The Role of Prohibitin 1 in Ethanol's Sensitizing Effect on Colorectal Liver Metastasis - ABSTRACT Prohibitin 1 (PHB1) is an evolutionary conserved protein with multiple functions depending on its subcellular location, post-translational modifications (PTMs) and interactome. Mitochondrial PHB1 is a chaperone, nuclear PHB1 regulates gene expression. While the role of PHB1 in cancer is conflicting, our preliminary data show nuclear and mitochondrial PHB1 exert opposing actions, with nuclear PHB1 being tumor suppressive. We found normal hepatocyte PHB1 expression defends against colorectal liver metastasis (CRLM) as Alb-Cre Phb1+/- mice are markedly sensitized to CRLM via induction in matrix metalloproteinase-7 (MMP-7) transcription since the heightened sensitivity to CRLM was abolished if hepatic MMP-7 was silenced prior to intrasplenic injection of colorectal cancer (CRC) cells. Interestingly, once CRC cells reach the liver, signal(s) are sent out that lower hepatocytes’ PHB1 expression and increase MMP-7, which is a novel feedforward mechanism to promote CRLM. Excessive alcohol intake increases the risk of primary cancers and CRLM in humans and mice. Interestingly, we found in murine and human alcohol-associated liver disease a dramatic depletion of nuclear and cytosolic PHB1 while mitochondrial PHB1 is protected, and increased MMP-7 expression. Hepatocyte nuclear PHB1 depletion is responsible for EtOH-mediated MMP-7 induction and activated hepatic stellate cells (HSCs) and Kupffer cells (KCs) through its secretome. In the cytosol PHB1 interacts with histone deacetylase 4 (HDAC4) so depletion of cytosolic PHB1 increased HDAC4 nuclear level. HDAC4 is implicated in cancer metastasis but its role in the sensitizing effect of ethanol (EtOH) on CRLM is unknown. Taken together, our results support the central hypothesis that EtOH sensitizes the liver to CRLM in part by depleting hepatocyte’s nuclear and cytosolic PHB1 pool. We will examine this central hypothesis in four specific aims: 1. Examine mechanisms and consequences of EtOH-mediated depletion of nuclear PHB1. Investigate whether EtOH impairs import and/or accelerates export, whether this is mediated via altered PHB1 PTMs and/or interactome and define PHB1 target genes impacted, and how EtOH- mediated hepatocyte nuclear PHB1 depletion activates HSCs and KCs. 2. Examine mechanisms and consequences of EtOH-mediated depletion of cytosolic PHB1. Examine whether this is due to increased export in extracellular vesicles (EVs), and if it leads to higher nuclear HDAC4 content and associated changes. If so, we will examine blocking increased nuclear HDAC4 on CRLM under control and EtOH diets. 3. Examine the role of PHB1-MMP-7 axis on EtOH’s sensitizing effect on CRLM. We will test the effect of silencing hepatic MMP-7 on CRLM under control and EtOH diets and examine if preserving nuclear PHB1 with a specific nuclear export peptide will protect from EtOH-induced CRLM. 4. Examine the EtOH-CRC crosstalk in modulating hepatoyte’s PHB1-MMP-7 axis. We will determine how CRC cells lower hepatocytes’ PHB1 while increasing MMP-7 expression and how EtOH enhances this response using proteomics and RNA-Seq.
