Elevated Expression of RIT1 Contributes to Hyper-activated RAS/MAPK Signal and Metastasis in Hepatocellular Carcinoma

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.

肝细胞癌发病率全球第六，死亡率全球第四。其病因多样化，潜在的分子机制尚不清楚。RAS/MAPK信号的过度激活在肝癌中非常普遍，但是常见的RAS突变在肝癌中并不常有。RAS/MAPK信号在肝癌中是怎么被激活的目前尚不清楚。我们利用TCGA数据库中的377个病人的测序数据，分析了10个RAS 亚家族，152个RAS 成员的突变以及基因组拷贝数变化。 发现13%的肝癌病人中存在RIT1拷贝数的扩增。除此以外RIT1的表达还受到CREB的调控。GSEA分析发现RIT1高表达的病人中，RAS/MAPK存在过度激活的现象。临床相关性分析发现，RIT1高表达病人总生存期(P=0.021)，无病生存期相对较短(P=0.013)，转移率更高(P<0.05)，血管浸润更多(P<0.001)。通过体内外的实验发现，RIT1可以通过抵抗氧化应激以及促进血管生成来促进转移，具体机制为，RIT1可以直接结合MAP3K11， MAP3K12, 和 CRAF ，通过MEK/ERK/EIF4E/HIF1-α/VEGFA来促进血管生成，通过p38 MAPK/AKT来抵抗氧化应激。基于以上发现，我们发现联合使用索拉菲尼和AKT抑制剂对RIT1高标的的肝癌患者或许有更好地效果，敲低RIT1可以进一步强化这个效果。在老鼠实验中我们发现5只实验鼠中有4只肿瘤完全消除。 总之，RIT1基因组的扩增是肝癌中最常见的RAS 突变。RIT1的高表达可以促进血管生成以及对抗氧化应激，此外RIT可以作为一个适用于索拉菲尼和AKT抑制剂联合使用的标志物。