Activated c-Met receptor tyrosine kinase: A novel molecular therapeutic target for the treatment of malignant pleural mesothelioma
Authors: Ramasamy Jagadeeswaran, Patrick C. Ma, Hedy L. Kindler, Sujatha Jagadeeswaran, Tanguy Seiwert, Osvaldo Zumba, Gavin J. Gordon, Robert A. Kratzke, David J. Sugarbaker, Raphael Bueno, Ravi Salgia
Journal: Cancer Research (2005)

Proc Amer Assoc Cancer Res, Volume 46, 2005

2570 

Malignant pleural mesothelioma is an aggressive malignancy, which is associated with asbestos exposure. Although standard chemotherapy has a modest impact on quality of life and survival, novel treatments are still needed. We have recently identified that the c-Met receptor tyrosine kinase is an important molecule in a variety of malignancies, including mesothelioma. c-Met is a proto-oncogene product consisting of an α-chain of 50 kDa and a β-chain of 145 kDa. The ligand for c-Met has been identified as hepatocyte growth factor (HGF). We examined the c-Met expression in mesothelioma cell lines (H513, H2596, H2373, H28, H2052, H2452 and MSTO211H) and normal mesothelial cell line (Met 5A) by immunoblotting. We found that c-Met was expressed at various levels in all the mesothelioma cell lines tested and predominantly with high level expression in the H28 and H2596 cells but not in Met 5A cells. Using H2596 cells as model, we further showed that there was enhanced cell proliferation with HGF (40ng/ml) stimulation, enhanced activation of c-Met itself (pY1230/pY1234/pY1235 and pY1003) and increased formation of reactive oxygen species (ROS). There was activation of ERK1/2 and AKT notably with HGF stimulation. Time-lapse phase contrast video microscopy (TLVM) studies validate the importance of the HGF-triggered cell migration in mesothelioma cells. We examined the potency of a small molecule inhibitor (SU11274) of c-Met against cell viability using MTT assay and downregulation of c-Met signaling using immunoblotting with specific antibodies. Inhibition of activated c-Met at both the autocatalytic site (pY1230/pY1234/pY1235) and the c-Cbl binding site (pY1003) was achieved at 5 μM of SU11274. Further inhibition of downstream effectors molecules such as activated phospho-ERK1/2 and phospho-AKT with reduction of cell viability was achieved in mesothelioma cells treated with the SU11274 in a dose dependent manner (0-10μM). IC50 of SU11274 at 48h for H28 and MST0 211H cells was found to be ∼ 2.5 μM. Interestingly, abrogation of activated c-Met signaling and high reduction of cell viability were achieved with low dose levels of SU11274 only at H28 and MSTO 211H cells, which have overexpressed and endogenously activated c-Met. As measured by immunoflorescence microscopy, there was more extensive ligand-induced internalization of c-Met with HGF in the c-Met overexpressing H28 cells than in H2052 cells which express less c-Met. Importantly, immunoblot analysis with survivin and phospho AKT antibodies demonstrated that the overexpression of survivin and activation of AKT might play a protective role against the apoptotic effect of the small molecule inhibitor SU11274. The development of c-Met-targeted therapeutic agents, and those that target the downstream signaling pathways, would provide new possibilities in the treatment of malignant pleural mesothelioma.