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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

Metastatic melanoma offers an ongoing clinical challenge. According to the American Cancer Society, the 5-year survival rate in 2012 for stage IV melanoma is only about 15% even with therapies. To improve outcomes, it is important to investigate new pathways involved in the regulation of metastasis as possible targets for prevention or treatment, such as autophagy. Autophagy (self-eating pathway) destroys a cell's damaged proteins and organelles through lysosomal degradation and has been shown to play roles in a wide variety of normal physiological processes including energy metabolism, nutrient or stress responses, growth regulation, and aging. The role of dysregulated autophagy in melanoma has not been well characterized. Preliminary research found cutaneous malignant melanoma cells display high levels of autophagy suggesting that this cellular process may provide an active metabolic state for invasive and metastatic melanoma. Chloroquine (CLQ), a classic antimalarial agent, is a potent inhibitor of autophagy and has been used safely in human patients for decades. This study is designed to dissect the role of autophagy in melanoma using melanoma cell lines (e.g. SK-Mel 19, 103, and 147), and primary melanocytes as controls to determine whether these cell types respond to CLQ as measured by cell viability . Similar to pancreatic and prostate cancer cell lines, our preliminary results indicate that metastatic melanoma cells require autophagy for growth and show high sensitivity to CLQ at doses as low as 20uM which resulted in greater than 60% decreases in cell viability. These findings suggest that metastatic melanoma cell lines rely on lysosomal degradation to a greater extent than what has been reported for other cell types. In addition, both N-ras mutation and androgen deprivation mitigate the effects of CLQ on melanoma cells. Ongoing experiments will confirm the expression of autophagy related genes, as well as imaging to determine the rate of autophagy in these cell lines. Finally, we will introduce stripped hormonal factors including vitamin D and estrogen, to evaluate whether this addition will progressively restore the original chloroquine sensitive phenotype. Findings from this work have the potential to be immediately translatable to patients as CLQ is already approved for therapeutic use in other human diseases. Citation Format: Kirsten A. White, Salina M. Torres, Natalia J. Gurule, Marianne Berwick, Chien-An A. Hu. Inhibition of autophagy in melanoma: using cell lines to model chloroquine sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2041. doi:10.1158/1538-7445.AM2013-2041