RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma.
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ABSTRACT:
SUBMITTER: Chen X
PROVIDER: S-EPMC5499527 | biostudies-literature | 2017 May
REPOSITORIES: biostudies-literature
Publications
RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma.
Chen Xu X Wu Qiuxia Q Depeille Philippe P Chen Peirong P Thornton Sophie S Kalirai Helen H Coupland Sarah E SE Roose Jeroen P JP Bastian Boris C BC
Cancer cell 20170501 5
Constitutive activation of Gαq 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 δ and ɛ 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 o ...[more]
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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:Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma (UM). Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions. In search of genetic vulnerability for UM, we found that inhibition of euchromatic histone lysine methyltransferase 2 (EHMT2) expression or activity significantly reduced the proliferation and migration capacity of cancer cells. Notably, elevated expression of EHMT2 had been validated in UM samples. Furthermore, Kaplan-Meier survival analysis indicated high EHMT2 protein level was related to poor recurrence-free survival and a more advanced T stage. Chromatin immunoprecipitation sequencing analysis and the following mechanistic investigation showed that ARHGAP29 was a downstream target of EHMT2. Its transcription was suppressed by EHMT2 in a methyltransferase-dependent pattern in GNAQ/11-mutant UM cells, leading to elevated RhoA activity. Rescuing constitutively active RhoA in UM cells lacking EHMT2 restored oncogenic phenotypes. Simultaneously blocking EHMT2 and GNAQ/11 signaling in vitro and in vivo showed a synergistic effect on UM growth, suggesting the driver role of these two key molecules. In summary, our study shows evidence for an epigenetic program of EHMT2 regulation that influences UM progression and indicates inhibiting EHMT2 and MEK/ERK simultaneously as a therapeutic strategy in GNAQ/11-mutant UM.
| S-EPMC10935147 | biostudies-literature
Project description:Cutaneous melanoma (CM) and uveal melanoma (UM) both originate from the melanocytic lineage but are primarily driven by distinct oncogenic drivers, BRAF/NRAS or GNAQ/GNA11, respectively. The melanocytic master transcriptional regulator, MITF, is essential for both CM development and maintenance, but its role in UM is largely unexplored. Here, we use zebrafish models to dissect the key UM oncogenic signaling events and establish the role of MITF in UM tumors. Using a melanocytic lineage expression system, we showed that patient-derived mutations of GNAQ (GNAQQ209L) or its upstream CYSLTR2 receptor (CYSLTR2L129Q) both drive UM when combined with a cooperating mutation, tp53M214K/M214K. The tumor-initiating potential of the major GNAQ/11 effector pathways, YAP, and phospholipase C-β (PLCβ)–ERK was also investigated in this system and thus showed that while activated YAP (YAPAA) induced UM with high potency, the patient-derived PLCβ4 mutation (PLCB4D630Y) very rarely yielded UM tumors in the tp53M214K/M214K context. Remarkably, mitfa deficiency was profoundly UM promoting, dramatically accelerating the onset and progression of tumors induced by Tg(mitfa:GNAQQ209L);tp53M214K/M214K or Tg(mitfa:CYSLTR2L129Q);tp53M214K/M214K. Moreover, mitfa loss was sufficient to cooperate with GNAQQ209L to drive tp53–wild type UM development and allowed Tg(mitfa:PLCB4D630Y);tp53M214K/M214K melanocyte lineage cells to readily form tumors. Notably, all of the mitfa−/− UM tumors, including those arising in Tg(mitfa:PLCB4D630Y);tp53M214K/M214K;mitfa−/− zebrafish, displayed nuclear YAP while lacking hyperactive ERK indicative of PLCβ signaling. Collectively, these data show that YAP signaling is the major mediator of UM and that MITF acts as a bona fide tumor suppressor in UM in direct opposition to its essential role in CM.
| S-EPMC9172632 | biostudies-literature
Project description:PurposeEarly/initiating oncogenic mutations have been identified for many cancers, but such mutations remain unidentified in uveal melanoma (UM). An extensive search for such mutations was undertaken, focusing on the RAF/MEK/ERK pathway, which is often the target of initiating mutations in other types of cancer.MethodsDNA samples from primary UMs were analyzed for mutations in 24 potential oncogenes that affect the RAF/MEK/ERK pathway. For GNAQ, a stimulatory alpha(q) G-protein subunit which was recently found to be mutated in UMs, resequencing was expanded to include 67 primary UMs and 22 peripheral blood samples. GNAQ status was analyzed for association with clinical, pathologic, chromosomal, immunohistochemical, and transcriptional features.ResultsActivating mutations at codon 209 were identified in GNAQ in 33 (49%) of 67 primary UMs, including 2 (22%) of 9 iris melanomas and 31 (54%) of 58 posterior UMs. No mutations were found in the other 23 potential oncogenes. GNAQ mutations were not found in normal blood DNA samples. Consistent with GNAQ mutation being an early or initiating event, this mutation was not associated with any clinical, pathologic, or molecular features associated with late tumor progression.ConclusionsGNAQ mutations occur in about half of UMs, representing the most common known oncogenic mutation in this cancer. The presence of this mutation in tumors at all stages of malignant progression suggests that it is an early event in UM. Mutations in this G-protein-coupled receptor provide new insights into UM pathogenesis and could lead to new therapeutic possibilities.
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Project description:Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo. Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.
| S-EPMC6633893 | biostudies-literature
Project description:The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1? (HIF-1?) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1? protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1? protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1? mutant into Mel285 cells with low HIF-1? baseline levels. In contrast, HIF-1? knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1? baseline levels. Pharmacologic blockade of HIF-1? protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1? signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1? pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.
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Project description:Cutaneous melanoma (CM) and uveal melanoma (UM) both originate from the melanocytic lineage but are primarily driven by distinct oncogenic drivers, BRAF/NRAS or GNAQ/GNA11 respectively. The melanocytic master transcriptional regulator, MITF, is essential for both CM development and maintenance, but its role in UM is largely unexplored. Here, we use zebrafish models to dissect the key UM oncogenic signaling events, and establish the role of MITF in UM tumors. Using a melanocytic lineage expression system, we showed that patient-derived mutations of GNAQ (GNAQQ209L) or its upstream CYSLTR2 receptor (CYSLTR2L129Q) both drive UM when combined with a cooperating mutation, tp53M214K/M214K. The tumor-initiating potential of the major GNAQ/11 effector pathways, YAP and PLCβ-ERK, was also investigated in this system, and thus showed that while activated YAP (YAPAA) induced UM with high potency, the patient-derived PLC4 mutation (PLCB4D630Y) very rarely yielded UM tumors in the tp53M214K/M214K context. Remarkably, mitfa deficiency was profoundly UM promoting, dramatically accelerating the onset and progression of tumors induced by Tg(GNAQQ209L);tp53M214K/M214K or Tg(CYSLTR2L129Q);tp53M214K/M214K. Moreover, mitfa loss was sufficient to cooperate with GNAQQ209L to drive tp53-wildtype UM development, and allowed Tg(PLCB4D630Y);tp53M214K/M214K melanocyte lineage cells to readily form tumors. Notably, all of the mitfa-/- UM tumors, including those arising in Tg(mitfa:PLCB4D630Y);tp53M214K/M214K;mitfa-/- fish, displayed nuclear YAP while lacking hyperactive ERK indicative of PLCβ signaling. Collectively, these data show that YAP signaling is the major mediator of UM, and that MITF acts as bona fide tumor suppressor in UM, in direct opposition to its essential role in CM.
2022-04-27 | GSE190802 | GEO
Project description:BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures, and lead to constitutive activation of the mitogen-activated protein (MAP) kinase pathway. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown. Here we report frequent somatic mutations in the heterotrimeric G protein alpha-subunit, GNAQ, in blue naevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the Ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP kinase activation in melanocytic neoplasia, providing new opportunities for therapeutic intervention.
| S-EPMC2696133 | biostudies-literature
Project description:PURPOSE:Activating Q209L/P mutations in GNAQ or GNA11 (GNAQ/11) are present in approximately 80% of uveal melanomas. Mutant GNAQ/11 are not currently therapeutically targetable. Inhibiting key down-stream effectors of GNAQ/11 represents a rational therapeutic approach for uveal melanomas that harbor these mutations. The mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase (MEK/MAPK) and PI3K/AKT pathways are activated in uveal melanoma. In this study, we test the effect of the clinically relevant small molecule inhibitors GSK1120212 (MEK inhibitor) and GSK2126458 (pan class I PI3K inhibitor) on uveal melanoma cells with different GNAQ/11 mutation backgrounds. EXPERIMENTAL DESIGN:We use the largest set of genetically annotated uveal melanoma cell lines to date to carry out in vitro cellular signaling, cell-cycle regulation, growth, and apoptosis analyses. RNA interference and small molecule MEK and/or PI3K inhibitor treatment were used to determine the dependency of uveal melanoma cells with different GNAQ/11 mutation backgrounds on MEK/MAPK and/or PI3K/AKT signaling. Proteomic network analysis was done to unveil signaling alterations in response to MEK and/or PI3K small molecule inhibition. RESULTS:GNAQ/11 mutation status was not a determinant of whether cells would undergo cell-cycle arrest or growth inhibition to MEK and/or phosphoinositide 3-kinase (PI3K) inhibition. A reverse correlation was observed between MAPK and AKT phosphorylation after MEK or PI3K inhibition, respectively. Neither MEK nor PI3K inhibition alone was sufficient to induce apoptosis in the majority of cell lines; however, the combination of MEK + PI3K inhibitor treatment resulted in the marked induction of apoptosis in a GNAQ/11 mutant-dependent manner. CONCLUSIONS:MEK + PI3K inhibition may be an effective combination therapy in uveal melanoma, given the inherent reciprocal activation of these pathways within these cells.
| S-EPMC3935730 | biostudies-literature
Project description:Uveal melanoma (UM) is characterized by mutually exclusive activating mutations in GNAQ, GNA11, CYSLTR2, and PLCB4, four genes in a linear pathway to activation of PLCβ in almost all tumors and loss of BAP1 in the aggressive subset. We generated mice with melanocyte-specific expression of GNA11Q209L with and without homozygous Bap1 loss. The GNA11Q209L mice recapitulated human Gq-associated melanomas, and they developed pigmented neoplastic lesions from melanocytes of the skin and non-cutaneous organs, including the eye and leptomeninges, as well as at atypical sites, including the lymph nodes and lungs. The addition of Bap1 loss increased tumor proliferation and cutaneous melanoma size. Integrative transcriptome analysis of human and murine melanomas identified RasGRP3 to be specifically expressed in GNAQ/GNA11-driven melanomas. In human UM cell lines and murine models, RasGRP3 is specifically required for GNAQ/GNA11-driven Ras activation and tumorigenesis. This implicates RasGRP3 as a critical node and a potential target in UM.
| S-EPMC5854482 | biostudies-literature