Hepatocellular cancer (HCC) is the 5th most common malignancy in the United States, and its incidence is equally high in the Veteran population. Although liver cancer resection has proven to be an effective treatment for localized disease, new therapeutic modalities are needed to treat unresectable disease. This is a 4-year grant proposal to study epigenetic aspects of hepatocellular cancer in order to study the pathophysiology of this deadly cancer and to begin to identify new therapeutic drug targets and/or define novel biomarkers for the disease. Metabolic reprogramming is a major hallmark of cancer cells. Due to the Warburg effect, tumor cells preferentially bypass the tricarboxylic acid cycle and switch to glycolytic energy metabolism despite available oxygen. Alterations in lipid and amino acid metabolism are also present. Currently, however, we know very little about how this malignant metabolic switch is regulated in cancer. Mitochondria are master regulators that modulate metabolic reprogramming. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and are characterized by overproduction of reactive oxygen species. Long noncoding RNAs (lncRNAs) are key regulators of metabolism that can modulate mitochondrial activity epigenetically. These lncRNAs may be transcribed from either the mitochondrial or the nuclear genomes, and the lncRNAs may be transported back and forth from the mitochondria to the cytoplasm to the nucleus. In Specific Aim 1, the mitochondrial lncRNAs in human normal liver and hepatocellular cancer cell lines will be extensively profiled, to learn which specific lncRNAs are involved in carcinogenesis. The molecular mechanisms whereby lncRNAs shuttle back and forth between the mitochondria and the nucleus will be determined. In Specific Aim 2, the roles of these lncRNAs will be assessed to gain a mechanistic understanding of how lncRNAs use epigenetic processes to initiate or propagate hepatocellular cancer in order to devise strategies to prevent or treat the disease. In addition to cultured cell lines, we will create tumoroids, organoids that are derived from hepatocellular cancer stem cells. These tumoroids recapitulate the development of malignant tumors in vitro, providing a model system to learn which lncRNAs initiate, propagate and maintain the malignant state. Innovative aspects of this grant include a new understanding of the role of lncRNAs in cancer, the development of several novel molecular epigenetic tools that have broad applicability in developmental and cancer biology, and the assembly of new databases of mitochondria-associated lncRNAs in cancer. The ultimate goal of the project is to learn more about the pathophysiology and oncogenesis of hepatocellular cancer so that new drug targets and/or biomarkers can be discovered to aid in the diagnosis and treatment of this deadly cancer in male and female Veterans.