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Chandrani Chattopadhyay, Nancy J. Poindexter, Scott E. Woodman, Bita Esmaeli, Elizabeth A. Grimm; Targeting c-Met/HGF Pathway In Uveal Melanoma Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1443.
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Uveal melanoma is the most frequent primary intraocular cancer in adults, and unique as metastases are blood borne and grow preferentially in the liver. The mechanism for this is largely unknown, but growth factors synthesized in the liver may play an important role. We have been exploring these factors and their receptors. The hepatocyte growth factor (HGF) and its receptor c-Met appear particularly attractive as preliminary data show that c-Met is expressed on uveal melanoma. This study has attempted to determine the role of c-Met/HGF system in growth/survival and migration of uveal melanoma cells. We also tested the effect of a specific small molecule c-met inhibitor in uveal melanoma cells.
RT PCR and Western blotting were used to detect c-Met mRNA and protein in a panel of 11 human uveal melanoma cell lines. These included 5 with GNAQ mutation and 1 expressing GNA11. The small molecule c-Met inhibitor MK-8033 from Merck was used to block the c-Met/HGF pathway. We determined its effect on proliferation by MTT assays, its effect on c-Met dependent downstream signaling by Western blots for chosen molecular markers, its effect on cell migration in a modified in vitro Boyden chamber migration assay and its induction of apoptosis via cell cycle analysis with PI staining and PARP processing by Western blots.
Detectable levels of c-Met mRNA were found in all 11 uveal melanoma cells lines tested, but only 8 of them expressed the protein. The activation of c-Met was constitutive in some cell lines while in others it was ligand induced. We found that Akt activation is downstream to c-Met activation. Using a small molecule c-Met inhibitor, MK8033, c-Met phosphorylation (activation) in cells expressing c-met protein was blocked in a dose-dependent fashion. Such inhibition also resulted in inhibition of cell proliferation, reduced Akt activation and reduced cell migration (60-85% inhibition on treatment with 1µM MK-8033) in c-Met positive cell lines. MK-8033 treatment led to induction of apoptosis (enhanced PARP processing) in uveal melanoma cell lines, and the number of cells gated in G0/G1 increased from 3-5% to 15-20% with the lowest dose of 2.5µM MK-8033.
Inhibition of c-Met in c-Met positive uveal melanoma cell lines decrease cell survival, Akt activation, cell migration, as well as the induction of apoptosis. Therefore we conclude that blocking the c-Met/HGF pathway in uveal melanoma may be a relevant targeted therapy option in this disease that may also be effective in the adjuvant setting to prevent liver metastasis.
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