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Boris C. Bastian - One of the best experts on this subject based on the ideXlab platform.
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rasgrp3 mediates mapk pathway activation in GNAQ mutant uveal melanoma
Cancer Cell, 2017Co-Authors: Xu Chen, Philippe Depeille, Jeroen P. Roose, Peirong Chen, Sophie Thornton, Helen Kalirai, Sarah E Coupland, Boris C. BastianAbstract: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 occurs via PKC δ- and ɛ-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.
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Abstract 2138: RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma
Molecular and Cellular Biology, 2015Co-Authors: Xu Chen, Philippe Depeille, Jeroen P. Roose, Boris C. BastianAbstract:Uveal melanoma (UM) is the most common intraocular malignancy in adults and no effective treatment options are available for metastatic disease. Over 80% of UM show mutations in the Gαq family members GNAQ and GNA11. MAP-kinase pathway activation in part mediated by protein kinase C (PKC) has been shown as one critical contributing factor to GNAQ-mediated oncogenesis. However PKC inhibition alone does not completely suppress MAPK signaling. A more refined understanding of the signaling cascade linking MAPK signaling to mutant GNAQ or GNA11 is required to develop more effective strategies for targeted therapy. Among more than 10 different PKC isoforms, we identified both PKC δ and PKC e to be required and sufficient to activate MAPK pathway in GNAQ mutant melanomas by using siRNA mediated knock-down and co-transfection of GNAQ Q209L and specific PKC isoform cDNAs. Overexpression of GNAQ Q209L in 293FT cells increased Ras-GTP level and knock down of three Ras isoforms in GNAQ mutant uveal melanoma cell lines decreased MAPK signaling. Microarray analysis of 5 different GNAQ/11 mutant and 5 NRAS/BRAF-mutant melanoma cells revealed RasGRP3, a Ras-guanyl nucleotide exchange factor (RasGEF), ranks at the top of 487 differentially expressed genes between (cut off : p 2 or 100 fold) elevated RasGRP3 levels in GNAQ/11 mutant melanoma cells, while other RasGEFs were not significantly altered. Knock down of RasGRP3 decreased MAPK signaling and proliferation in GNAQ mutant melanoma cells, while it no effect on BRAF mutant cells. Mutating the PKC phosphorylation site of RasGRP3 (T133) partially attenuated RasGRP3-mediated MAPK signaling. Overexpression of PKC δ and PKC e increased RasGRP3 T133 phosphorylation but not PKC α and PKC ζ. While PKC inhibition completely abrogated RasGRP3 T133 phosphorylation, it did not fully suppress RasGRP3-mediated MAPK signaling, indicating that its activating effect only partly requires PKC. We found that RasGRP3 activation mediated by its diacylglycerol(DAG) binding C1 domain provides an independent conduit of activation, which is augmented by PKC-mediated phosphorylation at T133. DAG is a second messenger that is released by phospholipase C, a direct effector of activated GNAQ/11. Finally, we found that the markedly elevated RasGRP3 expression in GNAQ mutant melanoma cell lines is a direct consequence of the oncogenic signaling and is mediated by PKC δ and e. Hence, our data identify RasGRP3 as a critical signaling node linking oncogenic signaling downstream of GNAQ/11 to the Ras/Raf/MEK/ERK pathway through three mechanisms: phosphorylation by PKC δ and e, binding of the second messenger DAG, and by upregulation of its protein level as a consequence of oncogenic GNAQ-mediated activation of PKC δ and PKC e. The findings nominate RasGRP3 as a possible therapeutic target for cancers driven by oncogenic GNAQ/11. Citation Format: Xu Chen, Qiuxia Wu, Philippe Depeille, Jeroen P. Roose, Boris C. Bastian. RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2015-2138
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Abstract 2345: Combined inhibition of protein kinase C and MEK is a rational therapeutic approach for melanomas with GNAQ or GNA11 mutations.
Experimental and Molecular Therapeutics, 2013Co-Authors: X Chen, Boris C. BastianAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. There currently is no effective treatment for metastatic UM. UM differs from cutaneous melanoma in that it lacks BRAF, NRAS, or KIT mutations but instead harbors mutations in the Gαq family members GNAQ and GNA11 in over 80 to 90% of cases. Understanding the downstream effector pathways of these oncoproteins is of considerable interest to identify opportunities for targeted therapy. Using 293T cells, immortalized mouse and human melanocytes, we find that GNAQQ209L or GNA11Q209L induce activation of the protein kinase C (PKC) and MAPK pathways. ShRNA mediated knock-down of mutant GNAQ abrogates both PKC and MAPK activation in human melanoma cell lines or model systems. Using two different PKC inhibitors we found selective extinction of PKC and MAPK signalling and induction of G1 arrest in panels of melanoma cell lines carrying GNAQ or GNA11 mutations, whereas no such effect was observed in melanoma cell lines with other mutations. MAPK activation occurred downstream of activated PKC in cells with GNAQ or GNA11 mutations. In contrast to PKC inhibitors, which selectively inhibited the growth of melanoma cell lines with GNAQ or GNA11 mutations, the MEK inhibitor PD0325901 inhibited melanoma cell lines irrespective of their GNAQ/11 mutation status. In vivo the PKC inhibitor AEB071 slowed the growth of tumors in an allograft model of GNAQQ209L transduced melanocytes, but did not induce tumor shrinkage. Analysis of tumor lysates identified suppression of PKC signaling but not of MAP-kinase pathway signaling under chronic PKC inhibition. The rebound of MAP-kinase activation could be overcome using a combination of AEB071 with the MEK inhibitor PD0325901, which showed strong synergy in halting proliferation and in inducing apoptosis. Our data suggest that PKC is a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations and indicate that a combination therapy with MEK inhibitor is superior to treatment with either approach alone. Citation Format: Xu Chen, Qiuxia Wu, Boris C. Bastian. Combined inhibition of protein kinase C and MEK is a rational therapeutic approach for melanomas with GNAQ or GNA11 mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2345. doi:10.1158/1538-7445.AM2013-2345
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GNAQ and GNA11 mutations in melanocytomas of the central nervous system
Acta neuropathologica, 2012Co-Authors: Rajmohan Murali, Thomas Wiesner, Marc K. Rosenblum, Boris C. BastianAbstract:Melanocytic tumors in the central nervous system (CNS) include metastatic melanoma and less commonly primary melanocytic tumors, which usually arise from melanocytes of the leptomeninges. The latter include, in order of increasing biologic potential, melanocytomas, melanocytic neoplasms of intermediate grade/differentiation and primary melanomas.(2) Primary leptomeningeal melanocytic neoplasms belong to a larger taxonomic group of benign and malignant tumors arising from melanocytes that are not associated with epithelia. The group also includes blue nevi, melanomas arising in association with blue nevi (so-called “malignant blue nevi”), uveal nevi and uveal melanomas (4, 5, 7). Neoplasms in this group share morphologic features such as predominance of spindled and epithelioid cells, conspicuous pigmentation and absence of epithelial involvement, along with frequent mutations of GNAQ or GNA11, two closely related G-proteins of the Gq family that encode critical amino acids required for the GTPase function of the proteins. GNAQ or GNA11 mutations occur in a mutually exclusive pattern and affect codon 183 in exon 4 or codon 209 in exon 5 of either gene.(7) Mutations at these sites cripple enzymatic function and lead to a constitutively activated GTP-bound state. When in this state, GNAQ and GNA11 act as dominant-acting oncogenes that activate several critical signalling pathways including the MAP-kinase pathway (5, 7). A recent study reported mutations in exon 5 of GNAQ in 6/12 (50%) melanocytomas, 5 of which were in paraspinal locations.(3) We extended this analysis to include exons 4 and 5 of both GNAQ and GNA11. Five cases of CNS melanocytoma were identified from the consultation files of one of the authors (M.K.R.). Sections were cut from formalin-fixed, paraffin-embedded tumor tissue, and stained with hematoxylin-eosin; immunohistochemistry was performed. Tumor DNA was extracted from tissue carefully microdissected from deparaffinized, unstained sections. Direct (Sanger) sequencing for exons 4 and 5 of each of GNAQ and GNA11 was performed using methods described previously.(7) The clinical and pathologic features of the tumors are summarized in Table 1. The tumors occurred in 3 males and 2 females, with a median age of 42 years (range 29-61 years). They were composed of spindle-shaped, oval or epithelioid cells arranged in nests and vague whorls. Tumor cells ranged from amelanotic (Fig. 1a) to conspicuously pigmented (Fig. 1c). Immunohistochemically, all tumors were diffusely positive for S-100 and HMB-45, and negative for epithelial membrane antigen. Mutations were identified in exon 4 of GNAQ (1 case, Fig. 1b) and exon 5 of GNA11 (1 case, Fig. 1d). The mutations [GNAQ:(c.548G>A, p.(Arg183Gln) and GNA11:c.626A>C, p.(Gln209Pro)] are identical to those seen in uveal melanoma and intradermal melanocytic proliferations (6, 7). Our results expand the spectrum of GNAQ and GNA11 mutations that may occur in melanocytomas. To our knowledge, this is the first description of mutations in GNA11 and in exon 4 of GNAQ in melanocytomas. Figure 1 a) Case 1 – A monomorphous population of slightly spindled, amelanotic cells in vaguely whorling array is present (hematoxylin-eosin, x100). Inset - Diffuse cytoplasmic immunolabeling for HMB-45 confirms the melanocytic nature of the tumor cells ... TABLE Clinical and pathologic features of melanocytomas The occurrence of GNAQ and GNA11 mutations in melanocytomas,(3) dermal melanocytic tumors(6, 7) and uveal melanomas(6, 7) suggests the possibility of a developmental link between the cells of origin of these tumors. Indeed, this thesis is supported by the recent description of a developmental pathway in which a subgroup of melanocytes derives from Schwann cell precursors.(1) Although we identified a GNAQ exon 4 mutation in 1 of 5 cases of melanocytoma, exon 4 mutations in either GNAQ or GNA11 are rare in uveal melanomas (7/145, 4.8%) and blue nevi (2/96, 2.1%) (7). Mutational analysis of larger numbers of tumors is required to determine the true incidence of the different mutations in GNAQ and GNA11, and to investigate associations of specific mutations with clinical features, pathologic characteristics, and biologic behavior of melanocytomas.
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oncogenic GNAQ mutations are not correlated with disease free survival in uveal melanoma
British Journal of Cancer, 2009Co-Authors: Jurgen Bauer, Boris C. Bastian, Emine Kilic, Jolanda Vaarwater, Claus Garbe, A De KleinAbstract:Recently, oncogenic G protein alpha subunit q (GNAQ) mutations have been described in about 50% of uveal melanomas and in the blue nevi of the skin. GNAQ exon 5 was amplified from 75 ciliary body and choroidal melanoma DNAs and sequenced directly. GNAQ mutation status was correlated with disease-free survival (DFS), as well as other clinical and histopathological factors, and with chromosomal variations detected by FISH and CGH. Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. Univariate and multivariate analysis showed that GNAQ mutation status was not significantly correlated with DFS. The GNAQ mutation status is not suitable to predict DFS. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.
Gary K Schwartz - One of the best experts on this subject based on the ideXlab platform.
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the phosphoinositide 3 kinase α selective inhibitor byl719 enhances the effect of the protein kinase c inhibitor aeb071 in GNAQ gna11 mutant uveal melanoma cells
Molecular Cancer Therapeutics, 2014Co-Authors: Elgilda Musi, Grazia Ambrosini, Elisa De Stanchina, Gary K SchwartzAbstract:G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.
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The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.
Molecular cancer therapeutics, 2014Co-Authors: Elgilda Musi, Grazia Ambrosini, Elisa De Stanchina, Gary K SchwartzAbstract:G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.
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inhibition of mutant GNAQ signaling in uveal melanoma induces ampk dependent autophagic cell death
Molecular Cancer Therapeutics, 2013Co-Authors: Grazia Ambrosini, Elgilda Musi, Elisa De Stanchina, Gary K SchwartzAbstract:Oncogenic mutations in GNAQ and GNA11 genes are found in 80% of uveal melanoma. These mutations result in the activation of the RAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. In this study, using a siRNA strategy, we show that mutant GNAQ signals to both MEK and AKT, and that combined inhibition of these pathways with the MEK inhibitor selumetinib (AZD6244) and the AKT inhibitor MK2206 induced a synergistic decrease in cell viability. This effect was genotype dependent as autophagic markers like beclin1 and LC3 were induced in GNAQ-mutant cells, whereas apoptosis was the mechanism of cell death of BRAF-mutant cells, and cells without either mutation underwent cell-cycle arrest. The inhibition of MEK/ATK pathways induced activation of AMP-activated protein kinase (AMPK) in the GNAQ-mutant cells. The downregulation of AMPK by siRNA or its inhibition with compound C did not rescue the cells from autophagy, rather they died by apoptosis, defining AMPK as a key regulator of mutant GNAQ signaling and a switch between autophagy and apoptosis. Furthermore, this combination treatment was effective in inhibiting tumor growth in xenograft mouse models. These findings suggest that inhibition of MEK and AKT may represent a promising approach for targeted therapy of patients with uveal melanoma. Mol Cancer Ther; 12(5); 768–76. ©2013 AACR .
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Identification of Unique MEK-Dependent Genes in GNAQ Mutant Uveal Melanoma Involved in Cell Growth, Tumor Cell Invasion, and MEK Resistance
Clinical cancer research : an official journal of the American Association for Cancer Research, 2012Co-Authors: Grazia Ambrosini, Christine A. Pratilas, Li-xuan Qin, Madhavi Tadi, Oliver Surriga, Richard D. Carvajal, Gary K SchwartzAbstract:Purpose: Metastatic uveal melanoma represents the most common intraocular malignancy with very poor prognosis and no effective treatments. Oncogenic mutations in the G-protein α-subunit q and 11 have been described in about 85% of uveal melanomas and confer constitutive activation. Multiple signaling pathways are induced as a consequence of GNAQ/11 activation, which include the MEK/ERK kinase cascade. We analyzed the transcriptional profile of cell lines treated with a mitogen-activated protein (MAP)/extracellular signal–regulated (ERK) kinase (MEK) inhibitor to identify gene targets of activated GNAQ and to evaluate the biologic importance of these genes in uveal melanoma. Experimental Design: We conducted microarray analysis of uveal melanoma cell lines with GNAQ mutations treated with the MEK inhibitor selumetinib. For comparison, we used cells carrying BRAF V600E and cells without either mutation. Changes in the expression of selected genes were then confirmed by quantitative real-time PCR and immunoblotting. Results: We found that GNAQ mutant cells have a MEK-dependent transcriptional output and identified a unique set of genes that are downregulated by MEK inhibition, including the RNA helicase DDX21 and the cyclin-dependent kinase regulator CDK5R1 whereas Jun was induced. We provide evidence that these genes are involved in cell proliferation, tumor cell invasion, and drug resistance, respectively. Furthermore, we show that selumetinib treatment regulates the expression of these genes in tumor tissues of patients with metastatic GNAQ/11 mutant uveal melanoma. Conclusions: Our findings define a subset of transcriptionally regulated genes by selumetinib in GNAQ mutant cells and provide new insights into understanding the biologic effect of MEK inhibition in this disease. Clin Cancer Res; 18(13); 3552–61. ©2012 AACR .
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Abstract 5035: The MEK inhibitor AZD6244 is active in GNAQ mutant ocular melanoma cells
Cellular and Molecular Biology, 2010Co-Authors: Grazia Ambrosini, Gary K SchwartzAbstract:Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Uveal melanoma represents the most common intraocular malignancy in the United States. However, there are no effective treatments for this aggressive metastatic disease. While activating mutations in B-RAF are rare in ocular melanoma, oncogenic G protein alpha subunit q (GNAQ) mutations at codon 209 have been described in about 50% of uveal melanomas. Knock-down of GNAQ by siRNA inhibits p-ERK and the MEK inhibitor U0126 has been shown to inhibit growth of a GNAQ mutant cell line (Van Raamsdonk CD et al, Nature 2009). AZD6244 (ARRY-142886) is a MEK inhibitor now in clinical trials. Published phase I results have indicated clinical benefit in a patient with ocular melanoma (Adjei AA et al, J Clin Oncol. 2008). In view of this, we elected to test AZD6244 across a panel of ocular melanoma cell lines that differ in their GNAQ mutational status. Treatment with AZD4244 inhibited cell proliferation in a time- and dose-dependent manner, with IC50s in the 100-150nM range. This corresponded to the inhibition ERK phosphorylation and down-regulation of cyclin D1 in GNAQ mutant (both Q209L and P209L), but not in GNAQ wild-type cells. Over-expression of mutant GNAQ(Q209L) in wild type OCM290 cells, which are highly resistant to AZD6244, resulted in drug sensitization with inhibition of ERK phosphorylation. Depletion of GNAQ by siRNA in the GNAQ mutant cells resulted in inhibition of ERK phosphorylation and growth suppression. Interestingly, GNAQ depletion had no effect on ocular melanoma cells with wild type GNAQ or mutant B-RAF. We conclude that the GNAQ activating mutation in ocular melanoma can mediate sensitivity to AZD6244. This data provide a rationale for the clinical evaluation of AZD6244 in uveal melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5035.
Renbing Jia - One of the best experts on this subject based on the ideXlab platform.
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the oncolytic virus h101 combined with GNAQ sirna mediated knockdown reduces uveal melanoma cell viability
Journal of Cellular Biochemistry, 2019Co-Authors: Chun Qiu, Qingfeng Shang, Guanxiang Qian, Xianqun Fan, Renbing JiaAbstract:Background Uveal melanoma (UM) is a severe human malignancy with a high mortality rate, as well as high metastasis and recurrence potential. The active mutation of G protein subunit alpha q (GNAQ) or G protein subunit alpha 11 (GNA11) is a major trigger for UM. Oncolytic adenovirus H101 (H101) is the first oncolytic virus approved for clinical applications in cancer therapy by the China Food and Drug Administration. We investigated whether combining H101 with the downregulation of GNAQ expression would act synergistically in UM therapy. Methods Three UM cell lines OMM2.3 and 92.1, harboring GNAQ mutation, and OCM1, harboring B-Raf proto-oncogene mutation, were chosen for our research. The cellular toxicity of adenoviral infection and the cell growth rate were measured with a Cell Counting Kit-8. Western blot analysis was used to detect GNAQ, p-MEK1/2, YAP, and p-YAP expression. The apoptosis and cell-cycle distribution of cells were evaluated with annexin-V and propidium iodide staining. Results Our results revealed that OMM2.3 and 92.1 cells were more sensitive to H101 infection than OCM1 cells. GNAQ expression was markedly reduced by small interfering RNA, siGNAQ. Combined treatment of siGNAQ and H101 inhibited the proliferation and activated the apoptosis of OMM2.3 and 92.1 cells by blocking the phosphorylation of MEK1/2 and increasing the phosphorylation of YAP. Conclusions In summary, a therapy combining H101 and siGNAQ is feasible, with potential utility as a novel targeted molecular therapy for UM, especially those carrying a GNAQ mutation.
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The oncolytic virus H101 combined with GNAQ siRNA‐mediated knockdown reduces uveal melanoma cell viability
Journal of cellular biochemistry, 2018Co-Authors: Chun Qiu, Qingfeng Shang, Guanxiang Qian, Xianqun Fan, Renbing JiaAbstract:Background Uveal melanoma (UM) is a severe human malignancy with a high mortality rate, as well as high metastasis and recurrence potential. The active mutation of G protein subunit alpha q (GNAQ) or G protein subunit alpha 11 (GNA11) is a major trigger for UM. Oncolytic adenovirus H101 (H101) is the first oncolytic virus approved for clinical applications in cancer therapy by the China Food and Drug Administration. We investigated whether combining H101 with the downregulation of GNAQ expression would act synergistically in UM therapy. Methods Three UM cell lines OMM2.3 and 92.1, harboring GNAQ mutation, and OCM1, harboring B-Raf proto-oncogene mutation, were chosen for our research. The cellular toxicity of adenoviral infection and the cell growth rate were measured with a Cell Counting Kit-8. Western blot analysis was used to detect GNAQ, p-MEK1/2, YAP, and p-YAP expression. The apoptosis and cell-cycle distribution of cells were evaluated with annexin-V and propidium iodide staining. Results Our results revealed that OMM2.3 and 92.1 cells were more sensitive to H101 infection than OCM1 cells. GNAQ expression was markedly reduced by small interfering RNA, siGNAQ. Combined treatment of siGNAQ and H101 inhibited the proliferation and activated the apoptosis of OMM2.3 and 92.1 cells by blocking the phosphorylation of MEK1/2 and increasing the phosphorylation of YAP. Conclusions In summary, a therapy combining H101 and siGNAQ is feasible, with potential utility as a novel targeted molecular therapy for UM, especially those carrying a GNAQ mutation.
Grazia Ambrosini - One of the best experts on this subject based on the ideXlab platform.
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abstract b29 differential effects of brd4 inhibition on uveal melanoma cells with GNAQ 11 mutations
Cancer Research, 2015Co-Authors: Grazia AmbrosiniAbstract:Uveal melanoma (UM) represents the most common intraocular malignancy, and there are no effective treatments for this aggressive disease. Nearly 80% of UM is characterized by oncogenic mutations in G protein subunits GNAQ and Gna11, and 70% have extra copies of the oncogene MYC. Chromatin regulators have become attractive targets for cancer therapy. In particular, the BET-bromodomain inhibitor JQ1 has shown selective inhibition of c-Myc expression, followed by the suppression of c-Myc-dependent target genes. Treatments of UM cell lines with JQ1 showed that the GNAQ/Gna11 mutant cells were the most sensitive to BET inhibition with induction of cell death, while the cells without the mutations underwent cell cycle arrest in G1. Microarray analysis of UM cells revealed that several genes were differentially regulated by JQ1 treatment in GNAQ/11 mutant cells, with the downregulation of c-Myc, of the anti-apoptotic protein Bcl-2, and DNA repair proteins Rad51 and Brca1. While depletion of each of these genes did not affect cell viability, the concomitant silencing of Rad51 and Bcl-2 represented the minimal requirement to mimic the apoptotic effects of JQ1. As opposed to hematopoietic cancer cell lines, c-Myc depletion did not seem to play a major role in inhibiting the growth of these cells. Furthermore, the treatment with JQ1 resulted in significant antitumor activity in a mouse xenograft model of uveal melanoma with GNAQ mutation. Our results suggest the potential therapeutic use of BET bromodomain inhibitors for treating uveal melanoma with GNAQ/Gna11 mutations. Citation Format: Grazia Ambrosini. Differential effects of BRD4 inhibition on uveal melanoma cells with GNAQ/11 mutations. [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 B29.
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Abstract B29: Differential effects of BRD4 inhibition on uveal melanoma cells with GNAQ/11 mutations
Principles of Response and Resistance to Therapy, 2015Co-Authors: Grazia AmbrosiniAbstract:Uveal melanoma (UM) represents the most common intraocular malignancy, and there are no effective treatments for this aggressive disease. Nearly 80% of UM is characterized by oncogenic mutations in G protein subunits GNAQ and Gna11, and 70% have extra copies of the oncogene MYC. Chromatin regulators have become attractive targets for cancer therapy. In particular, the BET-bromodomain inhibitor JQ1 has shown selective inhibition of c-Myc expression, followed by the suppression of c-Myc-dependent target genes. Treatments of UM cell lines with JQ1 showed that the GNAQ/Gna11 mutant cells were the most sensitive to BET inhibition with induction of cell death, while the cells without the mutations underwent cell cycle arrest in G1. Microarray analysis of UM cells revealed that several genes were differentially regulated by JQ1 treatment in GNAQ/11 mutant cells, with the downregulation of c-Myc, of the anti-apoptotic protein Bcl-2, and DNA repair proteins Rad51 and Brca1. While depletion of each of these genes did not affect cell viability, the concomitant silencing of Rad51 and Bcl-2 represented the minimal requirement to mimic the apoptotic effects of JQ1. As opposed to hematopoietic cancer cell lines, c-Myc depletion did not seem to play a major role in inhibiting the growth of these cells. Furthermore, the treatment with JQ1 resulted in significant antitumor activity in a mouse xenograft model of uveal melanoma with GNAQ mutation. Our results suggest the potential therapeutic use of BET bromodomain inhibitors for treating uveal melanoma with GNAQ/Gna11 mutations. Citation Format: Grazia Ambrosini. Differential effects of BRD4 inhibition on uveal melanoma cells with GNAQ/11 mutations. [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 B29.
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the phosphoinositide 3 kinase α selective inhibitor byl719 enhances the effect of the protein kinase c inhibitor aeb071 in GNAQ gna11 mutant uveal melanoma cells
Molecular Cancer Therapeutics, 2014Co-Authors: Elgilda Musi, Grazia Ambrosini, Elisa De Stanchina, Gary K SchwartzAbstract:G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.
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The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.
Molecular cancer therapeutics, 2014Co-Authors: Elgilda Musi, Grazia Ambrosini, Elisa De Stanchina, Gary K SchwartzAbstract:G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.
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inhibition of mutant GNAQ signaling in uveal melanoma induces ampk dependent autophagic cell death
Molecular Cancer Therapeutics, 2013Co-Authors: Grazia Ambrosini, Elgilda Musi, Elisa De Stanchina, Gary K SchwartzAbstract:Oncogenic mutations in GNAQ and GNA11 genes are found in 80% of uveal melanoma. These mutations result in the activation of the RAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. In this study, using a siRNA strategy, we show that mutant GNAQ signals to both MEK and AKT, and that combined inhibition of these pathways with the MEK inhibitor selumetinib (AZD6244) and the AKT inhibitor MK2206 induced a synergistic decrease in cell viability. This effect was genotype dependent as autophagic markers like beclin1 and LC3 were induced in GNAQ-mutant cells, whereas apoptosis was the mechanism of cell death of BRAF-mutant cells, and cells without either mutation underwent cell-cycle arrest. The inhibition of MEK/ATK pathways induced activation of AMP-activated protein kinase (AMPK) in the GNAQ-mutant cells. The downregulation of AMPK by siRNA or its inhibition with compound C did not rescue the cells from autophagy, rather they died by apoptosis, defining AMPK as a key regulator of mutant GNAQ signaling and a switch between autophagy and apoptosis. Furthermore, this combination treatment was effective in inhibiting tumor growth in xenograft mouse models. These findings suggest that inhibition of MEK and AKT may represent a promising approach for targeted therapy of patients with uveal melanoma. Mol Cancer Ther; 12(5); 768–76. ©2013 AACR .
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the phosphoinositide 3 kinase α selective inhibitor byl719 enhances the effect of the protein kinase c inhibitor aeb071 in GNAQ gna11 mutant uveal melanoma cells
Molecular Cancer Therapeutics, 2014Co-Authors: Elgilda Musi, Grazia Ambrosini, Elisa De Stanchina, Gary K SchwartzAbstract:G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.
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The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.
Molecular cancer therapeutics, 2014Co-Authors: Elgilda Musi, Grazia Ambrosini, Elisa De Stanchina, Gary K SchwartzAbstract:G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.
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inhibition of mutant GNAQ signaling in uveal melanoma induces ampk dependent autophagic cell death
Molecular Cancer Therapeutics, 2013Co-Authors: Grazia Ambrosini, Elgilda Musi, Elisa De Stanchina, Gary K SchwartzAbstract:Oncogenic mutations in GNAQ and GNA11 genes are found in 80% of uveal melanoma. These mutations result in the activation of the RAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. In this study, using a siRNA strategy, we show that mutant GNAQ signals to both MEK and AKT, and that combined inhibition of these pathways with the MEK inhibitor selumetinib (AZD6244) and the AKT inhibitor MK2206 induced a synergistic decrease in cell viability. This effect was genotype dependent as autophagic markers like beclin1 and LC3 were induced in GNAQ-mutant cells, whereas apoptosis was the mechanism of cell death of BRAF-mutant cells, and cells without either mutation underwent cell-cycle arrest. The inhibition of MEK/ATK pathways induced activation of AMP-activated protein kinase (AMPK) in the GNAQ-mutant cells. The downregulation of AMPK by siRNA or its inhibition with compound C did not rescue the cells from autophagy, rather they died by apoptosis, defining AMPK as a key regulator of mutant GNAQ signaling and a switch between autophagy and apoptosis. Furthermore, this combination treatment was effective in inhibiting tumor growth in xenograft mouse models. These findings suggest that inhibition of MEK and AKT may represent a promising approach for targeted therapy of patients with uveal melanoma. Mol Cancer Ther; 12(5); 768–76. ©2013 AACR .