The gastric cancer-associated mutations R5W and Y42C in the RAS homologous RHOA protein cause distinct biochemical alterations, exhibit gain-of-function signaling and oncogenic activities.

Missense mutations of the RAS homologous RHOA gene have recently been identified in ~25% of diffuse gastric cancer (DGC). Unexpectedly, in contrast to the gain-of-function hotspot mutations found in RAS in cancer, DGC-associated RHOA mutations (e.g., R5W, Y42C) are localized at different hotspots that instead suggest loss-of-function alterations. First, we utilized bacterially expressed recombinant proteins and determined the biochemical consequences of these mutations on RHOA function. We found that RHOA WT and Y42C, but not R5W nucleotide exchange, was catalyzed by the ECT2 RhoGEF. Similarly, WT and Y42C, but not R5W GTP hydrolysis activity, was stimulated by the p190RhoGAP. Surprisingly, Y42C and to a lesser degree R5W exhibited impaired intrinsic GTP hydrolysis activity. Interestingly, Y42C showed both loss- and gain-of-function interaction with effectors. Second, we ectopically expressed WT and mutant RHOA proteins in NIH/3T3 fibroblast, and surprisingly, found that Y42C and R5W, similar to the lab-generated constitutively GTP-bound Q63L mutant (analogous to RAS Q61L), stimulated actin stress fiber formation and focal adhesion assembly. Finally, when ectopically expressed in beta-catenin-deficient (another mutation found in gastric cancer) mouse gastric organoids, Y42C disrupted the 3D architecture and induced a DGC-like phenotype in vitro and in vivo. In summary, our studies suggest mutation-specific biochemical defects that act as gain-of-function oncogenic RHOA mutations. Citation Format: Antje Schaefer, Richard G. Hodge, Devon R. Blake, Haisheng Zhang, Adam J. Bass, Channing J. Der. The gastric cancer-associated mutations R5W and Y42C in the RAS homologous RHOA protein cause distinct biochemical alterations, exhibit gain-of-function signaling and oncogenic activities [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A29.