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Minireview: Epigenetic Changes in Ovarian Cancer Curt Balch, Fang Fang, Daniela E. Matei, Tim H.-M. Huang, and Kenneth P. Nephew Medical Sciences (C.B., F.F., K.P.N.), School of Medicine, Indiana University, Bloomington, Indiana 47405; Melvin and Bren Simon Cancer Center (C.B., D.E.M., K.P.N.), Departments of Cellular and Integrative Physiology (C.B., K.P.N.) and Obstetrics and Gynecology (K.P.N.), and Division of Hematology/Oncology (D.E.M., K.P.N.), Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202; and The Human Cancer Genetics Program (T.H.-M.H.), Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210

Epigenetic aberrations, including DNA methylation, histone modifications, and micro-RNA dysregulation, are now well established in the development and progression of ovarian cancer, and their gradual accumulation is associated with advancing disease stage and grade. Epigenetic aberrations are relatively stable, associated with distinct disease subtypes, and present in circulating serum, representing promising diagnostic, prognostic, and pharmacodynamic biomarkers. In contrast to DNA mutations and deletions, aberrant gene-repressive epigenetic modifications are potentially reversible by epigenetic therapies, including inhibitors of DNA methylation or histone-modifying enzymes. Although epigenetic monotherapies have not shown activity against solid tumors, including ovarian cancer, preclinical studies suggest they will be effective when used in combination with one another or with conventional chemotherapeutics, and combinatorial epigenetic therapy regiments are being examined in cancer clinical trials. A greater understanding of the role of epigenetics in ovarian neoplasia will provide for improved interventions against this devastating malignancy. (Endocrinology 150: 4003– 4011, 2009)

varian cancer is the most lethal gynecological cancer, causing an estimated 15,520 U.S. deaths in 2008 (1). Due to few early symptoms, most (⬎70%) patients are diagnosed with advanced-stage disease and 5-yr survival rates are less than 20%, with only modestly improved survival over the past 40 yr (1). Although most advancedstage patients respond to standard chemotherapies, relapse occurs in over 70% of patients, resulting in chemoresistant, fatal disease (2). Altered epigenetic states are intimately associated with ovarian tumorigenesis. Epigenetics is defined as a heritable change in gene expression without alteration of the DNA sequence itself and includes DNA methylation, histone modification, nucleosome repositioning, and posttranscriptional gene regulation by micro-RNAs (miRNAs) (3, 4). The most studied epigenetic alteration is DNA meth-

O

ylation, the addition of a methyl moiety to the cytosine-5 position within the context of a CpG dinucleotide, mediated by DNA methyltransferases (DNMTs) (3). Although most CpG sites in the human genome are methylated, CpG-dense regions known as CpG islands (often geneassociated) are typically unmethylated in normal tissue (Fig. 1, top). Additionally, DNA-associated histone proteins are subject to extensive modifications that mediate the assembly of transcriptionally permissive or repressive (i.e. open or closed) chromatin (Fig. 1). It is now recognized that DNA methylation and histone modifications are intimately linked (3). The overall epigenetic state (e.g. DNA methylation, histone modification, and miRNA expression) corresponding to a specific cell phenotype is now referred to as the epigenome (5). Although repressive epigenetic modifications (including DNA methylation) reg-

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2009-0404 Received March 31, 2009. Accepted June 23, 2009. First Published Online July 2, 2009

Abbreviations: CSC, Cancer stem cell; DNMT, DNA methyltransferase; HDAC, histone deacetylase; HDACI, HDAC inhibitor; miRNA, micro-RNA.

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Normal:

RNA P o II Pol

DNA

activated

T F

Repeat DNA heterochromatin

CpG island

Tumor suppressor gene

Chromatin

T F

Cancer:

RNA Pol II

repressed

DNA

DNA methyltransferase

DNMT

Repeat DNA heterochromatin

CpG island

T F

transcrption factor

Tumor suppressor gene RNA Pol II

Chromatin

RNA polymerase II

methylcytosine cytosine H3K4me1/2/3 H3/H4 acetylation H3K9me2/3 H3K27me2/3

FIG. 1. Altered epigenetic states are intimately associated with ovarian tumorigenesis.

ulate genes in normal tissues (e.g. imprinted genes and female X-chromosome inactivation), these are significantly altered in cancer (3, 6). Specifically, in cancer cells, global DNA hypomethylation and localized hypermethylation of promoter-associated CpG islands occur (Fig. 1, bottom), with the latter serving as a surrogate for point mutations or deletions to cause transcriptional silencing of tumor suppressor genes (3). The most recently discovered epigenetic phenomenon is posttranscriptional gene down-regulation by small (21–23 nucleotides in length), non-protein-coding RNA molecules known as miRNAs (4, 7). About 1000 miRNA genes have been computationally predicted in the human genome, with each miRNA targeting multiple proteincoding transcripts (7). Although miRNAs are vital to normal cell physiology, their misexpression has been linked to cancer development, and miRNA profiles have now been used to classify human cancers (8). The influence of miRNAs on the epigenetic machinery and the reciprocal epigenetic regulation of miRNA expression (9) strongly suggests that miRNA deregulation during tumorigenesis has important implications for global regulation of epigenetics (10).

Epigenetic Aberrations in Ovarian Cancer (Table 1) Similar to all malignancies, aberrant DNA methylation, including global hypomethylation of heterochromatin and local CpG island methylation, occurs in ovarian cancer (11). Specific examples of hypomethylation include chromosome 1 satellite 2 and LINE-1 repetitive elements (11, 12). A number of genes, including the classical tumor suppressors BRCA1 (breast cancer susceptibility gene-1) (13), p16 (14), and MLH1 (15) as well as putative tumor suppressor (RASSF1A and OPCMLI) (16, 17), imprinted (ARH1 and PEG3I) (18) proapoptotic (LOT1, DAPK, TMS1/ASC, and PAR-4) (19 –21) and cell adhesion (ICAM-1 and CDH1) (22, 23) are hypermethylated and down-regulated in ovarian cancer. A newly identified gene, HSulf-1, encoding an arylsulfatase that acts on cell surface heparin sulfate proteoglycans and inhibits growth factor signaling and angiogenesis (24) was found methylated in over 50% of ovarian tumors and cell lines (25). PALB2 (partner and localizer of BRCA2) was reported to be hypermethylated similarly to BRCA1 (26) in inherited and sporadic breast and ovarian cancer, and hypermeth-

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Endocrinology, September 2009, 150(9):4003– 4011

TABLE 1.

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Altered epigenetic regulation in ovarian cancer

Epigenetic regulator DNA methylation

Modification Hypermethylation

Hypomethylation

Histone modifications

Lysine acetylation (gene-activating)

Type

Targets

Tumor suppressor genes

BRCA1 p16 hMLH1 RASSF1A OPCML ARLTS1 MYO18B SPARC CTGF ANGPTL2 Imprinted genes ARH1 PEG3 Cell adhesion genes ICAM-1 CDH1 Proapoptotic genes LOT1 DAPK TMS1/ASC PAR-4 Arylsulfatase gene Hsulf-1 DNA damage repair gene PALB2 TUBB3 Class III ␤-tubulin Lipid phosphatase PTEN Development genes HOXA10, HOXA11 Ribosomal genes 18S, 28S rDNAs Tumor promoter SNCG MCJ IGF2 BORIS Claudin-4 Acetylated histones H3 and H4 GATA4, GATA6 p21/WAF1 Cyclin B1 Trimethylated H3K4 GATA4, GATA6

Lysine methylation (gene-activating) Lysine methylation Dimethylated H3K9 (gene-repressing)

Trimethylated H3K27

miRNAs

4005

Up-regulation

Down-regulation

miR-200a miR-299-5p miR-135b miR-141 miR-200c miR-200b miR-214 miR-302d miR-373 miR-199a miR-140 miR-145 let-7i miR-15/16

ylation of the class III ␤-tubulin TUBB3 gene may contribute to taxane resistance (27). Recently discovered, candidate tumor suppressor genes hypermethylated in ovarian cancer include SPARC (secreted protein acidic

Refs. 42 14 15 16 17 87 86 28 30 29 18 18 22 23 19 15 21 20 24, 25 26 27 85 31 92 33 32 35 34 36 46 48 47 46

GATA4, GATA6

46

Adam19 GATA4, GATA6 Adam19 RASSF1 BAP1, SIP1, ZEB1/2 DLK1 MSX2 BAP1 BAP1, ZEB1/2, SIP1 BAP1, ZEB1/2, SIP1 PTEN VEGFA VEGFA c-SRK, MMP13, and FGF2 c-SRK, MMP13, and FGF2, VEGFA c-SRK, MMP13, and FGF2,PARP8, IRS1 Unknown BCL2

49 46 49 50 8, 54 8 8 8 8, 54 8, 54 8, 52 88 88 8 8, 88 8, 89, 90 53 91

and rich in cysteine) (28), ANGPTL2 (angiopoietin-like protein 2) (29), and CTGF (connective tissue growth factor) (30), whereas methylation of the embryonic developmentally regulated genes HOXA10 and HOXA11 was

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also found to be highly discriminative between normal and malignant ovarian tissues (31). In addition to repetitive elements and DNA satellites, a number of protein-coding genes are overexpressed in ovarian cancer, in association with promoter hypomethylation. These include MCJ (methylation-controlled DNAJ gene), associated with chemoresistance (32); SNCG (synuclein-␥), encoding an activator of the MAPK and Elk-1 signal cascades (33); and BORIS (brother of the regulator of imprinted sites), a cancer testis antigen-family candidate oncogene (34). Other ovarian cancer-hypomethylated genes include IGF2, an imprinted gene implicated in numerous malignancies (35), and claudin-4, whose overexpression leads to disrupted tight junctions between epithelial ovarian cancer cells (36). Although candidate gene studies (as described above) have successfully identified a number of important epigenetically regulated genes in ovarian cancer, allowing greater insight into disease progression, we have also globally examined DNA hypermethylation (using methylation microarrays) to demonstrate that ovarian tumors contain a large number of hypermethylated loci (37). Such disease stage-specific methylated loci represent possible methylation signatures for classification and possible targets for therapy (38, 39), and these and other (40) studies demonstrate that CpG island methylation is cumulative with ovarian cancer progression. Although the relationship between gene hypermethylation in ovarian cancer and altered DNMT RNA levels is not straightforward (41, 42), functional validation of promoter DNA hypermethylation in ovarian carcinogenesis was further shown by Huang and co-workers (41) who, by downregulating two DNA methyltransferase enzymes, demonstrated that extensive loss of CpG hypermethylation correlates significantly with ovarian cancer cell growth inhibition. One of the most studied genes in ovarian cancer is BRCA1, due to its role in both inherited and sporadic forms of this disease (42). The clinical outcome for ovarian cancer patients having tumor-hypermethylated BRCA1 has recently been compared with patients with germline BRCA1 mutations or wild-type BRCA1 (43). BRCA1 hypermethylation occurs in 10 –15% of sporadic disease cases, associates strongly with loss of BRCA1 RNA and protein (13), and significantly correlates with poor patient outcome (42). These studies suggest that BRCA1 hypermethylation, which has also been reported in ovarian cancer patient serum (16), may represent a minimally invasive approach for predicting patient response to standard therapies. Although histone modifications (in addition to DNA methylation) regulate numerous normal ovarian func-

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tions, including estrogen synthesis, folliculogenesis, and luteal phase activity (44), ovarian cancer cells significantly alter their expression of chromatin-modifying proteins (45). Additionally, specific histone modifications (but not promoter DNA methylation) act to regulate the differentiation genes GATA4 and GATA6 (46) and the cell cycle regulatory proteins cyclinB1 (47) and p21WAF1/CIP1 (48). Similarly, we recently showed that in ovarian cancer cells refractory to TGF-␤1, two repressive histone modifications (trimethyl-H3K27 and dimethyl-H3K9), in concert with histone deacetylase (HDAC) enzymes, down-regulate ADAM19 (a disintegrin and metalloprotease domain 19) in the absence of significant CpG island methylation (49). Those results demonstrate that aberrant TGF-␤1 signaling can result in formation of a repressive chromatin environment, without DNA methylation, in ovarian cancer cells. Similarly, using a dominant-negative histone overexpression approach, our group showed that genome-wide loss of the repressive trimethyl-H3K27 mark associated with reduced global DNA methylation, allowing platinum resensitization of chemoresistant ovarian cancer cells, with one of the affected genes, RASSF1 shown to be a direct target of H3K27 methylation-mediated silencing (50). Because loss of H3K27 trimethylation has also been associated with poor prognosis in ovarian and other malignancies (51), and gene promoter DNA methylation can be maintained in the absence of this repressive mark (50), the above findings collectively demonstrate that complex epigenetic patterns, involving DNA methylation and histone modifications, contribute to ovarian cancer progression and drug resistance. miRNAs represent the most recently discovered epigenetic phenomenon, and ovarian tumors were recently found to significantly up-regulate miR-199a, miR-200a, and miR-214 and down-regulate miR-100, and specifically, miR-214 was demonstrated to target the tumor suppressor PTEN and associate with platinum resistance (8, 52). The miRNA let-7i was recently found to be a tumor suppressor significantly down-regulated in platinumresistant ovarian tumors, and let-7i gain-of-function restored drug sensitivity of chemoresistant ovarian cancer cells, thus representing a candidate biomarker and therapeutic target (53). miR-429, miR-200a, and miR-200b were found to be clustered on a single primary transcript regulated by the epithelial-to-mesenchymal transition (EMT, a metastatic phenotype) repressor ZEB1/SIP1, with miR-200a and miR-200b negatively regulating ZEB1/SIP1 and creating a double-negative feedback cycle (54). In another study, 27 miRNAs significantly associated with chemotherapy response (55), showing that (similar to DNA methylation) miRNAs represent

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possible prognostic and diagnostic biomarkers for ovarian cancer. With respect to miRNA gene regulation, a group of six miRNAs clustered on chromosome 19, and seven clustered on chromosome 14, were up-regulated by the DNMT inhibitor decitabine (described below), demonstrating that miRNAs can be regulated by DNA methylation (56). Moreover, an overall, collective tumor-suppressive effect of miRNAs was suggested by down-regulation of Drosha and Dicer, two enzymes involved in miRNA processing, being significantly associated with advanced ovarian cancer stage and poor prognosis (57).

Epigenetic Therapies and Biomarkers for Ovarian Cancer As mentioned above, ovarian cancer tumor progression is well characterized by a number of combinatorial epigenetic aberrations distinct to this malignancy, including (but not limited to) DNA methylation of RASSF1A, DAPK, H-Sulf-1, BRCA1, and HOXA10. Consequently, these methylated DNA sequences represent potential biomarkers for diagnosis, staging, prognosis (i.e. prognostic biomarkers), and monitoring of response to therapy (predictive biomarkers) (15). DNA methylation biomarkers hold a number of advantages over other biomarker types, such as proteins, gene expression, and DNA mutations, including their stability, ability to be amplified (thus greatly enhancing detection sensitivity), relatively low cost of assessment, and restriction to limited regions of DNA (CpG islands) (58). In the future, it is highly likely that DNA methylation analyses of resected ovarian tumors will be used to individually tailor treatment, similar to recently discovered predictive markers in stage I non-small-cell lung cancer (59). Although methylation assessment of single genes lacks sufficient specificity for ovarian cancer diagnostics, it is believed that panels of multiple methylation biomarkers may achieve the accuracy required for widespread population screening (58, 60). Toward that objective, a panel of 112 methylated DNA markers was found to associate with ovarian cancer progression-free survival (39). Similarly, methylation of MCJ and hMLH1 correlates with chemotherapy response (38), and methylation of a four-gene panel was found to significantly predict overall survival and relapse (61). In ovarian cancer particularly, methylation biomarkers could likely augment the specificity of CA-125, similar to ongoing prostate cancer studies examining various prostate-specific antigen/ biomarkers (62). In addition to tissue analysis, methylated DNA has been detected in the serum and peritoneal fluid of ovarian cancer patients (15, 16, 38, 61). Methylated DNA found

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in cancer patient serum correlated reasonably well with methylation levels in tumor tissue (16, 61, 63), and it is also believed that the source of serum DNA is necrotic tumor cells. Consequently, detection of methylated DNA biomarkers in body fluids has significant potential as a minimally invasive tool to regularly assess ovarian cancer patient response. A major impediment to improving survival of ovarian cancer patients is the development of chemoresistance, and specifically, platinum resistance is strongly associated with methylation-induced silencing of various drug response genes and pathways. Whereas genetic mutations, deletions, or allelic losses are fixed and irreversible, epigenetic abnormalities can potentially be corrected (3, 6). In this regard, several drugs that inhibit DNMT activity are now in clinical use. These drugs act by covalently and irreversibly binding to the DNMT enzyme active site, resulting in genomic hypomethylation (64). Although monotherapy of these agents is effective against hematological malignancies, their activity against solid tumors has been disappointing, suggesting greater promise in drug combinations (60, 64). In preclinical studies, various DNMT inhibitors were found to elicit DNA hypomethylation and reverse chemoresistance of platinum-resistant ovarian cancer cells (65– 67) and mouse xenografts (68), laying the foundation for the clinical evaluation of DNMT inhibitors for chemotherapy resensitization in ovarian cancer patients (69). To that end, we are currently conducting a phase I/II trial (NCT00477386, Study ID 0704-07, www.clinicaltrials.gov) of one DNMT inhibitor, decitabine (Dacogen; Eisai, Inc., Tokyo, Japan) paired with carboplatin, hypothesizing that low-dose decitabine derepresses silenced tumor suppressors to resensitize platinum-resistant ovarian tumors to carboplatin. The phase I component of this study is now complete, demonstrating safety of the combined regimen and biological activity in vivo, as assessed by decreased methylation of genome-wide repetitive elements and specific genes (70). Because histone deacetylation is another transcriptional silencing mechanism in ovarian cancer, HDAC inhibitors (HDACIs) can relieve epigenetic gene repression and exert anticancer effects by inhibiting the deacetylation of nonhistone proteins (71). Similar to DNMT inhibitors, however, poor single-agent activity of HDACIs against solid tumors has been demonstrated. In a single-agent ovarian cancer trial of the HDACI vorinostat (Zolinza; Merck & Co., Inc.), only one of 27 patients experienced a partial response (72), suggesting that HDACIs may be more effective when used in combination with other agents (60, 71). In one preclinical ovarian cancer study, the third-generation HDACI belinostat (CuraGen Corp.,

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Endocrinology, September 2009, 150(9):4003– 4011

OCICs survive

OCICs proliferate regenerate tumor

Patient relapses; tumor now highly drug resistant

Future Directions

Recent findings suggest a role for tumor progenitors, known as cancer-initiating or cancer stem cells (CSCs), in the propagation of a drug-resistant phenotype in numerous malignancies (77), including ovarian cancer (78 – 81) (Fig. 2). The Ta rg cancer stem cell hypothesis posits e us ted Tumor ing th that conventional chemotherapies, e m O ra e bulk 1, .g., O arke CIC py Na rs c targeted to highly mitotic cells, fail to no t4, N g, oth otc Tumor will shrink due destroy quiescent or slowly dividing ers hto its inability to ) OCICs CSCs, which then regrow the tumor regenerate killed (77). Because epigenetic therapies are FIG. 2. Targeting ovarian cancer-initiating/stem cells (OCIC). well-established differentiating agents (60, 64), they may also target poorly Branford, CT) resensitized platinum-resistant xenografts differentiated CSCs (3) (Fig. 2). Moreover, the realizain mice (73). Likewise, our group also demonstrated that tion of the Human Epigenome Project, an exhaustive another rationally designed HDACI, AR-42 (Arno Therannotation of all deoxcytosine and histone modificaapeutics, Parsippany, NJ; www.arnothera.com), could retions throughout the human genome (82), could allow sensitize platinum-resistant ovarian cancer cells and xenofor the establishment of epigenetic ovarian cancer digrafts, even more potently than vorinostat (74); AR-42 is agnostic, prognostic, and pharmacodynamic biomarkscheduled to begin clinical trials this year. Because additive ers (15, 83, 84) (Fig. 3). In summary, a greater underor synergistic effects of HDAC and DNMT inhibitor comstanding of the role of epigenetics in ovarian cancer will binations on silenced gene reexpression have been demallow for improved interventions against this devastatonstrated (75), combining these two classes of epigenetic ing malignancy. drugs with conventional therapies may be the most effective approach to use in the clinic (3, 60, 64). In support of this possibility, one preclinical study showed that a comAcknowledgments bination of decitabine with belinostat elicited greater platinum resensitization of resistant ovarian cancer xeno- Address all correspondence and requests for reprints to: Professor Kenneth P. Nephew, Medical Sciences, Indiana University grafts than decitabine alone (76). OCICs

ap y ther of d ar d lk Stan ates bu ICs in OC elim but not r tumo

)

FIG. 3. A, Normal cell epigenome; B, ovarian cancer cell epigenome.

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School of Medicine, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, Indiana 47405-4401. E-mail: [email protected]. This work was supported by National Institutes of Health, National Cancer Institute Grants CA085289 (to K.P.N.), CA113001 (to T.T.-M.H), CA133877 (to D.E.M), Ovar’coming Together (Indianapolis, IN; to C.B.), the Walther Cancer Institute (Indianapolis, IN; to K.P.N.), and Phi Beta Psi Sorority (Brownsburg, IN; to K.P.N.). Disclosure Summary: C.B., F.F., D.E.M., T.H.-M.H., and K.P.N. have nothing to disclose.

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