Phase 1b study to assess the safety, tolerability, and clinical activity of pamiparib in combination with temozolomide in patients with locally advanced or metastatic solid tumors.

BACKGROUND: Pamiparib is a potent, selective, poly (ADP-ribose) polymerase 1/2 inhibitor that demonstrates synthetic lethality in cells with breast cancer susceptibility gene mutations or other homologous recombination deficiency. This two-stage phase 1b study (NCT03150810) assessed pamiparib in combination with temozolomide (TMZ) in adult patients with histologically confirmed locally advanced and metastatic solid tumors. METHODS: Oral pamiparib 60 mg was administered twice daily. During the dose-escalation stage, increasing doses of TMZ (40-120 mg once daily pulsed or 20-40 mg once daily continuous) were administered to determine the recommended dose to be administered in the dose-expansion stage. The primary objectives were to determine safety and tolerability, maximum tolerated/administered dose, recommended phase 2 dose and schedule, and antitumor activity of pamiparib in combination with TMZ. Pharmacokinetics of pamiparib and TMZ and biomarkers were also assessed. RESULTS: Across stages, 139 patients were treated (dose escalation, n = 66; dose expansion, n = 73). The maximum tolerated dose of TMZ, which was administered during dose expansion, was 7-day pulsed 60 mg once daily. The most common treatment-emergent adverse events (TEAEs) were anemia (dose escalation, 56.1%; dose expansion, 63.0%), nausea (dose escalation, 54.5%; dose expansion, 49.3%), and fatigue (dose escalation, 48.5%; dose expansion, 47.9%). In the dose-escalation stage, four patients experienced dose-limiting toxicities (three neutropenia and one neutrophil count decreased). No TEAEs considered to be related to study drug treatment resulted in death. Antitumor activity was modest, indicated by confirmed overall response rate (dose escalation, 13.8%; dose expansion, 11.6%), median progression-free survival (3.7 and 2.8 months), and median overall survival (10.5 and 9.2 months). Administration of combination therapy did not notably impact pamiparib or TMZ pharmacokinetics. CONCLUSIONS: Pamiparib in combination with TMZ had a manageable safety profile. Further investigation of the efficacy of this combination in tumor types with specific DNA damage repair deficiencies is warranted.

Pamiparib in patients with locally advanced or metastatic HER2-negative breast cancer with germline BRCA mutations: a phase II study.

PURPOSE: To evaluate the efficacy and safety of pamiparib in patients with locally advanced or metastatic human epidermal growth factor receptor 2-negative (HER2-) breast cancer, with deleterious or suspected deleterious germline BRCA1/2 mutations (gBRCA1/2 m). METHODS: In this open-label, phase II, multicenter study in China (NCT03575065), patients with triple-negative breast cancer (TNBC cohort) or hormone receptor-positive (HR+)/HER2- breast cancer (HR+/HER2- cohort) and ≤ 2 prior lines of chemotherapy received pamiparib 60 mg orally twice daily in 28-day, continuous cycles. The primary endpoint was objective response rate (ORR; RECIST v1.1) by independent review committee. RESULTS: In total, 88 patients were enrolled (TNBC cohort: 62; HR+/HER2- cohort: 26). Median age was 45.5 (range: 27-67) years, and 60 patients (68.2%) had received 1 or 2 prior lines of chemotherapy; 42 patients (47.7%) had previously received platinum chemotherapy. In the TNBC cohort, ORR was 38.2% (95% confidence interval [CI] 25.4-52.3) and median duration of response (DoR) was 7.0 months (95% CI 3.9-not estimable). In the HR+/HER2- cohort, ORR was 61.9% (95% CI 38.4-81.9) and median DoR was 7.5 months (95% CI 5.6-14.8). The most common treatment-emergent adverse events (TEAEs), treatment-related TEAEs, and ≥ Grade 3 TEAEs were hematologic (including anemia, decreased neutrophil count, and decreased white blood cell count). Overall, 64.8% of patients had TEAEs leading to dose reduction and 2.3% had TEAEs leading to treatment discontinuation. CONCLUSION: Pamiparib showed encouraging efficacy and an acceptable safety profile in patients with locally advanced and metastatic HER2- breast cancer with gBRCA1/2 m. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03575065; July 2, 2018.

Pamiparib dose escalation in Chinese patients with non-mucinous high-grade ovarian cancer or advanced triple-negative breast cancer.

BACKGROUND: The recommended phase 2 dose (RP2D) of pamiparib, an investigational PARP1/2 inhibitor, was established as 60 mg twice daily (BID) in a first-in-human (FIH) study (NCT02361723). METHODS: Chinese patients with advanced non-mucinous high-grade ovarian cancer (HGOC) or triple-negative breast cancer (TNBC) whose disease either progressed despite standard therapy, or for which there is no standard therapy were enrolled in the dose-escalation (DE) portion of a phase 1/2 study (NCT03333915). The primary endpoint was safety/tolerability; secondary objectives were pharmacokinetics and antitumor activity. BRCA1/2 mutation status was retrospectively evaluated. RESULTS: Nine HGOC and six TNBC patients (N = 15; n = 4, 20 mg; n = 4, 40 mg; n = 7, 60 mg) were enrolled; as of 30 September 2019, one HGOC patient remained on treatment. Seven patients (n = 5, HGOC; n = 2, TNBC) had germline BRCA1/2 mutation (gBRCAmut ); all HGOC patients were resistant/refractory to platinum. Asthenia and nausea (n = 12 each) were the most common treatment-related adverse events (TRAEs). Decreased hemoglobin was the most common grade 3 TRAE (n = 3); no grade ≥4 AEs were observed. No dose-limiting toxicities (DLTs) were reported. Pamiparib plasma exposure was similar to exposure observed in the FIH study after a single-dose administration, albeit slightly higher at steady state. Among 13 RECIST-evaluable patients, two with HGOC (gBRCAmut , n = 1) achieved a confirmed partial response and six with HGOC (gBRCAmut , n = 4) achieved stable disease; all TNBC RECIST-evaluable patients (n = 5) reported progressive disease. CONCLUSIONS: Pamiparib was generally well tolerated in Chinese patients, with durable responses observed in patients with HGOC. Based on these results, pamiparib 60 mg BID was confirmed as the RP2D.

Global DNA Hypomethylation in Epithelial Ovarian Cancer: Passive Demethylation and Association with Genomic Instability.

A hallmark of human cancer is global DNA hypomethylation (GDHO), but the mechanisms accounting for this defect and its pathological consequences have not been investigated in human epithelial ovarian cancer (EOC). In EOC, GDHO was associated with advanced disease and reduced overall and disease-free survival. GDHO (+) EOC tumors displayed a proliferative gene expression signature, including FOXM1 and CCNE1 overexpression. Furthermore, DNA hypomethylation in these tumors was enriched within genomic blocks (hypomethylated blocks) that overlapped late-replicating regions, lamina-associated domains, PRC2 binding sites, and the H3K27me3 histone mark. Increased proliferation coupled with hypomethylated blocks at late-replicating regions suggests a passive hypomethylation mechanism. This hypothesis was further supported by our observation that cytosine DNA methyltransferases (DNMTs) and UHRF1 showed significantly reduced expression in GDHO (+) EOC after normalization to canonical proliferation markers, including MKI67. Finally, GDHO (+) EOC tumors had elevated chromosomal instability (CIN), and copy number alterations (CNA) were enriched at the DNA hypomethylated blocks. Together, these findings implicate a passive DNA demethylation mechanism in ovarian cancer that is associated with genomic instability and poor prognosis.

Epigenetic activation of POTE genes in ovarian cancer.

The POTE gene family consists of 14 homologous genes localized to autosomal pericentromeres, and a sub-set of POTEs are cancer-testis antigen (CTA) genes. POTEs are over-expressed in epithelial ovarian cancer (EOC), including the high-grade serous subtype (HGSC), and expression of individual POTEs correlates with chemoresistance and reduced survival in HGSC. The mechanisms driving POTE overexpression in EOC and other cancers is unknown. Here, we investigated the role of epigenetics in regulating POTE expression, with a focus on DNA hypomethylation. Consistent with their pericentromeric localization, Pan-POTE expression in EOC correlated with expression of the pericentromeric repeat NBL2, which was not the case for non-pericentromeric CTAs. POTE genomic regions contain LINE-1 (L1) sequences, and Pan-POTE expression correlated with both global and POTE-specific L1 hypomethylation in EOC. Analysis of individual POTEs using RNA-seq and DNA methylome data from fallopian tube epithelia (FTE) and HGSC revealed that POTEs C, E, and F have increased expression in HGSC in conjunction with DNA hypomethylation at 5' promoter or enhancer regions. Moreover, POTEs C/E/F showed additional increased expression in recurrent HGSC in conjunction with 5' hypomethylation, using patient-matched samples. Experiments using decitabine treatment and DNMT knockout cell lines verified a functional contribution of DNA methylation to POTE repression, and epigenetic drug combinations targeting histone deacetylases (HDACs) and histone methyltransferases (HMTs) in combination with decitabine further increased POTE expression. In summary, several alterations of the cancer epigenome, including pericentromeric activation, global and locus-specific L1 hypomethylation, and locus-specific 5' CpG hypomethylation, converge to promote POTE expression in ovarian cancer.

Expression of the POTE gene family in human ovarian cancer.

The POTE family includes 14 genes in three phylogenetic groups. We determined POTE mRNA expression in normal tissues, epithelial ovarian and high-grade serous ovarian cancer (EOC, HGSC), and pan-cancer, and determined the relationship of POTE expression to ovarian cancer clinicopathology. Groups 1 & 2 POTEs showed testis-specific expression in normal tissues, consistent with assignment as cancer-testis antigens (CTAs), while Group 3 POTEs were expressed in several normal tissues, indicating they are not CTAs. Pan-POTE and individual POTEs showed significantly elevated expression in EOC and HGSC compared to normal controls. Pan-POTE correlated with increased stage, grade, and the HGSC subtype. Select individual POTEs showed increased expression in recurrent HGSC, and POTEE specifically associated with reduced HGSC OS. Consistent with tumors, EOC cell lines had significantly elevated Pan-POTE compared to OSE and FTE cells. Notably, Group 1 & 2 POTEs (POTEs A/B/B2/C/D), Group 3 POTE-actin genes (POTEs E/F/I/J/KP), and other Group 3 POTEs (POTEs G/H/M) show within-group correlated expression, and pan-cancer analyses of tumors and cell lines confirmed this relationship. Based on their restricted expression in normal tissues and increased expression and association with poor prognosis in ovarian cancer, POTEs are potential oncogenes and therapeutic targets in this malignancy.

PRAME expression and promoter hypomethylation in epithelial ovarian cancer.

PRAME is a cancer-testis antigen (CTA) and potential immuno-therapeutic target, but has not been well-studied in epithelial ovarian cancer (EOC) or its high grade serous (HGSC) subtype. Compared to normal ovary, PRAME expression was significantly increased most EOC, regardless of stage and grade. Interestingly, PRAME mRNA expression was associated with improved survival in the HGSC subtype. The PRAME locus was a frequent target for copy number alterations (CNA) in HGSC but most changes were heterozygous losses, indicating that elevated PRAME expression is not typically due to CNA. In contrast, PRAME promoter DNA hypomethylation was very common in EOC and HGSC and correlated with increased PRAME expression. PRAME expression and promoter hypomethylation both correlated with LINE-1 hypomethylation, a biomarker of global DNA hypomethylation. Pharmacologic or genetic disruption of DNA methyltransferase (DNMT) enzymes activated PRAME expression in EOC cells. Immunohistochemistry (IHC) of PRAME in EOC revealed frequent, but low level, protein expression, and expression was confined to epithelial cells and localized to the cytoplasm. Cytoplasmic PRAME expression was positively associated with PRAME mRNA expression and negatively associated with promoter methylation, but the latter correlation was not statistically significant. PRAME protein expression did not correlate with EOC clinicopathology or survival. In summary, PRAME is frequently expressed in EOC at the mRNA and protein levels, and DNA methylation is a key mechanism regulating its expression. These data support PRAME as an immunotherapy target in EOC, and suggest treatment with DNMT inhibitors as a means to augment PRAME immunotherapy.

DNA Methylome Analyses Implicate Fallopian Tube Epithelia as the Origin for High-Grade Serous Ovarian Cancer.

UNLABELLED: High-grade serous ovarian cancer (HGSC) is the most common and lethal form of epithelial ovarian cancer (EOC). Two distinct tissues have been suggested as the tissue of origin: ovarian surface epithelia (OSE) and fallopian tube epithelia (FTE). We hypothesized that the DNA methylome of HGSC should more closely resemble the methylome of its tissue of origin. To this end, we profiled HGSC (n = 10), and patient-matched OSE and FTE (n = 5) primary fresh-frozen tissues, and analyzed the DNA methylome using Illumina 450K arrays (n = 20) and Agilent Sure Select methyl-seq (n = 7). Methylomes were compared using statistical analyses of differentially methylated CpG sites (DMC) and differentially methylated regions (DMR). In addition, methylation was evaluated within a variety of different genomic contexts, including CpG island shores and Homeobox (HOX) genes, due to their roles in tissue specification. Publicly available HGSC methylome data (n = 628) were interrogated to provide additional comparisons with FTE and OSE for validation. These analyses revealed that HGSC and FTE methylomes are significantly and consistently more highly conserved than are HGSC and OSE. Pearson correlations and hierarchal clustering of genes, promoters, CpG islands, CpG island shores, and HOX genes all revealed increased relatedness of HGSC and FTE methylomes. Thus, these findings reveal that the landscape of FTE more closely resembles HGSC, the most common and deadly EOC subtype. IMPLICATIONS: DNA methylome analyses support the hypothesis that HGSC arise from the fallopian tube and that due to its tissue-specificity and biochemical stability, interrogation of the methylome may be a valuable approach to examine cell/tissue lineage in cancer. Mol Cancer Res; 14(9); 787-94. ©2016 AACR.

Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression.

The FOXM1 transcription factor network is frequently activated in high-grade serous ovarian cancer (HGSOC), the most common and lethal subtype of epithelial ovarian cancer (EOC). We used primary human EOC tissues, HGSOC cell lines, mouse and human ovarian surface epithelial (OSE) cells, and a murine transgenic ovarian cancer model to investigate genetic determinants of FOXM1 overexpression in EOC, and to begin to define its functional contribution to disease pathology. The Cancer Genome Atlas (TCGA) data indicated that the FOXM1 locus is amplified in ~12% of HGSOC, greater than any other tumor type examined, and that FOXM1 amplification correlates with increased expression and poor survival. In an independent set of primary EOC tissues, FOXM1 expression correlated with advanced stage and grade. Of the three known FOXM1 isoforms, FOXM1c showed highest expression in EOC. In murine OSE cells, combined knockout of Rb1 and Trp53 synergistically induced FOXM1. Consistently, human OSE cells immortalized with SV40 Large T antigen (IOSE-SV) had significantly higher FOXM1 expression than OSE immortalized with hTERT (IOSE-T). FOXM1 was overexpressed in murine ovarian tumors driven by combined Rb1/Trp53 disruption. FOXM1 induction in IOSE-SV cells was partially dependent on E2F1, and FOXM1 expression correlated with E2F1 expression in human EOC tissues. Finally, FOXM1 functionally contributed to cell cycle progression and relevant target gene expression in human OSE and HGSOC cell models. In summary, gene amplification, p53 and Rb disruption, and E2F1 activation drive FOXM1 expression in EOC, and FOXM1 promotes cell cycle progression in EOC cell models.

DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.

Epigenetic potentiation of NY-ESO-1 vaccine therapy in human ovarian cancer.

The cancer-testis/cancer-germline antigen NY-ESO-1 is a vaccine target in epithelial ovarian cancer (EOC), but its limited expression is a barrier to vaccine efficacy. As NY-ESO-1 is regulated by DNA methylation, we hypothesized that DNA methyltransferase (DNMT) inhibitors may augment NY-ESO-1 vaccine therapy. In agreement, global DNA hypomethylation in EOC was associated with the presence of circulating antibodies to NY-ESO-1. Pre-clinical studies using EOC cell lines showed that decitabine treatment enhanced both NY-ESO-1 expression and NY-ESO-1-specific CTL-mediated responses. Based on these observations, we performed a phase I dose-escalation trial of decitabine, as an addition to NY-ESO-1 vaccine and doxorubicin liposome (doxorubicin) chemotherapy, in 12 patients with relapsed EOC. The regimen was safe, with limited and clinically manageable toxicities. Both global and promoter-specific DNA hypomethylation occurred in blood and circulating DNAs, the latter of which may reflect tumor cell responses. Increased NY-ESO-1 serum antibodies and T cell responses were observed in the majority of patients, and antibody spreading to additional tumor antigens was also observed. Finally, disease stabilization or partial clinical response occurred in 6/10 evaluable patients. Based on these encouraging results, evaluation of similar combinatorial chemo-immunotherapy regimens in EOC and other tumor types is warranted.

LINE1 and Alu repetitive element DNA methylation in tumors and white blood cells from epithelial ovarian cancer patients.

OBJECTIVE: We determined whether DNA methylation of repetitive elements (RE) is altered in epithelial ovarian cancer (EOC) patient tumors and white blood cells (WBC), compared to normal tissue controls. METHODS: Two different quantitative measures of RE methylation (LINE1 and Alu bisulfite pyrosequencing) were used in normal and tumor tissues from EOC cases and controls. Tissues analyzed included: i) EOC, ii) normal ovarian surface epithelia (OSE), iii) normal fallopian tube surface epithelia (FTE), iv) WBC from EOC patients, obtained before and after treatment, and v) WBC from demographically-matched controls. RESULTS: REs were significantly hypomethylated in EOC compared to OSE and FTE, and LINE1 and Alu methylation showed a significant direct association in these tissues. In contrast, WBC RE methylation was significantly higher in EOC cases compared to controls. RE methylation in patient-matched EOC tumors and pre-treatment WBC did not correlate. CONCLUSIONS: EOC shows robust RE hypomethylation compared to normal tissues from which the disease arises. In contrast, RE are generally hypermethylated in EOC patient WBC compared to controls. EOC tumor and WBC methylation did not correlate in matched patients, suggesting that RE methylation is independently controlled in tumor and normal tissues. Despite the significant differences observed over the population, the range of RE methylation in patient and control WBC overlapped, limiting their specific utility as an EOC biomarker. However, our data demonstrate that DNA methylation is deranged in normal tissues from EOC patients, supporting further investigation of WBC DNA methylation biomarkers suitable for EOC risk assessment.

DNA methylation and nucleosome occupancy regulate the cancer germline antigen gene MAGEA11.

MAGEA11 is a cancer germline (CG) antigen and androgen receptor co-activator. Its expression in cancers other than prostate, and its mechanism of activation, has not been reported. In silico analyses reveal that MAGEA11 is frequently expressed in human cancers, is increased during tumor progression, and correlates with poor prognosis and survival. In prostate and epithelial ovarian cancers (EOC), MAGEA11 expression was associated with promoter and global DNA hypomethylation, and with activation of other CG genes. Pharmacological or genetic inhibition of DNA methyltransferases (DNMTs) and/or histone deacetylases (HDACs) activated MAGEA11 in a cell line specific manner. MAGEA11 promoter activity was directly repressed by DNA methylation, and partially depended on Sp1, as pharmacological or genetic targeting of Sp1 reduced MAGEA11 promoter activity and endogenous gene expression. Importantly, DNA methylation regulated nucleosome occupancy specifically at the -1 positioned nucleosome of MAGEA11. Methylation of a single Ets site near the transcriptional start site (TSS) correlated with -1 nucleosome occupancy and, by itself, strongly repressed MAGEA11 promoter activity. Thus, DNA methylation regulates nucleosome occupancy at MAGEA11, and this appears to function cooperatively with sequence-specific transcription factors to regulate gene expression. MAGEA11 regulation is highly instructive for understanding mechanisms regulating CG antigen genes in human cancer.

BORIS/CTCFL mRNA isoform expression and epigenetic regulation in epithelial ovarian cancer.

Cancer germline (CG) genes are normally expressed in germ cells and aberrantly expressed in a variety of cancers; their immunogenicity has led to the widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL is an autosomal CG antigen and promising cancer vaccine target. BORIS is the only known paralog of CTCF, a gene intimately involved in genomic imprinting, chromatin insulation, and nuclear regulation. We have previously shown that BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression coincides with promoter and global DNA hypomethylation. Recently, 23 different BORIS mRNA variants have been described, and have been functionally grouped into six BORIS isoform families (sf1-sf6). In the present study, we have characterized the expression of BORIS isoform families in normal ovary (NO) and EOC, the latter of which were selected to include two groups with widely varying global DNA methylation status. We find selective expression of BORIS isoform families in NO, which becomes altered in EOC, primarily by the activation of BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we find that BORIS sf1 and sf2 isoform families are selectively activated in globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform families was induced to varying extents by epigenetic modulatory drugs in EOC cell lines, particularly when DNMT and HDAC inhibitors were used in combination.

Coordinated cancer germline antigen promoter and global DNA hypomethylation in ovarian cancer: association with the BORIS/CTCF expression ratio and advanced stage.

PURPOSE: Cancer germline (CG) antigens are frequently expressed and hypomethylated in epithelial ovarian cancer (EOC), but the relationship of this phenomenon to global DNA hypomethylation is unknown. In addition, the potential mechanisms leading to DNA hypomethylation, and its clinicopathologic significance in EOC, have not been determined. EXPERIMENTAL DESIGN: We used quantitative mRNA expression and DNA methylation analyses to determine the relationship between expression and methylation of X-linked (MAGE-A1, NY-ESO-1, XAGE-1) and autosomal (BORIS, SOHLH2) CG genes, global DNA methylation (5mdC levels, LINE-1, Alu, and Sat-α methylation), and clinicopathology, using 75 EOC samples. In addition, we examined the association between these parameters and a number of mechanisms proposed to contribute to DNA hypomethylation in cancer. RESULTS: CG genes were coordinately expressed in EOC and this was associated with promoter DNA hypomethylation. Hypomethylation of CG promoters was highly correlated and strongly associated with LINE-1 and Alu methylation, moderately with 5mdC levels, and rarely with Sat-α methylation. BORIS and LINE-1 hypomethylation, and BORIS expression, were associated with advanced stage. GADD45A expression, MTHFR genotype, DNMT3B isoform expression, and BORIS mRNA expression did not associate with methylation parameters. In contrast, the BORIS/CTCF expression ratio was associated with DNA hypomethylation, and furthermore correlated with advanced stage and decreased survival. CONCLUSIONS: DNA hypomethylation coordinately affects CG antigen gene promoters and specific repetitive DNA elements in EOC, and correlates with advanced stage disease. The BORIS/CTCF mRNA expression ratio is closely associated with DNA hypomethylation and confers poor prognosis in EOC.

Lack of evidence for green tea polyphenols as DNA methylation inhibitors in murine prostate.

Green tea polyphenols (GTP) have been reported to inhibit DNA methylation in cultured cells. Here, we tested whether oral consumption of GTPs affects normal or cancer-specific DNA methylation in vivo, using mice. Wild-type (WT) and transgenic adenocarcinoma of mouse prostate (TRAMP) mice were given 0.3% GTPs in drinking water beginning at 4 weeks of age. To monitor DNA methylation, we measured 5-methyl-deoxycytidine (5mdC) levels, methylation of the B1 repetitive element, and methylation of the Mage-a8 gene. Each of these parameters were unchanged in prostate, gut, and liver from WT mice at both 12 and 24 weeks of age, with the single exception of a decrease of 5mdC in the liver at 12 weeks. In GTP-treated TRAMP mice, 5mdC levels and the methylation status of four loci hypermethylated during tumor progression were unaltered in TRAMP prostates at 12 or 24 weeks. Quite surprisingly, GTP treatment did not inhibit tumor progression in TRAMP mice, although known pharmacodynamic markers of GTPs were altered in both WT and TRAMP prostates. We also administered 0.1%, 0.3%, or 0.6% GTPs to TRAMP mice for 12 weeks and measured 5mdC levels and methylation of B1 and Mage-a8 in prostate, gut, and liver tissues. No dose-dependent alterations in DNA methylation status were observed. Genome-wide DNA methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR assay also revealed no significant hypomethylating effect of GTP. These data indicate that oral administration of GTPs does not affect normal or cancer-specific DNA methylation in the murine prostate.