Elevated expression of RIT1 contributes to hyper-activated RAS/MAPK signal and metastasis in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the 4th leading cause of cancer death and the 6th most common cancer in the world. HCC pathogenesis is heterogeneous, and the underlying biological and molecular mechanisms still obscure. Hyperactivation of RAS/MAPK signaling is commonly observed in HCC. However, gain-of-function mutations of canonical RAS genes are rarely detected in HCC and it remains unclear how the activity of this pathway is turned on during hepatocarcinogenesis. We performed a comprehensive analysis of RAS superfamily genetic alterations across 10 sub-family, 152 members in 377 HCC patients from the Cancer Genome Atlas (TCGA) database to dig out the potential driver of the hyper-activated RAS/MAPK signal in HCC. RIT1 (Ras-like without CAAX 1) was the most frequently altered RAS member and amplified in 13% of the HCC cohort. Besides, we found oxidative stress could upregulate RIT1 expression via activating CREB. Gene set enrichment analysis (GSEA) showed an enrichment of the RAS/MAPK pathway in these tumors with high-level RIT1 expression. Clinical correlation analysis showed that RIT1 overexpression correlated with poor overall survival (P=0.021), disease-free survival (P=0.013), metastasis (P<0.05), and vascular invasion (P<0.001) in HCC. Moreover, functional characterization and mechanistic investigation of RIT1 were verified both in vitro and in vivo. The functional study found that RIT1 promotes HCC metastasis by promoting angiogenesis and survival under oxidative stress. The potential downstream pathways of RIT1 were investigated by RNA interfering screen, Co-immunoprecipitation (Co-IP), and western blots. Then we found that RIT1 induced angiogenesis via the MEK/ERK/EIF4E/HIF1-α/VEGFA axis. MAP3K11 and MAP3K12, in addition to CRAF, could mediate this process by binding to RIT1. Moreover, RIT1 increased the phosphorylation of p38 MAPK and AKT to promote cell survival. Based on this mechanistic understanding, we treated RIT1-overexpressing HCC with combined regimen sorafenib plus AKT inhibitor, and achieved enhanced antitumor effects in vivo. Of note, knockdown RIT1 furtherly improved the sorafenib plus AKT inhibitor combination treatment, 4/5 mice released from the tumor burden.

In summary, Copy number amplification of RIT1 is the most common genetic alteration of the RAS family in HCC. Its overexpression promotes HCC growth and metastasis by enhancing tumor vascularization and anti-stress survival. Besides, RIT1 is a useful biomarker for the combination treatment of sorafenib and AKT inhibitor. Targeting RIT1 therapy will be a promising therapeutic strategy in HCC.