GNA11 Q209L Mouse Model Reveals RasGRP3 as an Essential Signaling Node in Uveal Melanoma.
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ABSTRACT:
SUBMITTER: Moore AR
PROVIDER: S-EPMC5854482 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Uveal melanoma is the most common intraocular cancer. There are no effective therapies for metastatic disease. Mutations in GNAQ, the gene encoding an alpha subunit of heterotrimeric G proteins, are found in 40% of uveal melanomas.We sequenced exon 5 of GNAQ and GNA11, a paralogue of GNAQ, in 713 melanocytic neoplasms of different types (186 uveal melanomas, 139 blue nevi, 106 other nevi, and 282 other melanomas). We sequenced exon 4 of GNAQ and GNA11 in 453 of these samples and in all coding exons of GNAQ and GNA11 in 97 uveal melanomas and 45 blue nevi.We found somatic mutations in exon 5 (affecting Q209) and in exon 4 (affecting R183) in both GNA11 and GNAQ, in a mutually exclusive pattern. Mutations affecting Q209 in GNA11 were present in 7% of blue nevi, 32% of primary uveal melanomas, and 57% of uveal melanoma metastases. In contrast, we observed Q209 mutations in GNAQ in 55% of blue nevi, 45% of uveal melanomas, and 22% of uveal melanoma metastases. Mutations affecting R183 in either GNAQ or GNA11 were less prevalent (2% of blue nevi and 6% of uveal melanomas) than the Q209 mutations. Mutations in GNA11 induced spontaneously metastasizing tumors in a mouse model and activated the mitogen-activated protein kinase pathway.Of the uveal melanomas we analyzed, 83% had somatic mutations in GNAQ or GNA11. Constitutive activation of the pathway involving these two genes appears to be a major contributor to the development of uveal melanoma. (Funded by the National Institutes of Health and others.).
| S-EPMC3107972 | biostudies-literature
Project description:Constitutive activation of G?q signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the mechanistic details remained unclear. We identified PKC ? and ? as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC ?- and ?-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11.
| S-EPMC5499527 | biostudies-literature
Project description:Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. Eighty percent of UMs harbor mutations in the G?q family members GNAQ and GNA11. Understanding the effector pathways downstream of these oncoproteins is important to identify opportunities for targeted therapy. We report consistent activation of the protein kinase C (PKC) and MAPK pathways as a consequence of GNAQ or GNA11 mutation. PKC inhibition with AEB071 or AHT956 suppressed PKC and MAPK signalling and induced G1 arrest selectively in melanoma cell lines carrying GNAQ or GNA11 mutations. In contrast, treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status, indicating that in the context of GNAQ or GNA11 mutation MAPK activation can be attributed to activated PKC. AEB071 significantly slowed the growth of tumors in an allograft model of GNAQ(Q209L)-transduced melanocytes, but did not induce tumor shrinkage. In vivo and in vitro studies showed that PKC inhibitors alone were unable to induce sustained suppression of MAP-kinase signaling. However, combinations of PKC and MEK inhibition, using either PD0325901or MEK162, led to sustained MAP-kinase pathway inhibition and showed a strong synergistic effect in halting proliferation and in inducing apoptosis in vitro. Furthermore, combining PKC and MEK inhibition was efficacious in vivo, causing marked tumor regression in a UM xenograft model. Our data identify PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations and demonstrate that combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone.
| S-EPMC4524511 | biostudies-literature
Project description:Activating mutations in G?q proteins, which form the ? subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic G?q signaling to induce all of these downstream pathways as well as ?-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and ?-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for G?-mediated diseases.
| S-EPMC5027844 | biostudies-literature
Project description:Approximately 10% of melanoma cases are familial, but only 25-40% of familial melanoma cases can be attributed to germ-line mutations in the CDKN2A - the most significant high-risk melanoma susceptibility locus identified to date. The pathogenic mutation(s) in most of the remaining familial melanoma pedigrees have not yet been identified. The most common mutations in nevi and sporadic melanoma are found in BRAF and NRAS, both of which result in constitutive activation of the MAPK pathway. However, these mutations are not found in uveal melanomas or the intradermal melanocytic proliferations known as blue nevi. Rather, multiple studies report a strong association between these lesions and somatic mutations in Guanine nucleotide-binding protein G(q) subunit alpha (GNAQ), Guanine nucleotide-binding protein G(q) subunit alpha-11 (GNA11), and BRCA1-associated protein-1 (BAP1). Recently, germ-line mutations in BAP1, the gene encoding a tumor suppressing deubiquitinating enzyme, have been associated with predisposition to a variety of cancers including uveal melanoma, but no studies have examined the association of germ-line mutations in GNAQ and GNA11 with uveal melanoma and blue nevi. We have now done so by sequencing exon 5 of both of these genes in 13 unique familial melanoma pedigrees, members of which have had either uveal or cutaneous melanoma and/or blue nevi. Germ-line DNA from a total of 22 individuals was used for sequencing; however no deleterious mutations were detected. Nevertheless, such candidate gene studies and the discovery of novel germ-line mutations associated with an increased MM susceptibility can lead to a better understanding of the pathways involved in melanocyte transformation, formulation of risk assessment, and the development of specific drug therapies.
| S-EPMC3695489 | biostudies-literature
Project description:Mutations in GNAQ and GNA11, encoding the oncogenic G-protein alpha subunit q and 11, respectively, occur frequently in the majority of uveal melanomas.Exons 4 and 5 from GNAQ and GNA11 were amplified and sequenced from 92 ciliary body and choroidal melanomas. The mutation status was correlated with disease-free survival (DFS) and other parameters.None of the tumours harboured a GNAQ exon 4 mutation. A GNAQ mutation in exon 5 codon 209 was found in 46 out of 92 (50.0%) of the tumours. Only 1 out of 92 (1.1%) melanomas showed a mutation in GNA11 exon 4 codon 183, whereas 39 out of 92 (42.4%) harboured a mutation in exon 5 of GNA11 codon 209. Six tumours did not show any mutations in exons 4 and 5 of these genes. Univariate analyses showed no correlation between DFS and the mutation status.GNAQ and GNA11 mutations are, in equal matter, not associated with patient outcome.
| S-EPMC3721402 | biostudies-literature
Project description:BackgroundUveal melanoma (UM) is the most common primary intraocular malignancy with a strong tendency to metastasize. The prognosis is poor once metastasis occurs. The treatment remains challenging for metastatic UM, even though our understanding of UM has advanced, mostly because the complexity of the genetic and immunologic background has not been fully explored.MethodsSingle-cell sequencing data were acquired from a healthy dataset and three UM datasets. The differentially expressed genes between primary and metastatic UM in The Cancer Genome Atlas (TCGA) data were attributed to specific cell types and explained with functional annotation. The analysis for cell-cell communication was conducted by "CellChat" to understand the cell crosstalk among the cell clusters and to delineate the dysfunctional signaling pathways in metastatic UM. CCK-8, EdU and transwell assays were performed to verify the function of the genes of interest.ResultsWe revealed aberrant signaling pathways with distinct functional statuses between primary and metastatic UM by integrating multiple datasets. The crucial signals contributing most to outgoing or incoming signaling of metastasis were identified to uncover the potential targeting genes. The association of these genes with disease risk was estimated based on survival data from TCGA. The key genes associated with proliferation and metastasis were verified.ConclusionsConclusively, we discovered the potential key signals for occurrence and metastasis of UM and provided a theoretical basis for potential clinical application.
| S-EPMC9258069 | biostudies-literature
Project description:In this article, we first briefly outline the function of G protein coupled receptors in cancer, and then specifically examine the roles of the seven transmembrane G protein coupled Endothelin B receptor (Ednrb) and the G proteins, GNAQ and GNA11, in both melanocyte development and melanoma. Ednrb plays an essential role in melanocyte development. GNAQ and GNA11 are oncogenes when mutated in certain types of melanocytic lesions, being extremely frequent in uveal melanoma, which forms from melanocytes located in the eye. Previously, we reported that in mice, Schwann cell precursor derived melanocytes colonize the dermis and hair follicles, while the inter-follicular epidermis is populated by other melanocytes. A pattern has emerged whereby melanocytes whose activities are affected by gain-of-function mutations of the Endothelin 3 ligand and G?q/11 are the same subset that arise from Schwann cell precursors. Furthermore, the forced expression of the constitutively active human GNAQ(Q209L) oncogene in mouse melanocytes only causes hyper-proliferation in the subset that arise from Schwann cell precursors. This has led us to hypothesize that in Schwann cell precursor derived melanocytes, Ednrb signals through G?q/11. Ednrb is promiscuous and may signal through other G protein alpha subunits in melanomas located in the inter-follicular epidermis.
| S-EPMC4837292 | biostudies-literature
Project description:PurposeThe activation of the mitogen-activated protein kinase (MAPK) pathway has been suggested as the major downstream target when GNAQ and GNA11 (GNAQ/11) are mutated in uveal melanoma (UM). However, clinical trials with single agent MEK inhibitor showed no clinical significance in altering the overall outcome of the disease in UM; therefore, we investigated the correlation between naturally occurring mutations in GNAQ/11 and activation of MAPK pathway in vivo in primary UM.MethodsScreening for activating mutations in codons 183 and 209 of GNAQ/11 was carried out by sequencing and restriction fragment length polymorphism (RFLP) in a cohort of 42 primary UM. Activation of the MAPK pathway and other potential downstream signals was assessed by immunohistochemistry and/or Western blot analysis. Potential downstream signaling of mutant and wild type GNAQ/11 was studied by transient transfection assay in nonmutant cell lines.ResultsSomatic mutations in GNAQ/11 were observed in 35/42 (83.3%) of primary UM. Tumors with GNAQ/11 mutations showed variations in the activation of ERK1/2 with significant tumor heterogeneity. Weak and undetectable ERK1/2 activation was observed in 4/35 (11.4%) and 8/35 (22.9%) of the GNAQ/11 mutant UM, respectively. Tumor heterogeneity of GNAQ/11 mutations was also observed in a subset of tumors.ConclusionsOur results indicate that there is marked variation in MAPK activation in UM with GNAQ/11 mutations. Thus, GNAQ/11 mutational status is not a sufficient biomarker to adequately predict UM patient responses to single-agent selective MEK inhibitor therapy.
| S-EPMC6557618 | biostudies-literature
Project description:BackgroundMetastatic uveal melanoma is a highly fatal disease; most patients die from their hepatic metastasis within 1 year. A major drawback in the development of new treatments for metastatic uveal melanoma is the difficulty in obtaining appropriate cell lines and the lack of appropriate animal models. Patient-derived xenograft (PDX) tumor models, bearing ectopically implanted tumors at a subcutaneous site, have been developed. However, these ectopically implanted PDX models have obstacles to translational research, including a low engraftment rate, slow tumor growth, and biological changes after multiple passages due to the different microenvironment. To overcome these limitations, we developed a new method to directly transplant biopsy specimens to the liver of immunocompromised mice.ResultsBy using two metastatic uveal melanoma cell lines, we demonstrated that the liver provides a more suitable microenvironment for tumor growth compared to subcutaneous sites and that surgical orthotopic implantation (SOI) of tumor pieces allows the creation of a liver tumor in immunocompromised mice. Subsequently, 10 of 12 hepatic metastasis specimens from patients were successfully xenografted into the immunocompromised mice (83.3% success rate) using SOI, including 8 of 10 needle biopsy specimens (80%). Additionally, four cryopreserved PDX tumors were re-implanted to new mice and re-establishment of PDX tumors was confirmed in all four mice. The serially passaged xenograft tumors as well as the re-implanted tumors after cryopreservation were similar to the original patient tumors in histologic, genomic, and proteomic expression profiles. CT imaging was effective for detecting and monitoring PDX tumors in the liver of living mice. The expression of Ki67 in original patient tumors was a predictive factor for implanted tumor growth and the success of serial passages in PDX mice.ConclusionsSurgical orthotopic implantation of hepatic metastasis from uveal melanoma is highly successful in the establishment of orthotopic PDX models, enhancing their practical utility for research applications. By using CT scan, tumor growth can be monitored, which is beneficial to evaluate treatment effects in interventional studies.
| S-EPMC5481921 | biostudies-literature