Abstract 5372: Target proteins involved in resistance to c-Met inhibitors and their therapeutic role in human melanoma
Authors: Fong Jason T. , Puri Neelu 
Published: June 2012

Abstract
c-Met is activated by binding with its ligand, hepatocyte growth factor (HGF), and is an attractive target for molecular therapeutic inhibition. c-Met is over-expressed and mutated in human melanoma and plays a role in tumorigenecity and metastasis. c-Met overexpression could be one of the factors responsible for the severe invasiveness of metastatic melanoma resulting in poor patient prognosis. We found overexpression of c-Met in 6 out of 7 melanoma cell lines studied by immunoblotting. MU, WK, RU, MC, EP and LH over-expressed c-Met, however, in AN we found minimal c-Met expression. Small molecule tyrosine kinase inhibitors (TKI's), such as SU11274, which is an ATP-competitive small molecule inhibitor, can inhibit phosphorylation, activation and tumorigenecity of c-Met. We found that SU11274 inhibited c-Met phosphorylation and cell growth in all cell lines by 85-98% at 96 hrs with an IC50 between 1 and 2.5 μM and caused apoptosis (12-58%) in all cell lines except AN. To test the therapeutic effects of SU11274 in vivo, five million RU melanoma cells were injected subcutaneously into the hind flanks of RAG (−/-) mice. Tumors were allowed to develop for a week after which daily intratumoral injections of 50μg of SU11274 or equal volumes of vehicle control were given. We found that SU11274 decreased the size of MU Melanoma tumor xenografts in RAG (−/-) mice by 6.2 fold compared to mice treated with a vehicle control.
While c-Met inhibitors are currently in clinical trials, resistance to TKI's has been observed in cancer patients. To determine the mechanism of c-Met inhibitor resistance, four melanoma lines (MU, RU, WK, and EP) were exposed to increasing concentrations of SU11274 to develop drug resistance. The resistant melanoma cell lines proliferated in the presence of 7-9 fold higher concentration of SU11274. After generating cell lines resistant to SU11274, the expression levels of AKT and ERK were determined by immunoblotting to compare expression in parental vs. resistant cells. It was discovered that MU and EP drug resistant melanoma cells exhibited upregulation of p-AKT (up to 2.8 fold) and ERK (up to1.5 Fold) in the presence of SU11274. This upregulation may be through a c-Met independent pathway, possibly through the PI3K or HER-2/neu pathway, or there may be multiple pathways mediating resistance to c-Met TKI's. Conversely, RU and WK resistant lines showed down regulation of p-AKT (up to 7.3), however these cell lines showed upregulation of p-S6-kinase (up to1.3 fold) indicating that cell survival in these cell lines maybe mediated by p-S6- kinase. Additionally, WK and EP lines showed upregulation of p-4E-BP1 (up to 5.8 fold). Since both p-4E-BP1 and p-S6-Kinase are phosphorylated by mTOR, in human melanoma these results indicate that the mTOR pathway may play a key role in mediating resistance and further studies are being conducted to break SU11274 resistance in melanoma by rapamycin which targets the mTOR pathway.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5372. doi:10.1158/1538-7445.AM2011-5372