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 posttreatment 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. Recently, our group described a heretofore unknown component of melanoma pathogenesis. A transgenic murine model of melanoma was developed by the ectopic expression of metabotropic glutamate receptor 1 (GRM1) in melanocytes (1-3). These mice spontaneously develop melanocytic lesions indistinguishable from human melanoma. We have expanded these original studies and have now shown that more than 60% of human melanomas express GRM1 and that activation of this receptor results in activation of the mitogen-activated protein kinase (MAPK) pathway in a B-Raf-and N-Rasindependent fashion (1). In preclinical studies, we have shown that the ectopic expression of GRM1 in melanocytes is transforming and that inhibition of GRM1 signaling in vitro and in vivo results in cell cycle arrest and subsequent apoptosis in human melanoma (2).We have now translated our findings into the clinic and have completed a phase 0 trial of riluzole in patients with stage III and IV melanoma. Riluzole (2-amino-6-trifluoromethoxybenzothiazole) is a noncompetitive GRM1 receptor antagonist that has been shown to be safe and effective in patients with amylotropic lateral sclerosis (ALS; refs. 4-7). Rilu...

The goal of this study was to examine the effects of 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 noncompetitive GRM1 antagonist BAY36-7620 we were able to induced considerable inhibition of colony formation and invasion in GRM1-expressing melanoma lines. Neither pharmacological agent induced a 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 74μm nylon mesh. Both Riluzole and BAY36-7620 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 (BAY36-7620) 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.

Previously, by a yeast 2-hybrid screen, we identified signal transducer and activator of transcription 5b (Stat5b) as a substrate of the insulin receptor (IR). We demonstrated that refeeding of fasted mice leads to rapid activation of Stat5 proteins in liver, skeletal muscle, and fat, suggesting that Stat5b is a physiological target of insulin. Here, we show that injection of glucose or insulin into fasted mice leads to robust activation of both Stat5a and Stat5b in skeletal muscle. In C2C12 myotubes, we find that insulin stimulates tyrosine phosphorylation of Stat5a and Stat5b by 3-5-fold. This degree of Stat5 activation in vitro is significantly lower than what we observe in vivo and inversely correlates with IRS-1/2 levels. We can recapitulate robust insulin activation of Stat5 in C2C12 cells by stable overexpression of the human IR (hIR). To identify insulin-activated genes that are Stat5 targets, we also overexpressed an IR mutant (LA-hIR) that signals normally for mitogenactivated protein kinase-and phosphatidylinositol 3-kinase-dependent pathways but is deficient in Stat5 signaling in response to insulin. We demonstrate that insulin induces the expression of SOCS-2 mRNA in the wild type hIR but not in the LA-hIR-overexpressing cells. The induction of SOCS-3 by insulin is reduced but not lost in the LA-hIR cells. Therefore, our results suggest that insulin induction of SOCS-2, and in part SOCS-3 mRNA expression, is mediated by Stat5 and can be independent of mitogen-activated protein kinase and phosphatidylinositol 3-kinase-signaling pathways.Insulin plays a pivotal role in the regulation of glucose homeostasis and exerts numerous metabolic and proliferative responses in insulin-sensitive tissues (1). These effects are mediated by the binding of insulin and subsequent activation of the insulin receptor (IR) 1 tyrosine kinase (1). The activated receptor phosphorylates itself as well as several other intracellular substrates. Unlike several members of the receptor tyrosine kinase superfamily, which directly recruit and phosphorylate signaling/adapter proteins, the IR mainly signals by recruiting and phosphorylating members of the insulin receptor substrate family, IRS-1, -2, -3, and -4, Gab1 and -2, and p62dok-1, -2, and -3 (2, 3). These proteins then serve as multivalent docking sites for the recruitment of other Src homology domain 2-containing signaling proteins. Subsequently, at least two major signal transduction cascades are initiated including the mitogen-activated protein kinase-and phosphatidylinositol 3Ј-kinase-signaling pathways, which propagate the signal to various cytoplasmic and nuclear effectors (1, 4).Unlike the IR, cytokine receptors do not possess intrinsic tyrosine kinase activity. Instead, they depend upon receptorassociated Janus kinase tyrosine kinases to initiate signaling after ligand binding (5-7). These Janus kinases become activated and phosphorylate themselves, the cytokine receptor to which they are bound, and Src homology domain 2-containing signaling/adapter proteins, includin...

Using loss-of-function mutants of Ros and inducible epidermal growth factor receptor-Ros chimeras we investigated the role of various signaling pathways in Rosinduced cell transformation. Inhibition of the mitogenactivated protein kinase (MAPK) pathway with the MEK (MAP/extracellular signal-regulated kinase kinase) inhibitor PD98059 had little effect on the Ros-induced monolayer and anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells even though more than 70% of the MAPK was inhibited. In contrast, inhibiting the phosphatidylinositol 3-kinase (PI3K) pathway with the drug LY294002, a dominant negative mutant of PI3K, ⌬p85, or the phosphatidylinositol phosphatase PTEN (phosphatase and tensin homologue deleted in chromosome ten) resulted in a dramatic reduction of v-Ros-and epidermal growth factor receptor-Ros-promoted anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells, respectively. Parallel and downstream components of PI3K signaling such as the Rho family GTPases (Rac, Rho, Cdc42) and the survival factor Akt were all shown to contribute to Ros-induced anchorage-independent growth, although Rac appeared to be less important for Ros-induced colony formation in NIH3T3 cells. Furthermore, the transformation-attenuated v-Ros mutants F419 and DI could be complemented by constitutively active mutants of PI3K and Akt. Finally, we found that overexpressing a constitutively active mutant of STAT3 (STAT3C) conferred a resistance to the inhibition of Ros-induced anchorage-independent growth by LY294002, suggesting a possible overlap of functions between PI3K and STAT3 signaling in mediating Ros-induced anchorage-independent growth.The proto-oncogene c-Ros encodes a receptor protein-tyrosine kinase (RPT kinase) 1 that shares structural homology with the sevenless protein of Drosophila melanogaster (1). In addition, the protein-tyrosine kinase (PT kinase) domain of c-Ros shares greater sequence and structural homology with those of the insulin receptor family members than any other RPT kinases (1, 2). Although its ligand remains unknown, c-Ros expression is specific and highly regulated. c-Ros has been found to be expressed in a variety of organs/tissues, with relatively high levels in the kidney and intestine. c-Ros protein has been shown to be localized in the epithelial cells of the kidney collecting duct and intestinal villi in chicken (3). v-Ros is the oncogenic form of c-Ros and was identified as the transforming gene of the avian sarcoma virus UR2 (4). Comparison of the v-Ros oncogene with c-Ros revealed that all but 21 nucleotides immediately upstream of the transmembrane domain of c-Ros were deleted and replaced by the 150 amino acids of the retroviral p19 gag sequence (5) (Fig. 1A). v-Ros codes for a gag-Ros fusion protein with the p19 gag sequence protruding extracellularly (6). Aside from enhanced monolayer and anchorageindependent growth, UR2-infected chicken embryo fibroblasts (CEF) display a distinct transforming characteristic with an extremely elongated morph...

Objective. There is evidence that interleukin-4 (IL-4) plays a major role in the induction of extracellular matrix protein synthesis in fibrotic disease. We therefore examined the effect of IL-4 on collagen synthesis in primary fibroblasts isolated from normal and TSK/؉ mice, which spontaneously develop a scleroderma-like syndrome characterized by diffuse cutaneous hyperplasia.Methods. Expression of the IL-4 receptor was determined by flow cytometry and Western blotting. The IL-4 signal transduction cascade was analyzed by Western blotting. We assessed the role of signal transducer and activator of transcription 6 (STAT-6) in IL-4 induction of ␣2(I) collagen promoter activity and message levels via luciferase reporter assay and real-time polymerase chain reaction. The activation status of the transcription factors activator protein 1 (AP-1) and Sp-1 upon stimulation with IL-4 in normal and TSK/؉ fibroblasts was examined by electrophoretic mobility shift assay.Results. Flow cytometry and Western blotting showed that IL-4 receptor ␣ expression was elevated in TSK/؉ fibroblasts compared with normal fibroblasts. After IL-4 stimulation, janus-activated kinase 1 (JAK-1) and JAK-2 were phosphorylated to a greater degree in TSK/؉ fibroblasts than in C57BL/6 fibroblasts. TSK/؉ fibroblasts appeared to be hyperresponsive to IL-4, displaying increased synthesis of ␣1(I) collagen messenger RNA (mRNA), collagen protein, and activity of a luciferase reporter construct containing the -300 to ؉54 murine ␣2(I) collagen promoter. Overexpression of STAT-6 enhanced this effect, whereas expression of a dominant-negative STAT-6 abrogated the ability of IL-4 to induce ␣1(I) collagen mRNA in TSK/؉ fibroblasts. Moreover, IL-4 induced increased DNA binding activity of transcription factors that are important for collagen synthesis.Conclusion. Our observations indicate that IL-4 has a profound effect on several factors that have been identified as playing major roles in the regulation of collagen synthesis and suggest that IL-4 increases the expression of type I collagen through a mechanism involving the activation of transcription factors that bind to and activate collagen promoter.

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.

Background Follicular lymphoma (FL) is the most common indolent lymphoma and the 2nd most common non- Hodgkin lymphoma, accounting for 10%–20% of all lymphomas in the Western world. Epidemiologic and geographic trends of FL in Canada have not been investigated. Our study’s objective was to analyze incidence and mortality rates and the geographic distribution of FL patients in Canada for 1992–2010.Methods Demographic and geographic patient data for FL cases were obtained using the Canadian Cancer Registry, the Registre quebecois du cancer, and the Canadian Vital Statistics database. Incidence and mortality rates and 95% confidence intervals were calculated per year and per geographic area. Rates were plotted using linear regression models to assess trends over time. Overall data were mapped using Microsoft Excel mapping software (Redmond, WA, U.S.A.) to identify case clusters across Canada.Results Approximately 22,625 patients were diagnosed with FL during 1992–2010. The age-standardized incidence rate of this malignancy in Canada was 38.3 cases per million individuals per year. Geographic analysis demonstrated that a number of Maritime provinces and Manitoba had the highest incidence rates, and that the provinces of Nova Scotia and Quebec had the highest mortality rates in the nation. Regional data demonstrated clustering of FL within cities or regions with high herbicide use, primary mining, and a strong manufacturing presence.Conclusions Our study provides a comprehensive overview of the FL burden and its geographic distribution in Canada. Regional clustering of this disease in concentrated industrial zones strongly suggests that multiple environmental factors might play a crucial role in the development of this lymphoma.

Background Microsatellite instability (MSI) is a marker of chemoresistance, but it is associated with improved survival when compared to microsatellite-stable (MSS) colon cancers. We hypothesized that MSI tumors over-express chemoresistance-associated genes and under-express DNA damage/repair genes. We used ultra high-throughput sequencing (UHTS) to assess the expression of representative genes in MSI and MSS colon cancer cell lines. Methods Solexa UHTS was used to examine gene expression in HCT116 (MSI) and HT29 (MSS) cells, and normal colonic mucosa (NCM). We compared expression of 40 genes involved in chemoresistance, DNA repair, DNA damage, and drug metabolism pathways. Results We observed gene expression differences between MSI and MSS cell lines in 8 out of 40 genes involved in mismatch repair (MMR), DNA repair, drug metabolism and chemoresistance. MMR gene expression was lower in MSI cells, which is consistent with the MSI phenotype, whereas DNA repair genes were highly expressed in these cells. Genes associated with chemoresistance and drug metabolism also had increased expression in MSI cells. No difference in expression of DNA damage genes was observed between MSI and MSS cell lines. Conclusion Using UHTS gene expression analysis, we identified differential expression of genes between MSI and MSS cell lines which may account for resistance to chemotherapy in MSI tumors. UHTS expression analysis has the potential to identify genome-wide predictors of response or resistance to chemotherapy.