interacting with the zinc binding site within the HDAC1/2 pocket 133,134 .
DNA methyltransferase 1 (DNMT1) inhibitors can remove methylation resulting in expression Regulator of G protein signaling 10 (RGS10), a regulator of cell survival and chemo resistance. These events increase susceptibility of ovarian cancer cells to cisplatin 135 . DNMT1 can be broken down by DNMT1 inhibitors resulting in CPG demethylation, causing re-expression of tumor suppressor genes p21, p16, and retinoic acid receptor beta 2 (RARB2) 136 . DNMT1 inhibitors in tandem with TNF-related apoptosis inducing ligand (TRAIL) inhibitory antibodies were found to reduce breast tumor size in a xenograft mouse model 137 . DMT1 inhibitor, when combined with telomere inhibitor GT-oligo, caused a significant reduction in ovarian cancer proliferation 137,138 . Growth of several types of cancer, such as breast, ovarian, prostate, and leukemia, were affected when DNMT1 inhibitors were combined with calpain protease inhibitor 139,140 . Calpain, a ubiquitous protease, regulates multiple signaling proteins and their pathways and disruption in calpain expression is known to be a pathological event in cancer formation. Lapinska et al. studied the effects of two structurally different DNMT1 inhibitors: Class 1 sodium butyrate, and class 11 sub eranilo-hydroxamic acid (SAHA) both combined with the calpain protease inhibitor, calpeptin.
In the study, two distinct ovarian cancer cell lines, CAOV-3 and SKOV3, were used. Results showed significant growth inhibition of both ovarian cancer cell lines 140 . DNMT1 inhibitors when combined with calpeptin resulted in the inhibition of cell-cycle progression, a decrease in metastatic activity, programmed cell death, autophagy, and re-expression of tumor-suppressor genes such as ADP-ribosylarginine hydrolase (ARH1), p21 and RARB2 140 . Therefore, combination therapy, when used effectively, can be a potential treatment option for ovarian cancer.

Bromodomain and Extra-terminal Domain (BET) Bromodomain Inhibition
BET bromodomain inhibitors interrupt acetyl lysine recognition by replacing BET bromodomain proteins from transcriptional complexes, resulting in gene inhibition 141 . BRd4, a member of BET (bromodomain and extra-terminal domain) family, activity is known to be important for tumor progression. Inhibition of BRd4 was found to interfere with transcription of oncogenes, causing tumor regression and apoptosis 142,143 . Targeting using BET bromodomain inhibitors was shown to affect kinome dynamics in ovarian cancer cell lining 144 . Baratta et al. found that resistance to BET bromodomain inhibitors involved the downstream signaling pathways of P13K, AKT, and MAPK/ERK alongside the activation of receptor tyrosine kinases and prolonged contact with a BET bromodomain inhibitor JQ1 resulted in accelerated susceptibility of ovarian cancer cells to combination therapies targeting the pathways mentioned above 142,143 . Unfortunately, combination therapies involving inhibitors such as BET bromodomain inhibitors attack kinases that are involved in other critical and beneficial pathways 142,144 . BET bromodomain inhibitor-mediated apoptosis by JQ1 showed cell-specific effects on different ovarian cancer cell lines 144 . Gene-expression studies using RNA sequencing in A1847 and OVCAR5 ovarian cancer cells following JQ1 therapy were performed to see how it affected RNA expression of BRd4 targets 145 . According to the results, RNA expression was reduced for Interleukin-7 receptor (IL7R), FOXM1, CDK4, CDK6, MYC and FOSlike antigen (FOSL), which were established to be BRd4 targets 145 . The majority of studies concluded that BET bromodomain inhibitor therapies may not be suitable to provide long-term treatment benefits due to acquired drug resistance of ovarian cancer 146 .

Epigenetic Therapies to Overcome Chemotherapy Resistance
Epigenetic therapies largely target histone deacetylation or DNA methylation as means of reversing these damaging epigenetic DNA changes 147 . Hypermethylation of CpG-rich promoter regions can result in gene silencing, and when tumor-suppressor genes are silenced, destabilization of the cell cycle may occur. More specifically, these epigenetic changes can prevent cell apoptosis and permit uncontrolled proliferation, leading to the formation of a tumor 147 . Histone deacetylation is also damaging, as it can destabilize tumor-suppressor genes and result in tumor formation. The two drugs which focus on these epigenetic issues are DNMTs and HDAC inhibitors 148 . Over the past few decades, researchers have focused on these epigenetic therapies as a means of treating multiple cancer types 147,148 . Hydralazine, an oral antihypertensive and non-nucleoside DNMT inhibitor, inhibits DNMT1 activity by reducing DNMT1 and DNMT3A expression 148 . In cervical cancer, hydralazine demethylation and gene-reactivating activity were observed 149 . Valproic acid, an anti-epileptic fatty acid, resulted in N-terminal hyperacetylation of H3 and H4, resulting in blocking substrate access in ovarian cancer cells 150 .

Complementary and Alternative Interventions and Targeting Epigenetic Pathways
Human beings have traditionally utilized medicinal plants against cancer worldwide. Lawsonia intermis (henna) is used as an agent for blood cancer in Ayurveda and Unani medicine, and used as anticancer drug in Sudan 151,152 . Trigonella foenum-graecum (fenugreek) is traditionally utilized to treat high cholesterol, diabetes, wound inflammation, and cancer [153][154][155] . Ambrosa maritima, a Sunanese traditional medicine, is widely used for several kinds of diseases including malignancies 156 . Compounds in plants have been reported to inhibit migration, invasion, and proliferation of cancer cells. Indeed, many dietary components are linked to affecting DNA methylation or regulating histone modifications such as isothiocyanates, genistein, resveratrol, epigallocatechin-3-gallate and quercetin. Many studies were performed to examine these types of compounds for preventing and treating cancer. Quercetin a representative flavonoid, ameliorates ROSmediated cellular damage, and prevents tumor invasion and metastasis by reducing expression of MMP2 and -9 157,158 . Quercetin-3-O-glucuronide also inhibits MMP2 and 9 expression and cell invasion via suppressing beta 2 adrenergic receptor (β2-AR) signaling in noradrenalinestimulated breast cancer cells 159 .
DNA methylation is catalyzed by DNA methyltransferase, which is known to silence tumorsuppressing genes. DNA methyltransferase has been an attractive target to epigenetically prevent or treat malignancies 160 . Cytosine analogs such as 5-azacytidine and 5-aza-2'-deoxycytidine can act as demethylating agents by replacing cytosines in DNA thereby inhibiting DNA methyltransferase 161,162 . Some efficacy has been reported for the use of hypomethylating agents in blood cancer such as myelodysplastic syndromes but there are fewer data for solid tumors. There is some evidence that treatment with decitabine may reinvigorate the sensitization of platinumresistant ovarian tumors 163 . 2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl) propanoic acid (RG 1 08) was identified and used as a different type of potent inhibitor of DNA methyltransferase but has not been tested for ovarian cancer. RG 1 08 suppresses DNA transferase via combining with its active moiety 163,164 .
Studies on pharmacological interventions against ovarian cancer have also been reported. Genistein, 4',5,7-trihydroxyisoflavone, a type of flavonoid found in soybeans, peas, lentils, and other beans 165 , was reported to inhibit EMT in ovarian cancer cells. Apicidin, a fungal metabolite, is a cyclic tetrapeptide that inhibits migration and proliferation of SKOV-3 cell by down-regulating HDAC 4 and MMP2 166 . Guadecitabine (SGI-110) acts as a demethylating agent and up-regulates tumor-suppressor genes 167 . Advanced stages of ovarian cancer can negate the effects of certain inhibitors of demethylating agents, such as fazarabine 168 .

Alternative Cancer Therapies
Alternative forms of chemotherapy are becoming progressively popular in cancer treatment. The health hazards of radiation therapy and increasing tolerance of cancer cells to platinum-based drugs has geared research towards different phytochemicals and novel drug delivery methods 169 . Afroze et al. studied the differential effects of a cardiotonic steroid, cinobufotalin on three different ovarian cancer cells and found that cinobufotalin affected the growth and metastatic capacity of ovarian cancer cells 170 . Some common natural compounds that are being explored to investigate their anticancer properties are flavonoids, garlic, turmeric, stinging nettle, green tea, and many more. Yamauchi et al. investigated the effect of a modified quercetin derivative known as 3'4'7-O-trimetheylquercetin (347TMQ) which inhibited the invasion and migration of three types of ovarian cancer cell 171 . Ashraf et al. followed up on this investigation and determined that 347TMQ resulted in expression of pro-apoptotic proteins causing apoptosis in three different ovarian cancer cell lines 172 . Therapies using these compounds address some of the pervasive difficulties associated with cancer diagnosis, treatment, and survival 169,173 .
Cancer cells take advantage of the Warburg effect whereby glycolysis is upregulated resulting in an increased lactate accumulation 174 . In cancer cells, NADH that is produced by glyceraldehyde phosphate dehydrogenase (GAPDH) and must be used up to regenerate nicotinamide adenine dinucleotide (NAD + ) to continue glycolysis. This high rate of glycolysis supports a vigorous metabolic environment that allows for the rapid growth of cancer cells 175 . Coverdale et al. was able to increase the lipophilicity of cancer cells by delivering intact active organoosmium catalysts inside cells. This made cancer cells more susceptible to metallodrugs therefore, maximizing anticancer effects 176 . Coverdale et al. used a synthesized osmium molecule in tandem with sodium formate to manipulate pyruvate breakdown in cancer cells, resulting in decreased proliferation and increased apoptosis 174 . The combination of the sodium formate and osmium reduced NAD + to NADH inside cancer cells using non-chiral osmium catalysts 176 . This novel approach targets the inherent redox vulnerability of cancer cells, which results from dysfunctional mitochondria 174 . A study by Wargovich et al. showed that phase I and phase II metabolic processes by natural compounds may have similar anticancer activity 177 . Using osmium complex with higher concentrations of sodium formate significantly reduced the proliferation of A2780 cancer cells by causing G 1 cell-cycle arrest. This mechanism of action prevents DNA damage by preventing nuclear accumulation of osmium and causing S/G 2 M cell cycle arrest 174,178 .
Thus, many of the epigenetic foci related to the pathogenesis of ovarian cancer have been reviewed along with types of epigenetic modifications that have been elicited in the available research literature. In addition, the role of miRNA and cancer stem cells are discussed in this growing area of research related to oncogenic pathways related to ovarian neoplasms. This is followed by a summary of research regarding the targeting of epigenetic pathways with alternative and combination related therapies. Given that ovarian cancer is still a leading cause of cancer-related death and is commonly diagnosed at latter stages and with continued issues of treatment resistance, more research regarding the underlying mechanisms and possible treatment modalities are warranted.