Abstract 4283: NAMPT-enriched small extracellular vesicle promotes liver cancer via activation of SLC27A4-mediated glycolysis

Abstract Introduction: Tumor-derived small extracellular vesicles (sEV) act as a major mediator of the tumor microenvironment (TME) and are reported to regulate various metabolic pathways. Methods: Small extracellular vesicles (sEV) were collected from normal human hepatocyte and metastatic HCC cell lines using differential ultracentrifugation. Mass Spectrometry proteomic analysis was employed to identify NAMPT to be enriched in metastatic HCC-sEV. The glycolytic effect of NAMPT-sEV was analyzed by seahorse glycolytic assay. Result: Mass spectrometry protein analysis revealed nicotinamide phosphoribosyl transferase (NAMPT) to be upregulated in metastatic HCC-sEV. NAMPT is a rate-limiting enzyme essential for maintaining cellular level of nicotinamide dinucleotide (NAD+). However, the underlying pathway of sEV-NAMPT-induced metabolic consequence in HCC remains largely unknown. This study elucidated sEV-NAMPT’s carcinogenic role by establishing NAMPT-knockdown (NAMPT-KD) stable clone in metastatic HCC cell lines, which significantly hampered the promoting effect of HCC-sEV in glycolysis. sEV-mediated in vitro and in vivo tumor growth was also inhibited. Through sEV treatment, we found that sEV-NAMPT significantly elevate SLC27A4 in both transcription and protein level of recipient cells. Mechanistically, it is demonstrated that SLC27A4 expression was enhanced by NF-κB transcription factor, which is activated upon binding of sEV-NAMPT to toll-like receptor 4 (TLR4). SLC27A4 mainly function as a long-chain fatty acid transporter or acyl-CoA synthetase. Lipidomic and metabolomic analysis unveiled SLC27A4 to be positively correlated with intracellular triacylglycerol (TG) and dihydroxyacetone phosphate (DHAP) level. Elevation of TG aggravates lipolysis by hepatic lipase and promotes conversion of glycerol-3-P to DHAP, which is an intermediate metabolite that transit between lipid metabolism and glycolysis. This study uncovers a regulatory axis of sEV-NAMPT-mediated SLC27A4 in glycolysis, apart from conventional fatty acid-related metabolisms. Clinically, we explored the therapeutic potential of targeting sEV-NAMPT by inhibitor FK866. Treatment of NAMPT-inhibitor significantly hampered tumor growth in various HCC in vivo models, which suggested an alternative therapeutic strategy for HCC. Conclusion: NAMPT is enriched in metastatic HCC sEV. sEV-NAMPT regulates the promoting effect of HCC-sEV in glycolysis and oncogenesis. SLC27A4 function as the downstream target that promotes HCC progression via glycolysis. NAMPT inhibitor FK866 are proven to reduce in vitro and in vivo tumorigenic ability of metastatic HCC-sEV, which may serve as potential HCC treatment option. Citation Format: Lot Sum Cherlie Yeung, Tu Him ng, Judy Wai Ping Yam. NAMPT-enriched small extracellular vesicle promotes liver cancer via activation of SLC27A4-mediated glycolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4283.