Muscle RAS oncogene homolog is a novel therapeutic target gene contributing to hepatocellular carcinoma progression and sorafenib-resistance

Introduction:

Mutations in RAS proteins are an important cause of cancer genesis and development. The only approved drug for treatment of advanced HCC is sorafenib. Unfortunately, drug resistance to sorafenib limits survival in almost every patient. The RAS/RAF/ERK-pathway is the main cellular target of sorafenib in HCC. Other studies from our group newly show that the wildtype forms of canonical RAS genes (e.g. NRAS) are major therapeutic targets in HCC. Apart from canonical RAS proteins (i.e. KRAS, HRAS, NRAS), the roles of non-canonical RAS genes like Muscle RAS oncogene homolog (MRAS) are completely unknown in HCC. Therefore, the aim of this study was to characterize the expression, regulation and function of MRAS in HCC progression and sorafenib-resistance.

Methods:

Different HCC cell lines (PLC, Hep3B, HepG2 and Huh-7) were used for expression and functional analysis. Sorafenib-resistant cell clones (Hep3B-SR) were created by stepwise elevation of sorafenib treatment in cell culture. Quantitative RT-PCR and Western blot analysis were used for quantification of RNA and protein levels, respectively. Immunofluorescence analysis was used for confirmation of cellular localization of MRAS protein. MRAS function was explored after specific gene knockdown using single si-RNAs or si-RNA-Pools. Boyden chamber analysis was used for quantification of migration and invasion. Real-time cell analysis and Clonogenicity assays were used to quantify proliferation and stem cell properties.

Results:

MRAS was overexpressed in HCC cell lines and patient tissues. Knockdown of MRAS showed strong reduction of cell proliferation, clonogenicity, migration, and pAKT-signaling in HCC cell lines. Fluorescence-activated cell sorting (FACS) analysis and LDH-quantification in cell supernatents revealed that MRAS knockdown revealed no toxic effects on primary human hepatocytes. In silico analysis of The Cancer Genome Atlas (TCGA) derived datasets revealed that elevated MRAS expression is correlated with a poor patient outcome. MRAS expression was also upregulated, and MRAS knockdown was functional in acquired resistance to sorafenib.

Conclusions:

In this study, both in vitro and in vivo derived data show that the non-canonical RAS isoform MRAS is a novel therapeutic target gene contributing to HCC progression and sorafenib-resistance.