Advertisement

In uveal melanoma Gα-protein GNA11 mutations convey a shorter disease-specific survival and are more strongly associated with loss of BAP1 and chromosomal alterations than Gα-protein GNAQ mutations

      Highlights

      • GNA11 mutations are associated with increased risk of death in uveal melanoma.
      • GNA11 mutated uveal melanoma shows high risk cytogenetics and gene expression.
      • GNAQ but not GNA11 protein physically interacts with the demethylating enzyme TET2.
      • GNAQ and GNA11 mutated uveal melanoma shows different DNA-methylation patterns.

      Abstract

      Background and aim of the study

      Mutations in the Gα-genes GNAQ and GNA11 are found in 85–90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms.

      Methods

      We analyzed the association between GNAQ and GNA11 mutations with disease-specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation.

      Results

      GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1-associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12–3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High-risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression.

      Conclusions

      GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to European Journal of Cancer
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Chang A.E.
        • Karnell L.H.
        • Menck H.R.
        The national cancer data base report on cutaneous and noncutaneous melanoma A summary of 84,836 cases from the past decade.
        1998
        • Singh A.D.
        • Topham A.
        Incidence of uveal melanoma in the United States: 1973-1997.
        Ophthalmology. 2003; 110: 956-961https://doi.org/10.1016/S0161-6420(03)00078-2
        • Virgili G.
        • Gatta G.
        • Ciccolallo L.
        • Capocaccia R.
        • Biggeri A.
        • Crocetti E.
        • et al.
        Incidence of uveal melanoma in Europe.
        Ophthalmology. 2007; 114https://doi.org/10.1016/J.OPHTHA.2007.01.032
        • Singh A.D.
        • Borden E.C.
        Metastatic uveal melanoma.
        Ophthalmol Clin North Am. 2005; 18 (ix. https://doi.org/S0896-1549(04)00086-0 [pii]10.1016/j.ohc.2004.07.003): 143-150
        • Group∗ COMS
        Development of metastatic disease after enrollment in the COMS trials for treatment of choroidal melanoma: collaborative ocular melanoma study group report No. 26.
        Arch Ophthalmol. 2005; 123: 1639-1643https://doi.org/10.1001/ARCHOPHT.123.12.1639
        • Amaro A.
        • Gangemi R.
        • Piaggio F.
        • Angelini G.
        • Barisione G.
        • Ferrini S.
        • et al.
        The biology of uveal melanoma.
        Cancer Metastasis Rev. 2017; 36: 109-140https://doi.org/10.1007/s10555-017-9663-3
        • Jager M.J.
        • Shields C.L.
        • Cebulla C.M.
        • Abdel-Rahman M.H.
        • Grossniklaus H.E.
        • Stern M.H.
        • et al.
        Uveal melanoma.
        Nat Rev Dis Prim. 2020; 6: 1-25https://doi.org/10.1038/s41572-020-0158-0
        • Rossi E.
        • Croce M.
        • Reggiani F.
        • Schinzari G.
        • Ambrosio M.
        • Gangemi R.
        • et al.
        Uveal melanoma metastasis.
        Cancers. 2021; 13 (2021;13:5684): 5684https://doi.org/10.3390/CANCERS13225684
        • Van Raamsdonk C.D.
        • Bezrookove V.
        • Green G.
        • Bauer J.
        • Gaugler L.
        • O'Brien J.M.
        • et al.
        Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.
        Nature. 2009; 457: 599-602https://doi.org/10.1038/nature07586
        • Van Raamsdonk C.D.
        • Griewank K.G.
        • Crosby M.B.
        • Garrido M.C.
        • Vemula S.
        • Wiesner T.
        • et al.
        Mutations in GNA11 in uveal melanoma.
        N Engl J Med. 2010; 363: 2191-2199https://doi.org/10.1056/NEJMoa1000584
        • Dono M.
        • Angelini G.
        • Cecconi M.
        • Amaro A.
        • Esposito A.I.
        • Mirisola V.
        • et al.
        Mutation frequencies of GNAQ, GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single case of uveal melanoma.
        Br J Cancer. 2014; 110: 1058-1065https://doi.org/10.1038/bjc.2013.804
        • Onken M.D.
        • Worley L.A.
        • Long M.D.
        • Duan S.
        • Council M.L.
        • Bowcock A.M.
        • et al.
        Oncogenic mutations in GNAQ occur early in uveal melanoma.
        Investig Ophthalmol Vis Sci. 2008; 49: 5230-5234https://doi.org/10.1167/iovs.08-2145
        • Koopmans A.E.
        • Vaarwater J.
        • Paridaens D.
        • Naus N.C.
        • Kilic E.
        • de Klein A.
        • et al.
        Patient survival in uveal melanoma is not affected by oncogenic mutations in GNAQ and GNA11.
        Br J Cancer. 2013; 109: 493-496https://doi.org/10.1038/bjc.2013.299
        • Robertson A.G.
        • Shih J.
        • Yau C.
        • Gibb E.A.
        • Oba J.
        • Mungall K.L.
        • et al.
        Integrative analysis identifies four molecular and clinical subsets in uveal melanoma.
        Cancer Cell. 2018; 33: 151https://doi.org/10.1016/j.ccell.2017.12.013
        • Markby D.W.
        • Onrust R.
        • Bourne H.R.
        Separate GTP binding and GTPase activating domains of a Gα subunit.
        Science. 1993; 262 (901): 1895https://doi.org/10.1126/science.8266082
        • O'Hayre M.
        • Degese M.S.
        • Gutkind J.S.
        Novel insights into G protein and G protein-coupled receptor signaling in cancer.
        Curr Opin Cell Biol. 2014; 27C: 126-135https://doi.org/10.1016/j.ceb.2014.01.005
        • Feng X.
        • Degese M.S.
        • Iglesias-Bartolome R.
        • Vaque J.P.
        • Molinolo A.A.
        • Rodrigues M.
        • et al.
        Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated Rho GTPase signaling circuitry.
        Cancer Cell. 2014; 25: 831-845https://doi.org/10.1016/j.ccr.2014.04.016
        • Yu F.X.
        • Luo J.
        • Mo J.S.
        • Liu G.
        • Kim Y.C.
        • Meng Z.
        • et al.
        Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP.
        Cancer Cell. 2014; 25: 822-830https://doi.org/10.1016/j.ccr.2014.04.017
        • Griewank K.G.
        • van de Nes J.
        • Schilling B.
        • Moll I.
        • Sucker A.
        • Kakavand H.
        • et al.
        Genetic and clinico-pathologic analysis of metastatic uveal melanoma.
        Mod Pathol. 2014; 27: 175-183https://doi.org/10.1038/modpathol.2013.138
        • Staby K.M.
        • Gravdal K.
        • Mørk S.J.
        • Heegaard S.
        • Vintermyr O.K.
        • Krohn J.
        Prognostic impact of chromosomal aberrations and GNAQ, GNA11 and BAP1 mutations in uveal melanoma.
        Acta Ophthalmol. 2018; 96: 31-38https://doi.org/10.1111/aos.13452
        • Wall M.A.
        • Coleman D.E.
        • Lee E.
        • Iñiguez-Lluhi J.A.
        • Posner B.A.
        • Gilman A.G.
        • et al.
        The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2.
        Cell. 1995; 83: 1047-1058https://doi.org/10.1016/0092-8674(95)90220-1
        • Herlihy N.
        • Dogrusöz M.
        • Van Essen T.H.
        • William Harbour J.
        • Van Der Velden P.A.
        • Van Eggermond M.C.J.A.
        • et al.
        Skewed expression of the genes encoding epigenetic modifiers in high-risk uveal melanoma.
        Investig Ophthalmol Vis Sci. 2015; 56: 1447-1458https://doi.org/10.1167/iovs.14-15250
        • Amaro A.
        • Parodi F.
        • Diedrich K.
        • Angelini G.
        • Götz C.
        • Viaggi S.
        • et al.
        Analysis of the expression and single-nucleotide variant frequencies of the butyrophilin-like 2 gene in patients with uveal melanoma.
        JAMA Ophthalmol. 2016; 134: 1125-1133https://doi.org/10.1001/jamaophthalmol.2016.2691
        • Amaro A.
        • Mirisola V.
        • Angelini G.
        • Musso A.
        • Tosetti F.
        • Esposito A.I.
        • et al.
        Evidence of epidermal growth factor receptor expression in uveal melanoma: inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity.
        Eur J Cancer. 2013; 49: 3353-3365https://doi.org/10.1016/j.ejca.2013.06.011
        • Robertson A.G.
        • Shih J.
        • Yau C.
        • Gibb E.A.
        • Oba J.
        • Mungall K.L.
        • et al.
        Integrative analysis identifies four molecular and clinical subsets in uveal melanoma.
        Cancer Cell. 2017; 32 (e15): 204-220https://doi.org/10.1016/j.ccell.2017.07.003
        • Koopmans A.E.
        • Verdijk R.M.
        • Brouwer R.W.
        • van den Bosch T.P.
        • van den Berg M.M.
        • Vaarwater J.
        • et al.
        Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma.
        Mod Pathol. 2014; 27: 1321-1330https://doi.org/10.1038/modpathol.2014.43
        • Scholz S.L.
        • Moller I.
        • Reis H.
        • Susskind D.
        • van de Nes J.A.P.
        • Leonardelli S.
        • et al.
        Frequent GNAQ, GNA11, and EIF1AX mutations in Iris melanoma.
        Invest Ophthalmol Vis Sci. 2017; 58: 3464-3470
        • Van Essen T.H.
        • Van Pelt S.
        • Versluis M.
        • Bronkhorst I.H.G.
        • Van Duinen S.G.
        • Marinkovic M.
        • et al.
        Prognostic parameters in uveal melanoma and their association with BAP1 expression.
        Br J Ophthalmol. 2014; 98: 1738-1743https://doi.org/10.1136/BJOPHTHALMOL-2014-305047
        • Patrone S.
        • Maric I.
        • Rutigliani M.
        • Lanza F.
        • Puntoni M.
        • Banelli B.
        • et al.
        Prognostic value of chromosomal imbalances, gene mutations, and BAP1 expression in uveal melanoma.
        Genes Chromosom Cancer. 2018; 57: 387-400https://doi.org/10.1002/gcc.22541
        • Shah A.A.
        • Bourne T.D.
        • Murali R.
        BAP1 protein loss by immunohistochemistry: a potentially useful tool for prognostic prediction in patients with uveal melanoma.
        Pathology. 2013; 45: 651-656https://doi.org/10.1097/PAT.0000000000000002
        • Piaggio F.
        • Tozzo V.
        • Bernardi C.
        • Croce M.
        • Puzone R.
        • Viaggi S.
        • et al.
        Secondary somatic mutations in g-protein-related pathways and mutation signatures in Uveal melanoma.
        Cancers (Basel). 2019; 11https://doi.org/10.3390/cancers11111688
        • Harbour J.W.
        A prognostic test to predict the risk of metastasis in uveal melanoma based on a 15-gene expression profile.
        Methods Mol Biol. 2014; 1102: 427-440https://doi.org/10.1007/978-1-62703-727-3_22
        • Daulat A.M.
        • Maurice P.
        • Froment C.
        • Guillaume J.L.
        • Broussard C.
        • Monsarrat B.
        • et al.
        Purification and identification of G proteincoupled receptor protein complexes under native conditions.
        Mol Cell Proteomics. 2007; 6: 835-844
        • Field M.G.
        • Kuznetsov J.N.
        • Bussies P.L.
        • Cai L.Z.
        • Alawa K.A.
        • Decatur C.L.
        • et al.
        BAP1 loss is associated with DNA methylomic repatterning in highly aggressive class 2 uveal melanomas.
        Clin Cancer Res. 2019; 25: 5663-5673https://doi.org/10.1158/1078-0432.CCR-19-0366
        • Van Raamsdonk C.D.
        • Fitch K.R.
        • Fuchs H.
        • De Angelis M.H.
        • Barsh G.S.
        Effects of G-protein mutations on skin color.
        Nat Genet. 2004; 36: 961-968https://doi.org/10.1038/NG1412
        • Damato B.
        • Dopierala J.
        • Klaasen A.
        • van Dijk M.
        • Sibbring J.
        • Coupland S.E.
        Multiplex ligation-dependent probe amplification of uveal melanoma: correlation with metastatic death.
        Invest Ophthalmol Vis Sci. 2009; 50 (https://doi.org/iovs.08-3165 [pii]10.1167/iovs.08-3165): 3048-3055
        • Kalirai H.
        • Dodson A.
        • Faqir S.
        • Damato B.E.
        • Coupland S.E.
        Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing.
        Br J Cancer. 2014; 111: 1373-1380https://doi.org/10.1038/bjc.2014.417
        • Naus N.C.
        • Verhoeven A.C.A.
        • van Drunen E.
        • Slater R.
        • Mooy C.M.
        • Paridaens D.A.
        • et al.
        Detection of genetic prognostic markers in uveal melanoma biopsies using fluorescence in situ hybridization.
        Clin Cancer Res. 2002; 8
        • White V.A.
        • Chambers J.D.
        • Courtright P.D.
        • Chang W.Y.
        • Horsman D.E.
        Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma.
        Cancer. 1998; 83: 354-359https://doi.org/10.1002/(SICI)1097-0142(19980715)83:2<354::AID-CNCR20>3.0.CO;2-R
        • de Lange M.J.
        • van Pelt S.I.
        • Versluis M.
        • Jordanova E.S.
        • Kroes W.G.
        • Ruivenkamp C.
        • et al.
        Heterogeneity revealed by integrated genomic analysis uncovers a molecular switch in malignant uveal melanoma.
        Oncotarget. 2015; 6: 37824-37835https://doi.org/10.18632/oncotarget.5637
        • Ehlers J.P.
        • Worley L.
        • Onken M.D.
        • Harbour J.W.
        DDEF1 is located in an amplified region of chromosome 8q and is overexpressed in uveal melanoma.
        Clin Cancer Res. 2005; 11: 3609-3613https://doi.org/10.1158/1078-0432.CCR-04-1941
        • Biasini M.
        • Bienert S.
        • Waterhouse A.
        • Arnold K.
        • Studer G.
        • Schmidt T.
        • et al.
        SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information.
        Nucleic Acids Res. 2014; 42https://doi.org/10.1093/NAR/GKU340
        • Waldo G.L.
        • Ricks T.K.
        • Hicks S.N.
        • Cheever M.L.
        • Kawano T.
        • Tsuboi K.
        • et al.
        Kinetic scaffolding mediated by a phospholipase C-beta and Gq signaling complex.
        Science. 2010; 330: 974-980https://doi.org/10.1126/SCIENCE.1193438
        • Dogrusöz M.
        • Trasel A.R.
        • Cao J.
        • Ҫolak S.
        • van Pelt S.I.
        • Kroes W.G.M.
        • et al.
        Differential expression of DNA repair genes in prognostically-favorable versus unfavorable uveal melanoma.
        Cancers (Basel). 2019; 11https://doi.org/10.3390/cancers11081104
        • Irizarry R.A.
        • Bolstad B.M.
        • Collin F.
        • Cope L.M.
        • Hobbs B.
        • Speed T.P.
        Summaries of Affymetrix GeneChip probe level data.
        Nucleic Acids Res. 2003; 31: e15https://doi.org/10.1093/nar/gng015
        • Irizarry R.A.
        • Hobbs B.
        • Collin F.
        • Beazer-Barclay Y.D.
        • Antonellis K.J.
        • Scherf U.
        • et al.
        Exploration, normalization, and summaries of high density oligonucleotide array probe level data.
        Biostatistics. 2003; 4: 249-264https://doi.org/10.1093/biostatistics/4.2.249
        • Nannya Y.
        • Sanada M.
        • Nakazaki K.
        • Hosoya N.
        • Wang L.
        • Hangaishi A.
        • et al.
        A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays.
        Cancer Res. 2005; 65: 6071-6079https://doi.org/10.1158/0008-5472.CAN-05-0465
        • de Lange M.J.
        • Razzaq L.
        • Versluis M.
        • Verlinde S.
        • Dogrusoz M.
        • Bohringer S.
        • et al.
        Distribution of GNAQ and GNA11 mutation signatures in uveal melanoma points to a light dependent mutation mechanism.
        PLoS One. 2015; 10e0138002https://doi.org/10.1371/journal.pone.0138002
        • Ksander B.R.
        • Rubsamen P.E.
        • Olsen K.R.
        • Cousins S.W.
        • Streilein J.W.
        Studies of tumor-infiltrating lymphocytes from a human choroidal melanoma.
        Invest Ophthalmol Vis Sci. 1991; 32: 3198-3208
        • De Waard-Siebinga I.
        • Blom D.R.
        • Griffioen M.
        • Schrier P.I.
        • Hoogendoorn E.
        • Beverstock G.
        • et al.
        Establishment and characterization of an uveal-melanoma cell line.
        Int J Cancer. 1995; 62: 155-161https://doi.org/10.1002/IJC.2910620208
        • Nareyeck G.
        • Zeschnigk M.
        • Bornfeld N.
        • Anastassiou G.
        Novel cell lines derived by long-term culture of primary uveal melanomas.
        Ophthalmologica. 2009; 223: 196-201https://doi.org/10.1159/000201566
        • Kulak N.A.
        • Pichler G.
        • Paron I.
        • Nagaraj N.
        • Mann M.
        Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.
        Nat Methods. 2014; 11: 319-324https://doi.org/10.1038/nmeth.2834
        • Marino A.
        • Camponovo A.
        • Degl'Innocenti A.
        • Bartolucci M.
        • Tapeinos C.
        • Martinelli C.
        • et al.
        Multifunctional temozolomide-loaded lipid superparamagnetic nanovectors: dual targeting and disintegration of glioblastoma spheroids by synergic chemotherapy and hyperthermia treatment.
        Nanoscale. 2019; 11: 21227-21248https://doi.org/10.1039/C9NR07976A
        • Cox J.
        • Mann M.
        MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
        Nat Biotechnol. 2008; 26: 1367-1372https://doi.org/10.1038/NBT.1511
        • Perez-Riverol Y.
        • Csordas A.
        • Bai J.
        • Bernal-Llinares M.
        • Hewapathirana S.
        • Kundu D.J.
        • et al.
        The PRIDE database and related tools and resources in 2019: improving support for quantification data.
        Nucleic Acids Res. 2019; 47: D442-D450https://doi.org/10.1093/NAR/GKY1106
        • Free R.B.
        • Hazelwood L.A.
        • Sibley D.R.
        Identifying novel protein-protein interactions using co-immunoprecipitation and mass spectroscopy.
        Curr Protoc Neurosci. 2009; Chapter 5: Unit 5.28https://doi.org/10.1002/0471142301.ns0528s46
        • Tusher V.G.
        • Tibshirani R.
        • Chu G.
        Significance analysis of microarrays applied to the ionizing radiation response.
        Proc Natl Acad Sci USA. 2001; 98: 5116-5121https://doi.org/10.1073/pnas.091062498
        • Chen E.Y.
        • Tan C.M.
        • Kou Y.
        • Duan Q.
        • Wang Z.
        • Meirelles G.V.
        • et al.
        Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.
        BMC Bioinf. 2013; 14https://doi.org/10.1186/1471-2105-14-128
        • Kuleshov M.V.
        • Jones M.R.
        • Rouillard A.D.
        • Fernandez N.F.
        • Duan Q.
        • Wang Z.
        • et al.
        Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.
        Nucleic Acids Res. 2016; 44 (7): W90https://doi.org/10.1093/nar/gkw377
        • Pathan M.
        • Keerthikumar S.
        • Ang C.-S.S.
        • Gangoda L.
        • Quek C.Y.J.
        • Williamson N.A.
        • et al.
        FunRich: An open access standalone functional enrichment and interaction network analysis tool. 2015; 15: 2597-2601
      1. FunRich:: Functional enrichment analysis tool:: Home n.d. http://www.funrich.org/(accessed July 17, 2021).

      2. Maxime Meylan, Etienne Becht, Catherine Sautès-Fridman, Aurélien de Reyniès, Wolf H. Fridman, Florent Petitprez bioRxiv 2020.12.03.400754; doi: https://doi.org/10.1101/2020.12.03.400754.

        • Becht E.
        • Giraldo N.A.
        • Lacroix L.
        • Buttard B.
        • Elarouci N.
        • Petitprez F.
        • Selves J.
        • Laurent-Puig P.
        • Sautès-Fridman C.
        • Fridman W.H.
        • de Reyniès A.
        Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression.
        Genome Biol. 2016 Oct 20; 17: 218https://doi.org/10.1186/s13059-016-1070-5
      3. Erratum in: Genome Biol. 2016 Dec 1; 17 (PMID: 27765066; PMCID: PMC5073889): 249