Epigenomics of ovarian cancer and its chemoprevention.

Epigenetic regulation of gene expression plays a fundamental role in normal tissue as well as diseases. Previous epigenetic studies focused on individual genes have provided us with a wealth of insights into the mechanisms of ovarian cancer. In this review, we summarized most of the related progress on epigenomics of ovarian cancer, including its development, potential biomarker, chemoresistance, and possible treatment on its resensitization. To understand this complex disease comprehensively, there is an urgent need to reveal the epigenetic status genome-wide (epigenomics) in ovarian cancer. Numerous pathways involved in basic cell metabolism have been demonstrated to be aberrantly regulated by epigenetic mechanisms during the progression of ovarian cancer, such as Wnt, TGF-beta, cell cycle regulation, and the apoptosis pathway. Although considerable progress has been made on the understanding of the nature of ovarian cancer epigenomics, especially of DNA methylation, more effort is still needed, specifically on histone modifications and miRNA-related epigenomics underlying the development of different stages and chemoresistance of ovarian cancer. Knowledge in this regard will undoubtedly promote the development of novel biomarkers and lead to the design of more effective epigenetically based chemotherapy. As ovarian cancer patients have already begun to benefit from studies in this field, continued efforts in this direction would be justified.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Currently, the gold standard treatment for ovarian cancer is surgical excision followed by cytotoxic chemotherapy. Cisplatin and carboplatin have often been mainstays for treatment of ovarian cancer. Cisplatin can form interstrand and intrastrand adducts once it bound to DNA double strands. The adducts are characterized by DNA bending toward the major groove, unwinding of the double helix, and alterations of the grooves and of the puckering of the sugars (Takahara et al., 1995 ). Adducts can further be recognized by several proteins of the DNA damage repair system of the cell and eventually leads to apoptosis. Although most ovarian cancers respond to first-line chemotherapy, recurrence of ovarian cancer occurs in up to 75% of the patients (Agarwal and Kaye, 2003 ). This resistance can be intrinsic or acquired. Intrinsic resistance means that the resistant cell already exists in the tumor. The hypothesis is that the pretreatment tumors contain certain gene expression signatures that lead to the resistance. By contrast, acquired resistance occurs in tumors that generate resistance after chemotherapy by gene mutation or epigenetic modulation. The nature of cisplatin-mediated chemotherapy (targeted at DNA) is based on the potential of acquired resistance (caused by gene mutation) occurring. Cisplatin resistance could be caused by multiple factors. Generally, those factors could be pharmacokinetics-related, including cisplatin uptake and efflux, inactivation by thiol-containing molecules, such as glutathione (gamma-glutamylcysteinylglycine, GSH), or could be caused by the sensitivity of the target cell to DNA damage caused by cisplatin, including an aberrant DNA repair system, change in apoptosis pathway, or hyperactivation of oncogenes (Borst et al., 2008 ; Figure 2 ). Potential pathways involved in cisplatin resistance in ovarian cancer cells . Cisplatin resistance can occur due to the decrease of proteins that are responsible for its uptake and an increase of proteins that can transport cisplatin to the outside of a cell. Cisplatin in the cell can also be inactivated by thiol-containing molecules (GSH) and cisplatin could cause DNA damage. The DNA repair system ( hMLH1 ) can then work together with p53 and cell cycle regulating proteins (p16, p21) to engage G1 and G2 cell cycle arrest. Depending on the result of DNA repair and the balance between pro-apoptosis and anti-apoptosis proteins, the cell can either survive or undergo apoptosis. Targets that could be regulated by epigenetic mechanisms had been highlighted in red. Apparently, genetic and epigenetic mechanisms both play essential roles in these processes. Due to the reversible nature of epigenetic modulation, it has become a promising target for cancer treatment. With the consideration that multiple factors may be involved in the resistance of ovarian cancer cells to cisplatin treatment, a strategy of targeting those multiple factors simultaneously may hold the key for this refractory disease. Potential targets for epigenetic mechanisms on chemotherapy possibly include most of the key genes involved in the metabolism pathway of chemotherapy drugs and apoptosis pathway. Epigenomic studies thus may provide us valuable information on the aberrant epigenetic status of those genes which could guide specialized therapy. Several studies had indicated that epigenetic inactivation of genes involved in trigging an apoptotic response would result in drug resistance. For instance, MLH1 can recognize certain damages in DNA which can further activate a cascade response that can induce apoptosis. Methylation of MLH1 and further transcriptional silencing occurs in cisplatin-resistant ovarian cancer (Gifford et al., 2004 ). DAPK, which is a gene involved in apoptosis, has also been indicated to be silenced in drug resistant cancer due to methylation (Lehmann et al., 2002 ). In a recent study by Li et al. ( 2009 ) DNA methylation and gene expression were integrated together to analyze the pathways involved in platinum resistance in ovarian cancer. This study indicates disruption of tumor-suppressive functions by hypermethylation, such as cell adhesion molecules, tight junction formation, PPAR signaling, and leukocyte transendothelial migration pathways. Significant up-regulation of tumor-promoting cascades by hypomethylation, such as PIK3K , PDGFRA , E2F1 , and TGFBR2 , which represents PI3K/Akt, cell cycle progression, and TGF-beta pathways, was also reported. A number of studies have been performed to investigate the ability of epigenetic modulating drugs on resensitizing resistant ovarian cancer to chemotherapy. DNMT inhibitors (DNMTIs) are analogs of deoxycytosine with different substitutions at their 5-carbons. They can prevent transfer of a methyl moiety by trapping the methyltransferases once they are incorporated into DNA (Lyko and Brown, 2005 ). These inhibitors have been used to induce re-expression of silenced TSGs caused by hypermethylation. It is hypothesized that reactivation of those genes could sensitize the apoptotic pathway mediated by chemotherapy. RASSF1A and hMLH1 up-regulation by decitabine and zebularine, two DNMTIs, has contributed to the resensitization of cisplatin-resistant ovarian cancer cells to platinum (Balch et al., 2005 ). Histone deacetylation is another recognized mechanism that contributes to silencing of TSGs in cancer. Histone deacetylation could lead to a compact conformation of DNA which is not accessible by DNA transcription factors. HDACIs could inhibit deacetylation by HDACs, thereby releasing genes from epigenetic repression. HDACIs have been indicated to induce cell cycle arrest, differentiation, or apoptosis in cancer cells (Secrist et al., 2003 ). The combination of a DNMTI and HDACI to re-sensitize resistant cancer cells has also been investigated. It is hypothesized that there should be an additive or synergestic effect on silenced TSGs expression through this approach (Karpf and Jones, 2002 ). In studies conducted by Steele et al. ( 2009 ) it was shown that a combination of decitabine (a DNMTI) with belinostat (an HDACI) is more effective in resensitizing ovarian cancer xenografts than decitabine alone. Several studies have also demonstrated that miRNAs act as potential targets for resensitization of ovarian cancer. Liang et al. ( 2010 ) showed miRNA-related genetic polymorphisms may impact ovarian cancer predisposition and clinical outcome both individually and jointly. Others showed that low tumoral miR-200 expression was significantly associated with high β-tubulin III protein content, a trend toward poor PFS (Leskelä et al., 2011 ). In a more recent study, Boyerinas found that Let-7 can sensitize ovarian cancer with acquired resistance to Taxanes via targeting IMP-1, a protein known to stabilize mRNAs of a number of genes, which can lead to degradation of the drug resistance gene MDR1 (Boyerinas et al., 2011 ). Although conventional epigenetic drugs are promising in treating cancer, their non-specific effect could be detrimental to cancer prevention. DNMTIs could lead to genomic hypomethylation which could result in GI. They could also activate potential oncogenes or drug resistant genes due to their demethylation effect on the CGIs of these genes. HDACIs could have a similar effect due to their non-specific targets. A number of natural compounds extracted from plants, including epigallocatechin-3-gallate (EGCG) from green tea, sulforaphane (SFN) from cruciferous vegetables, and genistein from soybean have been demonstrated to effectively inhibit proliferation of cancer cells via their epigenetic modulating effects. EGCG has been found to function as a DNMTI to reactivate TSGs (Fang et al., 2003 ). It has also been shown to inhibit telomerase expression in cancer cells through epigenetic remodeling of the hTERT promoter (Mittal et al., 2004 ; Berletch et al., 2008 ; Chen et al., 2009 ; Meeran et al., 2011 ). The advantage of these natural compounds compared with conventional epigenetic modulators is their low toxic effect to normal cells. Furthermore, a combination of these natural compounds may exert additive or synergistic effects in killing cancer cells which would allow yet lower and safer doses to be applied. Epigenomic studies on the epigenetic alterations of genes in response to treatment with natural compounds may reveal potential mechanisms for how these compounds exert their extra benefits and which could guide design of future anticancer drugs.

Ovarian cancer has the highest mortality rate among reproductive cancers and is the leading cause of gynecologic cancer death. The ovaries contain three types of tissue: germ, stromal, and epithelial cells. Germ cells and stromal cells are inside of the ovary, while epithelial cells are on the surface of the ovary. Germ cells can make eggs, and stromal cells produce most of the female hormones (estrogen and progesterone). The epithelium is the tissue where most ovarian cancers arise. Ovarian cancer is classified into several stages according to the American Joint committee on Cancer/Tumor Node Metastasis (AJCC/TNM) and International Federation of Gynecology and Obstetrics (FIGO) staging systems which are based on how far the cancer has spread. In stages I and II, the tumor is confined to the ovaries, while there is local metastasis (usually lymph) in stage III and there is distal organ metastases in stage IV (Yarbro et al., 1999 ). The etiology of ovarian cancer is still not clear. However, previous epidemiology studies indicate that for sporadic ovarian cancer, the younger the individuals are when they have their first child and the more children they have, the less the risk of suffering from ovarian cancer (Chen and Karlan, 1998 ). A hypothesis has been proposed based on these epidemiology studies, that is, too much wound repair after ovulation may contribute to the ovarian tumorigenesis processes (Roett and Evans, 2009 ). Due to the atypical syndrome of the early stage of ovarian cancer, it is difficult to diagnose in its early stages. By the time most ovarian cancers are diagnosed, they are already at stage III or IV. The two most significant obstacles to the effective treatment of ovarian cancers are the lack of early diagnostic markers and the development of drug resistance after therapeutic treatment of advanced disease. Ovarian cancer screening with transvaginal ultrasound (TVU) and CA125 was evaluated in the Prostate, Lung, Colorectal, and Ovarian (PLCO) trial, however, it was revealed that the predictive value of both tests was relatively low (Buys et al., 2005 ). Increasing evidence indicates that epigenetic mechanisms may play a major role in the development of ovarian cancer. The epigenome is defined as the mechanisms that regulate all the genes expression without alteration of the DNA sequence (Bernstein et al., 2007 ). These epigenetic mechanisms include DNA methylation, histone modification, and microRNA (miRNAs) expression which are known to regulate gene expression in cancer cells. DNA methylation is characterized by transferring of a methyl moiety to cytosine at the 5-carbon (C 5 ) position by enzymes known as DNA methyltransferases (DNMTs). DNA methylation can regulate gene transcription by recruiting methyl CpG-binding proteins and therefore regulate chromatin status. Typically, DNA hypermethylation could silence genes (e.g., tumor suppressors) due to recruitment of repressors and further lead to chromatin condensation (Herceg, 2007 ). Histone modifications are characterized by covalent modification of the N-terminal tails of core of histones (H2A, H2B, H3, and H4) including acetylation, methylation, phosphorylation, biotinylation, and ubiquitination (Feinberg et al., 2006 ; Hassan and Zempleni, 2006 ; Doi et al., 2009 ). Based on studies focused on the biological significance of these modifications, concepts like “histone code” and “histone language” have been proposed (Oliver and Denu, 2011 ). Acetylation has been the most studied in cancer among all these histone modifications. Histone acetyltransferases (HATs) are responsible for the acetylation of the lysine residues of histones (Turner, 2000 ), while histone deacetylases (HDACs) can remove the acetyl moieties (Mottet and Castronovo, 2008 ; Choudhuri et al., 2010 ). Histones acetylation generates an open conformation of chromatin which is accessible to transcription regulators, leading to the promotion of gene expression. Moreover HDACs can remove acetyl moieties from acetylated sites in non-histone proteins thereby adding another layer of regulation on cellular activities (Marson, 2009 ). Non-coding miRNAs are regulatory RNAs that are 20–30 nucleotides in length that match the 3′ untranslated regions of target mRNAs. miRNAs can inhibit mRNA translation or degrade mRNA (Ducasse and Brown, 2006 ; Guil and Esteller, 2009 ; Lujambio et al., 2010 ). miRNAs target multiple protein-coding transcripts and are known to cause posttrancriptional gene down-regulation and regulate a number of genes (Jones and Baylin, 2007 ). While miRNAs are tightly regulated in normal cells, in cancer cells miRNAs are vastly deregulated (Veeck and Esteller, 2010 ). Additionally, miRNA profiles are being used to classify cancers and the misexpression of miRNAs is associated with cancer development (Iorio et al., 2010 ; Veeck and Esteller, 2010 ). A global down-regulation of miRNAs in multiple tumor tissues has also been observed (Lu et al., 2005 ).