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Naturally occurring hotspot cancer mutations in Gα13 promote oncogenic signaling

2020; Elsevier BV; Volume: 295; Issue: 49 Linguagem: Inglês

10.1074/jbc.ac120.014698

ISSN

1083-351X

Autores

Marcin Maziarz, Anthony Federico, Jingyi Zhao, Lorena Dujmusic, Zhiming Zhao, Stefano Monti, Xaralabos Varelas, Mikel Garcia‐Marcos,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

Heterotrimeric G-proteins are signaling switches broadly divided into four families based on the sequence and functional similarity of their Gα subunits: Gs, Gi/o, Gq/11, and G12/13. Artificial mutations that activate Gα subunits of each of these families have long been known to induce oncogenic transformation in experimental systems. With the advent of next-generation sequencing, activating hotspot mutations in Gs, Gi/o, or Gq/11 proteins have also been identified in patient tumor samples. In contrast, patient tumor-associated G12/13 mutations characterized to date lead to inactivation rather than activation. By using bioinformatic pathway analysis and signaling assays, here we identified cancer-associated hotspot mutations in Arg-200 of Gα13 (encoded by GNA13) as potent activators of oncogenic signaling. First, we found that components of a G12/13-dependent signaling cascade that culminates in activation of the Hippo pathway effectors YAP and TAZ is frequently altered in bladder cancer. Up-regulation of this signaling cascade correlates with increased YAP/TAZ activation transcriptional signatures in this cancer type. Among the G12/13 pathway alterations were mutations in Arg-200 of Gα13, which we validated to promote YAP/TAZ-dependent (TEAD) and MRTF-A/B-dependent (SRE.L) transcriptional activity. We further showed that this mechanism relies on the same RhoGEF-RhoGTPase cascade components that are up-regulated in bladder cancers. Moreover, Gα13 Arg-200 mutants induced oncogenic transformation in vitro as determined by focus formation assays. In summary, our findings on Gα13 mutants establish that naturally occurring hotspot mutations in Gα subunits of any of the four families of heterotrimeric G-proteins are putative cancer drivers. Heterotrimeric G-proteins are signaling switches broadly divided into four families based on the sequence and functional similarity of their Gα subunits: Gs, Gi/o, Gq/11, and G12/13. Artificial mutations that activate Gα subunits of each of these families have long been known to induce oncogenic transformation in experimental systems. With the advent of next-generation sequencing, activating hotspot mutations in Gs, Gi/o, or Gq/11 proteins have also been identified in patient tumor samples. In contrast, patient tumor-associated G12/13 mutations characterized to date lead to inactivation rather than activation. By using bioinformatic pathway analysis and signaling assays, here we identified cancer-associated hotspot mutations in Arg-200 of Gα13 (encoded by GNA13) as potent activators of oncogenic signaling. First, we found that components of a G12/13-dependent signaling cascade that culminates in activation of the Hippo pathway effectors YAP and TAZ is frequently altered in bladder cancer. Up-regulation of this signaling cascade correlates with increased YAP/TAZ activation transcriptional signatures in this cancer type. Among the G12/13 pathway alterations were mutations in Arg-200 of Gα13, which we validated to promote YAP/TAZ-dependent (TEAD) and MRTF-A/B-dependent (SRE.L) transcriptional activity. We further showed that this mechanism relies on the same RhoGEF-RhoGTPase cascade components that are up-regulated in bladder cancers. Moreover, Gα13 Arg-200 mutants induced oncogenic transformation in vitro as determined by focus formation assays. In summary, our findings on Gα13 mutants establish that naturally occurring hotspot mutations in Gα subunits of any of the four families of heterotrimeric G-proteins are putative cancer drivers. Heterotrimeric G-proteins are critical transducers of signaling triggered by a large family of G-protein–coupled receptors (GPCRs). Essentially, GPCRs promote GTP loading on the α-subunits of G-proteins (1Oldham W.M. Hamm H.E. Heterotrimeric G protein activation by G-protein-coupled receptors.Nat. Rev. 2008; 9 (18043707): 60-7110.1038/nrm2299Crossref Scopus (807) Google Scholar, 2Gilman A.G. G proteins: transducers of receptor-generated signals.Annu. Rev. Biochem. 1987; 56 (3113327): 615-64910.1146/annurev.bi.56.070187.003151Crossref PubMed Scopus (4713) Google Scholar), which triggers signaling downstream. Heterotrimeric G-proteins are composed of a nucleotide-binding Gα subunit and an obligatory Gβγ dimer, and they are classified into four families based on the nature of the Gα subunits. These four families are Gs, Gi/o, Gq/11, and G12/13, and Gα subunits of each one of them have distinct actions on specific effectors. For example, Gs members stimulate adenylyl cyclase activity, whereas Gi/o family members tend to inhibit it; Gq/11 members stimulate phospholipase C enzymes and a subgroup of RhoGEFs; and G12/13 members stimulate a different subgroup of RhoGEFs (3Neves S.R. Ram P.T. Iyengar R. G protein pathways.Science. 2002; 296 (12040175): 1636-163910.1126/science.1071550Crossref PubMed Scopus (983) Google Scholar, 4Dorsam R.T. Gutkind J.S. G-protein-coupled receptors and cancer.Nat. Rev. Cancer. 2007; 7 (17251915): 79-9410.1038/nrc2069Crossref PubMed Scopus (1011) Google Scholar). Signaling is terminated upon GTP hydrolysis mediated by the intrinsic GTPase of Gα subunits. The role of heterotrimeric G-proteins in cancer-related signaling has been documented for decades. Early studies identified cancer-associated mutations in Gαs that disrupted its GTPase activity, rendering the G-protein constitutively active (5Landis C.A. Masters S.B. Spada A. Pace A.M. Bourne H.R. Vallar L. 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Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.Nature. 2009; 457 (19078957): 599-60210.1038/nature07586Crossref PubMed Scopus (1148) Google Scholar, 23Van Raamsdonk C.D. Griewank K.G. Crosby M.B. Garrido M.C. Vemula S. Wiesner T. Obenauf A.C. Wackernagel W. Green G. Bouvier N. Sozen M.M. Baimukanova G. Roy R. Heguy A. Dolgalev I. et al.Mutations in GNA11 in uveal melanoma.N. Engl. J. Med. 2010; 363 (21083380): 2191-219910.1056/NEJMoa1000584Crossref PubMed Scopus (1080) Google Scholar). Similarly, activating mutations in Gαs (GNAS) can be as frequent as 70% in certain subtypes of pancreatic ductal carcinomas (24Ideno N. Yamaguchi H. Ghosh B. Gupta S. Okumura T. Steffen D.J. Fisher C.G. Wood L.D. Singhi A.D. Nakamura M. Gutkind J.S. Maitra A. 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Molinolo A.A. et al.Inactivating mutations in GNA13 and RHOA in Burkitt's lymphoma and diffuse large B-cell lymphoma: a tumor suppressor function for the Gα13/RhoA axis in B cells.Oncogene. 2016; 35 (26616858): 3771-378010.1038/onc.2015.442Crossref PubMed Scopus (54) Google Scholar, 28Muppidi J.R. Schmitz R. Green J.A. Xiao W. Larsen A.B. Braun S.E. An J. Xu Y. Rosenwald A. Ott G. Gascoyne R.D. Rimsza L.M. Campo E. Jaffe E.S. Delabie J. et al.Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.Nature. 2014; 516 (25274307): 254-25810.1038/nature13765Crossref PubMed Scopus (216) Google Scholar). This suggests that, at least in these lymphomas, Gα13 activity is tumor-suppressive. This is the opposite of the oncogene function previously suggested from experiments in vitro with a constitutively active artificial Gα13 mutation (7Xu N. Voyno-Yasenetskaya T. Gutkind J.S. Potent transforming activity of the G13 α subunit defines a novel family of oncogenes.Biochem. Biophys. Res. Commun. 1994; 201 (8002992): 603-60910.1006/bbrc.1994.1744Crossref PubMed Scopus (88) Google Scholar, 9Voyno-Yasenetskaya T.A. Pace A.M. Bourne H.R. Mutant α subunits of G12 and G13 proteins induce neoplastic transformation of Rat-1 fibroblasts.Oncogene. 1994; 9 (8058319): 2559-2565PubMed Google Scholar). Activation of Gα12 or Gα13 proteins leads to activation of RhoA-dependent transcriptional programs, including those mediated by the activation of the Hippo pathway effectors YAP and TAZ. The cascade of events involves the direct activation of a subgroup of RhoGEFs, composed of p115-RhoGEF, PDZ-RhoGEF, and LARG, by active, GTP-loaded Gα subunits of G12/13 proteins, which in turn activates RhoA, RhoB, and RhoC GTPases (29Aittaleb M. Boguth C.A. Tesmer J.J. Structure and function of heterotrimeric G protein-regulated Rho guanine nucleotide exchange factors.Mol. Pharmacol. 2010; 77 (19880753): 111-12510.1124/mol.109.061234Crossref PubMed Scopus (118) Google Scholar). Through mechanisms that involve the remodeling of the actin cytoskeleton, Rho GTPases induce transcriptional responses that include those regulated by YAP/TAZ, which serve as co-factors for the TEA domain–containing transcription factor family (TEADs), and via myocardin-related transcription factors A and B (MRTF-A/B), which serve as co-activators for the transcriptional factor SRF (30Miralles F. Posern G. Zaromytidou A.I. Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL.Cell. 2003; 113 (12732141): 329-34210.1016/s0092-8674(03)00278-2Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar, 31Cen B. Selvaraj A. Prywes R. Myocardin/MKL family of SRF coactivators: key regulators of immediate early and muscle specific gene expression.J. Cell. Biochem. 2004; 93 (15352164): 74-8210.1002/jcb.20199Crossref PubMed Scopus (136) Google Scholar, 32Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.-Y. Chinnaiyan A.M. Lai Z.-C. Guan K.-L. 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We found that mutations in the Arg-200 of Gα13, a residue required to hydrolyze GTP, lead to activation of YAP/TAZ-dependent and MRTF-A/B-dependent transcription through a RhoGEF–Rho GTPase cascade and that they promote oncogenic transformation in vitro. This implies that naturally occurring hotspot mutations in Gα subunits of any of the four families of heterotrimeric G-proteins are putative cancer drivers. We mined data from the Cancer Genome Atlas (TCGA) through cBioportal to explore genomic alterations in components of a G12/13-YAP/TAZ pathway (Fig. 1A). More specifically, we queried the G-proteins Gα12 (GNA12) and Gα13 (GNA13); the RhoGEFs p115-RhoGEF (ARHGEF1), PDZ-RhoGEF (ARHGEF11), and LARG (ARHGEF12); the Rho GTPases RhoA (RHOA), RhoB (RHOB), and RhoC (RHOC); and the Hippo pathway effectors YAP (YAP1) and TAZ (WWTR1). We found that these genes were altered in a large portion (∼40%) of the TCGA bladder cancers (TCGA-BLCA) (Fig. 1B). The alterations appeared to be largely mutually exclusive and trending toward up-regulation. For example, both heterotrimeric G-proteins, two of the three RhoGEFs, and both Hippo effectors displayed amplifications as the dominant feature. For RhoA (RHOA) and RhoB (RHOB), the main feature was that they were mutated, and several of these mutations are classified as putative drivers in cBioportal (38Cerami E. Gao J. Dogrusoz U. Gross B.E. Sumer S.O. Aksoy B.A. Jacobsen A. Byrne C.J. Heuer M.L. Larsson E. Antipin Y. Reva B. Goldberg A.P. Sander C. Schultz N. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.Cancer Discov. 2012; 2 (22588877): 401-40410.1158/2159-8290.CD-12-0095Crossref PubMed Scopus (9496) Google Scholar). Although not all RhoA/RhoB mutations have been characterized, some of them have been previously proposed to lead to signaling activation, like Ala-161 mutations in RhoA (39Nagata Y. Kontani K. Enami T. Kataoka K. Ishii R. Totoki Y. Kataoka T.R. Hirata M. Aoki K. Nakano K. Kitanaka A. Sakata-Yanagimoto M. Egami S. Shiraishi Y. Chiba K. et al.Variegated RHOA mutations in adult T-cell leukemia/lymphoma.Blood. 2016; 127 (26574607): 596-60410.1182/blood-2015-06-644948Crossref PubMed Scopus (75) Google Scholar) or the E172K mutation in RhoB (40Hess J.M. Bernards A. Kim J. Miller M. Taylor-Weiner A. Haradhvala N.J. Lawrence M.S. Getz G. Passenger hotspot mutations in cancer.Cancer Cell. 2019; 36 (31526759): 288-301.e1410.1016/j.ccell.2019.08.002Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Thus, although LARG (ARHGEF12) and RhoC (RHOC) are exceptions to the overall trend, these observations suggest that the G12/13-YAP/TAZ pathway might be up-regulated in bladder cancer. Motivated by these observations, we carried out a bioinformatic analysis of gene expression data to establish a potential correlation between up-regulation of the G12/13 pathway and YAP/TAZ activation. For this, we turned to a previously characterized 24-gene signature that depends on YAP/TAZ (41Wang Y. Xu X. Maglic D. Dill M.T. Mojumdar K. Ng P.K. Jeong K.J. Tsang Y.H. Moreno D. Bhavana V.H. Peng X. Ge Z. Chen H. Li J. Chen Z. et al.Comprehensive molecular characterization of the Hippo signaling pathway in cancer.Cell Rep. 2018; 25 (30380420): 1304-1317.e510.1016/j.celrep.2018.10.001Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) and analyzed its relationship to the expression levels of the rest of the upstream components of the proposed G12/13 pathway. We used single-sample gene set enrichment analysis (ssGSEA) to quantify relative enrichment of each pathway across over 400 primary tumors in the TCGA-BLCA RNA-Seq data set. We found a strong correlation between the activation scores of the G12/13 pathway and the activation scores for YAP/TAZ (Fig. 1C). We then tested the observed correlation coefficient against a null distribution of correlations between ssGSEA-quantified activity of the G12/13 pathway and 10,000 random 24-gene signatures, resulting in a significant p value of 1e−4 (Fig. 1D). Taken together, these observations indicate that up-regulation of the G12/13 pathway in bladder cancer correlates with increased transcriptional output of the downstream effectors YAP/TAZ. Although overexpression of WT G12/13 family Gα proteins has been found before to be sufficient to promote transformation (7Xu N. Voyno-Yasenetskaya T. Gutkind J.S. Potent transforming activity of the G13 α subunit defines a novel family of oncogenes.Biochem. Biophys. Res. Commun. 1994; 201 (8002992): 603-60910.1006/bbrc.1994.1744Crossref PubMed Scopus (88) Google Scholar, 8Xu N. Bradley L. Ambdukar I. Gutkind J.S. A mutant α subunit of G12 potentiates the eicosanoid pathway and is highly oncogenic in NIH 3T3 cells.Proc. Natl. Acad. Sci. U. S. 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The presence of an arginine in this position is absolutely conserved across Gα subunits (Fig. 2A), and its mutation in several other Gα subunits leads to increased activity and favors oncogenic transformation (5Landis C.A. Masters S.B. Spada A. Pace A.M. Bourne H.R. Vallar L. GTPase inhibiting mutations activate the α chain of Gs and stimulate adenylyl cyclase in human pituitary tumours.Nature. 1989; 340 (2549426): 692-69610.1038/340692a0Crossref PubMed Scopus (1225) Google Scholar, 11Pace A.M. Wong Y.H. Bourne H.R. A mutant α subunit of Gi2 induces neoplastic transformation of Rat-1 cells.Proc. Natl. Acad. Sci. U. S. A. 1991; 88 (1651490): 7031-703510.1073/pnas.88.16.7031Crossref PubMed Scopus (136) Google Scholar, 13Gupta S.K. Gallego C. Lowndes J.M. Pleiman C.M. Sable C. Eisfelder B.J. Johnson G.L. Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes.Mol. Cell. 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Because mutation of this arginine to any other residue is expected to have similar consequences (5Landis C.A. Masters S.B. Spada A. Pace A.M. Bourne H.R. Vallar L. GTPase inhibiting mutations activate the α chain of Gs and stimulate adenylyl cyclase in human pituitary tumours.Nature. 1989; 340 (2549426): 692-69610.1038/340692a0Crossref PubMed Scopus (1225) Google Scholar, 43Freissmuth M. Gilman A.G. Mutations of GS α designed to alter the reactivity of the protein with bacterial toxins: substitutions at ARG187 result in loss of GTPase activity.J. Biol. Chem. 1989; 264 (2557345): 21907-21914Abstract Full Text PDF PubMed Google Scholar), we focused our efforts on characterizing Gα13 R200K and Gα13 R200G because these are the two mutants most frequently found in bladder cancer. Before assessing the impact of these mutants in cell signaling assays, we validated that they adopted an active conformation by using a well-validated assay that relies on protection from trypsin hydrolysis (Fig. S1) (44Kleuss C. Raw A.S. Lee E. Sprang S.R. Gilman A.G. Mechanism of GTP hydrolysis by G-protein α subunits.Proc. Natl. Acad. Sci. U. S. A. 1994; 91 (7937899): 9828-983110.1073/pnas.91.21.9828Crossref PubMed Scopus (94) Google Scholar, 46Leyme A. Marivin A. Casler J. Nguyen L.T. Garcia-Marcos M. Different biochemical properties explain why two equivalent Gα subunit mutants cause unrelated diseases.J. Biol. Chem. 2014; 289 (24982418): 21818-2182710.1074/jbc.M114.549790Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Next, we expressed Gα13 R200K and Gα13 R200G in HEK293T cells and assessed activation of YAP/TAZ using a TEAD reporter assay (Fig. 2C). We compared the effect of expressing these two mutants with that of Gα13 WT as well as with that of Gα13 Q226L, an artificial mutant previously shown to enhance downstream signaling including YAP/TAZ-dependent TEAD transcriptional activity (34Yu F.X. Zhao B. Panupinthu N. Jewell J.L. Lian I. Wang L.H. Zhao J. Yuan H. Tumaneng K. Li H. Fu X.D. Mills G.B. Guan K.L. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.Cell. 2012; 150 (22863277): 780-79110.1016/j.cell.2012.06.037Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar, 37Park H.W. Kim Y.C. Yu B. Moroishi T. Mo J.S. Plouffe S.W. Meng Z. Lin K.C. Yu F.X. Alexander C.M. Wang C.Y. Guan K.L. Alternative Wnt signaling activates YAP/TAZ.Cell. 2015; 162 (26276632): 780-79410.1016/j.cell.2015.07.013Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Whereas expression of Gα13 WT led to a modest increase of TEAD activity, expression of Gα13 R200K and Gα13 R200G led to a significantly larger increase comparable with that observed in cells expressing the control mutant Gα13 Q226L (Fig. 2C). To determine whether the observed increase in TEAD activity by Gα13 mutants was mediated by YAP/TAZ, we knocked down both proteins simultaneously using a previously validated siRNA sequence (47Chaulk S.G. Lattanzi V.J. Hiemer S.E. Fahlman R.P. Varelas X. The Hippo pathway effectors TAZ/YAP regulate dicer expression and microRNA biogenesis through Let-7.J. Biol. Chem. 2014; 289 (24324261): 1886-189110.1074/jbc.C113.529362Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 48Yang C.S. Stampouloglou E. Kingston N.M. Zhang L. Monti S. Varelas X. Glutamine-utilizing transaminases are a metabolic vulnerability of TAZ/YAP-activated cancer cells.EMBO Rep. 2018; 19 (29661856)e43577 10.15252/embr.201643577Crossref PubMed Scopus (51) Google Scholar). As expected, depletion of YAP and TAZ led to a large suppression of TEAD activation by Gα13 R200K, R200G, or Q226L (Fig. 2D). To further map the cascade of events leading to YAP/TAZ activation by Gα13 mutants, we blocked the pathway that putatively operates in bladder cancer at different levels. First, inhibition of the Rho GTPases RhoA, RhoB, and RhoC by expression of Clostridium botulinum C3 toxin efficiently suppressed TEAD activation by Gα13 R200K, R200G, or Q226L (Fig. 2E). Then we tested the effect of a fragment of p115-RhoGEF that works as a dominant-negative by preventing the binding of active Gα13 to its target RhoGEFs that operate upstream of Rho GTPases in the pathway (49Reinhard N.R. Mastop M. Yin T. Wu Y. Bosma E.K. Gadella Jr., T.W.J. Goedhart J. Hordijk P.L. The balance between Gαi-Cdc42/Rac and Gα12/13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate.Mol. Biol. Cell. 2017; 28 (28954861): 3371-338210.1091/mbc.E17-03-0136Crossref PubMed Google Scholar). Expression of this dominant-negative construct, consisting of p115-RhoGEF's RGS homology (RH) domain (p115RH), but not a control construct, inhibited TEAD activation by Gα13 R200K, R200G, or Q226L (Fig. 2F). To further validate the specificity of these manipulations, we tested their impact on Gα13-mediated activation of another transcriptional output not controlled by YAP/TAZ but still dependent on Rho GTPase activation (i.e. the transcriptional activation of SRF via MRTF-A/B) (Fig. 2B). As expected, bladder cancer–associated mutants Gα13 R200K and R200G led to robust activation of the SRF reporter, comparable with that of the positive control Gα13 Q226L, which was suppressed by inhibition of the activation of RhoGEFs or Rho GTPases but not upon YAP/TAZ depletion (Fig. 2, G–I). Taken together, these results demonstrate that Gα13 hotspot mutations in Arg-200 found in bladder cancer are bona fide activating mutations that lead to induction of YAP/TAZ-dependent transcription via a RhoGEF–Rho GTPase cascade. Finally, we sought to determine whether the Gα13 hotspot mutations in Arg-200 described above would be sufficient to promote oncogenic transformation in vitro. For this, we used focus formation assays with NIH3T3 cells. This widely used system is particularly well-suited to analyze the putative oncogenic activity of Gα13 Arg-200 mutants because it has been used for the vast majority of Gα oncogenic mutants reported to date as a good proxy for tumor growth in mice, including for the oncogenic activity of artificial activating mutations introduced in Gα13 (7Xu N. Voyno-Yasenetskaya T. Gutkind J.S. Potent transforming activity of the G13 α subunit defines a novel family of oncogenes.Biochem. Biophys. Res. Commun. 1994; 201 (8002992): 603-60910.1006/bbrc.1994.1744Crossref PubMed Scopus (88) Google Scholar). First, we assessed whether Gα13 R200K and Gα13 R200G mutants also lead to increased signaling activity in NIH3T3 cells. Surprisingly, we found that whereas Gα13 R200K and Gα13 R200G led to robust increases in the MRTF-A/B-dependent SRE.L reporter, they had no significant effect on the activity of the YAP-TAZ–dependent TEAD reporter (Fig. S2). These results confirm that Gα13 R200K and Gα13 R200G behave as active G-proteins but that the downstream signaling consequences are cell type–specific. Next, we generated NIH3T3 cell lines stably expressing Gα13 WT, Gα13 R200K, and Gα13 R200G at comparable levels by lentiviral transduction and selection with the appropriate agents (Fig. 3A). Both Gα13 R200K and Gα13 R200G induced the formation of numerous foci, whereas Gα13 WT only had a modest effect (Fig. 3, B and C). Recent reports have suggested that mutations in Gα13 are putative oncogene drivers in bladder cancer based on bioinformatics predictions (17Wu V. Yeerna H. Nohata N. Chiou J. Harismendy O. Raimondi F. Inoue A. Russell R.B. Tamayo P. Gutkind J.S. Illuminating the Onco-GPCRome: novel G protein-coupled receptor-driven oncocrine networks and targets for cancer immunotherapy.J. Biol. Chem. 2019; 294 (31171722): 11062-1108610.1074/jbc.REV119.005601Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 42Robertson A.G. Kim J. Al-Ahmadie H. Bellmunt J. Guo G. Cherniack A.D. Hinoue T. Laird P.W. Hoadley K.A. Akbani R. Castro M.A.A. Gibb E.A. Kanchi R.S. Gordenin D.A. Shukla S.A. TCGA Research Network et al.Comprehensive molecular characterization of muscle-invasive bladder cancer.Cell. 2017; 171 (28988769): 540-556.e2510.1016/j.cell.2017.09.007Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 50Bailey M.H. Tokheim C. Porta-Pardo E. Sengupta S. Bertrand D. Weerasinghe A. Colaprico A. Wendl M.C. Kim J. Reardon B. Kwok-Shing Ng P. Jeong K.J. Cao S. Wang Z. Gao J. et al.Comprehensive characterization of cancer driver genes and mutations.Cell. 2018; 174 (30096302): 1034-103510.1016/j.cell.2018.07.034Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), but no other experimental evidence to support the predictions has been provided. The results presented here provide the missing experimental evidence that supports the idea of Gα13 hotspot mutations as putative drivers in bladder cancer and suggest that pharmacological blockade of the pathway activated downstream might be a viable therapeutic avenue. Moreover, our findings on Gα13 mutants establish that naturally occurring hotspot mutations in Gα subunits of any of the four families of heterotrimeric G-proteins (i.e. in Gs, Gi/o, Gq/11, and, now, G12/13) are putative cancer drivers, thereby providing definitive confirmation of a long-held tenet.

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