Integrative analysis reveals early epigenetic alterations in high-grade serous ovarian carcinomas

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological malignancy. To date, the profiles of gene mutations and copy number alterations in HGSOC have been well characterized. However, the patterns of epigenetic alterations and transcription factor dysregulation in HGSOC have not yet been fully elucidated. In this study, we performed integrative omics analyses of a series of stepwise HGSOC model cells originating from human fallopian tube secretory epithelial cells (HFTSECs) to investigate early epigenetic alterations in HGSOC tumorigenesis. Assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) methods were used to analyze HGSOC samples. Additionally, protein expression changes in target genes were confirmed using normal HFTSECs, serous tubal intraepithelial carcinomas (STICs), and HGSOC tissues. Transcription factor motif analysis revealed that the DNA-binding activity of the AP-1 complex and GATA family proteins was dysregulated during early tumorigenesis. The protein expression levels of JUN and FOSL2 were increased, and those of GATA6 and DAB2 were decreased in STIC lesions, which were associated with epithelial-mesenchymal transition (EMT) and proteasome downregulation. The genomic region around the FRA16D site, containing a cadherin cluster region, was epigenetically suppressed by oncogenic signaling. Proteasome inhibition caused the upregulation of chemokine genes, which may facilitate immune evasion during HGSOC tumorigenesis. Importantly, MEK inhibitor treatment reversed these oncogenic alterations, indicating its clinical effectiveness in a subgroup of patients with HGSOC. This result suggests that MEK inhibitor therapy may be an effective treatment option for chemotherapy-resistant HGSOC.

Error bars represent mean ± standard deviation (SD).
xenograft experiments and integrative analysis of stepwise HGSOC model cells.a-b Subcutaneous mouse xenograft experiments using HF1/TP53/KRAS/AKT (a) and HF1/TP53/KRAS/MYC (b) cells, respectively.The confirmation of the HGSOC-like phenotype in the mouse xenograft experiments was carried out by a pathologist who examined the results of hematoxylin eosin (HE) staining and PAX8 staining, which is a positive marker for HGSOC.Scale bars, 100 μm.Detailed information of mouse xenograft experiments is previously described 11 .c A heatmap with hierarchical clustering of gene expression levels calculated using RNA-seq.d A heatmap with hierarchical clustering of TF motif enrichment scores calculated using ATAC-seq.e mRNA expression levels (RT-qPCR; n = 3) of CCND1, VEGFA, and ribosomal protein genes in the indicated HGSOC model cells.Error bars represent mean ± standard deviation (SD).Statistical analysis was performed using unpaired Student's t-test.* p < 0.05.Supplementary Fig. 2 AP-1 family and GATA family genes are dysregulated in HGSOCs.a-b Copy number alteration (CNA) of AP-1 family genes (a) and GATA family genes (b) in HGSOC samples.Each sample was segregated according to their CNA status: amplification (CNA = +2); gain (CNA = +1); duplicate (CNA = 0); deletion (CNA = -1); deep deletion (CNA = -2).Data are sourced from TCGA project of HGSOC.c mRNA expression levels (RNA-seq, n = 3) of GATA6 and DAB2 are downregulated in HGSOC model cells.
analysis identifies targetable genes in HGSOCs.a mRNA expression levels of MAF in two independent microarray datasets.GSE18521 and GSE26712 contain microarray data of human ovarian surface epithelial cell (HOSE) samples and HGSOC samples [GSE18521(HOSE: n = 10, HGSOC: n = 53) and GSE26712 (HOSE: n = 10, HGSOC: n = 185)].Statistical analysis was performed using unpaired Student's t-test.Error bars represent mean ± standard deviation (SD).b mRNA expression levels of MAF are plotted against those of DAB2 and WWOX (n = 489).The data were analyzed using Pearson's correlation coefficients.c mRNA expression levels (RT-qPCR, n = 3) of epithelial-mesenchymal transition (EMT)-related genes.Downregulation of epithelial markers, including CDH1 and GSK3B, are observed during oncogenic transformation.Error bars represent mean ± standard deviation (SD).d mRNA expression levels (RNA-seq, n = 3) of DANCR, EZH2, HDAC1 and HDAC2 are upregulated in HGSOC model cells.Error bars represent mean ± SD. e Colony formation assay with DANCR siRNA knockdown experiments in HF1/TP53/KRAS/AKT, HF1/TP53/KRAS/MYC, OVCAR3, and CaOV3 cells.Error bars represent mean ± SD of three biological replicates.Statistical analysis was performed using unpaired Student's t-test.f Cell viability assay with TSA treatment.The cytotoxic effects of TSA are amplified in tumorigenic HGSOC model cells.Relative cell number shows the relative ratio between the number of cells in the absence of the inhibitors and the number of cells in the presence of various concentrations of TSA.Error bars represent mean ± SD of three biological replicates.Page 10 of 15 Supplementary Fig. 4 Inhibition of MAF, GATA6 and DAB2 upregulates epithelial-mesenchymal transition (EMT)-related genes and poor prognostic genes of HGOSCs.a mRNA expression levels (RNAseq; n = 3) of EMT-related genes upon siRNA knockdown of MAF, GATA6 and DAB2 in HF1 cells.Inhibition of MAF, GATA6 and DAB2 upregulates EMT-related genes.Error bars represent mean ± standard deviation (SD).b mRNA expression levels (RNA-seq; n = 3) of KRT7 and KRT19 genes upon siRNA knockdown of MAF, GATA6 and DAB2 in HF1 cells.Error bars represent mean ± SD. c Kaplan-Meier survival curves classified by high or low KRT7 or KRT19 mRNA expression levels in the TCGA HGSOC cohort.High KRT7 and high KRT19 group exhibits poor overall survival.d mRNA expression levels (RNA-seq; n = 3) of PSMB8 and PSMB9 genes upon siRNA knockdown of MAF, GATA6 and DAB2 in HF1 cells.Error bars represent mean ± SD. e Kaplan-Meier survival curves classified by high or low PSMB8 or PSMB9 mRNA expression levels in the TCGA HGSOC cohort.Low PSMB8 and Low PSMB9 group exhibits poor overall survival.

Page 2 of 15 Supplementary Table 1
Information of certified cell lines.

Table 2
Information of primers.