Seung-Shick Shin

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.

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.

AKT2 is a downstream target of metabotropic glutamate receptor 1 (Grm1)

We reported earlier on the oncogenic properties of Grm1 by demonstrating that stable Grm1‐mouse‐melanocytic clones proliferate in the absence of growth supplement and anchorage in vitro. In addition, these clones also exhibit aggressive tumorigenic phenotypes in vivo with short latency in tumor formation in both immunodeficient and syngeneic mice. We also detected strong activation of AKT in allograft tumors specifically AKT2 as the predominant isoform involved. In parallel, we assessed several human melanoma biopsy samples and found again that AKT2 was the predominantly activated AKT in these human melanoma biopsies. In cultured stable Grm1‐mouse‐melanocytic clones, as well as an metabotropic glutamate receptor 1 (Grm1) expressing human melanoma cell line, C8161, stimulation of Grm1 by its agonist led to the activation of AKT, while preincubation with Grm1‐antagonist abolished Grm1‐agonist‐induced AKT activation. In addition, a reduction in tumor volume of Grm1‐mouse‐melanocytic‐allografts was detected in the presence of small interfering AKT2 RNA (siAKT2). Taken together, these results showed that, in addition to the MAPK pathway previously reported being a downstream target of stimulated Grm1, AKT2 is another downstream target in Grm1 mediated melanocyte transformation.

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.

Activation of the Glutamate Receptor GRM1 Enhances Angiogenic Signaling to Drive Melanoma Progression

Glutamate-triggered signal transduction is thought to contribute widely to cancer pathogenesis. In melanoma, overexpression of the metabotropic glutamate receptor (GRM)-1 occurs frequently and its ectopic expression in melanocytes is sufficient for neoplastic transformation. Clinical evaluation of the GRM1 signaling inhibitor riluzole in patients with advanced melanoma has demonstrated tumor regressions that are associated with a suppression of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathways. Together, these results prompted us to investigate the downstream consequences of GRM1 signaling and its disruption in more detail. We found that melanoma cells with enhanced GRM1 expression generated larger tumors in vivo marked by more abundant blood vessels. Media conditioned by these cells in vitro contained relatively higher concentrations of interleukin-8 and VEGF due to GRM1-mediated activation of the AKT-mTOR-HIF1 pathway. In clinical specimens from patients receiving riluzole, we confirmed an inhibition of MAPK and PI3K/AKT activation in posttreatment as compared with pretreatment tumor specimens, which exhibited a decreased density of blood vessels. Together, our results demonstrate that GRM1 activation triggers proangiogenic signaling in melanoma, offering a mechanistic rationale to design treatment strategies for the most suitable combinatorial use of GRM1 inhibitors in patients.

Targeting Glutamatergic Signaling and the PI3 Kinase Pathway to Halt Melanoma Progression

Our group has previously reported that the majority of human melanomas (> 60%) express the metabotropic glutamate receptor 1 (GRM1) and that the glutamate release inhibitor riluzole, a drug currently used to treat amyotrophic lateral sclerosis, can induce apoptosis in GRM1-expressing melanoma cells. Our group previously reported that in vitro riluzole treatment reduces cell growth in three-dimensional (3D) soft agar colony assays by 80% in cells with wildtype phosphoinositide 3-kinase (PI3K) pathway activation. However, melanoma cell lines harboring constitutive activating mutations of the PI3K pathway (PTEN and NRAS mutations) showed only a 35% to 40% decrease in colony formation in soft agar in the presence of riluzole. In this study, we have continued our preclinical studies of riluzole and its effect on melanoma cells alone and in combination with inhibitors of the PI3 kinase pathway: the AKT inhibitor, API-2, and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. We modeled these combinatorial therapies on various melanoma cell lines in 3D and 2D systems and in vivo. Riluzole combined with mTOR inhibition is more effective at halting melanoma anchorage-independent growth and xenograft tumor progression than either agent alone. PI3K signaling changes associated with this combinatorial treatment shows that 3D (nanoculture) modeling of cell signaling more closely resembles in vivo signaling than monolayer models. Riluzole combined with mTOR inhibition is effective at halting tumor cell progression independent of BRAF mutational status. This makes this combinatorial therapy a potentially viable alternative for metastatic melanoma patients who are BRAF WT and are therefore ineligible for vemurafenib therapy.

Metabotropic glutamate receptor 1 mediates melanocyte transformation via transactivation of insulin-like growth factor 1 receptor

Our laboratory previously described the oncogenic properties of metabotropic glutamate receptor 1 (mGluR1) in melanocytes. mGluR1 transformed immortalized mouse melanocytes in vitro and induced vigorous tumor formation in vivo. Subsequently, we observed the activation of PI3K/AKT in mGluR1‐mediated melanocytic tumorigenesis in vivo. In particular, we identified AKT2 being the predominant isoform contributing to the activation of AKT. Suppression of Grm1 or AKT2 using an inducible Tet‐R siRNA system resulted in a 60 or 30% reduction, respectively, in in vivo tumorigenesis. We show that simultaneous downregulation of Grm1 plus AKT2 results in a reduction of approximately 80% in tumor volumes, suggesting that both mGluR1 and AKT2 contribute to the tumorigenic phenotype in vivo. The discrepancy between the mild in vitro transformation characteristics and the aggressive in vivo tumorigenic phenotypes of these stable mGluR1‐melanocytic clones led us to investigate the possible involvement of other growth factors. Here, we highlight a potential crosstalk network between mGluR1 and tyrosine kinase, insulin‐like growth factor 1 receptor (IGF‐1R).

Glutamate Signaling in Human Cancers

G-protein coupled receptors (GPCRs) represent a class of therapeutic targets that have been widely exploited for drug designs and development. Metabotropic glutamate receptors (mGluRs) belong to Class C GPCRs and are predominantly involved in maintaining cellular homeostasis in the central nervous system (CNS). The natural ligand of mGluRs, glutamate, interacts with recep‐ tor proteins leading to the activation of multiple signaling pathways. More recently, aberrant glutamate signaling has been shown to play a role in the transformation and maintenance of various cancer types, including melanoma. Glutamate secretion from these cells has been found to stimulate regulatory pathways that control tumor growth, proliferation and survival. In addition to synaptic transmissions, accumulating evidence suggesting other functional roles of glutamatergic signaling in human malignancies has presented intriguing possibilities to make mGluRs putative, novel targets for human cancer treatments. To this end, the aberrant expres‐ sion of metabotropic glutamate receptor 1 (mGluR1) was found as the driving force in inducing melanomagenesis in transgenic mouse models. Since then, other subtypes of mGluRs have been implicated in the pathogenesis of various cancer types such as malignant gliomas and medullo‐ blastomas. As such, increased efforts have been generated to elucidate the mechanisms by which mGluRs confer oncogenic potentials. This chapter summarizes our current knowledge on the participation of glutamate signaling in human cancers. Given that mGluRs are “druggable” members of the GPCR superfamily and their oncogenic implications in cancer, further under‐ standing on anti-mGluR signaling pathways will be beneficial.

Abstract A28: Apoptosis induction with the MEK inhibitor AZD6244 and BH3 mimetic ABT-737 in melanoma.

Over sixty percent of patients with melanoma have the activating B-RAFV600E mutation leading to increased activation of the mitogen activated protein kinase (MAPK) pathway. The downstream effects of MAPK pathway activation are increased proliferation, invasiveness, and survival through the regulation of down-stream targets such as the pro-apoptotic protein BIM. Prior studies have shown that inhibiting MAPK kinase (MEK), downstream of constitutively active B-RAF, can up-regulate BIM, leading to increased apoptosis in melanoma cell lines. However, single agent MEK inhibition has had limited effectiveness in the clinical setting for late-stage melanoma patients, likely due to an inadequate induction of apoptosis. We hypothesized that combining the clinically relevant MEK inhibitor, AZD6244 (ARRY142886) with a BCL-2 inhibitor, ABT-737, would lead to increased apoptosis over single agent therapies in melanoma cells via BIM activation and pan-BCL-2 inhibition. We compared single treatments of AZD6244 and ABT737 to the combination of these agents in a panel of melanoma tumor cell lines chosen by mutational status in both a monolayer system and an anchorage independent system. The combination of AZD6244 and ABT-737 had a synergistic effect on decreasing cell viability and increasing apoptosis compared to single agent treatments regardless of B-RAF mutational status. Western blotting revealed dramatic increases in cleaved caspase 3 and PARP with combinational treatment compared to single agent therapy. These early pre-clinical experiments indicate that AZD6244 combined with ABT-737 may be an effective combination therapy for patients with late-stage melanoma. Currently, we have plans to conduct in vivo studies to translate these results into the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A28.