Abstract 1244: An in vitro biological assay to identify small molecule upregulators of let-7 miRNA in LIN28- positive ovarian cancer cells
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AbstractOnco-fetal LIN28, present in two isoforms A and B and expressed in cancer cells, promotes tumorigenesis by suppressing the biogenesis of the tumor suppressor microRNA, let-7, thereby activating an array of oncogenes including RAS, MYC, HMGA2 and Cyclin D1. Studies have demonstrated a positive correlation between LIN28 A/B expression and advanced epithelial ovarian cancers (Histological grade 2 or 3). Correlation of LIN28 expression to a poor progression-free survival and resistance to platinum-based therapies makes LIN28-let-7 pathway an attractive target for ovarian cancer chemotherapy. The overall goal of this study was to identify small molecules that upregulate let-7 levels in LIN28A or LIN28B positive ovarian cancer cells. We hypothesize that induction of let-7 levels would potentially improve prognosis for ovarian cancer patients bearing LIN28 positive tumors. To test this hypothesis, we developed a dual luciferase let-7 reporter assay in which let-7 binding sites found in the 3 UTR of the HMGA2 gene were cloned downstream of the nanoluciferase reporter gene. In this assay, elevated let-7 levels would result in repression of the nanoluciferase signal. Data obtained was normalized to Firefly luciferase activity. We tested efficacy of the assay system using let-7 mimics and inhibitors in LIN28A (OVK-18) and LIN28B (TOV-112D) positive ovarian cancer cell lines stably expressing the reporter cassette that were generated in house. Transfection of the let-7 mimic resulted in a 0.5 fold decrease in both cell lines relative to the negative controls, whereas transfection of the let-7 inhibitor resulted in a two-fold increase in relative luminescence. We miniaturized the assay to 384-well format for small molecule screening. The Z prime (Z) scores for both cell lines was 0.58 and 0.84 respectively, indicating that the assay was robust to carry out the screens. We performed a primary screen using the Prestwick and LOPAC1280 libraries for each cell line in duplicate as well as a counter screen using a reporter cassette that lacked let-7 binding sites. A compound was identified as a hit if it ranked within the top 100 compounds in the primary screen and outside the top 250 compounds in the counter screen. The hit rate for LIN28A positive cell line was 80 % for the Prestwick and 74 % for the LOPAC1280 libraries. For the LIN28 B positive cell line, the hit rate was 73 % and 53 % for the Prestwick and LOPAC libraries. The hits were validated by assaying for let-7 levels using qRTPCR. The hits identified included compounds that inhibited PI3K-mTOR, nFkB, c-MYC, Cyclin D1, BET1 as well as aurora kinase. These targets have been reported in the literature to downregulate let-7 levels. In conclusion, our assay-system is the first to utilize a biological assay of let-7 levels for small molecule library screening thus serving as a valuable tool for cancer drug discovery.Citation Format: Miriam-Rose Menezes, Goeun Bae, Yong Sung Park, Julien Balzeau, Clifford C. Stephan, John P. Hagan. An in vitro biological assay to identify small molecule upregulators of let-7 miRNA in LIN28- positive ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1244. doi:10.1158/1538-7445.AM2017-1244
