Jin Namkoong

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.

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.

Metabotropic Glutamate Receptor 1 and Glutamate Signaling in Human Melanoma

Recently, several laboratories have started to investigate the involvement of glutamate signaling in cancer. In previous studies, we reported on a transgenic mouse model that develops melanoma spontaneously. Subsequent studies in these mice identified that the aberrant expression of metabotropic glutamate receptor 1 (GRM1) in melanocytes played a critical role in the onset of melanoma. Confirmation of the etiologic role of GRM1 in melanoma development was shown in a second transgenic line with GRM1 expression under the regulation of a melanocyte-specific dopachrome tautomerase promoter. Ectopic expression of GRM1 was also detected in a subset of human melanoma cell lines and biopsies, suggesting that aberrant expression of GRM1 in melanocytes may contribute to the development of human melanoma. GRM1, a seven-transmembrane domain G protein-coupled receptor, is normally expressed and functional in neuronal cells, and its ligand, glutamate, is the major excitatory neurotransmitter. Human melanoma cells are shown here to release elevated levels of glutamate, implying a possible autocrine loop. Treatment of GRM1-expressing human melanoma cells with a GRM1 antagonist (LY367385 or BAY36-7620) or a glutamate release inhibitor (riluzole) leads to a suppression of cell proliferation as well as a decrease in levels of extracellular glutamate. Treatment of human melanoma cell xenografts with riluzole for 18 days via p.o. gavage or i.v. injection leads to inhibition of tumor growth by 50% in comparison with controls. These data suggest the importance of glutamate signaling in human melanoma and imply that the suppression of glutamate signaling may be a new target for melanoma therapy.

Curcumin downregulates the constitutive activity of NF-κB and induces apoptosis in novel mouse melanoma cells

Melanoma, the most deadly form of skin cancer, is very aggressive and resistant to present therapies. The transcription factor nuclear factor-kappa B (NF-κB) has been reported to be constitutively active in many types of cancer. Constitutively active NF-κB 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-κB in these melanoma-derived cells, as shown by electrophoretic mobility shift assay and reporter assays. We hypothesized that agents that inhibit NF-κB may also inhibit cell proliferation and may induce apoptosis in such melanoma cells. Curcumin has been shown to inhibit NF-κB 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-κB-driven reporter activity correlated with decreased levels of phospho-IκB&agr;, and decreased expression of NF-κB-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-κB 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.

Oncogenic activities of metabotropic glutamate receptor 1 (Grm1) in melanocyte transformation.

Previously, we reported a transgenic mouse line, TG‐3, that develops spontaneous melanoma with 100% penetrance. We demonstrated that ectopic expression of Grm1 in melanocytes was sufficient to induce melanoma in vivo. In this present study, the transforming properties of Grm1 in two cultured immortalized melanocytes were investigated. We showed that, in contrast to parental melanocytes, these Grm1‐clones have lost their requirement of TPA supplement for proliferation and have acquired the ability to form colonies in semi‐solid medium. Xenografts of these cells formed robust tumors in both immunodeficient nude and syngeneic mice with a short latency (3–5 days). The malignancy of these cells was demonstrated by angiogenesis and invasion to the muscle and the intestine. The requirement of Grm1 expression for the maintenance of transformation was demonstrated by an inducible siRNA system. Induction of expression of siRNA for Grm1 reduced the number of proliferating/viable cells in vitro and suppressed in vivo xenografted tumor growth in comparison with control. Taken together, these results showed that expression of exogeneously introduced Grm1 is sufficient to induce full transformation of immortalized melanocytes.

Glutamatergic Pathway Targeting in Melanoma; Single Agent and Combinatorial Therapies

Purpose: Melanoma is a heterogeneous disease where monotherapies are likely to fail due to variations in genomic signatures. B-RAF inhibitors have been clinically inadequate but response might be augmented with combination therapies targeting multiple signaling pathways. We investigate the preclinical efficacy of combining the multikinase inhibitor sorafenib or the mutated B-RAF inhibitor PLX4720 with riluzole, an inhibitor of glutamate release that antagonizes metabotropic glutamate receptor 1 (GRM1) signaling in melanoma cells. Experimental Design: Melanoma cell lines that express GRM1 and either wild-type B-RAF or mutated B-RAF were treated with riluzole, sorafenib, PLX4720, or the combination of riluzole either with sorafenib or with PLX4720. Extracellular glutamate levels were determined by glutamate release assays. MTT assays and cell-cycle analysis show effects of the compounds on proliferation, viability, and cell-cycle profiles. Western immunoblotting and immunohistochemical staining showed apoptotic markers. Consequences on mitogen-activated protein kinase pathway were assessed by Western immunoblotting. Xenograft tumor models were used to determine the efficacy of the compounds in vivo. Results: The combination of riluzole with sorafenib exhibited enhanced antitumor activities in GRM1-expressing melanoma cells harboring either wild-type or mutated B-RAF. The combination of riluzole with PLX4720 showed lessened efficacy compared with the combination of riluzole and sorafenib in suppressing the growth of GRM1-expressing cells harboring the B-RAFV600E mutation. Conclusions: The combination of riluzole with sorafenib seems potent in suppressing tumor proliferation in vitro and in vivo in GRM1-expressing melanoma cells regardless of B-RAF genotype and may be a viable therapeutic clinical combination. Clin Cancer Res; 17(22); 7080–92. ©2011 AACR.

Cloning and characterization of a novel gene, SHPRH, encoding a conserved putative protein with SNF2/helicase and PHD-finger domains from the 6q24 region☆ ☆

Here we report the identification of a novel transcript containing SNF2, PHD-finger, RING-finger, helicase, and linker histone domains mapping to the q24 band region of human chromosome 6. These domains are characteristic of several DNA repair proteins, transcription factors, and helicases. We have cloned both human and mouse homologs of this novel gene using interexon PCR and RACE technologies. The human cDNA, termed SHPRH, is 6018 bp and codes for a putative protein of 1683 amino acids. The mouse cDNA, termed Shprh, is 7225 bp and codes for a putative protein of 1616 amino acids. The deduced amino acid sequences of the two proteins share 86% identity. Both genes are expressed ubiquitously, with a transcript size of approximately 7.5 kb. Mapping of this gene to 6q24, a region reported to contain a tumor suppressor locus, prompted us to evaluate SHPRH by mutation analysis in tumor cell lines. We have identified one truncating and three missense mutations, thus suggesting SHPRH as a possible candidate for the tumor suppressor gene.

Grm5 expression is not required for the oncogenic role of Grm1 in melanocytes

Melanoma is the aberrant proliferation of melanocytes, the cells in the skin responsible for pigment (melanin) production. In its early stages, melanoma can be surgically removed with great success, however, advanced stages of melanoma have a high mortality rate due to the lack of responsiveness to currently available therapies. We have previously characterized a mouse melanoma model, TG-3, which has implicated the ectopic expression of metabotropic glutamate receptor 1 (Grm1, formerly mGluR1), in melanomagenesis and metastasis [Pollock et al., 2003. Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia. Nat Genet. 34, 108-112.]. Here we report the characterization of several in vitro cell lines derived from independent mouse melanoma tumors. These cell lines show characteristic phenotypes of transformed melanocytes, and express Grm1, and Grm5 (another metabotropic glutamate receptor), as well as melanocyte-specific protein markers. To investigate the possible role of Grm5 in vivo during melanoma development in our mice, we have crossed Grm5 null mice with TG-3, generating a new line of transgenic mice, TGM. TGMs, which are homozygote knockouts for Grm5 and carry the TG transgene, develop tumors with onset, progression, and metastasis very similar to that described for TG-3. Taken together, these results indicate that Grm1 can act as an oncogene in melanocytes independently of Grm5 expression.