Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi

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.

Figure 1. GNAQ^Q209L transforms melanocytes

a, GNAQ^Q209L induces anchorage independent growth in soft agar of hTERT/CDK4^R24C/p53^DD melanocytes in a TPA-independent manner with comparable efficiency as NRAS^Q61R. b, Cells expressing Flag-tagged GNAQ^Q209L showed enlarged nuclei with irregular contours after 5 days. c, Melan-a cells stably transduced with GNAQ^Q209L, 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 activation

a, Increased expression of pERK in hTERT/CDK4^R24C/p53^DD melanocytes transfected with GNAQ^Q209L compared to similar melanocytes transfected with GNAQ^WT or empty vector. b, Cumulative distribution of mean pixel fluorescence intensity per cell obtained from immunofluorescent detection of pERK (p-values: GNAQ^Q209L vs. vector). c, Western blot showing increased pERK levels in hTERT/CDK4^R24C/p53^DD melanocytes expressing Flag-tagged GNAQ^Q209L compared to cells transduced with Flag-tagged GNAQ^WT or vector control. NRAS^Q61R 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 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.