Cutaneous malignant melanoma is considered one of the most deadly human cancers, based on both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wild-type NRAS. NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here, we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine exchange factor (RalGEF), using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we discovered that constitutive RalGEF activation had a major impact on several malignant phenotypes, particularly anchorage-independent growth, indicating that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target.

Cutaneous malignant melanoma is considered one of the most deadly human cancers, based upon both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wildtype NRAS. RAS gene products (HRas, KRas and NRas) are 21kd G-proteins that serve as molecular switches converting cell-surface kinase activation events to nuclear events, thus influencing cell behavior. The major downstream effectors of Ras are the Rafs (ARaf, BRaf and CRaf), phosphatidylinositol 3-kinase (PI3K) and the Ral guanine exchange factors (RalGEF). NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, PI3K and RalGEF, using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Activation of each of the three major downstream pathways of NRas has overt phenotypic effects on immortalized mouse melanocytes, many distinct. Our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we found that constitutively activated RalGEF stimulated melanocyte invasiveness and anchorage-independent growth almost as impressively as NRasQ61K. The suppressive effects of expression of a dominant negative Ral corroborated the role of the RalGEF-Ral pathway in anchorage-independent growth. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, melanocytes expressing constitutively activated mutants of all three NRas downstream factors (RLF-CAAX plus p110α-CAAX plus BRafV600E) achieved soft agar colony forming efficiency that was fully comparable to those expressing NRasQ61K alone. In conclusion, our data suggested a distinct function for all three NRas downstream pathways - Raf, PI3K and Ral in melanocyte senescence, invasiveness and transformation, respectively. Importantly, our finding that constitutive RalGEF activation confers malignant phenotypes in melanocytes, particularly anchorage-independent growth, indicates that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5076.