Recurrent inactivating RASA2 mutations in melanoma

Abstract

Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.

Figure 1. Effects of RASA2 mutations on RAS activity, growth and patient survival.

(A) Human RASA2 protein, conserved domains indicated as blocks: C2 domain first repeat (C2 1); C2 domain second repeat (C2 2); Ras-GTPase activating domain (RAS-GAP); Plekstrin homology domain (PH); Bruton’s tyrosine kinase Cys-rich motif (BTK). Somatic mutations indicated with arrows and amino-acid changes. Red triangles indicate deleterious mutations. Underlined mutations were functionally assessed. (B) Distribution of somatic mutations in BRAF, NRAS, NF1 and RASA2 in melanoma (n=501). (C) Immunoblot of RAS-GTP levels in 501Mel cells expressing the indicated constructs (left). 501Mel cells (middle) and 108T (right) cells depleted for RASA2 using human RASA2 siRNAs. RAS-GTP levels were assessed by RAS pull-down assay and the RAS-GTP/RAS ratio of 2 independent experiments were calculated and normalized to the vector control (lower panel). Error bars, standard deviation (S.D.) (D) 501Mel clones expressing the indicated constructs were seeded in 96-well plates in 2.5% FBS and average cell number was measured by assessing DNA content using SYBR Green I from two independent experiments with six repeats each. Error bars, standard deviation (S.D.).(E) Anchorage-independent proliferation of 501Mel RASA2 clones expressing the indicated constructs was assessed by measuring colony formation in soft agar in 2.5% serum from two independent experiments four repeats each after 7 days. **P< 0.005 for WT vs. vector and ***P< 0.0001 for WT vs mutants (Students t-tests). (F) Kaplan-Meier curve showing overall survival of AJCC stage III melanoma patients with positive (n=54) or negative (n=27) RASA2 expression (log rank p = 0.0043).