Abstract A10: MicroRNA 27a is downreguated in cisplatin resistant bladder cancer cells, and contributes to resistance through the targeting of xCT, a cystine transporter involved with glutathione production

Abstract

Abstract Cisplatin resistance represents a serious obstacle to the chemotherapeutic treatment of many tumors. The purpose of this study was to create a cell culture model of cisplatin resistant bladder cancer (CRBC), then use this model to identify changes in the expression of microRNAs (miRs) that may influence resistance. Single cell clones resistant to cisplatin were generated from parental bladder cancer cell lines by continuous culture in increasing concentrations of cisplatin for several months. The resultant CRBC cell lines were able to tolerate high doses of cisplatin (typically > 10 μM). MicroRNA expression in parental and CRBC cell lines was measured using q-pcr based low-density tiling arrays and a panel of miRs which were consistently altered in all CRBC cell lines was established. In addition the induction and repair of specific cisplatin-DNA adducts in parental and CRBC cell lines was assessed using antibodies against the guanine–guanine (Pt-[GG]) intrastrand crosslink. Four fold more cisplatin was required to produce the equivalent damage in CRBC cell lines compared to the parental control, while the rate of repair, as assessed by measurement of the removal of cisplatin-DNA adducts over time, remained broadly similar between parental and resistant lines. This demonstrated that resistance was due to reduced adduct formation rather than any defect in cross-link repair. Glutathione (GSH) inactivates cisplatin by binding with it irreversibly to form Pt(SG)2 adducts, thus preventing the drug from forming cytotoxic adducts with DNA. One cause of decreased adduct formation is therefore deregulation of the GSH biosynthesis pathway. Upon examination intracellular levels of GSH and its oxidized form glutathione disulfide (GSSG) were found to be increased in resistant cells. The expression of a number of genes involved with the synthesis of GSH was then measured by rt-pcr and western blotting, and several key proteins involved with GSH biosynthesis were found to be upregulated in resistant cells, suggesting increased levels of intracelluar GSH could be responsible for the observed decrease in the levels of cisplatin-DNA adducts observed in resistant cells. The 3′ untranslated regions (3′ UTRs) of these mRNAs were examined using TargetScan, an online miR target prediction program, to find potential sites of miR regulation. The most upregulated protein, xCT (SLC7A11), responsible for cystine import (known to be the rate-limiting step in GSH synthesis) was found to contain target sites for three miRs which had been shown to be significantly downregulated in our model of CRBC, namely miRs 25, 27a and 32. Upregulation of miRs 25, 27a and 32 by transfection of resistant cells with the pri-miR precursor molecules revealed that restoring expression of miR-27a resensitized CRBC cells to the cytotoxic effects of cisplatin. Restoration of miR-27a expression in CRBC cells was also shown to result in a downregulation of the expression of xCT at both the protein and mRNA level. In summary, we have identified a panel of microRNAs that are consistently dysregulated in CRBC, and implicated one of these (miR-27a) in the modulation of cisplatin resistance via increased glutathione production. We also provide evidence that reversal of this change in miR expression results in a reversal of the effects on downstream target, and critically, a reversal of the cisplatin resistant phenotype. The contribution made by miR-27a to the development of cisplatin resistance might represent a potential site for therapeutic intervention in the treatment of CRBC. Measurement of miR-27a expression in tumor material or urine may also serve as a predictive biomarker for patients likely response to cisplatin chemotherapy. Citation Format: Ross M. Drayton, Ewa Dudziec, James W.F. Catto, Helen E. Bryant. MicroRNA 27a is downreguated in cisplatin resistant bladder cancer cells, and contributes to resistance through the targeting of xCT, a cystine transporter involved with glutathione production [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A10.