Abstract 2635: Differential inhibition of autophagic pathways in melanoma is oncogene-dependent

Abstract

Abstract One person dies from melanoma every hour worldwide. The American Cancer Society estimates that in 2013 the 5-year survival rate for stage IV melanoma will remain unchanged at about 15%, even with new therapies. Autophagy (self-eating pathway) is a cellular recycling pathway that has been shown to play a role in normal physiological processes including nutrient and/or stress responses, antigen presentation, and aging. In melanoma, basal levels of autophagy appear to vary across tumor types and higher levels of autophagy are associated with increased hypoxia and poor clinical prognosis. Understanding the relationships between autophagy, melanoma and oncogene status will elucidate the molecular mechanisms/pathways contributing to this disease. Our preliminary research indicates that cutaneous malignant melanoma (CMM) cell lines differ in their response to chloroquine (CLQ), a classic inhibitor of autophagy. This study was designed to determine how differences in the autophagy pathway are influenced by oncogene status and whether induction of autophagy is oncogene dependent. Using LC3-II puncta and western blot quantification we evaluated the differential rate of autophagy by oncogene status using CMM cell lines. The rate of autophagy was characterized in 6 melanoma cell lines (e.g. SK-Mel 2, 19, 29, 94,103, and 147) and primary melanocytes to determine the extent to which these cell lines responded to CLQ and determined any decrease in cellular viability caused by treatment. Two common cutaneous melanoma activating mutations, BRAF and NRAS, appear to have opposing roles on the autophagy pathway in our metastatic cell lines. Our data indicate that the targeting of autophagy using CLQ in the BRAF oncogenic cell lines increases apoptosis and leads a significant decrease in viability. In addition, BRAF cell lines have higher basal protein expression of ATG12-ATG5 and LC3-II and a higher number of basal LC3-II punctae. Finally, the induction of autophagy through serum starvation results in differential basal LC3-II kinetics as measured by number of punctae between the BRAF and NRAS cell lines. These results indicate that BRAF is more dependent on autophagy for survival than NRAS mutant melanoma and that oncogene status dictates the sensitivity of melanoma cells to CLQ by altering autophagic flux, subsequently increasing apoptosis. Ongoing experiments include the evaluation of autophagy in patient samples to quantify expression of known autophagy markers as well an evaluation of LC3-II expression correlated with oncogene status. These findings are immediately translatable for patients that would benefit from CLQ co-therapy, which is already approved for clinical use. Citation Format: Kirsten A. M. White, Salina M. Torres, Todd A. Thompson, Chien-An A. Hu, Natalia J. Gurule, Marianne Berwick. Differential inhibition of autophagic pathways in melanoma is oncogene-dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2635. doi:10.1158/1538-7445.AM2014-2635