c-Met, a potential personalized and therapeutic biomarker for mesenchymal and stemness characteristics in metastatic hepatocellular carcinoma

Background: c-Met, a high-affinity receptor for hepatocyte growth factor (HGF), is a tyrosine kinase that plays a critical role in cancer growth, invasion and metastasis. Recent work at the NCI has demonstrated that hepatocellular carcinoma (HCC) patients with an active HGF/c-Met gene expression profile have a significantly worse prognosis and high association with metastatic disease. Although targeting the HGF/c-Met pathway has been effective in the treatment of multiple cancers, the effect of c-Met inhibition in HCC and metastatic HCC remains unclear. In this study, we investigated the significance of c-Met as a biomarker for mesenchymal/metastatic and stemness properties and as a personalized therapeutic target in metastatic HCC.

Materials and Methods: Two human HCC cell lines, Huh7 and MHCC97-H were used in this study. Huh7 cells have no significant metastatic properties and MHCC97-H cells were initially isolated from a lung metastasis and have high metastatic potential. Using these cell lines, we examined the effect of a small molecule c-Met tyrosine kinase inhibitor, PHA665752. XTT assay and colony formation assay were used to investigate cell proliferation, annexin V/PI staining was conducted to determine cell apoptosis by flow cytometry, western-blot was performed to test protein expression, and quantitative RT-PCR was used to measure gene expression. Tumor xenograft model of MHCC97-H cells in nude mice was utilized to evaluate the effect of PHA665752 on tumor growth.

Results: Using gene and protein expression analysis, the metastatic HCC cell line MHCC97-H demonstrates a mesenchymal phenotype with decreased expression of E-Cadherin and increased expression of c-Met, fibronectin and Zeb2 compared to Huh7 cells, an epithelial line. The c-Met inhibitor, PHA665752 blocked c-Met phosphorylation in MHCC97-H cells. XTT and colony formation assay demonstrated that PHA665752 significantly inhibited cell proliferation and colony formation in MHCC97-H, but not in Huh7 cells. Additionally, 16-hour PHA665752 treatment induced a significant level of apoptosis in MHCC97-H cells (9.5% of PHA665752 treatment vs 0.02% of control, p<0.01) compared to Huh7 (0.4% of PHA665752 treatment vs 0.02% of control, p>0.05). An investigation of downstream signaling indicated that inhibiting c-Met phosphorylation in MHCC97-H cells with PHA665752 resulted in a significant reduction in Akt and Erk phosphorylation. PHA665752 did not affect Akt and Erk phosphorlylation in Huh7 cells. In the MHCC97-H cell xenograft model, daily administration of PHA665752 significantly inhibited tumor growth compared to vehicle treated mice (tumor volume 152.6mm³ of 12-day PHA665752 treatment vs 451.8mm³ of vehicle treatment at day 33 after inoculation of MHCC97-H cells, p<0.05) . Within the PHA665752 treated tumors, c-Met and Akt phosphorylation was reduced. In addition, PHA665752 induced E-cadherin expression and inhibit fibronectin expression in MHCC97-H cells, a change to a more epithelial phenotype, indicating a potential loss of metastasis ability. Finally, our data demonstrated that MHCC97-H cells displayed characteristics of stemness, such as self-renewal, chemo-resistance, elevated CD44 and ABCG2 expression, and PHA665752 treatment completely abolished sphere-formation of MHCC97-H cells.

Conclusion: C-met is a potential biomarker for mesenchymal/metastatic and stemness properties in HCC and c-Met represents a target for personalized treatment in HCC patients with an active HGF/c-Met pathway.