Abstract 1575: Knocking out alpha synuclein causes decreased release of extracellular vesicles in melanoma cells

Abstract The purpose of this study is to understand the role of α-synuclein in the regulation of the trafficking and release of modulators of metastasis in melanoma. Metastasis causes melanoma-induced death and 5-year survival rates, subsequent to metastasis to regional lymph nodes or distal organs, of 9% and 4%, respectively, hence novel therapeutic targets are required to control metastasis. Emerging evidence suggests that α-synuclein, a 14.4 KDa protein, which is a key player in Parkinson’s disease, is significantly correlated with melanoma progression. Our hypothesis is that α-synuclein positively regulates the release of extracellular vesicles. Extracellular vesicles (EVs) have been shown to be important in tumor metastasis, immune modulation and chemoresistance. The rationale for this hypothesis is that α-synuclein is a unique membrane-associated catalyst of endocytosis and exocytosis, and, specifically, synuclein regulates neurotransmitter release through SNARE complex assembly and the dilation of the fusion pore. The objective of the study is to measure the concentration of extracellular vesicles in the conditioned media from wild type SK-MEL-28 melanoma cells, SNCA knock out cells, and rescued SNCA-KO cells (where α-synuclein is re-expressed in KO cells via lentivirus) using Nanoparticle Tracking Analysis. The expected result was that knocking out SNCA would decrease the number of EVs released per cell relative to wild type control cells. To this end, we grew wild type, SNCA-KO and rescued SNCA-KO cells for 48 hours at 37°C and collected conditioned media. EVs were extracted from each conditioned medium using the ExoQuick-TC kit (Systems Bioscience) and stored at -80°C. EVs were characterized by PAGE followed by western blotting for common EV-markers and EV samples were shipped to the University of Colorado for Nanoparticle Tracking. Here we report a significant reduction in the concentration of EVs in SNCA-KO cell lines compared to control cells (P=0.0123), an effect that was rescued upon re-expression of α-synuclein in knock-out cells (P<0.01). Unpublished data from our lab suggests that invasion and migration are significantly reduced in SNCA-KO melanoma compared to control cells, a pathological outcome that returns in rescued SNCA-KO cells. These observations point to considering α-synuclein as a therapeutic target for regulating the release of pro-metastatic factors in extracellular vesicles, an intervention that would have important health implications as a novel therapeutic approach in melanoma. Work is ongoing in our lab to elucidate the mechanistic details of how α-synuclein promotes EV release. Citation Format: Nirjhar M. Aloy, Michael W. Graner, Christina Coughlan, Sergey V. Slepenkov, Sahar Shekoohi, Stephan N. Witt. Knocking out alpha synuclein causes decreased release of extracellular vesicles in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1575.