The Ras family of small GTP binding proteins are tightly regulated through a complex network of proteins. In melanoma the Ras signaling pathway is frequently activated by mutations in NRAS (20%), KRAS (2%) and HRAS (1%); alternatively, Ras can also be activated by the inactivation of Ras GTPase activating proteins (RasGAPs) such as NF1, RASA1, and RASA2. Recently, we observed that inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform and that R-Ras was required to promote anchorage-independent growth driven by RASA1 inactivation. Moreover, a low level of RASA1 mRNA expression is associated with decreased overall survival in melanoma patients with BRAF mutations. Based on these observations, we hypothesized that, although not mutated, R-Ras is activated in melanoma by inactivation of RasGAPs and that BRAF activation cooperates with this RasGAP/R-Ras pathway activation in melanoma tumorigenesis.
In this study, we addressed the importance of R-Ras, a previously less appreciated member of the Ras small GTPases family, in melanoma tumorigenesis. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines and human melanoma specimens. In addition, RNAi-mediated knockdown of R-Ras suppressed anchorage-independent colony growth and tumor growth. Of the 3 major RAS effector pathways, specifically MEK/ERK, AKT and Ral-A, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by R-Ras activation downstream of RASA1 inactivation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacological (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a Rasa1L/L; BRAFCA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in expression of constitutively activated mutant BRAF and deletion of Rasa1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in Rasa1L/L BRAFCA/CA mice compared to Rasa1+/+ BRAFCA/CA littermates; as well as, the development of cutaneous melanoma in Rasa1 deficient mice. Tumors generated in this animal model will be analyzed to determine the extent of R-Ras and Ral-A activity in vivo. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways.
Citation Format: Kristen S. Hill, Evan R. Roberts, Xue Wang, Youngchul Kim, Jane Messina, Minjung Kim. R-Ras activation cooperates with BRAF mutation in melanoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4364.