Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: transcription factor Snail represses let-7 and is associated with invasiveness phenotype

Abstract

Objectives: Metastatic progression in epithelial ovarian carcinoma (EOC) is in part driven by epithelial-mesenchymal transition (EMT) and cell reversion to cancer stem cell (CSC) phenotype. Decrease in let-7 microRNA(miRNA) levels correlates with reversion to mesenchymal phenotype and shorter survival. We aimed to 1) describe the mechanism for let-7 repression, 2) determine the effect of Snail knockdown (KD) on EOC cell sternness and invasiveness, and 3) determine whether Snail KD results in decreased tumor burden in an orthotopic patient-derived xenograft (PDX) model. Methods: EOC lines and patient-derived samples: Cells were treated with EGF to increase Snail expression. Snail KD was achieved via lentiviral delivery of small hairpin (sh)RNA. Quantitative RT-PCR was used to quantitate gene expression. Cell surface expression of CSC markers was analyzed by flow cytometry. Cell migration was determined by scratch assay and live-cell imaging. Data was analyzed by linear regression. Snail binding to let-7 promoters was demonstrated by chromatin immunoprecipitation (CHIP) assays. Snail overexpression was achieved via tamoxifen induction of estrogen receptor fusion protein. Let-7 transcription was measured by luciferase assays, analyzed by Student's t-test. PDX murine model: 6 week old nude (J:NU) mice underwent ovarian bursa injections. Mice were injected with luciferized EOC cells: control vs. Snail knockdown (125,000 cells per injection). Bioluminescence was quantified (1V1S Lumina) over 16 days to assess tumor burden and quantitated by ANOVA. Results: Cell lines: Overexpression of Snail was associated with CSC phenotype (increased expression of pluripotency factors Lin28 and Nanog, decreased let-7 expression). Cells with shRNA Snail knockdown exhibited CSC phenotype reversal. OVCAR8 cells with Snail KD exhibited slower hourly migration rates in migration assays (p<0.001). CHIP assays demonstrated that Snail bound promoters of let-7 miRNAs and promoter binding increased upon Snail overexpression. Luciferase assays demonstrated direct repression of let-7 by Snail. PDX murine model: Orthotopic xenografts injected with Snail KD cells resulted in decreased tumor burden (p<0.05). Conclusions: Snail directly represses let-7 transcription. Snail KD results in reversal of CSC phenotype in EOC cells. Further, knocking down Snail results in decreased tumor burden in murine orthotopic PDX. We propose that Snail is a potential pharmaceutical target for recurrent, metastatic EOC. Learning objective: Identify mechanisms of metastatic progression of ovarian cancer. Identify potential therapeutic targets for treating metastatic disease in ovarian cancer. Illustrate cancer stem phenotype of metastatic ovarian cancer.