PROJECT SUMMARY Approximately 35% of individuals with hepatocellular cancer (HCC) are on the US waitlist for a liver transplant. These patients initially had non-alcoholic steatohepatitis (NASH) and progressed to HCC. Yet, signaling mechanisms controlling cellular transformation from NASH to HCC are incompletely understood. Sirtuin 6 (SIRT6) is a protein deacetylase that limits the activity of lipogenic transcription factors. SIRT6 levels decrease in human fatty liver disease and altered in cancer. SIRT6 also protects against liver fibrosis by inhibiting TGF-β signaling via deacetylating SMAD2 and SMAD3. SMAD3, with its adaptor βII-spectrin (β2SP, encoded by the SPTBN1 gene), activates SIRT6 transcription, providing a regulatory loop in fibrosis and fatty liver disease. TGF-β induces fibrosis and promotes tumors, yet also has tumor suppressor functions that remain poorly understood. We explored these paradoxes and identified β2SP as a critical regulator of TGF-β signaling and a promoter of lipogenesis. In support of this observation, liver-specific knockout of β2SP (SPTBN1-LSKO) protects mice from diet-induced fatty liver and HCC and blocks NASH in human microfluidic cultures. Furthermore, we have identified in a human HCC a top “hit,” which reflected high expression of a β2SP mutation, D1089Y, which impairs SMAD3 activity- providing functional insight into β2SP-SMAD3 interactions. Based on these exciting findings, we hypothesize that disrupting the reciprocal regulation between SIRT6 and TGF-β signaling contributes to fatty liver disease, inflammation, and progression to HCC. We further posit that β2SP plays a pivotal role in these events. If the regulatory network connecting SIRT6, β2SP, and TGF-β signaling is impaired, lipid homeostasis and tumor suppression ability are compromised. In this renewal, we advance these studies and investigate the role of β2SP, the effects of acetylation and deacetylation, the functional consequences of D1089Y (β2SP) mutation, and the therapeutic efficacy of targeting β2SP in steatosis and HCC with the following three specific aims. AIM 1: We will examine how acetylation and SIRT6-mediated deacetylation regulate β2SP functions as a partner for SREBP1 and SMAD3. AIM 2: We will determine the functional effects of the HCC-associated mutation β2SP-D1089Y on SREBP1- regulated lipogenesis and HCC in the context of steatosis in SIRT6 global and cell-specific knockout mice. AIM 3: We will evaluate the therapeutic efficacy of targeting β2SP in the progression of steatosis and HCC in SIRT6 global and cell-specific knockout mice. Our studies aim to elucidate the central role of β2SP in the pathways involved in dysregulated lipogenesis and hepatocyte transformation and provide a new molecular target for preventing obesity-driven cancer. Since β2SP is critical in many human pathologies, including heart and neurogenerative diseases, our studies will open new avenues of research by setting a precedent.