The glutamate release inhibitor Riluzole decreases migration, invasion, and proliferation of melanoma cells.

The goal of this study was to examine the effects of metabotropic glutamate receptor-1 (GRM1) blockade on melanoma anchorage-independent growth and invasion. We performed colony and invasion assays using GRM1-expressing melanoma lines and the GRM1-negative UACC930 line. Using the glutamate-release inhibitor Riluzole or the non-competitive GRM1 antagonist BAY 36-7620 we were able to induce considerable inhibition of colony formation and invasion in GRM1-expressing melanoma lines. Neither pharmacological agent induced significant reduction in colony formation or invasion in the GRM1-negative melanoma line, UACC930. Additionally we assessed the efficacy of these inhibitors to inhibit the growth of fresh melanoma tumor samples cultured on a 74-mum nylon mesh. Both Riluzole and BAY 36-7620 significantly inhibited tumor cell growth into the interstitial spaces of the mesh. When repeated with normal mole samples both inhibitors were much less effective in preventing the outgrowth of cells. These experiments show that a specific antagonist of GRM1 (BAY 36-7620) or an inhibitor of glutamate release (Riluzole) can significantly suppress melanoma migration, invasion and colony formation as well as inhibit the proliferation of fresh melanoma cells. These findings, added to our previous work, strengthen the case that GRM1 is a valid therapeutic target in patients with melanoma.

A phase 0 trial of riluzole in patients with resectable stage III and IV melanoma.

PURPOSE: Ectopic expression of GRM1 in murine melanocytes results in transformation into a form of melanoma, and more than 60% of human melanoma samples tested ectopically express GRM1. Stimulation of this receptor in vitro results in up-regulation of activated extracellular signal-regulated kinase (ERK). Furthermore, a xenograft model of melanoma treated with riluzole, an oral GRM1 blocking agent, showed decreased tumor growth compared with the untreated controls. We have now completed a phase 0 trial of riluzole in patients with melanoma. EXPERIMENTAL DESIGN: Patients enrolled on this trial underwent a pretreatment biopsy, took 200 mg of oral riluzole per day for 14 days, and then underwent resection of their remaining tumor. We compared the levels of pERK and pAKT in the pretreatment and post-treatment samples and assessed the metabolic activity of pretreatment and post-treatment tumors using fluorodeoxyglucose positron emission tomography (FDG-PET) scanning. RESULTS: We accrued 12 patients and all expressed GRM1. We found a significant decrease in pAKT and/or pERK in post-treatment tumor samples as compared with pretreatment samples in 4 (34%) patients. These four patients had a significant decrease in FDG-PET intensity post-treatment as well. Two other patients had a clinical response with no corresponding metabolic response; five patients had similar pretreatment and post-treatment FDG-PET scan findings; and one patient had progressive disease. CONCLUSIONS: Our data show that glutamate blockade with riluzole can inhibit signaling through the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT pathways and suppress the metabolic activity of melanoma. The ectopic expression of metabotropic glutamate receptors may be important in the pathogenesis of human melanoma, and targeting this pathway may be an effective therapy.

Dual mechanism of signal transducer and activator of transcription 5 activation by the insulin receptor.

Insulin stimulates signal transducer and activator of transcription 5 (Stat5) activation in insulin receptor (IR)-overexpressing cell lines and in insulin target tissues of mice. Stat5b and insulin receptor substrate 1 (IRS-1) interact with the same autophosphorylation site in the IR [phosphotyrosine (pY) 972] in yeast two-hybrid assays, and the IR phosphorylates Stat5b in vitro. These data suggest that Stat5 proteins might be recruited to, and phosphorylated by, the activated IR in vivo. Nevertheless, insulin activates Janus kinases (JAKs) in IR-overexpressing cell lines and in insulin target tissues. To determine whether Stat5 proteins must be recruited to the pY972LSA motif in the IR for insulin-stimulated activation in mammalian cells, we generated and tested a series of IR mutants. The L973R/A975D mutation abolishes the ability of the IR to induce Stat5 activation, whereas IRS-1 phosphorylation is unaffected. In contrast, the N969A/P970A mutation in the IR has no effect on Stat5 activation but significantly reduces IRS-1 phosphorylation. In coimmunoprecipitation assays, insulin-stimulated Stat5 activation correlates with Stat5 recruitment to the IR. We also find that insulin stimulates tyrosine phosphorylation of JAKs that are constitutively associated with the IR. Expression of dominant-negative (DN) JAKs, the JAK inhibitor suppressor of cytokine signaling 1, or pretreatment with the JAK inhibitor, AG490, reduces, but does not eliminate, insulin-induced Stat5 activation. Expression of the appropriate pair of DN JAKs in each of the singly JAK-deficient cell lines further establishes a component of insulin-stimulated Stat5 activation that is JAK independent. This likely represents phosphorylation of Stat5 proteins by the IR, as we find that IR kinase domain phosphorylates Stat5b in vitro on Y699 as efficiently as JAK2. Increasing the concentration of Stat5 proteins in cells favors the direct phosphorylation of Stat5 by the IR kinase where the DN-JAK inhibition of insulin-stimulated Stat5 activation becomes insignificant. At physiological levels of Stat5 however, we propose that JAKs and the IR both contribute to the insulin-induced phosphorylation of Stat5.