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

Takashi Nishizawa,Masami Suzuki,Miwako Kakiuchi,Hiroyuki Aburatani,Kiyotaka Nakano,Shumpei Ishikawa,Aya Harada,Shin-Ichi Funahashi

doi:10.18632/oncotarget.25269

Takashi Nishizawa, Masami Suzuki + Show 6 more

Open Access

https://doi.org/10.18632/oncotarget.25269

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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.

Highlights

Diffuse-type gastric cancers (DGC) account for approximately 30% of all gastric cancers and are characterized by poorly differentiated adenocarcinoma with a worse prognosis than the intestinal type [1,2,3]
We revealed that RHOA mutations promoted cancer cell survival and migration activity by inactivating Rho-associated protein kinase (ROCK)
Contrary to our expectations, the knockdown of RHOA induced ROCK activation, and a ROCK inhibitor achieved cell survival similar to that seen in RHOA mutations; we concluded that functional RHOA mutations were lossof-function (LoF) mutations for ROCK activation

Summary

Introduction

Diffuse-type gastric cancers (DGC) account for approximately 30% of all gastric cancers and are characterized by poorly differentiated adenocarcinoma with a worse prognosis than the intestinal type [1,2,3]. DGC infiltrate into adjacent stromal tissues, spread without clear polyps or ulcers, and frequently show intraperitoneal metastasis [4, 5]. RHOA is a small GTPase that belongs to the RHO family and has various biological functions, such as cytokinesis, cell motility, and tissue development [9,10,11]. RHOA cycles between the GDP-bound inactive form and the GTP-bound active form under the control of regulatory proteins like guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). These regulatory proteins induce conformational change in RHOA to allow binding to substrates named effector proteins, one of which is Rho-associated protein kinase (ROCK). ROCK-LIMK-CFL1 signaling contributes to actin filament stabilization, while ROCK-MLCP-MLC signaling promotes actomyosin formation [12, 13]

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