Supplementary Figure 2. Effect of RhoC knock down in LuL2 bladder cancer cells A. Expression of RhoA and RhoC in LuL2 cells following stable knock down of RhoC by shRNA. Representative western blots shown. B. Invasion of LuL2 cells expressing shCont or shRhoC-1 through matrigel was determined using a transwell assay. Results expressed as number of invaded cells per well presented as the mean{plus minus} S.E. *P<0.05. Independent experiments (n=3) were performed in triplicate. C. Colony formation in soft agar was assessed in LuL2 cells expressing shCont or shRhoC-1. Bargraph indicates the number of colonies formed and is presented as the mean{plus minus} S.E. *P<0.05. Independent experiments (n=3) were performed in triplicate.
ARTICLE ABSTRACT
RhoGDI2 (ARHGDIB) suppresses metastasis in a variety of cancers but the mechanism is unclear, thus hampering development of human therapeutics. RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) for the Rho family of GTPases thought to primarily bind to Rac1; however, Rac1 activation was not decreased by RhoGDI2 expression in bladder cancer cells. To better understand the GTPase-binding partners for RhoGDI2, a mass spectrometry–based proteomic approach was used in bladder cancer cells. As expected, endogenous RhoGDI2 coimmunoprecipitates with Rac1 and unexpectedly also with RhoC. Further analysis demonstrated that RhoGDI2 negatively regulates RhoC, as knockdown of RhoGDI2 increased RhoC activation in response to serum stimulation. Conversely, overexpression of RhoGDI2 decreased RhoC activation. RhoC promoted bladder cancer cell growth and invasion, as knockdown increased cell doubling time, decreased invasion through Matrigel, and decreased colony formation in soft agar. Importantly, RhoC knockdown reduced in vivo lung colonization by bladder cancer cells following tail vein injection in immunocompromised mice. Finally, unbiased transcriptome analysis revealed a set of genes regulated by RhoGDI2 overexpression and RhoC knockdown in bladder cancer cells.Implications: RhoGDI2 suppresses bladder cancer metastatic colonization via negative regulation of RhoC activity, providing a rationale for the development of therapeutics that target RhoC signaling. Mol Cancer Res; 13(3); 483–92. ©2014 AACR.
