PROJECT SUMMARY The long-term goal of this proposal is to understand the molecular and cellular mechanisms in the tumor microenvironment that govern hepatocellular carcinoma (HCC) development, with a special focus on the role of hepatic stellate cells (HSCs) as central players in this process. While it is clear that activation of non-parenchymal cells is a key contributor to liver tumorigenesis, unfortunately, our knowledge of the molecular mechanisms whereby key stromal and immune cell populations regulated HCC initiation and progression still is fragmentary. Activated HSCs are key contributors to liver fibrosis and inflammation. The present proposal is based on our previously published results demonstrating that the autophagy and signaling adaptor p62 is reduced in HSCs from HCC patients and that its inactivation in mice, either globally or HSC-specific, promoted HCC due to the hyperactivation of the inflammatory and fibrotic activities of HSCs. Our new unpublished preliminary results demonstrate that the genetic ablation of NBR1, either globally or selectively in HSCs, completely reverts the pro- tumorigenic role of p62 deficiency in HSCs. However, this unexpected effect of NBR1 deficiency does not revert the pro-fibrotic, TGFb-driven effect of p62 loss and did not affect autophagy but resulted in the hyperactivation of the interferon (IFN) cascade that renders CD8+ T cells more active, increasing anti-tumor immunosurveillance. Our preliminary data also suggest that this hyperactivation of the IFN pathways is due to impaired chaperone- mediated autophagy (CMA). Therefore, in this proposal we intend to unravel the mechanisms whereby NBR1 promotes CMA to restrain IFN activation. We will also establish how reduced CMA due to the inactivation of NBR1 in HSCs results in the reprograming of the tumor microenvironment to repress tumorigenesis. To that end, we will build on our preliminary data to address the following critical questions: (Aim 1) Establish the molecular mechanisms whereby NBR1 regulates the IFN response in HSCs by (Aim 1.1) determining the effect of NBR1 and p62 inactivation on CMA activity in HSCs; (Aim 1.2) determining the functional contribution of CMA to NBR1- mediated IFN regulation in HSCs; and (Aim 1.3) determining the molecular mechanisms whereby NBR1 regulates CMA. We will also (Aim 2) establish the contribution of NBR1 to HSC activation and the creation of a tumor-suppressive microenvironment by (Aim 2.1) determining the impact that NBR1 deletion in HSCs has in the tumor microenvironment of HCC; (Aim 2.2) determining the functional contribution of the CMA-STING-IFN axis in NBR1-deficient HSCs to HCC progression in vivo; and (Aim 2.3) determining the impact of NBR1 loss in HSCs in therapy response and its relevance in human HCC. Results from these studies will serve to identify new biomarkers as well as non-parenchymal new therapeutic targets for the prevention and treatment of HCC.