RHOA^L57V drives the development of diffuse gastric cancer through IGF1R-PAK1-YAP1 signaling

Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr(42)-to-Cys (Y42C) and Leu(57)-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOA(Y42C) exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOA(L57V) promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOA(L57V), but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOA(L57V) also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOA(L57V) retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr(42) and Leu(57) in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOA(L57V) additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOA(Y42C). Our results reveal that RHOA(L57V) and RHOA(Y42C) drive the development of DGC through distinct biochemical and signaling mechanisms.