Abstract
PurposeOne major reason of the high mortality of epithelial ovarian cancer (EOC) is due to platinum-based chemotherapy resistance. Aberrant DNA methylation may be a potential mechanism underlying the development of platinum resistance in EOC. The purpose of this study is to discover potential aberrant DNA methylation that contributes to drug resistance.MethodsBy initially screening of 16 platinum-sensitive/resistant samples from EOC patients with reduced representation bisulfite sequencing (RRBS), the upstream region of the hMSH2 gene was discovered hypermethylated in the platinum-resistant group. The effect of hMSH2 methylation on the cellular response to cisplatin was explored by demethylation and knockdown assays in ovarian cancer cell line A2780. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was employed to examine the methylation levels of hMSH2 upstream region in additional 40 EOC patient samples. RT-qPCR and IHC assay was used to detect the hMSH2 mRNA and protein expression in extended 150 patients.ResultsRRBS assay discovered an upstream region from − 1193 to − 1125 of hMSH2 was significant hypermethylated in resistant EOC patients (P = 1.06 × 10−14). In vitro analysis demonstrated that global demethylation increased cisplatin sensitivity along with a higher expression of the hMSH2 mRNA and protein. Knockdown hMSH2 reduced the cell sensitivity to cisplatin. MALDI-TOF mass spectrometry assay validated the strong association of hypermethylation of hMSH2 upstream region with platinum resistance. Spearman’s correlation analysis revealed a significantly negative connection between methylation level of hMSH2 upstream region and its expression. The Kaplan-Meier analyses showed the high methylation of hMSH2 promoter region, and its low expressions are associated with worse survival. In multivariable models, hMSH2 low expression was an independent factor predicting poor outcome (P = 0.03, HR = 1.91, 95%CI = 1.85–2.31).ConclusionThe hypermethylation of hMSH2 upstream region is associated with platinum resistant in EOC, and low expression of hMSH2 may be an index for the poor prognosis.
Highlights
In the female reproductive system, epithelial ovarian cancer (EOC) is the third most common cancer and the first leading cause of cancer deaths [1]
The present study revealed that (1) 5aza-dC-induced demethylation can significantly increase the sensitivity of ovarian cancer cells to cisplatin as well as Human MutS homolog 2 (hMSH2) expression, (2) knockdown of hMSH2 expression could desensitize A2780 ovarian cancer cells to cisplatin, (3) the hMSH2 upstream region was significantly hypermethylated in the EOC tissue of platinumresistant patients, and (4) the lower expression of hMSH2 due to hypermethylation of the upstream region was associated with the clinical outcome of patients with EOC
The chemotherapeutic mechanism of platinum compounds involves its covalent binding to DNA to form DNA adducts, which results in a DNA replication block and promotes cell death. hMSH2, by itself [17] or as an hMSH2-hMSH6 complex [18], recognizes specific DNA damage caused by cisplatin and carboplatin
Summary
In the female reproductive system, epithelial ovarian cancer (EOC) is the third most common cancer and the first leading cause of cancer deaths [1]. Increasing evidence has shown that epigenetic changes may play an important role in chemotherapy resistance of ovarian cancer [5]. It has been suggested that DNA methylation, a well-studied epigenetic change, may serve as a potential biomarker for chemotherapy-resistant phenotypic screening [6]. Because chemotherapy is a mainstay of DNA-damaging agents in numerous cancer therapies, loss of MMR proteins renders cells resistant to DNAdamaging regents [9, 10]. A recent study using whole-genome CRISPR (clustered regularly interspaced short palindromic repeats) screen in a bladder cancer cell line identified that hMSH2 was the most significantly enriched gene that promotes resistance to cisplatin [13]. To date, there are no reports about the role of hMSH2 expression loss caused by aberrant methylation of the promoter region in platinum resistance
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