Abstract A15: mGlu1 Receptors and downstream signal transduction proteins as therapeutic targets for the treatment of metastatic melanoma

The purpose of this study was to elucidate the downstream signaling of mGlu1 receptors involved in melanoma cell proliferation. Previously, using melanoma cell lines expressing mGlu1 receptors, we demonstrated that glutamate stimulation of mGlu1 receptors increases melanoma cell growth in BRAF mutant, NRAS mutant, and BRAF/NRAS wild-type melanoma cells. Blockade of mGlu1 receptor activation through glutamate depletion, mGlu1 receptor knockdown, and mGlu1 receptor antagonism inhibited glutamate-stimulated melanoma cell growth, further identifying mGlu1 receptors as a therapeutic target for the treatment of metastatic melanoma. Because mGlu1 receptor antagonists blocked growth of both BRAF and NRAS mutant melanoma cells, it suggests that mGlu1 receptors stimulate melanoma cell proliferation either by a mechanism downstream of NRAS and BRAF or through a distinct and necessary pathway. Here we report that glutamate does not stimulate melanoma cell proliferation through activation of the canonical G alpha-q dependent pathway, as neither glutamate nor quisqualate stimulate phosphoinositide hydrolysis in the mGlu1 receptor-expressing melanoma cell lines SK-MEL-2 and SK-MEL-5 (as measured by scintillation proximity assay). Furthermore, the G protein biased ligand quisqualate does not stimulate SK-MEL-2 or SK-MEL-5 melanoma cell growth (as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay). In contrast, MEK inhibitors block glutamate stimulated growth of SK-MEL-2 and SK-MEL-5 melanoma cells, indicating that the MEK/ERK signal transduction cascade is involved in glutamate dependent growth. Our laboratory has shown in stably transfected CHO cells that mGlu1a receptors can also activate a G protein independent, Beta-arrestin-1 dependent pathway, and this Beta-arrestin-1 dependent signaling requires internalization of mGlu1 receptors. To assess the involvement of Beta-arrestin-1 dependent signaling in glutamate stimulated melanoma growth, cells were treated with a dynamin inhibitor, thus preventing internalization. Inhibition of dynamin selectively blocked glutamate stimulated growth of mGlu1 receptor expressing melanomas, but did not affect proliferation of melanoma cells lacking mGlu1 receptor expression. In summary, these data suggest that glutamate stimulates melanoma cell growth through activation of a dynamin-dependent pathway which may result in ERK phosphorylation. Understanding the signaling mechanism downstream of mGlu1 rece