PROJECT SUMMARY – PROJECT 3 The incidence of NASH-driven HCC is expected to increase worldwide due to its association with the obesity and type-2 diabetes epidemic. Chronic ER stress and overnutrition synergize to drive NASH-HCC and metabolic stress affects ER and mitochondrial function through mitochondria-associated membranes (MAM). SAB (SH3BP5) and STARD1, two mitochondrial-resident proteins induced by ER stress, contribute to ER- mitochondria crosstalk in acute liver injury. STARD1 transfers cholesterol to the mitochondrial inner membrane for metabolism to bile acids (BAs) in the alternative (acidic) pathway. SAB acts as a docking protein required for sustained p-JNK activation through mitochondrial dysfunction and an amplification of reactive oxygen species generation. Although basal levels of STARD1 and SAB are low, their expression increases in human NASH- HCC samples. We hypothesize that SAB and STARD1 act as complementary partners and downstream mediators of the NRF2/ATF6/FBP-1 loss network to induce mitochondrial dysfunction and metabolic reprogramming through the switch of BAs synthesis from the classic to the alternative pathway. Specific aims: Aim 1. How does STARD1-SAB/JNK promote NASH-driven HCC? We hypothesize that STARD1-SAB/JNK alters BA homeostasis leading to HCC development. 1.1. How does BAs generated through the alternative pathway lead to loss of FBP-1? 1.2 What is the effect of STARD1-SAB-JNK ablation on BAs and FBP-1 expression in human HCC PDXs/spheroids? 1.3 Determination of the tumorigenic potential of HCC cell lines from MUP- uPA/NRFAct-Hep mice. Aim 2. How does STARD1-SAB/JNK axis alter the lipid composition of MAMs/Mitochondria and cause ER stress to promote NASH-driven HCC? We hypothesize that STARD1 regulates mitochondrial cholesterol content and SAB/JNK changes MAM lipid composition via MFN-2 degradation, leading to metabolic reprogramming for HCC. 2.1 Determination of the lipidome of MAMs/Mitochondria from MUP-uPA/NRF2Act-Hep-derived HCC and the role of STARD1-SAB/JNK knockdown. 2.2 Analyze MAM/mitochondrial lipid composition in human HCC PDXs/spheroids. 2.3 How does ER stress induce STARD1-SAB expression to promote HCC? Aim 3. How does STARD1-SAB/JNK metabolic reprogramming lead to NASH-driven HCC? We postulate that STARD1-induced cholesterol trafficking and SAB-mediated JNK activation suppress oxidative phosphorylation, increase glycolysis and impair mitophagy to favor HCC development. 3.1 Determination of mitochondrial function, glycolysis and mitophagy in MUP- uPA/NRFAct-ΔHep mice. 3.2 Examination of mitochondrial function and glycolysis of HCC cell lines and human PDXs/spheroids. 3.3 Impact of STARD1-SAB/JNK in stabilization of HIF-1. Thus, it is anticipated that the integration of our work with Projects 1 and 2 and Cores 1 and 2 will advance our understanding of HCC pathogenesis and lead to new therapeutic approaches.