Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma

Abstract

Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C β4), the downstream effector of Gαq signaling1,2,3. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester–independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: CYSLTR2 mutation encoding p.Leu129Gln is a hotspot mutation and is mutually exclusive with known drivers in uveal melanoma.
Figure 2: Leu129Gln CysLT2R exhibits high basal coupling to Gαq.
Figure 3: Leu129Gln CysLT2R promotes TPA-independent growth in vitro and enforces a melanocyte-lineage-specific signature.
Figure 4: Leu129Gln CysLT2R promotes tumorigenesis in vivo and enforces a melanocyte-lineage-specific signature.
Figure 5: CYSLTR2 is required for the growth and maintenance of the melanocyte-lineage-specific signature in melan-a cells transformed to express Leu129Gln CysLT2R.

Similar content being viewed by others

Accession codes

Accessions

European Nucleotide Archive

NCBI Reference Sequence

Protein Data Bank

References

  1. Van Raamsdonk, C.D. et al. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 457, 599–602 (2009).

    Article  CAS  Google Scholar 

  2. Van Raamsdonk, C.D. et al. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 363, 2191–2199 (2010).

    Article  CAS  Google Scholar 

  3. Johansson, P. et al. Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4. Oncotarget 7, 4624–4631 (2016).

    PubMed  Google Scholar 

  4. Furney, S.J. et al. SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discov. 3, 1122–1129 (2013).

    Article  CAS  Google Scholar 

  5. Martin, M. et al. Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat. Genet. 45, 933–936 (2013).

    Article  CAS  Google Scholar 

  6. Chang, M.T. et al. Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity. Nat. Biotechnol. 34, 155–163 (2016).

    Article  CAS  Google Scholar 

  7. Leiserson, M.D., Wu, H.T., Vandin, F. & Raphael, B.J. CoMEt: a statistical approach to identify combinations of mutually exclusive alterations in cancer. Genome Biol. 16, 160 (2015).

    Article  Google Scholar 

  8. Harbour, J.W. et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 330, 1410–1413 (2010).

    Article  CAS  Google Scholar 

  9. Harbour, J.W. et al. Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat. Genet. 45, 133–135 (2013).

    Article  CAS  Google Scholar 

  10. Evans, J.F. Cysteinyl leukotriene receptors. Prostaglandins Other Lipid Mediat. 68-69, 587–597 (2002).

    Article  CAS  Google Scholar 

  11. Sakmar, T.P. Structure of rhodopsin and the superfamily of seven-helical receptors: the same and not the same. Curr. Opin. Cell Biol. 14, 189–195 (2002).

    Article  CAS  Google Scholar 

  12. Ballesteros, J.A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein–coupled receptors. in Methods in Neurosciences Vol. 25 (ed. Stuart, C.S.) 366–428 (Academic Press, 1995).

  13. Tao, Y.X. Constitutive activation of G protein–coupled receptors and diseases: insights into mechanisms of activation and therapeutics. Pharmacol. Ther. 120, 129–148 (2008).

    Article  CAS  Google Scholar 

  14. Zhang, C. et al. High-resolution crystal structure of human protease-activated receptor 1. Nature 492, 387–392 (2012).

    Article  CAS  Google Scholar 

  15. Bennett, D.C., Cooper, P.J. & Hart, I.R. A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth. Int. J. Cancer 39, 414–418 (1987).

    Article  CAS  Google Scholar 

  16. Prince, S., Wiggins, T., Hulley, P.A. & Kidson, S.H. Stimulation of melanogenesis by tetradecanoylphorbol 13-acetate (TPA) in mouse melanocytes and neural crest cells. Pigment Cell Res. 16, 26–34 (2003).

    Article  CAS  Google Scholar 

  17. Yaar, M. & Gilchrest, B.A. Human melanocyte growth and differentiation: a decade of new data. J. Invest. Dermatol. 97, 611–617 (1991).

    Article  CAS  Google Scholar 

  18. Griewank, K.G. et al. Genetic and molecular characterization of uveal melanoma cell lines. Pigment Cell Melanoma Res. 25, 182–187 (2012).

    Article  CAS  Google Scholar 

  19. Parma, J. et al. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365, 649–651 (1993).

    Article  CAS  Google Scholar 

  20. Liu, G. et al. Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. N. Engl. J. Med. 341, 1731–1736 (1999).

    Article  CAS  Google Scholar 

  21. Kan, Z. et al. Diverse somatic mutation patterns and pathway alterations in human cancers. Nature 466, 869–873 (2010).

    Article  CAS  Google Scholar 

  22. Prickett, T.D. et al. Exon capture analysis of G protein–coupled receptors identifies activating mutations in GRM3 in melanoma. Nat. Genet. 43, 1119–1126 (2011).

    Article  CAS  Google Scholar 

  23. Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333–339 (2013).

    Article  CAS  Google Scholar 

  24. Lawrence, M.G. et al. A preclinical xenograft model of prostate cancer using human tumors. Nat. Protoc. 8, 836–848 (2013).

    Article  CAS  Google Scholar 

  25. Garraway, L.A. et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 436, 117–122 (2005).

    Article  CAS  Google Scholar 

  26. Leff, J.A. et al. Montelukast, a leukotriene-receptor antagonist, for the treatment of mild asthma and exercise-induced bronchoconstriction. N. Engl. J. Med. 339, 147–152 (1998).

    Article  CAS  Google Scholar 

  27. Wunder, F. et al. Pharmacological characterization of the first potent and selective antagonist at the cysteinyl leukotriene 2 (CysLT2) receptor. Br. J. Pharmacol. 160, 399–409 (2010).

    Article  CAS  Google Scholar 

  28. Bond, R.A. & Ijzerman, A.P. Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery. Trends Pharmacol. Sci. 27, 92–96 (2006).

    Article  CAS  Google Scholar 

  29. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

  30. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  31. Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  Google Scholar 

  32. Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).

    Article  Google Scholar 

  33. Cheng, D.T. et al. Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J. Mol. Diagn. 17, 251–264 (2015).

    Article  CAS  Google Scholar 

  34. Refaeli, Y., Van Parijs, L., Alexander, S.I. & Abbas, A.K. Interferon γ is required for activation-induced death of T lymphocytes. J. Exp. Med. 196, 999–1005 (2002).

    Article  CAS  Google Scholar 

  35. Leduc, M. et al. Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands. J. Pharmacol. Exp. Ther. 331, 297–307 (2009).

    Article  CAS  Google Scholar 

  36. Martí-Renom, M.A. et al. Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29, 291–325 (2000).

    Article  Google Scholar 

  37. Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  Google Scholar 

  38. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38, 27–28 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the members of the Molecular Diagnostics Service in the Department of Pathology and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology. We thank T. Wiesner (MSKCC and Medical University of Graz) for the melan-a and MEL290 cell lines and intellectual input, D. Abramson for intellectual input, C. Kandoth and the TCGA for data sharing, M. Berger and N. Bouvier for obtaining patient DNA, Y.A. Berchiche for the flow cytometry analysis and scientific input, and M. Bouvier (Université de Montréal) for the kind gift of the EPAC construct. This work was supported in part by grants from the NIH to P.C. (P50CA140146, CDA; DP2CA174499; K08CA151660), the NIH to Y.C. (K08CA140946), the Sidney Kimmel Foundation to P.C. (Kimmel Scholar Award), the Cycle for Survival Fund to P.C. and Y.C., the Geoffrey Beene Award to P.C. and Y.C., the Prostate Cancer Foundation to Y.C., the STARR Cancer Consortium to P.C. and Y.C., and the François Wallace ç Monahan Fellowship to E.C.

Author information

Authors and Affiliations

Authors

Contributions

Project planning and experimental design: P.C., Y.C., A.R.M., T.P.S., M.A.K., and E.C. Bioinformatic analysis of exome sequencing data: Y.C., A.R.M., M.T.C., and B.S.T. Clinical specimen acquisition, annotation, and analysis: A.N.S. Immunoblots, growth curves, qPCR, and all cellular assays: A.R.M., E.C., and Y.G. Xenograft assays: A.R.M., J.J.S., and E.G.W. Generation of the expression vectors: J.Q.Z., E.C., and A.R.M. Molecular modeling: T.H. Manuscript writing: A.R.M., P.C., Y.C., E.C., and T.P.S. All authors reviewed the final manuscript.

Corresponding author

Correspondence to Yu Chen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Raw sequencing reads of CYSLTR2 mutations.

(a) IGV view and wild-type and mutant read count (right) of sequencing reads around the CYSLTR2 p.Leu129Gln (c.386T>A) mutation of germline DNA, tumor DNA, and tumor RNA (when available) of four mutant samples from next-generation sequencing. The wild-type T allele is in red, and the mutant A allele is in green. Note that the mutant reads are found in tumor DNA and RNA but not in germline DNA. (b) Sanger sequencing trace of tumor and germline DNA from an MSKCC patient with uveal melanoma and is wild-type for GNAQ and GNA11.

Supplementary Figure 2 CYSLTR2 mutations in TCGA data sets.

(a) Prevalence of CYSLTR2 mutations in TCGA cohorts. (b) Location of all CYSLTR2 mutations in TCGA cohorts. Two amino acids, Leu129 and Arg136, are mutated in more than one sample. There are three p.Leu129Gln mutations, all in uveal melanoma (red arrow), two p.Arg136His mutations (one colorectal and one adrenal cortical), and one p.Arg136Cys mutation (colorectal) (blue arrow). (c) Gene expression of CYSLTR2 in wild-type and mutant samples in selected TCGA cohorts. Data show that, other than p.Leu129Gln in uveal melanoma, CYSLTR2 is not highly expressed in mutated samples in other cancer types. A blue arrow indicates samples with Arg136 mutation. A red arrow indicates samples with p.Leu129Gln mutation. (d) Total mutational count of tumor samples in selected TCGA cohorts in CYSLTR2 wild-type and mutated samples. This highlights the low mutational burden of uveal melanoma. A blue arrow indicates samples with Arg136 mutation. A red arrow indicates samples with Leu129Gln mutation.

Supplementary Figure 3 HEK293T expression of wild-type and Leu129Gln CysLT2R.

(a) Immunoblotting for the N-terminal FLAG tag, C-terminal 1D4 tag, and -tubulin in HEK293T cells transfected with empty-vector, wild-type, or Leu129Gln CysLT2R. (b) Representative image of immunofluorescence for 1D4 (green) and FLAG (red) in HEK293T cells transfected with wild-type CysLT2R, or Leu129Gln CysLT2R. Hoechst (blue) was used to counterstain the nucleus. Scale bar, 10 m. (c) Flow cytometry analysis of live transfected HEK293T cells using FLAG antibody against the extracellular FLAG tag showing the respective cell surface expression of the receptor.

Supplementary Figure 4 Effect of Leu129Gln CysLT2R on tumorigenesis and TPA independent of cell growth.

(a) Tumor volume of mice implanted with NIH3T3 cells expressing empty-vector, wild-type CysLT2R, Leu129Gln CysLT2R, or Arg136His CysLT2R (n = 10). Error bars, ±s.e.m. *P < 0.005. (b) Photographs of cellular pellets of melan-a cells expressing emptyvector, wild-type CysLT2R or Leu129Gln CysLT2R grown in the absence of TPA for 4 and 10 d.

Supplementary Figure 5 Uncropped immunoblots.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1. (PDF 1325 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moore, A., Ceraudo, E., Sher, J. et al. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma. Nat Genet 48, 675–680 (2016). https://doi.org/10.1038/ng.3549

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.3549

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer