PDF file - 78K, Table S1. List of primer and siRNA sequences; Table S2. Intersection between Predict target of miR-200a and genes that regulate the apoptotic processes; Table S3. The number of zebrafish embryos with migrating tumor (Dil) cells transfected with miR-200a, YAP1 siRNA, miR-Ctrl , Scramble RNA, antagomiR-200a and antagomiR-Ctrl respectively; Table S4. The number of migrated cells per tail vein of zebrafish embryos that had migrating tumor (Dil) cells transfected with miR-200a, YAP1 siRNA, miR-Ctrl and Scramble RNA, antagomiR-200a and antagomiR-Ctrl respectively
ARTICLE ABSTRACT
Purpose: The process of metastases involves the dissociation of cells from the primary tumor, penetration into the basement membrane, invasion, and exiting from the vasculature to seed and colonize distant tissues. miR-200a is involved in this multistep metastatic cascade. This study aimed to test the hypothesis that miR-200a promotes metastasis through increased anoikis resistance in breast cancer.Experimental Design: Breast cancer cells transfected with mimic or inhibitor for miR-200a were assayed for anoikis in vitro. miR-200a expression was assessed by quantitative real-time PCR (qRT-PCR). Luciferase assays, colony formation assays, and animal studies were conducted to identify the targets of miR-200a and the mechanism by which it promotes anoikis resistance.Results: We found that overexpression of miR-200a promotes whereas inhibition of miR-200a suppresses anoikis resistance in breast cancer cells. We identified Yes-associated protein 1 (YAP1) as a novel target of miR-200a. Our data showed that targeting of YAP1 by miR-200a resulted in decreased expression of proapoptotic proteins, which leads to anoikis resistance. Overexpression of miR-200a protected tumor cells from anoikis and promoted metastases in vivo. Furthermore, knockdown of YAP1 phenocopied the effects of miR-200a overexpression, whereas restoration of YAP1 in miR-200a overexpressed breast cancer cells reversed the effects of miR-200a on anoikis and metastasis. Remarkably, we found that YAP1 expression was inversely correlated with miR-200a expression in breast cancer clinical specimens, and miR-200a expression was associated with distant metastasis in patients with breast cancer.Conclusions: Our data suggest that miR-200a functions as anoikis suppressor and contributes to metastasis in breast cancer. Clin Cancer Res; 19(6); 1389–99. ©2013 AACR.
