Abstract B12: the Mir-15 Family and Mir-155 Mediate WEE1 and CHEK1 Effects on Cisplatin-Resistance in Cancer Cells

Abstract A major challenge in the successful treatment of cancer is the ability of cancer cells to acquire a multi-drug resistant (MDR) phenotype, preventing response to chemotherapeutic agents. Cisplatin (cis-diamminedichloroplatinum(II)) is a chemotherapeutic drug commonly used against malignancies including ovarian, and cervical tumors. Cisplatin resistance (CP-r) studied in cultured cancer cells often results from a cellular defense mechanism that results in a highly complex pleiotropic phenotype that confers resistance by reducing apoptosis, up-regulating DNA damage repair mechanisms, altering cell cycle checkpoints, altering protein localization, redirecting cell surface transporters, and disrupting assembly of the cytoskeleton thereby changing protein distribution in the cell. This results in cells that are permanently resistant not only to cisplatin but also show cross-resistance to multiple toxic compounds that piggyback into cells on existing transporters. The highly pleiotropic CP-r phenotype occurs in a single step that has yet to be identified. miRNAs have crucial roles in diverse biological processes such as phenotypic stabilization, and have been shown to also be involved in tumor growth and response to chemotherapy. We hypothesized that miRNAs may play a role in mediating the phenotypic changes associated with a pleiotropic cisplatin resistance mechanism. KB-CP.5 (resistant to 0.5ug/ml CP) cells were screened with whole kinome siRNA (Ambion SilencerSelect, 3 siRNAs per gene) and miRNA mimics (Sanger miRBase 13.0). Cells were then challenged with cisplatin at IC5 and IC30 doses (normally causing minimal toxicity to CP-r cells). Cell viability was determined using the Promega CellTiter-Glo luminescent viability assay, and conditions that sensitized the CP-r cells were examined. The miR-15/16/195/424/497 family miRNA mimics were found to confer >2-fold increased sensitivity to CP.5 cells. miR-16 gave a 5-fold increase in sensitivity and its family member miR-15a caused a 3.4-fold increase, accounting for two of the top three hits in the primary screen. Follow-up dose-response analysis indicated that miR-15a and miR-16 mimics yielded a 20- and 10-fold shift in the cisplatin sensitivity of CP.5 cells, respectively. The two strongest kinome effects were elicited by silencing WEE1 and CHEK1. Using TargetScanHuman (available at www.targetscan.org), an algorithm program for the prediction of miRNA targets, we found that the miR-15 family is predicted to target WEE1 and CHEK1. For the miR-15 family, RT-PCR shows a 2 to 5-fold decrease in expression in the CP-r cells compared to KB-3-1 parental cells. In addition miR-155 has a 3-fold decrease in expression in KB-CP.5 compared to KB-3-1 parental cells. TargetScan analysis of WEE1 indicates that miR-155 is also a potential regulator of the WEE1 kinase. We confirmed the ability of the miR-15 family and miR-155 to sensitize resistant cells to cisplatin by targeting WEE1 and CHEK1 by performing loss-of -function and gain-of-function experiments. Using the MTT cytotoxicity assay and full cisplatin dose response curves, miRNA inhibitors of the miR15 family increased resistance while mimics sensitized resistant cells. miR-155 mimics resulted in sensitivity returning to parental levels and inhibitors of WEE1 and CHEK1 sensitized KB-CP.5 cells to CP. In conclusion, we have identified a miRNA family responsible for regulating the cell-cycle checkpoint kinases WEE1 and CHEK1 associated with cisplatin resistance. Citation Format: Lynn M. Pouliot, Jennifer Bai, Matthew D. Hall, Scott E. Martin, Michael M. Gottesman. The miR-15 family and miR-155 mediate WEE1 and CHEK1 effects on cisplatin-resistance in cancer cells [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 B12.