Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi

Catherine D Van Raamsdonk, Vladimir Bezrookove, Gary Green, Jürgen Bauer, Lona Gaugler, Joan M O'Brien, Elizabeth M Simpson, Gregory S Barsh, Boris C Bastian, Catherine D Van Raamsdonk, Vladimir Bezrookove, Gary Green, Jürgen Bauer, Lona Gaugler, Joan M O'Brien, Elizabeth M Simpson, Gregory S Barsh, Boris C Bastian

Abstract

BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures, and lead to constitutive activation of the mitogen-activated protein (MAP) kinase pathway. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown. Here we report frequent somatic mutations in the heterotrimeric G protein alpha-subunit, GNAQ, in blue naevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the Ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP kinase activation in melanocytic neoplasia, providing new opportunities for therapeutic intervention.

Figures

Figure 1. GNAQ Q209L transforms melanocytes
Figure 1. GNAQQ209L transforms melanocytes
a, GNAQQ209L induces anchorage independent growth in soft agar of hTERT/CDK4R24C/p53DD melanocytes in a TPA-independent manner with comparable efficiency as NRASQ61R. b, Cells expressing Flag-tagged GNAQQ209L showed enlarged nuclei with irregular contours after 5 days. c, Melan-a cells stably transduced with GNAQQ209L, but not with wild-type GNAQ (n=3) or vector control (n=4), induce highly pigmented tumors of spindled and epithelioid melanocytes after 10 weeks in four out of five animals.
Figure 2. GNAQ Q209L induces MAP kinase…
Figure 2. GNAQQ209L induces MAP kinase activation
a, Increased expression of pERK in hTERT/CDK4R24C/p53DD melanocytes transfected with GNAQQ209L compared to similar melanocytes transfected with GNAQWT or empty vector. b, Cumulative distribution of mean pixel fluorescence intensity per cell obtained from immunofluorescent detection of pERK (p-values: GNAQQ209L vs. vector). c, Western blot showing increased pERK levels in hTERT/CDK4R24C/p53DD melanocytes expressing Flag-tagged GNAQQ209L compared to cells transduced with Flag-tagged GNAQWT or vector control. NRASQ61R transduced melanocytes are shown as a positive control. * The band migrating just below the Flag band is non-specific reactive band in the lysate.
Figure 3. Knockdown of GNAQ in OMM1.3…
Figure 3. Knockdown of GNAQ in OMM1.3 cells results in MAP-kinase inhibition, reduced growth and apoptosis
a, Western blot after treatment with 2 pools of siRNAs against GNAQ shows decreased pERK levels compared to control treated cells: cyclophilin B and non-target siRNA. b, After 72 hours, GNAQ knockdown results in marked reduction of cell numbers, similar to the effect of MEK inhibitor U0126. Bars show means and standard error of five replicate assays. * p<0.05, t-test compared to mock or vehicle control, respectively. c, GNAQ knockdown reduces the number of colonies (upper left corner) formed in soft agar. d, Cell cycle profiles showing an increase of the sub-G0/G1 population after GNAQ knockdown.

References

    1. Davies H, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–54.
    1. Pollock PM, et al. High frequency of BRAF mutations in nevi. Nat Genet. 2002;25:25.
    1. Saldanha G, et al. High BRAF mutation frequency does not characterize all melanocytic tumor types. Int J Cancer. 2004;111:705–10.
    1. Horsman DE, White VA. Cytogenetic analysis of uveal melanoma. Consistent occurrence of monosomy 3 and trisomy 8q. Cancer. 1993;71:811–9.
    1. Singh AD, Bergman L, Seregard S. Uveal melanoma: epidemiologic aspects. Ophthalmol Clin North Am. 2005;18:75–84. viii.
    1. Zembowicz A, Mihm MC. Dermal dendritic melanocytic proliferations: an update. Histopathology. 2004;45:433–51.
    1. Singh AD, et al. Lifetime prevalence of uveal melanoma in white patients with oculo(dermal) melanocytosis. Ophthalmology. 1998;105:195–8.
    1. Van Raamsdonk CD, Fitch KR, Fuchs H, de Angelis MH, Barsh GS. Effects of G-protein mutations on skin color. Nat Genet. 2004;36:961–8.
    1. Markby DW, Onrust R, Bourne HR. Separate GTP binding and GTPase activating domains of a G alpha subunit. Science. 1993;262:1895–901.
    1. Landis CA, et al. GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature. 1989;340:692–6.
    1. Kalinec G, Nazarali AJ, Hermouet S, Xu N, Gutkind JS. Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells. Mol Cell Biol. 1992;12:4687–93.
    1. Lyons J, et al. Two G protein oncogenes in human endocrine tumors. Science. 1990;249:655–9.
    1. Garraway LA, et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature. 2005;436:117–122.
    1. Hubbard KB, Hepler JR. Cell signalling diversity of the Gqalpha family of heterotrimeric G proteins. Cell Signal. 2006;18:135–50.
    1. Zuidervaart W, et al. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS. Br J Cancer. 2005;92:2032–8.
    1. Shin MK, Levorse JM, Ingram RS, Tilghman SM. The temporal requirement for endothelin receptor-B signalling during neural crest development. Nature. 1999;402:496–501.
    1. Dissanayake SK, et al. The Wnt5A/protein kinase C pathway mediates motility in melanoma cells via the inhibition of metastasis suppressors and initiation of an epithelial to mesenchymal transition. J Biol Chem. 2007;282:17259–71.
    1. Sheldahl LC, Park M, Malbon CC, Moon RT. Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr Biol. 1999;9:695–8.
    1. Marin YE, et al. Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon. Cell Signal. 2006;18:1279–86.
    1. Mizushima J, Nogita T, Higaki Y, Horikoshi T, Kawashima M. Dormant melanocytes in the dermis: do dermal melanocytes of acquired dermal melanocytosis exist from birth? Br J Dermatol. 1998;139:349–50.
    1. Chang AE, Karnell LH, Menck HR. The National Cancer Data Base report on cutaneous and noncutaneous melanoma: a summary of 84,836 cases from the past decade. The American College of Surgeons Commission on Cancer and the American Cancer Society. Cancer. 1998;83:1664–78.
    1. Curtin JA, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353:2135–47.
    1. Bennett DC, Cooper PJ, Hart IR. A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth. Int J Cancer. 1987;39:414–8.

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe