Abstract 2145: Coupling S100A4 to Rhotekin alters Rho signaling output in breast cancer cells

Abstract

Listen

Translate article

Abstract S100A4 is a calcium binding protein and tumor metastasis associated factor that has been suggested to promote motility and invasiveness of different types of cancer. This migratory promoting effect, in part, is due to the interaction of S100A4 with actin and actin binding proteins, such as myosin IIA. We found that S100A4 interacts with Rho binding domain of Rhotekin (TRBD), one of the Rho effectors, thus suggesting a connection between the Rho and S100A4 pathways. To test whether this interaction is specific, we performed GST pull-down assays using Rho binding domain from different Rho effectors such as Rhotekin, Rhophilin, PKN, ROCK II, and Citron. These results showed that S100A4 specifically interacts with Rho binding domain of Rhotekin but not of other Rho effectors. We further determined that this interaction is direct and calcium-dependant by performing GST-TRBD pull-down assays with purified S100A4 in the presence and absence of calcium. The in vivo interaction of these two proteins was further confirmed by immunoprecipitation of exogenously expressed mutated forms of Rhotekin with endogenous S100A4 from MDA-MB-231 cells. Confocal microscopy showed that Rhotekin and S100A4 co-localize at the edge of membrane ruffles in response to EGF. We demonstrated that S100A4 did not bind TRBD using the same residues as Rho, as determined by using a triple-A mutant of Rhotekin. Interestingly, we found that active Rho, S100A4 and Rhotekin are in a complex by using constitutively active GST-RhoQ63L pull-down assay. To examine the function of this interaction, RNAi was used to suppress Rhotekin and/or S100A4 in MDA-MB-231. F-actin staining showed that suppression of both RTKN and S100A4 leads to the loss of Rho-dependent membrane ruffling in response to EGF stimulation, an increase in contractile F-actin “stress” fibers in the cell body and a reduction in invasive growth in three-dimensional culture. Taken together, we showed that S100A4 specifically and directly interacts with Rhotekin in a calcium-dependent manner. This coupling permits S100A4 to complex with RhoA and switch RhoA function from stress fiber formation to membrane ruffling. Based on the function of this interaction, we propose that interaction of S100A4 and Rhotekin alters the functional output of Rho signaling to confer an invasive phenotype in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2145. doi:1538-7445.AM2012-2145