Epithelial-mesenchymal transcription factor Snail leads to cancer stem cell phenotype via repression of tumor suppressor miRNA let-7: Snail knockdown restricts metastatic progression in epithelial ovarian carcinomas

Abstract

Objectives: Metastatic progression and disease recurrence in epithelial ovarian carcinomas (EOC) is in part driven by epithelialmesenchymal transition (EMT) and cell reversion to cancer stem cell (CSC) phenotype. Objectives of this study were to understand how EMT transcription factors (i.e., Snail) and tumor suppressor miRNAs (i.e., let-7) affect CSC phenotype, with the long-term goal of targeting CSCs to arrest disease progression. Method: EOC lines and patient-derived samples were analyzed; 6-week-old nude (J:NU) mice underwent ovarian bursa injections. In 6 cell lines, expression of epithelial and mesenchymal markers, let-7 miRNA levels, and pluripotency/CSC markers were characterized. Quantitative reverse transcriptase PCR was used to quantitate gene expression. Cell surface expression of CSC markers was analyzed by flow cytometry. Snail expression in EOC cells was induced. Small hairpin RNA (shRNA) was used to knock down Snail. Snail binding to let-7 promoters was demonstrated by chromatin immunoprecipitation assays. Let-7 transcription was measured by luciferase assays. Mice were injected with luciferized EOC cells: control versus Snail knockdown (250-500,000). Bioluminescence was quantified (IVIS Lumina) over 21-50 days. Results: For cell lines, overexpression of Snail was associated with CSC phenotype (increased expression of pluripotency factors Lin28 and Nanog and decreased let-7). Cells with shRNA Snail knockdown exhibited CSC phenotype reversal. Snail bound promoters of let-7 miRNAs. Luciferase assays demonstrated direct repression of let-7 expression by Snail. For the Murine model, orthotopic xenografts of cells in which Snail was knocked down resulted in decreased growth and metastasis of tumors. (See Fig. 1.) Conclusion: Snail knockdown results in reversal of CSC phenotype in EOC cell lines. Further, knocking down Snail results in decreased metastasis in murine orthotopic xenografts. We propose that Snail repression is a potential target for recurrent, metastatic EOC. (Figure Presented).