Clinical relevance and functional role of nuclear met in hepatocellular carcinoma

Abstract

AIMS: Met is a receptor tyrosine kinase which triggers a wide range of normal physiological signaling cascades. However, a perturbation of the Met pathway is commonly found in human cancers. Emerging evidence has shown the presence of nuclear Met in some cancerous tissues and cell lines, suggesting that nuclear Met could have unexplored functions in the nucleus. The present study aimed to assess the expression and functions of nuclear Met in hepatocellular carcinoma (HCC). METHODS: Nuclear Met expression of 103 clinicopathologically characterized HCC paired samples was examined by immunohistochemistry using an antibody against the carboxyl terminus of Met. Statistical analyses were applied to evaluate the association of nMet with different clinical parameters. Nuclear localization of Met was determined by western blot analysis and immunofluorescence microscopy. Met cytoplasmic fragments were characterized by in vitro functional assay such as migration, invasion and proliferation in HCC cells. Nude mice model was employed to investigate the in vivo functional impact of nuclear Met. RESULTS: Nuclear Met is overexpressed in nearly 90% of HCC paired samples and its expression is progressively increased along HCC development from non-tumorous liver tissue to advanced HCC. Nonetheless, nuclear Met overexpression is significantly associated with venous invasion and poorer overall survival. We found that nuclear Met, which has a lower molecular weight than Met, could only be detected using an antibody against the carboxyl terminus of Met (C28) in tumorous tissues. This finding strongly suggests that nuclear Met only comprises of the carboxyl cytoplasmic region of full length Met. Moreover, both western blot analysis of nuclear fraction of HCC cells and immunofluorescence confirmed the nuclear localization of Met. We designed construct J1, J3 and T2 that encode Met fragment truncated after tyrosine residues D972 and P1027 in the juxtamembrane region and after tyrosine kinase domain beginning at L1157, respectively. Immunofluorescence microscopy showed both J1 and J3 constructs are dominantly expressed in the nucleus whereas T2 construct is expressed in the cytoplasm. These observations indicated the region in between J1 and T2 as the important region that facilitates the nuclear localization of Met. In vitro functional assay showed that nMet significantly promoted HCC cell proliferation and anchorage independent growth. It also significantly augmented HCC cell migration and invasiveness. Besides that, nMet also enhanced HCC tumor formation in animal model. Furthermore, we showed that nMet promoted tumor invasiveness and aggressiveness through NF-κB/MMP2 pathway. CONCLUSIONS: Nuclear Met is overexpressed and associated with venous invasion and poorer overall survival in HCC. We found that nuclear Met is actually the carboxyl terminal fragment of Met and translocates into nucleus to promote invasiveness in HCC cells.