Methylomic Signatures of High Grade Serous Ovarian Cancer

Abstract

High-grade serous ovarian cancer (HGSOC) harbours aberrant epigenetic features, including DNA methylation. In this study we delineate pathways and networks altered by DNA methylation and associated with HGSOC initiation and progression to a platinum-resistant state. By including tumours from patients who had been treated with the hypomethylating agent (HMA) guadecitabine, we also addressed the role of HMAs in treatment of HGSOC. Tumours from patients with primary (platinum-naïve) HGSOC (n = 20) were compared to patients with recurrent platinum-resistant HGSOC and enrolled in a recently completed clinical trial (NCT01696032). Human ovarian surface epithelial cells (HOSE; n = 5 samples) served as normal controls. Genome-wide methylation profiles were determined. DNA methyltransferase (DNMT) expression levels were examined by immunohistochemistry and correlated with clinical outcomes. Cancer-related and tumorigenesis networks were enriched among differentially methylated genes (DMGs) in primary OC vs. HOSE. When comparing platinum-resistant and primary tumours, 452 CpG island (CGI)-containing gene promoters acquired DNA methylation; of those loci, decreased (P < 0.01) methylation after HMA treatment was observed in 42% (n = 189 CGI). Stem cell pluripotency and cytokine networks were enriched in recurrent platinum-resistant OC tumours, while drug metabolism and transport-related networks were downregulated in tumours from HMA-treated patients compared to HOSE. Lower DNMT1 and 3B protein levels in pre-treatment tumours were associated with improved progression-free survival. The findings provide important insight into the DNA methylation landscape of HGSOC tumorigenesis, platinum resistance and epigenetic resensitization. Epigenetic reprogramming plays an important role in HGSOC aetiology and contributes to clinical outcomes.

Keywords: Ovarian cancer; epigenetics; methylation; platinum; resistance.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.

Differential methylation between each OC group and HOSE. (a) workflow of data analysis. (b) Volcano plots depicting differentially methylated CpG sites in different genomic regions (All CpG sites, Gene Body, Promoter, CpG island) of three OC groups compared to HOSE. Differentially methylated CpG sites are depicted as blue dots. The number of sites at different regions are indicated in each volcano plot. HOSE is human ovarian surface epithelium, which served as the normal control

Figure 2.

Methylomic changes during acquired platinum resistance. (a) Venn diagram shows unique and common hypermethylated genes in all three ovarian tumour groups, in the blue ellipse, 452 genes identified hypermethylated during acquired platinum resistance (hypermethylated in recurrent but not in primary OC). (b) Gene ontology analysis of the 452 genes hypermethylated in recurrent OC, different shapes on the out layer are genes from 452 gene list, whereas the ones in the middle are different gene ontology terms enriched by those genes from the outer layer. (c) Pathways enriched by 452 hypermethylated genes during acquired platinum resistance

Figure 3.

Hypomethylated genes induced by the HMA guadecitabine. (a) Venn diagram shows unique and common hypomethylated genes in all three ovarian tumour groups, in the blue ellipse, 189 genes identified as hypomethylated and induced by guadecitabine (genes hypomethylated in guadecitabine-treated but not in recurrent OC). Networks of epithelial-mesenchymal transition (EMT) inhibition and reduced cell survival of cancer cells (b) and pathways (c) enriched by the189 guadecitabine-induced hypomethylated genes

Figure 4.

Effects of guadecitabine on expression of selected genes. (a–c) Basal mRNA expression levels of FXYD6, PDHX and UEB4A measured by real-time RT-PCR in platinum resistant (r) OVCAR5_R, A2780_R and SKOV3_R; (d–f) expression levels of IRF9, EVI2A and CCL19 on parental and cisplatin-resistant (R) A2780, SKOV3, and OVCAR5 treated with guadecitabine (100 nM) for 72 hours, and (g) OVCAR3 cells treated with the guadecitabine (100 nM) for 72 hours. Bars represent mean ± SD, n = 3 (* P < 0.05, ** P < 0.01)

Figure 5.

Expression levels of DNMT1, DNMT3A, and DNMT3B mRNAs in OC tumours. (a) DNMT1 and DNMT3B mRNA levels are positively correlated in HGSOC tumours. Box plots show medians and 25–75% quartiles of DNMT mRNAs levels measured by real-time RT-PCR in HGSOC primary tumours (n = 20, Sample/Patient Group 1) relative to fallopian tube epithelium. (b-d) Scatter plots and correlation coefficients between mRNA levels of DNMT1 and DNMT3B (b), DNMT1 and DNMT3A (c), or DNMT3A and DNMT3B (d) in HGSOC primary tumours (n = 20, Sample/Patient Group 1)

Figure 6.

DNMT1 and DNMT3B are associated with progression-free (PFS) and overall survival (OS) of ovarian cancer patients. (a) Examples of DNMT1, DNMT3A and DNMT3B IHC immunostaining in sections from the same recurrent HGSOC tumour (original magnification: 200X). (b) Box plots show medians and 25–75 quartiles of DNMT1, DNMT3A, and DNMT3B IHC scores in recurrent HGSOC tumours (n = 32, Sample/Patient Group 2) before patients received guadecitabine treatment (*P < 0.05). (c) Scatter plots and correlation coefficients of IHC scores between DNMT1 and DNMT3A, DNMT1 and DNMT3B, or DNMT3A and DNMT3B in recurrent HGSOC tumours (n = 32, Sample/Patient Group 2). (d–g) Kaplan-Meier plots of progression-free survival (PFS) (d–e) and overall survival (f–g) by IHC score level (low or high) of DNMT1, or DNMT3B in recurrent HGSOC tumours from patients included in C above, and subsequently treated with a combination of carboplatin and guadecitabine. HR, hazard ratio