Inactivation Of Rasa1 Promotes Melanoma Tumorigenesis Via R-Ras Activation

Hyperactivation of the Ras/Raf/Mitogen-activated protein kinase (MAPK) pathway has been commonly observed in melanoma via frequent activating mutations in NRAS and BRAF. Novel mutations in other components of this pathway such as MEK1, MEK2, MAP3K5, and MAP3K9 have been reported recently by high-throughput sequencing efforts. In addition, Ras GTPase activating proteins (RasGAPs) that negatively regulate Ras, such as NF1 (neurofibromatosis type 1) and RASA2, have been shown to be mutated or suppressed in melanoma. However, importance of other RasGAPs in melanoma has not been addressed. To obtain a comprehensive view of melanoma genomes, we conducted whole genome sequencing (WGS) of 15 metastatic melanomas and matched normal PBMC genomes from 13 melanoma patients. All melanoma genomes from these 13 patients contained at least one mutation in genes of Ras-Raf-MAPK pathway (MAPK1, MAP3K1, MAP4K2, MAP3K14, NRAS, and BRAF). In addition, we identified two novel, clustered somatic missense mutations (p.Tyr472His and p.Leu481Phe) in RASA1 (RAS p21 protein activator 1, p120RasGAP). In this study, we addressed functional roles of RASA1 in melanoma tumorigenesis. The RNAi-mediated down-regulation of RASA1 promoted, while ectopic expression of wild type RASA1 decreased, anchorage-independent colony formation, tumor growth, and RAS activation. Interestingly, RASA1 Y472H mutant enhanced soft agar colony formation and tumor growth, while RASA1 L481F mutant lost its tumor suppressive activity. Mechanistically, RASA1 required RasGAP activity to suppress colony formation and showed higher activity toward R-Ras (related RAS viral (r-ras) oncogene homolog) isoform among the Ras superfamily of small GTPases. Moreover, RASA1 consistently suppressed Ral-A among Ras downstream effectors. Reduced R-Ras or Ral-A expression via siRNAs suppressed anchorage-independent growth induced by RASA1 loss. Interestingly, RASA1 expression was frequently down-regulated in metastatic melanoma samples (11.4% (4/35) of lymph node metastasis and 3.4% (1/29) of distal metastases) compared to primary melanomas (33.3% (21/63)) and dysplastic nevi (44.1% (15/34)). We also observed significantly shorter overall survival of melanoma patients with BRAF mutations when RASA1 mRNA expression is low, which may be explained by possible cooperative interactions between activation of BRAF/MAPK/ERK and RASA1/R-Ras/Ral-A pathways. Taken together, these data support that RASA1 is a novel melanoma tumor suppressor that is inactivated by suppressed expression or by mutation. Citation Format: Kristen S. Hill, Hyeran Sung, Krishna L. Kanchi, Jane L. Messina, Ji-Hyun Lee, Youngchul Kim, Li Ding, Richard K. Wilson, Jeffrey S. Weber, Minjung Kim. Inactivation of RASA1 promotes melanoma tumorigenesis via R-Ras activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1863.