Abstract 1989: 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
Authors: Hojo Nozomi , Huisken Alyse , Wang Hanmin , Chirshev Evgeny , Nguyen Sang , Glackin Carlotta , Ioffe Yevgeniya , Unternaehrer Juli 
Published: August 2018

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 prevent recurrence.

Experimental Procedures:We determined the correlation between mesenchymal and stem cell phenotype in a panel of high grade serous ovarian cancer (HGSOC) cells, including patient-derived cells. Cell surface expression of cancer stem cell (CSC) markers was analyzed by flow cytometry. Expression at the protein level was detected by Western blot, at RNA level by q-RT-PCR. Epithelial-mesenchymal transition (EMT) was induced with epidermal growth factor (EGF). Snail knockdown (KD) was achieved by lentiviral small hairpin (sh)RNA expression and puromycin selection. Migration was determined by wound healing assay, chemoresistance by MTT assay. 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 miRNA 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. Bioluminescence was quantified by IVIS Lumina III.

Results:We characterized several cell lines that accurately model HGSOC for their epithelial (E) vs. mesenchymal (M) and stem cell properties using a novel index. OVSAHO, Kuramochi, COV318, and OVCAR8 were selected for further study in increasing order of M characteristics. M cell status correlated with morphology, stemness, and invasiveness. Increasing the tumor suppressor miRNA let-7 levels or decreasing Snail levels disrupted CSC phenotype, reduced cells' migratory ability, and sensitized cells to cisplatin. Snail bound promotors of let-7 , and 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 KD animals.

Conclusions:The EMT factor Snail represses let-7, placing Snail at the nexus of dedifferentiation via loss of let-7, and invasiveness via EMT. Orthotopic PDX demonstrate that Snail targeting reduces tumor burden. Cell line and patient-derived data supports the relationship between Snail expression, let-7 downregulation, and the induction of CSC. We hypothesize that Snail expression destabilizes differentiation and introduces the CSC state in part via let-7 repression. We propose that Snail is a potential target for recurrent, metastatic EOC.

Citation Format: Nozomi Hojo, Alyse Huisken, Hanmin Wang, Evgeny Chirshev, Sang Nguyen, Carlotta Glackin, Yevgeniya Ioffe, Juli Unternaehrer. 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 American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1989.