Abstract/Project Summary The Ras homologous (Rho) proteins comprise a major branch of the Ras superfamily of small GTPases. My studies are focused on RhoA. Since RhoA shares significant structural and biochemical identities with the Ras oncoproteins, early studies addressed the possibility that RhoA may also function as an oncogene and drive cancer growth. Since RhoA regulates the actin cytoskeleton, cell migration and motility, and cell cycle progression, it seems logical that aberrant RhoA function can indeed impact the biology of cancer cells. Supporting an oncogene role for RhoA, early studies designed activated mutants of RhoA based on the cancer-associated mutants found in Ras. These studies in rodent fibroblast models made observations that supported mutant RhoA function in cancer. Therefore, it was disappointing when early cancer genome sequencing studies failed to identify RHOA mutations in the most common cancer types. This changed in 2014 when sequencing studies of T cell lymphomas and gastric cancers found recurrent missense mutations in RHOA. However, the mutations found were unexpected and suggested that loss rather than gain of RhoA function was responsible for driving the growth of these cancer types. My studies will address this apparent paradox in the field: is it a gain or loss of function in RhoA that is important to drive cancer? I propose comprehensive biochemical and cellular evaluation of the cancer-associated RhoA mutants to complete three aims to (1) determine the biochemical defect caused by cancer-associated mutations in RhoA; (2) evaluate the cellular activities of these RhoA mutants to assess gain or loss of function; and (3) determine if different RhoA mutants can drive cancer-associated growth phenotypes. In summary, my studies will provide a better mechanistic understanding of how aberrant RhoA function may drive cancer growth, an important first step to guide the development of pharmacologic approaches for the treatment of RHOA-mutant cancers. Furthermore, these studies will expose me to a wide variety of techniques and instruments and will enhance my development as a cancer researcher.