Nasopharyngeal cancer (NPC) is highly prevalent in Southeast Asia. Although NPC is highly curable at an early stage, the mortality rate of recurrent and metastatic NPC remains high even with the most aggressive radiochemotherapy. The mechanism governing the high metastatic potential of NPC is largely unexplored. Previous clinical studies implicated that c-Met may be crucial for the malignant progression of NPC. In this study, we investigate the contribution of c-Met signaling to NPC tumorigenesis. c-Met protein is overexpressed in all NPC cell lines examined (CNE-1, CNE-2, HK1, HONE-1 and C666-1) when compared to normal epithelial control cell line, Het-1A (immortalized by SV40 large T antigen, from ATCC, USA). HGF, the ligand of c-Met, is expressed at detectable levels in NPC cells (high levels in HONE-1 and low levels in CNE-1) by RT-PCR. In two representative NPC cell lines, HONE-1 and CNE-2, HGF (20ng/ml) induced a rapid phosphorylation of c-Met (as early as 2 mins; by immunoprecipitation), and subsequent activation of MAPK (p-p42/44 MAPK) and STAT3 (p-STAT3-Tyr705). Sustained HGF-induced MAPK activation was observed up to 48 hrs. In both NPC cell lines, HGF induced a dose-dependent increase in cell proliferation at 48 hrs. HGF seems to be a stronger mitogen than EGF at equal concentration of 50ng/ml (by cell counting experiment at 48 hrs) in both cell lines. HGF enhanced HONE-1 proliferation by 128.8 ± 7.17 % and EGF by 48.11 ± 7.37 %, while in CNE-2, HGF enhanced cell growth by 44.06 ± 7.93 % and EGF by 28.05 ± 7.12 %, respectively. The effects of c-Met activation by HGF on NPC invasiveness and metastatic potential were then examined. HGF treatment induced a dose-dependent increase cell-cell dissociation (by cell scattering assay at 24 hrs) and cellular motility (by wound healing assay at 24 hrs) in HONE-1 and CNE-2. Moreover, specific inhibition of c-Met expression by a c-Met shRNA expression vector abrogated HONE-1 cell growth by 70% at 3 days after transfection when compared to control vector. The invasiveness (assayed by Matrigel Invasion assay) of c-Met shRNA transfected HONE-1 cells was significantly reduced by 46 % and 92 % (p=0.0017) in the absence of HGF and presence of HGF (20 ng/ml) respectively when compared with the control shRNA transfectant. Significant abrogation of HGF-induced cell scattering and migration (by wound healing assay at 24 hrs) was observed with the c-Met shRNA, but not the control shRNA transfectants. In summary, our results suggest that c-Met signaling contributes to the malignant phenotype of NPC.

Financial Support: Endowment Fund Research Grant for 2006/2007, United College, CUHK (Grant awarded to VWYL).

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA