PROJECT SUMMARY -PROJECT 2 Hepatocellular carcinoma is (HCC) is the most common primary liver cancer and 3rd leading cause of cancer deaths worldwide. HCC etiology is rapidly changing toward metabolic drivers and is a growing medical threat. Cellular insults cause accumulation of misfolded protein in the endoplasmic reticulum (ER), which activates the unfolded protein response (UPR) in human non-alcoholic steatohepatitis (NASH) and HCC and is a negative prognosticator for outcome in solid tumors. The UPR causes cleavage and activation of the transcription factor ATF6 to produce the ATF6p50 fragment that migrates to the nucleus for transcription of ER chaperones and metabolic genes. Our new findings show that ATF6 promotes HCC: a) Nuclear ATF6p50 is elevated in human HCC tumor samples compared to non-tumor tissues and is associated with downregulation of the tumor suppressor fructose 1,6, bisphosphatase 1 (FBP1); b) Expression of ATF6p50 promotes HCC in mice; and c) Deletion of ATF6 reduces tumor incidence in mice. We hypothesize that ATF6p50 in hepatocytes promotes HCC through ER stress, UPR induction, and increased cholesterol synthesis leading to a metabolic imbalance favoring a malignant state which we will test: 1) Can discoveries from the mouse be translated to human tissues and preclinical human HCC models for insight into mechanism and potential intervention? We will analyze human HCC tissue samples by digital spatial profiling and next generation sequencing to define ATF6p50-specific transcriptome signatures, associating ATF6p50 positivity with genes encoding ER, mitochondria, or cytosolic functions in human HCC. We will decipher targetable mechanisms and assess the therapeutic benefit of targeting ATF6 in xenografts of surgically resected tumor specimens obtained from NASH- related HCC patients; 2) How does ATF6 exert its oncoprotein role in HCC? The role of ATF6 cleavage/activation in driving HCC will focus on: a) Can UPR activation and cell death promote HCC in combination with oncogenic drivers; b) Does ATF6 synergizes with NRF2 and/or play a parallel role through common downstream targets to promote HCC, such as FBP1 (with Project 1); Activated NRF2 and ATF6 have overlapping signatures correlating with human HCC omics datasets (with Core 2) and both suppress FBP1 to increase TCA cycle-related oncometabolites (with Core 1); c) Metabolic reprogramming via increased cholesterol synthesis (with Project 3); and 3) Does genetic or pharmacological inactivation of ATF6 prevent NASH-HCC? We will perform transcriptional, translational, and metabolomic analyses to define the impact of altered ATF6 signaling in HCC cell lines and PDXs that will be orthotopically transplanted into syngeneic high-fat/fructose diet-fed mice to monitor HCC progression. We will test whether ER protein misfolding drives NASH to HCC progression, which may be rescued by ATF6 antisense oligonucleotides or small molecule ATF6 inhibitors. Projects 1-3 provide synergy for t...