Tara Gelb

Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism

The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, β-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function.

Metabotropic glutamate receptor 1 acts as a dependence receptor creating a requirement for glutamate to sustain the viability and growth of human melanomas

Metabotropic glutamate 1 (mGlu) receptor has been proposed as a target for the treatment of metastatic melanoma. Studies have demonstrated that inhibiting the release of glutamate (the natural ligand of mGlu1 receptors), results in a decrease of melanoma tumor growth in mGlu1 receptor-expressing melanomas. Here we demonstrate that mGlu1 receptors, which have been previously characterized as oncogenes, also behave like dependence receptors by creating a dependence on glutamate for sustained cell viability. In the mGlu1 receptor-expressing melanoma cell lines SK-MEL-2 (SK2) and SK-MEL-5 (SK5), we show that glutamate is both necessary and sufficient to maintain cell viability, regardless of underlying genetic mutations. Addition of glutamate increased DNA synthesis, whereas removal of glutamate not only suppressed DNA synthesis but also promoted cell death in SK2 and SK5 melanoma cells. Using genetic and pharmacological inhibitors, we established that this effect of glutamate is mediated by the activation of mGlu1 receptors. The stimulatory potential of mGlu1 receptors was further confirmed in vivo in a melanoma cell xenograft model. In this model, subcutaneous injection of SK5 cells with short hairpin RNA-targeted downregulation of mGlu1 receptors resulted in a decrease in the rate of tumor growth relative to control. We also demonstrate for the first time that a selective mGlu1 receptor antagonist JNJ16259685 ((3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone) slows SK2 and SK5 melanoma tumor growth in vivo. Taken together, these data suggest that pharmacological inhibition of mGlu1 receptors may be a novel approach for the treatment of metastatic melanoma.

Hedgehog Signaling Inhibitors Fail to Reduce Merkel Cell Carcinoma Viability

TO THE EDITOR Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. Surgical resection and radiotherapy can successfully treat localized disease, but treatments for advanced MCC are needed. Aberrant activation of the Hedgehog (Hh) signaling pathway plays a role in several cancers, including basal cell carcinoma (BCC) and medulloblastoma (MDB) (Gupta et al., 2010). One study has reported increased immunohistochemical staining of Hh pathway components (Hh ligands, PTCH, SMO, and Gli family members) in MCC tumors, suggesting that Hh pathway activation plays a role in MCC pathogenesis (Brunner et al., 2010). This resulted in speculation that Hh inhibitors may be effective in treating MCC (Li et al., 2011). However, this hypothesis conflicts with mouse studies in which genetic activation of Hh signaling in the Merkel cell lineage failed to produce neuroendocrine skin tumors (Peterson et al., 2015; Xiao et al., 2015). Additionally, the loss-of-function PTCH1 mutations associated with Hh-driven cancers are largely absent in MCC (Cimino et al., 2014; Harms et al., 2015); the rare PTCH1 variants that have been reported in MCC (Goh et al., 2016) have unknown functional significance. To characterize the extent of Hh pathway activation in MCC, we analyzed mRNA microarray expression data from MCC tumor samples compared with BCC and Sonic Hedgehog (Shh)-subgroup MDB samples (see Supplementary Materials and Methods online). All 23 MCC samples showed low expression levels of target genes indicative of Hh pathway activity, including GLI1 and PTCH1, and obligate mediators of Hh signaling, such as SMO and GLI2. In comparison, Hhdriven BCC and MDB tumors showed marked up-regulation of these genes (Figure 1). This contrast, also noted in another comparison of MCC and BCC (Harms et al., 2013), suggests that active Hh signaling is not present in MCC. To further investigate the potential role of Hh signaling in MCC, we treated the MCC cell lines Mkl-1 and WaGa with the SMO inhibitors cyclopamine and itraconazole and the Gli inhibitor GANT61. The Hh-driven BCC cell line UW-BCC1 (Noubissi et al., 2014) and the HeLa cell line, shown to be insensitive to Hh-pathway inhibition (Samarzija and Beard, 2012), were used as positive and negative controls, respectively. All three drugs showed a similar dose-dependent inhibition of BCC viability (Figure 2). In contrast to its effect on BCC cells, the classic SMO inhibitor cyclopamine failed to decrease MCC cell viability. The alternative SMO antagonist itraconazole showed a dose-independent reduction of cell viability in the Mkl-1 cell line, but it similarly inhibited HeLa cells, suggesting its effects were not mediated by antagonizing Hh signaling. Itraconazole did not significantly affect WaGa viability. The Gli inhibitor GANT61 reduced the viability of both WaGa and HeLa cells at higher concentrations, again suggesting offtarget toxicity. In Mkl-1 cells, GANT61 failed to alter viability. Taken together, these data suggest that Hh pathway activation is not necessary for MCC viability and that antagonists of Hh signaling are unlikely to be effective treatments for MCC. These findings argue against testing Hh signaling inhibitors in patients with MCC. However, we encountered a patient who was diagnosed with MCC while taking a SMO inhibitor. A 73-year-old man developed a mass in his left axilla 2 months after beginning vismodegib for multiple locally advanced BCC tumors on his bilateral trunk. The patient did not have nevoid basal cell carcinoma syndrome, and his past medical history was most significant for seizure disorder, multiple skin cancers, and mesothelioma. His medications included once-daily 150 mg oral vismodegib, phenytoin, propranolol, triamterene/hydrochlorothiazide, atorvastatin, gabapentin, docusate, and a multivitamin. While taking vismodegib, the patient experienced mild muscle cramps and had reductions in the sizes of his BCC lesions. However, a semiannual

Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells

The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.

Abstract A15: mGlu1 Receptors and downstream signal transduction proteins as therapeutic targets for the treatment of metastatic melanoma

The purpose of this study was to elucidate the downstream signaling of mGlu1 receptors involved in melanoma cell proliferation. Previously, using melanoma cell lines expressing mGlu1 receptors, we demonstrated that glutamate stimulation of mGlu1 receptors increases melanoma cell growth in BRAF mutant, NRAS mutant, and BRAF/NRAS wild-type melanoma cells. Blockade of mGlu1 receptor activation through glutamate depletion, mGlu1 receptor knockdown, and mGlu1 receptor antagonism inhibited glutamate-stimulated melanoma cell growth, further identifying mGlu1 receptors as a therapeutic target for the treatment of metastatic melanoma. Because mGlu1 receptor antagonists blocked growth of both BRAF and NRAS mutant melanoma cells, it suggests that mGlu1 receptors stimulate melanoma cell proliferation either by a mechanism downstream of NRAS and BRAF or through a distinct and necessary pathway. Here we report that glutamate does not stimulate melanoma cell proliferation through activation of the canonical G alpha-q dependent pathway, as neither glutamate nor quisqualate stimulate phosphoinositide hydrolysis in the mGlu1 receptor-expressing melanoma cell lines SK-MEL-2 and SK-MEL-5 (as measured by scintillation proximity assay). Furthermore, the G protein biased ligand quisqualate does not stimulate SK-MEL-2 or SK-MEL-5 melanoma cell growth (as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay). In contrast, MEK inhibitors block glutamate stimulated growth of SK-MEL-2 and SK-MEL-5 melanoma cells, indicating that the MEK/ERK signal transduction cascade is involved in glutamate dependent growth. Our laboratory has shown in stably transfected CHO cells that mGlu1a receptors can also activate a G protein independent, Beta-arrestin-1 dependent pathway, and this Beta-arrestin-1 dependent signaling requires internalization of mGlu1 receptors. To assess the involvement of Beta-arrestin-1 dependent signaling in glutamate stimulated melanoma growth, cells were treated with a dynamin inhibitor, thus preventing internalization. Inhibition of dynamin selectively blocked glutamate stimulated growth of mGlu1 receptor expressing melanomas, but did not affect proliferation of melanoma cells lacking mGlu1 receptor expression. In summary, these data suggest that glutamate stimulates melanoma cell growth through activation of a dynamin-dependent pathway which may result in ERK phosphorylation. Understanding the signaling mechanism downstream of mGlu1 receptors may lead to the identification of novel targets that could be used either separately or in combination with mGlu1 receptor antagonists for the treatment of metastatic melanoma. NIH Grant NS37436 PhRMA Pre Doctoral Fellowship in Pharmacology/Toxicology Citation Format: Tara Gelb, Sergey Pshenichkin, Hannah A. Hathaway, Ewa Grajkowska, Barry B. Wolfe, Jarda T. Wroblewski. mGlu1 Receptors and downstream signal transduction proteins as therapeutic targets for the treatment of metastatic melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A15.

Triple threat treatment: Exploiting the dependence receptor properties of metabotropic glutamate receptor 1 against melanoma.

Melanoma cells that express metabotropic glutamate 1 (mGlu1) receptors depend on glutamate for their survival and proliferation. The dependence receptor properties of mGlu1 allow us to propose and justify three promising approaches for melanoma treatment: glutamate depletion, mGlu1 receptor antagonism, and targeting of mGlu1 receptor signaling.