DGC-specific RHOA mutations maintained cancer cell survival and promoted cell migration via ROCK inactivation

// Takashi Nishizawa 1 , Kiyotaka Nakano 1 , Aya Harada 1 , Miwako Kakiuchi 2 , Shin-Ichi Funahashi 1 , Masami Suzuki 1 , Shumpei Ishikawa 2, 3 and Hiroyuki Aburatani 2 1 Department for Research, Forerunner Pharma Research Co., Ltd., Tokyo, Japan 2 Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan 3 Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Correspondence to: Takashi Nishizawa, email: nishizawatks@chugai-pharm.co.jp Keywords: RHOA; ROCK; diffuse gastric cancer; mutation; ARHGAP Received: November 26, 2017      Accepted: April 06, 2018      Published: May 01, 2018 ABSTRACT RHOA missense mutations exist specifically in diffuse type gastric cancers (DGC) and are considered one of the DGC driver genes, but it is not fully understood how RHOA mutations contribute to DGC development. Here we examined how RHOA mutations affect cancer cell survival and cell motility. We revealed that cell survival was maintained by specific mutation sites, namely G17, Y42, and L57. Because these functional mutations suppressed MLC2 phosphorylation and actin stress fiber formation, we realized they act in a dominant-negative fashion against the ROCK pathway. Through the same inactivating mechanism that maintained cell survival, RHOA mutations also increased cell migration activity. Cell survival and migration studies on CLDN18-ARHGAP ( CLG ) fusions, which are known to be mutually exclusive to RHOA mutations, showed that CLG fusions complemented cell survival under RHOA knockdown condition and also induced cell migration. Site-directed mutagenesis analysis revealed the importance of the GAP domain and indicated that CLG fusions maintained RHOA in the inactive form. Taken together, these findings show that the inactivation of ROCK would be a key step in DGC development, so ROCK activation might provide novel therapeutic opportunities.