Short interfering RNA directed against Slug blocks tumor growth, metastasis formation, and vascular leakage in bladder cancer

Abstract

In a previous study, we have identified Slug as selectively overexpressed in the highly invasive bladder cancer cells. Furthermore, Slug overexpression was correlated with metastasis in bladder cancer tissues. Here, we investigated the role and underlying mechanism of Slug in the process of growth, invasion and metastasis formation, and vascular leakage in a bladder cancer cell line T24. We knocked down in a bladder cancer cell line T24 by Slug siRNA, and the effects on invasion, metastasis, and proliferation were evaluated in vitro. A pseudometastatic model of bladder cancer in severe combined immunodeficient mice was used to assess the effects of Slug silencing on metastasis and angiogenesis development. Slug-specific siRNA in T24 cells led to a significant decrease in invasiveness and metastasis, proliferation rates, and angiogenesis in vitro. Slug-specific siRNA induced strong changes in cell invasiveness mainly through a mechanism of up-regulation of epithelial markers E-cadherin expression. Interestingly, E-cadherin-specific siRNA attenuated Slug siRNA-induced Matrigel invasion. Moreover, Slug-specific siRNA induced strong changes in microvessel counts in angiogenesis mainly through decreased activity of MMP-2, but not through cadherin expression and decreased activity of MMP-9. Restoration of MMP-2 expression in the Slug-silenced T24 cells resulted in an increased cell angiogenesis. Moreover, Slug-specific siRNA significantly reduced tumor growth by approximately 60% and inhibited metastasis and angiogenesis in a xenograft mouse model. This was accompanied by an increased expression of E-cadherin and a decreased expression of MMP-2 in tumor tissues. These data suggest that Slug regulates tumor growth, invasion and metastasis, and angiogenesis through E-cadherrin and MMPS passway. Blocking Slug signaling in bladder cancer may represent a novel strategy in the future to reduce metastatic disease burden in bladder cancer patients.