DNA methylation and Transcriptome Changes Associated with Cisplatin Resistance in Ovarian Cancer

Abstract

High-grade serous ovarian cancer is the most common ovarian cancer type. Although the combination of surgery and platinum-taxane chemotherapy provide an effective treatment, drug resistance frequently occurs leading to poor outcome. In order to clarify the molecular mechanisms of drug resistance, the DNA methylation and transcriptomic changes, associated with the development of drug resistance in high-grade serous ovarian cancer, were examined from patient derived malignant ascites cells. In parallel with large-scale transcriptome changes, cisplatin resistance was associated with loss of hypermethylation at several CpG sites primarily localized in the intergenic regions of the genome. The transcriptome and CpG methylome changes in response to cisplatin treatment of both sensitive and resistant cells were minimal, indicating the importance of post-translational mechanisms in regulating death or survival of the cells. The response of resistant cells to high concentrations of cisplatin revealed transcriptomic changes in potential key drivers of drug resistance, such as KLF4. Among the strongest changes was also induction of IL6 in resistant cells and the expression was further increased in response to cisplatin. Also, several other components of IL6 signaling were affected, further supporting previous observations on its importance in malignant transformation and development of drug resistance in ovarian cancer.

Figure 1

CpG methylome and transcriptome differences between cisplatin sensitive and resistant ovarian cancer lines. DNA methylomes of the cells sensitive or resistant to cisplatin were profiled with Reduced Representation Bisulfite Sequencing and transcriptomes with mRNA-sequencing. In (a) are the CpG sites with coverage ≥ 10 and minimum methylation difference of 20% (qval ≤ 0.05) in M019i cells, (see also Supplementary Table S1), (b) the distribution of differentially methylated cites in genomic regions, (c) distance of the differentially methylated sited from the closest transcription start sites, (d) the transcriptome differences (minimum absolute FC = 1.5, FDR ≤ 0.05) between cisplatin sensitive and resistant ovarian cancer cells (M019i), (e–h) qRT-PCR validation of AKR1C1, CYP4F11, CYP24A1, MIR205HG, and SLC6A14 differences in M019i and OC001 cells (y-axis: relative expression level).

Figure 2

Integrative analysis of DNA methylome, transcriptome and functional enrichment data. (a) In the figure are the numbers of differentially expressed genes (GE), differentially methylated CpG sites (meCpG) and their overlap (both) between cisplatin sensitive and resistant cells (M019i) before cisplatin treatment (0 μM), and changes detected in response to drug treatment. The putative upstream regulators with gene expression changes and canonical pathway enrichments common for both DNA methylome and transcriptome data are shown in the (b) Gene expression changes, DNA methylation changes or both are shown for the known direct target genes of KLF4, in different comparisons as indicated by the color codes in (a). (c) Comparison of differences observed before cisplatin treatment and in response to 7 μM cisplatin treatment of resistant cells as indicated in the figure. *indicates the number of differentially methylated sites: in integrative comparisons, the number of overlapping genes closest to differentially methylated sites is shown. The functional analyses and networks in the figure were generated by using Ingenuity Pathway Analysis (IPA®, Qiagen).

Figure 3

Identification of genes potentially associated with the drug resistance in ovarian cancer cells through integrative analysis. (a) Normalized relative gene expression counts and clustering of the genes differentially expressed by the cisplatin sensitive and resistant cells and regulated in response to 7 μM cisplatin treatment in resistant cells (M019i). (b,c) Normalized gene expression counts (RPKM) and statistics for CYP24A1 and IL6 genes in M019i cells. (d–f) Expression of KLF4, CYP24A1 and IL6 was validated with qRT-PCR in two platinum sensitive cell lines M019i and OC002 and their resistant variants (y-axis: relative expression level). (g) CYP24A1 protein expression and (h) IL6 protein concentrations in cell culture supernatants of the same cell lines as in (e and f), respectively).