Expression and function of neuroblastoma RAS viral oncogene homolog (NRAS) in hepatocellular carcinoma

Introduction:

RAS proteins represent desirable therapeutic targets in cancer. However, in hepatocellular carcinoma (HCC), the functional role of RAS proteins is mostly unexplored, likely because RAS genes are mutated only in approximately 5% in this cancer type. The aim of this study was to elucidate the role of wild-type Neuroblastoma RAS viral oncogene homolog (NRAS) in HCC progression and therapy-resistance.

Methods:

Human HCC cell lines (PLC, HepG2, Hep3B) and isolated primary human hepatocytes (PHH) were used for expression analysis and functional assays. Furthermore, human HCC and corresponding non-tumorous liver tissues were analysed. Sorafenib-resistant HCC cells were generated by long-term exposure of cells to sorafenib. Knockdown of RAS proteins was achieved by specific si-RNA-Pools. Functional analysis included real-time cell proliferation assays, clonogenicity assays and Boyden chamber cell migration assays. Additionally, in silico analysis of patient-derived datasets was performed to determine the impact of NRAS expression on HCC patient survival.

Results:

Wild-type NRAS mRNA and protein levels were strongly overexpressed in HCC cells and tissues as compared to non-tumorous cells and tissues. In silico analysis identified high NRAS expression as a predictor of poor outcome in HCC patients. NRAS-knockdown had no effect on migration and ERK-signaling and only slightly reduced proliferation, clonogenicity and AKT-activation in HCC cells. NRAS-knockdown lead to upregulation of KRAS, and KRAS-knockdown-mediated inhibition of cell proliferation was further enhanced by co-knockdown of NRAS. Moreover, co-upregulation of NRAS and KRAS were linked to poor patient outcome as compared to patients with only one upregulated RAS-protein.

Furthermore, NRAS expression was significantly upregulated in sorafenib-resistant cells as compared to non-resistant HCC cells. In contrast to non-resistant HCC cells, NRAS-knockdown (alone) was sufficient to inhibit cell proliferation and to restore chemoresistance in sorafenib-resistant HCC cells.

Conclusions:

Wild-type NRAS is strongly overexpressed in HCC cell lines and tissues and high levels of NRAS predict poor patient outcome. NRAS was further upregulated and functional in sorafenib-resistance in HCC. Moreover, the KRAS isoform could functionally rescue loss of NRAS in HCC cells. Our findings suggest that NRAS expression could serve as a prognostic marker in HCC and co-targeting of NRAS and KRAS might be an effective therapeutic approach to overcome sorafenib-resistance in HCC.