To develop effective therapies for advanced high grade serous ovarian cancer (HGSOC), understanding mechanisms of recurrence and metastasis is necessary. In this study, we define the epithelial/mesenchymal status of cell lines that accurately model HGSOC, and evaluate the therapeutic potential of targeting Snai1 (Snail), a master regulator of the epithelial/mesenchymal transition (EMT) in vitro and in vivo. The ratio of Snail to E-cadherin (S/E index) at RNA and protein levels was correlated with mesenchymal morphology in four cell lines. The cell lines with high S/E index (OVCAR8 and COV318) showed more CSC-like, motile, and chemoresistant phenotypes than those with low S/E index (OVSAHO and Kuramochi). We tested the role of Snail in regulation of malignant phenotypes including stemness, cell motility, and chemotherapy resistance: shRNA-mediated knockdown of Snail reversed these malignant phenotypes. Interestingly, the expression of let-7 tumour suppressor miRNA was upregulated in Snail knockdown cells. Furthermore, knockdown of Snail decreased tumour burden in an orthotopic xenograft mouse model. We conclude that Snail is important in controlling HGSOC malignant phenotypes and suggest that the Snail/Let-7 axis may be an attractive target for HGSOC treatment.
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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.