MicroRNA-20a-5p Inhibits the Autophagy and Cisplatin Resistance in Ovarian Cancer via Regulating DNMT3B-mediated DNA Methylation of RBP1

Poly (ADP-ribose) polymerase 1 inhibitors (PARPi) are used to treat recurrent ovarian cancer (OC) patients due to greater survival benefits and minimal side effects, especially in those patients with complete or partial response to platinum-based chemotherapy. However, acquired resistance of platinum-based chemotherapy leads to the limited efficacy of PARPi monotherapy in most patients. Twist is recognized as a possible oncogene and contributes to acquired cisplatin resistance in OC cells. In this study, we show how Twist knockdown cisplatin-resistant (CisR) OC cells blocked DNA damage response (DDR) to sensitize these cells to a concurrent treatment of cisplatin as a platinum-based chemotherapy agent and niraparib as a PARPi on in vitro two-dimensional (2D) and three-dimensional (3D) cell culture. To investigate the lethality of PARPi and cisplatin on Twist knockdown CisR OC cells, two CisR cell lines (OV90 and SKOV3) were established using step-wise dose escalation method. In addition, in vitro 3D spheroidal cell model was generated using modified hanging drop and hydrogel scaffolds techniques on poly-2-hydroxylethly methacrylate (poly-HEMA) coated plates. Twist expression was strongly correlated with the expression of DDR proteins, PARP1 and XRCC1 and overexpression of both proteins was associated with cisplatin resistance in OC cells. Moreover, combination of cisplatin (Cis) and niraparib (Nira) produced lethality on Twist-knockdown CisR OC cells, according to combination index (CI). We found that Cis alone, Nira alone, or a combination of Cis+Nira therapy increased cell death by suppressing DDR proteins in 2D monolayer cell culture. Notably, the combination of Nira and Cis was considerably effective against 3D-cultures of Twist knockdown CisR OC cells in which Endoplasmic reticulum (ER) stress is upregulated, leading to initiation of mitochondrial-mediated cell death. In addition, immunohistochemically, Cis alone, Nira alone or Cis+Nira showed lower ki-67 (cell proliferative marker) expression and higher cleaved caspase-3 (apoptotic marker) immuno-reactivity. Hence, lethality of PARPi with the combination of Cis on Twist knockdown CisR OC cells may provide an effective way to expand the therapeutic potential to overcome platinum-based chemotherapy resistance and PARPi cross resistance in OC.

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Hypermethylation of mismatch repair gene hMSH2 associates with platinum-resistant disease in epithelial ovarian cancer

Clinical Epigenetics ◽

10.1186/s13148-019-0748-4 ◽

2019 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Hua Tian ◽

Li Yan ◽

Li Xiao-fei ◽

Sun Hai-yan ◽

Chen Juan ◽

...

Keyword(s):

Mass Spectrometry ◽

Ovarian Cancer ◽

Dna Methylation ◽

Epithelial Ovarian Cancer ◽

Upstream Region ◽

Platinum Resistance ◽

Maldi Tof Mass Spectrometry ◽

Platinum Resistant ◽

Aberrant Dna Methylation ◽

Low Expression

Abstract Purpose One 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. Methods By 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. Results RRBS 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). Conclusion The 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.

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