To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

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Melanoma, the most deadly form of skin cancer, is very aggressive and resistant to present therapies. The transcription factor nuclear factor-kappa B (NF-kappaB) has been reported to be constitutively active in many types of cancer. Constitutively active NF-kappaB seen in melanoma likely plays a central role in cell survival and growth. We have established and characterized novel cell lines from our murine melanoma model. Here we report the constitutive activity of NF-kappaB in these melanoma-derived cells, as shown by electrophoretic mobility shift assay and reporter assays. We hypothesized that agents that inhibit NF-kappaB may also inhibit cell proliferation and may induce apoptosis in such melanoma cells. Curcumin has been shown to inhibit NF-kappaB activity in several cell types. In our system, curcumin selectively inhibited growth of melanoma cells, but not normal melanocytes. Curcumin induced melanoma cells to undergo apoptosis, as shown by caspase-3 activation, inversion of membrane phosphatidyl serine, and increases in cells in the sub-G1 phase. A curcumin dose-dependent inhibition of NF-kappaB-driven reporter activity correlated with decreased levels of phospho-IkappaBalpha, and decreased expression of NF-kappaB-target genes COX-2 and cyclin D1. This study demonstrates that the use of cells from our model system can facilitate studies of signaling pathways in melanoma. We furthermore conclude that curcumin, a natural and safe compound, inhibits NF-kappaB activity and the expression of its downstream target genes, and also selectively induces apoptosis of melanoma cells but not normal melanocytes. These encouraging in-vitro results support further investigation of curcumin for treatment of melanoma in vivo.

Non-neuronal expression of components of the glutamatergic system has been increasingly observed, and our laboratory previously had demonstrated the etiological role of ectopically expressed metabotropic glutamate receptor 1 (Grm1/mGluR1) in mouse models of melanoma. We hypothesize that inappropriate glutamatergic signaling in other cell types can dysregulate growth leading to transformation and tumorigenesis. As most cancers are carcinomas, we selected an immortalized primary baby mouse kidney (iBMK) cell model to assess whether Grm1 can transform epithelial cells. These iBMK cells, engineered to be immortal yet non-tumorigenic and retaining normal epithelial characteristics, were used as recipients for exogenous Grm1 cDNA. Several stable Grm1 expressing clones were isolated and the Grm1-receptors were shown to be functional, as evidenced by the accumulation of second messengers in response to Grm1 agonist. Additionally activated by agonist were MAPK and AKT signaling cascades, major intracellular pathways shown by many investigators to be critical in melanomagenesis and other neoplasms. These Grm1-iBMK cells exhibited enhanced cell proliferation in in vitro MTT assays and significant tumorigenicity in in vivo allografts. Persistent Grm1 expression was required for the maintenance of the in vivo tumorigenic phenotype as demonstrated by an inducible Grm1-silencing RNA. These are the first results that indicate Grm1 can be an oncogene in epithelial cells. Additionally, relevance to human disease in the corresponding tumor type of renal cell carcinoma (RCC) may be suggested by observed expression of GRM1/mGluR1 in a number of RCC tumor biopsy samples and cell lines, and the effects of GRM1 modulation on tumorigenicity therein. Moreover RCC cell lines exhibited elevated levels of extracellular glutamate, and some lines responded to drugs which modulate the glutamatergic system. These findings imply a possible role for glutamate signaling apparatus in RCC cell growth, and that the glutamatergic system may be a therapeutic target in renal cell carcinoma.

Although the glutamatergic system usually functions in the CNS, expression has been observed in non-neuronal tissues and a subset of cancers. Metabotropic glutamate receptors (mGlus) are highly "druggable" GPCRs and thus a priority for validation as therapeutic targets. We have previously reported that the aberrant expression of mGlu1 is sufficient to induce spontaneous melanoma development in vivo. We isolated and characterized several stable mGlu1-mouse melanocytic clones and demonstrated that these clones are transformed and tumorigenic. We hypothesize that expression of mGlus may not be uncommon in the pathogenesis of tumors other than melanoma, and that activity of an otherwise normal glutamate receptor in an ectopic cellular environment involves signaling pathways which dysregulate cell growth, ultimately leading to tumorigenesis. As most human cancers are of epithelial origin (carcinomas), in this review, the possibility that mGlu1 could function as a complete oncogene and transform epithelial cells is also discussed.