Abstract 6272: Loss of SIRT6 diverts TGF-β signaling to promote liver cancer

Abstract Background: In the US, ~35% of individuals on the waitlist for a liver transplant who had hepatocellular cancer (HCC) also had metabolic dysfunction-associated steatohepatitis (MASH). Yet, key signaling mechanisms controlling cellular transformation in this obesity-driven HCC remain only partially understood. SIRT6 is NAD+-dependent protein deacetylases that limits the activity of the lipogenic transcription factors SREBP1/2. Reduced SIRT6 is associated with human fatty liver and cancer. However, SIRT6 also limits liver fibrosis by inhibiting TGF-β signaling through the deacetylation of SMAD2 and SMAD3. We found that Smad3 driven TGF-β signaling is modulated by βII-Spectrin (encoded by SPTBN1), and that a hot mutation in HCC- D1089Y encompasses the SPTBN1-SMAD3 binding site (Gastro 2018; 154:195-210). In stressed hepatocytes, βII-Spectrin promotes lipogenesis through interactions between caspase-cleaved forms of both SREBP1 and βII-Spectrin (Sci Transl Med 2021;13:624). Liver-specific knockout of βII-Spectrin (LSKO) blocks fatty liver and HCC. Here we explored the extent of βII-spectrin-D1089Y regulation of SIRT6 expression through SMAD3, and the combined roles of SIRT6 and SPTBN1 in human HCC. Methods: We generated a SPTBN1-D1089Y mutant mouse and examined SIRT6 in the SPTBN1-D1089Y mutant mouse liver tissues and MEFs. TGF-β/Smad3 pathway activity was analyzed in SPTBN1-D1089Y mutant MEFs via western blot and RNA-seq. SIRT6 expression was examined in mouse MAFLD/MASH (GSE156059). Structure-based molecular docking simulations using three-dimensional structural coordinates of human SIRT6 (ID: Q8N6T7) and β2SP (ID: Q01082) from the AlphaFold database, followed by interactions between SIRT6 and SPTBN1. Results: SIRT6 levels are decreased in SPTBN1-D1089Y mutant liver cancer cell line and mouse liver tissues. TGF-β/SMAD3 pathway activity was altered in SPTBN1-D1089Y mutant MEF cells with decreased phospho-SMAD3 levels. The structural model predicted specific interactions between human SPTBN1 and SIRT6. Overexpression of Flag-SIRT6 reduced βII-Spectrin acetylation in HepG2 cells. TGF-β induced SMAD3, βII-Spectrin coimmunoprecipitation with SIRT6. SIRT6 knockdown increases nSREBP1, SCD1 (encoded by a nSREBP1 target gene), and βII-Spectrin in HepG2 cells. SIRT6 RNA expression is significantly decreased in liver macrophages from mice with NAFLD/NASH. Conclusions: Our data indicated a SMAD dependent as well as a SMAD3 independent role of SPTBN1 in regulating SIRT6. βII-spectrin-D1089Y together with SIRT6 deficiency may increase the susceptibility to MASH/HCC via regulating the crosstalk between macrophage and hepatocytes or regulating MASH and HCC. Citation Format: Kazufumi Ohshiro, Xiyan Xiang, Xiaochun Yang, Krishanu Bhowmick, Mohammad I. Hassan, Taj Mohammad, Lopa Mishra. Loss of SIRT6 diverts TGF-β signaling to promote liver cancer [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 6272.