A significant proportion of bladder cancer patients initially present with muscle invasive disease often having a high rate of metastasis which is frequently fatal. Understanding and preventing formation of metastases in bladder cancer is critical in decreasing its mortality and morbidity. The Theodorescu lab has shown that Rho GDP-dissociation inhibitor 2 (RhoGDI2) is a metastasis suppressor gene in bladder cancer. While target molecules that are important in the RhoGDI2 metastasis suppression pathway have been identified, regulation of the RhoGDI2 metastasis suppression pathway remains unclear. Through the use of mass spectroscopy and co-immunoprecipitation methods the Theodorescu lab has shown that cellular sarcoma tyrosine kinase (Src) is able to phosphorylate and bind RhoGDI2. The lab has also shown that Src phospho- mimetic RhoGDI2 (glutamic acid substitution for tyrosine 153 at Src phosphorylation site on RhoGDI2) suppresses experimental in vivo metastasis more than 10 fold when compared to WT RhoGD12. Additionally, it has been shown that constitutively active Src transfected into the aggressive bladder cancer cell line UMUC3 reduces migration in a cell motility assay. The latter two results are surprising considering the classic dogma of Src acting as a proto-oncogene in most cancers. The goal of this project is to elucidate the interaction of Src and RhoGDI2 in regards to the metastasis suppression pathway of bladder cancer.
Two aims have been proposed to evaluate the hypothesis that Src regulates RhoGDI2 in the bladder cancer metastasis suppression pathway.
The first aim will establish that Src is able to influence the formation of bladder cancer metastases in vivo and growth and migration of bladder cancer cells in vitro in relation to cellular RhoGDI2 levels. This will be accomplished by using both activating and inactivating mutations of Src kinase function in order to observe how these mutations affect both in vitro and in vivo activity of bladder cancer cells with varying levels of RhoGDI2, The second aim will establish that Src and RhoGDI2 bind and interact through distinct domains and that these interactions are pivotal in the bladder cancer metastasis suppression pathway. This will be accomplished by mutating domains/sites on both Src and RhoGDI2 and evaluating binding by co-immunoprecipitation and yeast two-hybrid assays, then studying their effects on in vitro and in vivo bladder cancer phenotypes. In summary, the specific aims are as follows: (1) Evaluate the role of Src on motility and migration of bladder cancer cells in vitro and formation of bladder cancer metastases in vivo and the relationship of cellular RhoGDI2 levels and phosphorylation on this effect, and (2) Determine the nature and functional consequence of Src and RhoGDI2 interaction domains.
|Guin, Sunny; Pollard, Courtney; Ru, Yuanbin et al. (2014) Role in tumor growth of a glycogen debranching enzyme lost in glycogen storage disease. J Natl Cancer Inst 106:|
|Pollard, Courtney; Smith, Steven C; Theodorescu, Dan (2010) Molecular genesis of non-muscle-invasive urothelial carcinoma (NMIUC). Expert Rev Mol Med 12:e10|