Epigenetic activation of POTE genes in ovarian cancer

ABSTRACT

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.

KEYWORDS:

• POTE
• Ovarian cancer
• high-grade serous ovarian cancer
• DNA hypomethylation
• pericentromeres
• LINE1

Introduction

Epithelial ovarian cancer (EOC) is the deadliest gynecologic malignancy [1]. High-grade serous EOC (HGSC), the most prevalent EOC subtype, is diagnosed at late stage and accounts for most EOC cases and deaths [2,3]. EOC is characterized by genetic and epigenetic alterations, the latter including DNA methylation changes [2,4-6]. For example, some tumors display BRCA1 hypermethylation, which can disrupt DNA repair [2]. In addition, a large group of EOC are characterized by global DNA hypomethylation, which may promote genomic instability [7,8].

POTE (Protein expressed in Prostate, Ovary, Testis, and Placenta) is a multigene family overexpressed in several human cancers [9]. There are 14 POTEs, which are classified into three groups based on phylogeny, and are dispersed on seven chromosomes (Table 1) [10-12]. POTEs originated from an ancestral version of ankyrin repeat domain 26 (ANKRD26) [11], a gene mutated in familial thrombocytopenia 2 (THC2) [13,14]. Genomically, POTEs localize to pericentromeres or ancestral pericentromeres [10] (Table 1), and contain a L1 element localized to the 3’ UTR [10,15]. The 3’ L1 may have promoted POTE dispersal through the primate genome via transposition. Interestingly, several POTEs contain a C-terminal in-frame fusion with Actin, resulting from L1 transposition [10,15]. Structurally, POTE proteins possess an N-terminal cysteine-rich region, central ankyrin repeats, and C-terminal spectrin-like α-helices, which suggests participation in protein-protein interactions and association with cell membranes [15,16]. In agreement, POTEs localize to the plasma membrane, and specific POTEs co-localize with actin filaments [15,17].

Table 1. POTE Gene Family.

HUGO Name	POTE Group	Chromosome band	Chromosome localization	CTA expression	Elevated in HGSC
POTEA	1	8p11.21	Pericentromeric	∏	∏
POTEB	2	15q11.2	Pericentromeric	∏	–
POTEB2	2	15q11.2	Pericentromeric	∏	∏
POTEB3	2	15q11.2	Pericentromeric	n/d	∏
POTEC	2	18p11.21	Pericentromeric	∏	∏
POTED	2	21q11.2	Pericentromeric	∏	–
POTEE	3	2q21.1	Ancestral Pericentromeric	–	∏
POTEF	3	2q21.1	Ancestral Pericentromeric	–	∏
POTEG	3	14q11.2	Pericentromeric	–	∏
POTEH	3	22q11.1	Pericentromeric	–	∏
POTEI	3	2q21.1	Ancestral Pericentromeric	–	∏
POTEJ	3	2q21.1	Ancestral Pericentromeric	–	–
POTEKP	3	2q21.1	Ancestral Pericentromeric	–	∏
POTEM	3	14q11.2	Pericentromeric	–	∏

n/d: not determined

Early studies of POTEs suggested that, based on expression, they should be classified as CTA genes [9]. CTAs [also known as cancer-germline (CG) genes] are repressed in somatic tissues other than testis, fetal ovary, and placenta, but are expressed in cancer [18,19]. The formal definition of CTA genes is based on expression pattern but, importantly, some CTAs are immunogenic, which has spurred the development of CTA-targeted cancer immunotherapies [18,20]. A recent report suggests that CTA expression correlates with a beneficial clinical response to anti-PD1 immune checkpoint therapy [21]. Notably, CTAs have additional importance in cancer biology because they promote oncogenic phenotypes [22-35].

Recently, we used RNA-seq data to determine the expression patterns of individual POTE genes in normal and tumor tissues, with a focus on ovarian cancer [36]. Our analyses revealed that Group 1 & 2 POTEs (POTEs A/B/C/D) have a characteristic CTA expression profile, with significant expression limited to testis and cancer, while Group 3 POTEs (POTEs E/F/G/H/I/J/KP/M) are expressed in several normal tissues as well as in cancer (Table 1). Notably, the majority (10/13) of POTEs were overexpressed in HGSC, including all three groups, regardless of whether they were CTAs [36].

The mechanisms responsible for increased POTE expression in cancer are unknown. In this context, several previous studies have implicated global and promoter-specific DNA hypomethylation as key mechanisms promoting CTA expression in cancer, including in EOC [7,18,37-46]. Related epigenetic mechanisms, including histone modifications and nucleosome occupancy, also regulate CTA genes, although these appear secondary to DNA methylation [18,42,47-51]. In addition, pericentromeric regions, on which POTEs reside, are epigenetically regulated [52]. Here, we tested whether epigenetic alterations promote POTE expression in ovarian cancer, focusing on DNA hypomethylation. Our findings lay initial groundwork for understanding mechanisms of POTE gene regulation in normal tissues and cancer.

Results

Pan-POTE expression in EOC is associated with expression of the pericentromeric repeat NBL2

All POTEs are localized to current or ancestral pericentromeres (Table 1) [10,11]. As pericentromeres are epigenetically regulated [52], we hypothesized that POTE expression in EOC may reflect, in part, the activation state of pericentromeric DNA. As a biomarker for the epigenetic status of pericentromeres, we measured expression of the NBL2 repetitive element [53-55]. Pan-POTE expression is significantly elevated in EOC compared to normal ovary (NO) (Figure 1(a)) [36]. NBL2 showed increased expression in EOC vs. NO, but the difference was not significant (Figure 1(b)). Notably, Pan-POTE and NBL2 expression showed a significant correlation in EOC (Figure 1(c)). To assess the biological significance of this association, we determined NBL2 correlations with non-pericentromeric CTA gene expression. NBL2 did not correlate with non-pericentromeric CTAs, with one exception (Table 2). These data suggest that increased POTE expression in EOC correlates with epigenetic activation of pericentromeric DNA.

Table 2. Pan-POTE and CTA correlations with NBL2 expression in EOC.

Gene	Chromosome	Position	N	Spearman r	P-value
Pan-POTE	Autosomes	Pericentromeres	87	0.415	<0.0001
CT45	Xq26.3	q arm	61	0.204	NS
CTCFL	20q13.31	q arm	43	0.086	NS
MAGE-A1	Xq28	telomeric	45	−0.208	NS
NY-ESO-1	Xq28	telomeric	43	−0.089	NS
PRAME	22q11.22	q arm	61	0.316	0.013
XAGE-1	Xp11.22	p arm	45	0.06	NS

Figure 1. Pan-POTE expression is associated with NBL2 expression in EOC. (a) PAN-POTE RNA expression in normal ovary (NO) and primary EOC. (b) NBL2 RNA expression in NO and primary EOC. (c) Pan-POTE RNA vs. NBL2 RNA in primary EOC. RNA expression was normalized to 18s rRNA. Panels A-B plot median values and two-tailed Mann-Whitney test results, and panel C shows Spearman correlation test results.

Pan-POTE expression in EOC is associated with global L1 and POTE-L1 DNA hypomethylation.

CTA expression correlates with global genomic DNA hypomethylation, which can be monitored using global L1 methylation [8,39,56]. Because some POTEs have a CTA expression pattern (Table 1) [36], we hypothesized that Pan-POTE expression may correlate with global L1 hypomethylation in EOC; our data verified this hypothesis (Figure 2(a)). To test this association with higher resolution, we used Affymetrix microarrays to profile POTE sub-group expression, as recently described [36]. We used two groups of primary EOC samples showing divergent L1 methylation (n = 20/group). In agreement with Pan-POTE data, several POTE sub-groups showed significantly elevated expression in L1 hypomethylated EOC, and no sub-group was significantly down-regulated (Figure S1).

Figure 2. Pan-POTE expression is associated with LINE-1 (L1) DNA hypomethylation in EOC. (a) Pan-POTE RNA vs. global L1 methylation in primary EOC. (b) POTE (GHM) L1 methylation in NO and primary EOC. (c) Pan-POTE RNA vs. POTE (GHM) L1 in primary EOC. Panels A & C show Spearman correlation test results, and panel B shows two-tailed Mann-Whitney test results.

POTE genes were originally reported to contain a conserved L1 element in their 3’ UTRs [10,15]. Based on the inverse relationship between Pan-POTE expression and global L1 methylation, we hypothesized that DNA methylation of POTE-L1 sequences may also associate with POTE expression. To test this, we first analyzed POTE genes for L1 sequence composition using human genomic data. In addition to the previously reported 3’ L1 sequences in POTEs [10], we observed several additional L1 sequences in POTE gene regions (Supplemental Information). Moreover, POTE genes were significantly enriched in L1 sequences compared to nuclear genes (Table S1). To test whether POTE-associated L1 methylation correlates with POTE expression, we designed a pyrosequencing assay specific for the 3’ UTR L1 region of a sub-set of POTEs (G/H/M). This assay (i.e., POTE-L1) demonstrated hypomethylation in EOC compared to NO, and revealed a significant indirect correlation of POTE-L1 methylation and Pan-POTE expression in EOC (Figure 2(b,c)).

POTE expression and 5’ POTE hypomethylation in HGSC

The high homology of POTE genes has hindered measurement of the expression of individual POTE genes [9,10]. However, RNA-seq can resolve individual POTEs [36]. In addition, interrogation of the DNA methylation status of individual POTEs is possible using Illumina 450 K arrays (450 K) (Supplemental Information). We thus obtained matched RNA-seq and 450 K data from normal FTE, and primary and recurrent HGSC, to test the relationship between individual POTE gene expression and DNA methylation [57]. Analyses of all relevant POTE data revealed that three POTEs (POTE C/E/F) are hypomethylated in HGSC compared to FTE, and show inversely correlated 5’ methylation and expression in HGSC (Figure S2; Figures 3–5). Information on the analyzed CpG sites is provided in Table S2. For POTEC & F, the hypomethylated sites lie in the promoter, while for POTEE the hypomethylated sites lie in a putative upstream enhancer. Interestingly, these CpG sites were located outside of L1 sequences and CpG islands (CGI) (Figures 3–5).

Figure 3. 5’ hypomethylation of POTEC in HGSC and correlation with gene expression. (a) UCSC Genome Browser data showing POTEC structure, LINE1 (L1) sequences, CpG islands (CGI), and Illumina 450K CpG sites. Asterisks indicate CpG sites with hypomethylation in HGSC and used for comparison with gene expression. Broken red arrow represents the direction of the transcript. (b) 450K methylation data for POTEC CpG sites in FTE (N = 6) and primary HGSC (N = 80). CpG sites selected for comparisons with gene expression indicated with asterisks. Shading indicates the extent of hypomethylation in HGSC (see key). (c) POTEC expression vs. 5’ methylation in HGSC (primary and recurrent), using the averaged methylation of the asterisked CpG sites shown in panels B and C. Spearman correlations and p-values are indicated (N = 89). mRNA expression values are FPKM normalized and log2 transformed.

Figure 4. 5’ hypomethylation of POTEE in HGSC and correlation with gene expression. (a) UCSC Genome Browser data showing POTEE structure, LINE1 (L1) sequences, CpG islands (CGI), and Illumina 450K CpG sites. Asterisks indicate CpG sites with hypomethylation in HGSC and used for comparison with gene expression. Broken red arrow represents the direction of the transcript. (b) 450K methylation data for POTEE CpG sites in FTE (N = 6) and primary HGSC (N = 80). CpG sites selected for comparisons with gene expression indicated with asterisks. Shading indicates the extent of hypomethylation in HGSC (see key). (c) POTEE expression vs. 5’ methylation in HGSC (primary and recurrent), using the averaged methylation of the asterisked CpG sites shown in panels B and C. Spearman correlations and p-values are indicated (N = 88). mRNA expression values are FPKM normalized and log2 transformed.

Figure 5. 5’ hypomethylation of POTEF in HGSC and correlation with gene expression. (a) UCSC Genome Browser data showing POTEF structure, LINE1 (L1) sequences, CpG islands (CGI), and Illumina 450K CpG sites. Asterisks indicate CpG sites with hypomethylation in HGSC and used for comparison with gene expression. Broken red arrow represents the direction of the transcript. (b) 450K methylation data for POTEF CpG sites in FTE (N = 6) and primary HGSC (N = 80). CpG sites selected for comparisons with gene expression indicated with asterisks. Shading indicates the extent of hypomethylation in HGSC (see key). (c) POTEF expression vs. 5’ methylation in HGSC (primary and recurrent), using the averaged methylation of the asterisked CpG sites shown in panels B and C. Spearman correlations and p-values are indicated (N = 90). mRNA expression values are FPKM normalized and log2 transformed.

Acquired resistance to chemotherapy is a major clinical problem in HGSC, and current efforts focus on understanding the mechanisms of chemoresistance and identification of therapeutic targets in these patients [58]. We recently reported that several POTEs, including POTEs C and F, are upregulated in recurrent HGSC [36]. Here we analyzed POTE 5’ methylation and expression in 10 patient-matched pairs of primary and recurrent HGSC [57], focusing on the POTE C/E/F CpG sites identified above (Table S2). Notably, several patients showed coordinate 5’ hypomethylation and increased expression of individual POTEs in recurrent HGSC (Figure 6). In addition, select individual patients showed hypomethylation and increased expression of multiple POTEs (e.g., AOCS-34, AOCS-64).

Figure 6. POTEC/E/F gene expression and 5’ DNA methylation in primary vs. recurrent HGSC. A. POTEC mRNA expression and averaged CpG methylation, using the CpG sites shown in Figure 3(c), in patient-matched primary vs. recurrent HGSC. B. POTEE mRNA expression and averaged CpG methylation, using the CpG sites shown in Figure 4(c), in patient-matched primary vs. recurrent HGSC. C. POTEF mRNA expression and averaged CpG methylation, using the CpG sites shown in Figure 5(c), in patient-matched primary vs. recurrent HGSC. Patient IDs are shown. Boxed patient IDs indicate samples with evidence for coordinate 5’ hypomethylation and increased gene expression. Blue dotted lines indicate the group average for the 10 primary HGSC samples. Orange dotted lines indicate the group average for the 10 recurrent HGSC samples.

DNA methylation represses POTE gene expression

To test whether DNA methylation represses POTEs, we utilized the DNMTi decitabine (DAC), which reactivates the expression of methylation-silenced genes, including CTAs [49,59]. In a panel of EOC and related cell lines, DAC treatment increased Pan-POTE regardless of the baseline level of expression, although induction was less substantial in cells with higher baseline expression (Figure 7(a)). To validate that DAC induction was not due to off-target effects, we measured Pan-POTE expression in cancer cells with DNA hypomethylation caused by DNMT knockout [60,61], and observed significant induction in DNMT1/3b double knockout cells (Figure 7(b)). In addition, pyrosequencing confirmed that both DAC treatment and DNMT knockout caused POTE gene DNA hypomethylation (Figure S3). We then conducted an exploratory analysis of epigenetic drug combinations to determine their effect on Pan-POTE expression. We treated a model HGSC cell line, Kuramochi, [62] with DAC, the pan-HDACi TSA, and three HMTi, including GSK343, which inhibits EZH2 (H3K27me) [63], BIX-01294, which inhibits G9a (H3K9me1/2) [64], and chaetocin, which inhibits SUV39H1 (H3K9me3) [65]. While HDAC and HMTi treatments alone had a minor effect on Pan-POTE expression, combinations of these agents with DAC increased Pan-POTE expression beyond the effect of DAC alone (Figure 7(c)). In particular, chaetocin, when combined with DAC, or DAC + TSA, led to the greatest induction of Pan-POTE.

Figure 7. DNA hypomethylation induces Pan-POTE expression. (a) Pan-POTE expression in a panel of ovarian cancer-related cell lines treated with 1µM DAC for 48 hours. (b) Pan-POTE expression in HCT116 colorectal cancer cells with DNMT gene knockout (genotype indicated). (c) Pan-POTE expression in Kuramochi cells treated with different combinations of epigenetic drugs for 72 hours. In all panels, data are plotted as mean + SD.

Discussion

Three genomic characteristics of POTE genes suggested they might be epigenetically regulated: 1) pericentromeric localization [52,66], 2) a conserved 3’ UTR L1 element [10,56], and 3) expression patterns (of some members) typical of CTA genes [18,36]. Our studies support that POTEs are epigenetically regulated. First, NBL2 expression correlated with POTE expression, which was not true for most non-pericentromeric CTAs, suggesting that POTE expression is promoted by epigenetic activation of pericentromeres. In agreement, NBL2 is resident at the pericentromeres of chromosomes 2, 13, 14, 15, and 22, which also contain POTEs [53-55,67] (Table 1). Second, POTE genes were enriched for L1 sequences, and POTE expression correlated with hypomethylation of global L1 sequences and POTE-embedded L1 sequences. L1 hypomethylation may promote POTE expression by impacting the regional chromatin environment, although additional studies are needed to test this. Third, selected POTEs have DNA hypomethylation at 5’ promoter or enhancer regions that correlate with increased expression. Interestingly, the hypomethylated regions did not overlap L1s or CpG islands (CGI). Non-CGI 5’ methylation can regulate gene expression, consistent with our data [68]. Thus, it appears that several different DNA methylation defects promote POTE family expression in EOC.

We used genetic and pharmacological approaches in EOC and related cell models to determine whether epigenetics functionally regulates POTE expression. Genetic knockout of DNMT1 and DNMT3b, which results in global hypomethylation in HCT116 cells, induced Pan-POTE expression. In agreement, DAC treatment induced Pan-POTE expression, while several other drugs that impact histone-modifying enzymes had a smaller effect, as single treatments. However, when used in combination with DAC, several drugs showed a significant augmenting effect. The HDACi TSA showed this effect, as observed previously for DNA methylation-regulated genes, including CTAs [49,59]. Notably, the SUV39H1 inhibitor chaetocin had a robust effect on Pan-POTE expression, when used in combination with DAC or DAC + TSA. As pericentromeric heterochromatin is repressed by DNA methylation and H3K9me3 [52]; we speculate that the impact of chaetocin on POTE expression may reflect euchromatization of pericentromeric DNA. Consistently, we observed that chaetocin treatment activates NBL2 expression (data not shown). Interestingly, chaetocin also was recently shown to enhance CTA expression in dendritic cells [69].

Although POTEs are overexpressed and epigenetically activated in EOC and HGSC, we do not yet know whether they play a functional role [9,36]. Mechanistically, recent data suggest that POTEs regulate cell survival [70-72]. In addition, POTE protein structure and cellular observations indicate a potential function in the cytoskeleton [10,15,17]. Our preliminary data suggest that POTEs promote oncogenic phenotypes in ovarian cancer cells (Sharma et al., unpublished). An oncogenic function in HGSC appears consistent with the link between Pan-POTE expression and advanced disease, and the association of POTEE expression with reduced HGSC survival [36]. In addition, several POTEs have increased expression in recurrent chemoresistant HGSC, which, in the case of POTEs C/E/F, is linked to 5’ hypomethylation. Also seemingly consistent with an oncogenic function for POTEs is our observation that several distinct cancer epigenetic alterations each promote POTE expression. Finally, recent data shows that other CTAs promote oncogenic phenotypes [28,31,34,35]. Future studies will examine the role of POTEs in EOC and other cancers, and their potential as therapeutic targets.

Materials and methods

Human EOC and normal ovary (NO) tissues

We obtained human primary EOC (n = 119) and NO (n = 17) (from patients without malignancy) using IRB-approved protocols at RPCCC, as described [73] (Figure S4). We performed biochemical extractions as described [46].

Cell culture

We have described all ovarian cancer and related cell lines used in this study elsewhere [36,74]. We obtained and cultured HCT116 and DNMT knockout cells as previously described [60,61].

Drug treatments

We treated a panel of cell lines with 1.0 µM decitabine (DAC; Sigma) and harvested RNA 48 hours post-treatment. For combination drug studies, we treated Kuramochi cells with 1.0 µM DAC for 48 hours, then added one or two additional drugs and continued the experiment for an additional 24 hours prior to RNA harvest. The additional drugs tested were: Trichostatin A (TSA; Sigma), GSK343 (Cayman Chemical), BIX-01294 (Sigma), and Chaetocin (Cayman Chemical). We treated cells with vehicle controls (PBS and/or DMSO) in parallel.

RT-qPCR

We extracted RNA using TRIzol (Invitrogen) and synthesized cDNA using iScript cDNA Synthesis Kit (BioRad). We sequenced RT-PCR products to confirm correct product amplification. We performed qPCR using the BioRad CFX Connect system, SYBR green master mix (Qiagen). We designed primers using Primer-Blast (NCBI) or Primer3, and we obtained primers from IDT. We amplified Pan-POTE as described [15]. We amplified NBL2 as described [67]. Primer sequences are shown in Table S3. We obtained tumor-matched expression data for other CTA genes (CTCFL, PRAME, MAGEA1, NY-ESO-1, CT45, XAGE1) from our previous studies [8,40,41].

Affymetrix microarrays

We performed Affymetrix HG 1.0ST arrays at the University at Buffalo Center of Excellence in Bioinformatics and Life Sciences. We normalized microarray probe cell intensity data (.cel) using the Affymetrix Expression Console (version 1.3.0.187) software running the RMA workflow. To determine significantly expressed genes we used a regularized t test analysis of control versus treatment comparisons using a Bayesian approach that estimates the within-treatment variation among replicates, using Cyber-T software.

POTE genomic organization and detection of embedded L1 sequences

We used the human genome assembly (GRCh38.p12, Ensembl Archive Release 92) to map stretches of L1 repetitive sequences (RepeatMasker annotations; http://www.repeatmasker.org) within 10 K upstream and downstream of the 14 POTE genes (Supplementary Information). We used a Python extension of BEDTools to identify L1 repeats that overlapped with the -/+10 K region of the POTEs [75,76]. We visualized L1 sequences using the UCSC Genome Browser.

POTE methylation and expression comparisons in FTE and HGSC

We utilized publically available data from the Australian Ovarian Cancer Study (AOCS) [57] (Figure S4). We retrieved Illumina Infinium (450 K) methylation data for normal FTE (n = 6) and HGSC samples (n = 90: 80 primary + 10 recurrent) from Gene Expression Omnibus (GEO) (GSE65820) [57]. We integrated POTE expression and methylation from patient-matched primary and recurrent HGSC (n = 10). The CpG sites used for analysis are presented in Table S2. We obtained RNA-seq data from the European Genome-phenome Archive (EGA) (EGAD00001000877) [57]. We downloaded and analyzed all data using Python. The python code is available at (https://goo.gl/R5w3bT).

Sodium bisulfite genomic DNA pyrosequencing

We performed pyrosequencing as described [7]. We amplified global L1 sequences as previously described [8]. We designed several new pyrosequencing assays that cover different regions of POTE genes; primers are presented in Table S3.

Acknowledgments

We thank Professors Nelly Auersperg, Francis Balkwill, Anirban Mitra, and Bert Vogelstein for generously providing cell lines, and other members of the Karpf Lab for helpful discussions and technical assistance. We thank the University of Buffalo Center of Excellence for microarray processing, and the UNMC DNA sequencing and Epigenomics Cores for sequencing and epigenomic analyses.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This project was supported by NIH RO1CA116674, The Betty J. and Charles D. McKinsey Ovarian Cancer Research Fund, the FPBCC Support Grant (NIH P30CA036727), and the RPCCC Support Grant (NIH P30CA016056).