Abstract A73: Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: The epithelial-mesenchymal transition factor Snail is associated with stemness and represses let-7 (2018)

Abstract Purpose: We aimed to understand how stemness is regulated by the Snail/let-7 axis in ovarian cancer cells, toward the goal of developing novel treatments that target stem-like cells to sensitize chemoresistant tumors. Background: Metastatic progression and disease recurrence in high-grade serous ovarian carcinoma (HGSOC) are in part driven by epithelial-mesenchymal transition (EMT) and cell reversion to cancer stem cell (CSC) phenotype. A contributor to the CSC phenotype is loss of miRNA let-7, due to increased expression of its targets, including oncogenes and pluripotency factors. Mechanisms leading to loss of let-7 are incompletely understood. Methods: Snail was overexpressed by estrogen receptor fusion, or EMT was induced with epidermal growth factor (EGF). Snail knockdown (KD) was by lentiviral small hairpin (sh)RNA expression and puromycin selection. Cell surface expression of CSC markers was analyzed by flow cytometry. Expression at the protein level was detected by immunofluorescence microscopy and Western blot. Gene expression of mesenchymal, epithelial, and pluripotency factors and miRNAs was detected by q-RT-PCR. Migration was determined by scratch assay and live-cell imaging. Data were analyzed by linear regression. Chromatin immunoprecipitation of Snail was followed by qPCR. Let-7i promoter luciferase with and without Snail were co-transfected into 293T cell lines and followed by luciferase assays for detection of bioluminescence. Let-7 overexpression was by transfection of mimics. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six-week-old nude (J:NU) mice, 5-8 per experiment, underwent ovarian bursa injections of luciferized HGSOC cells (OVCAR8 vs PDX) with control vs. Snail KD (250-500K cells). Bioluminescence was quantified by IVIS Lumina III over 21-50 days and analyzed by Living Image software. Results: We characterized several cell lines that accurately model HGSOC for their epithelial (E) vs. mesenchymal (M) and stem cell properties. OVSAHO, COV318, and OVCAR8 were selected for further study in increasing order of mesenchymal characteristics. Increasing let-7 levels or decreasing Snail levels disrupted the stem cell phenotype, reduced cells’ migratory ability, and sensitized cells to cisplatin. Snail bound promotors of let-7 in cell lines tested. Luciferase assays demonstrated direct repression of let-7 transcription by Snail. Metastatic tumor burden was significantly reduced in orthotopic xenografts using OVCAR8 cells expressing Snail shRNA. Similarly, in PDX derived from ascites or ovarian tumors, there was reduced tumor burden in Snail knockdown animals. Conclusions: We present preliminary data showing that the EMT factor Snail represses let-7, placing Snail at the nexus of dedifferentiation via loss of let-7, and invasiveness via EMT. PDX reproducibly phenocopy HGSOC. Cell line and patient-derived data support the relationship between Snail expression, let-7 downregulation, and the induction of CSC. We hypothesize that Snail KD disrupts the cancer stem cell state and disrupts disease progression. We propose that Snail is a potential pharmaceutical target for recurrent, metastatic EOC. Citation Format: Alyse Huisken, Nozomi Hojo, Hanmin Wang, Evgeny Chirshev, Carlotta Glackin, Yevgeniya Ioffe, Julia J. Unternaehrer-Hamm. Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: The epithelial-mesenchymal transition factor Snail is associated with stemness and represses let-7. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A73.