AASLD Abstracts Figure 1. Overexpression of ETV1 facilitates FAD synthesis, which increases LSD1 activity through transactivating RFK and FLAD1. (A) Heatmap of genes influenced by ETV1 knock- down. (B) Relative intracellular FAD levels of the indicated cells after treatment with 10 μM riboflavin for 72 hr. (C) Relative LSD1 activity in HCC cells with different concentrations of FAD. (D) Relative LSD1 activity in the indicated cells. (E) Relative LSD1 activity in the indicated cells with or without FAD (100 nM) treatment for 72 hr. (F-G) Relative RFK and FLAD1 mRNA and protein levels. (H-I) Deletion and selective mutation analysis identifies ETV1-responsive region in the RFK and FLAD1 promoters. (J) ChIP assays demonstrated the direct binding of ETV1 to the RFK and FLAD1 promoters. (K) Western blot analysis of RFK and FLAD1. (L) Relative intracellular FAD levels of the indicated cells after treatment with 10 μM riboflavin for 72 hr. Figure 2. LSD1 is critical for ETV1-mediated HCC metastasis and the LSD1 inhibitor ORY- 1001 suppresses ETV1-mediated HCC metastasis. (A-B) ETV1 upregulates LSD1 expression. (C) Transwell analysis of the indicated HCC cells. (D-H) In vivo metastasis assays. The S-1276 AASLD Abstracts indicated HCC cell lines were transplanted into the livers of nude mice. (D) Bioluminescent images and bioluminescence signals. (E) Incidence of lung colonization. (F) Number of lung-colonizing nodules. (G) Overall survival. (H) Representative HE staining of lung tissues from the different groups. (I) LSD1 activity after treatment with ORY-1001. (J) ORY-1001 inhibits ETV1-overexpressing HCC cell migration and invasion. (K-O) ORY-1001 suppresses ETV1-mediated HCC metastasis. (K) Bioluminescent images and bioluminescence signals. (L) Incidence of lung colonization. (M) Number of lung-colonizing nodules. (N) Overall survival. (O) Representative HE staining of lung tissues. (P) A schematic diagram of the role of ETV1-LSD1 signaling in HCC metastasis. 450 CLONAL DYNAMICS AND CELL-OF-ORIGIN OF NORMAL HEPATOCYTE EXPANSIONS IN HOMEOSTATIC HUMAN LIVERS AND THEIR ASSOCIATION WITH THE BILIARY EPITHELIUM Adam M. Passman, Magnus J. Haughey, Emanuela Carlotti, Mark Williams, Biancastella Cereser, Francesco P. Russo, Matthew Hoare, Hemant Kocher, Joanne Chin-Aleong, Trevor A. Graham, Nicholas A. Wright, Weini Huang, Malcolm Alison, Stuart A. McDonald In high turnover epithelial tissues such as the intestine, homeostatic cellular replacement is achieved via a pool of stem cells. By contrast, turnover in the normal human liver is slow and the location and necessity of liver stem cells has been hotly debated. Partial hepatectomy has demonstrated that new hepatocytes can be generated from pre-existing hepatocytes, without the requirement of a stem cell population. However, lineage tracing in chronic injury models supports the involvement of liver stem/progenitors. Further complexity has arisen from the demonstration of both periportal and centrilobular neo-hepatocyte generation. This collective knowledge has largely amassed from rodent studies such that far less is known regarding the dynamics of human liver turnover, particularly during normal homeostasis. We have explored hepatocyte expansion dynamics in normal human liver by marking mitochondrial cytochrome c oxidase deficiency; a phenomenon that initiates in long-lived cells such as stem/progenitor cells. Spatially, we show that cytochrome c oxidase deficient hepatocyte expansions are more commonly periportal than pericentral. Furthermore, by virtue of a common mitochondrial somatic variant, we have demonstrated that ductal epithelial cells and nearby hepatocytes share a common cell of origin. Additionally, by analysing the methylation status of non-expressed genes, we find an ancestral relationship exists between periportal, but not pericentral clonal hepatocytes. Lastly, utilising mtDNA next-generation sequencing from within clonal hepatocyte expansions, we find that greater genetic diversity exists in pericentral hepatocytes than in those located periportally. The periportal junction of the biliary epithelium and hepatocytes has long been suspected as a location of liver stem/progenitors, particularly in response to injury. Collectively, our data supports the existence of this periportal niche in homeostatic human liver, and the streaming of hepatocytes over time from the niche toward the central vein. 451 NCX1 CONTRIBUTES TO THE AUTOPHAGY REGULATION DURING TGF- β-INDUCED HEPATIC STELLATE CELL ACTIVATION VIA CALCIUM/ CAMKKβ/FOXO1 SIGNAL PATHWAY Qiushi Liao, Jingyu Xu, Biguang Tuo, Rui Xie Background and Aims: Activation of hepatic stellate cells(HSCs) plays a pivotal role in liver fibrogenesis, recent studies have comfirmed that HSCs activation is closely related to autophagy, but the regulated mechanism of autophagy during this process remains poorly understood. Previous studies demonstrated that the intracellular calcium ([Ca 2+ ]i) was increased during TGF-β induced HSCs activation, and Na + / Ca 2+ exchange protein 1 (NCX1) as a critical membrane transporter for the intracellular calcium signal might be involved in the regulation of autophagy and HSCs activation. Our study have showed that TGF- β induced an increase of intracellular calcium via the activity of NCX1, the increased [Ca 2+ ]i combined with CaMKK-β to regulate the dephosphorylation and nuclear translocation of FoxO1 through PI3K / AKT pathway and promote the autophagy genes expression. Methods: Immunohisto- chemistry was used to detect the genes expression in human tissues. The genes protein level were detected by the western blot analysis in HSCs cell lines, and the NCX1 function was examined through using cell calcium imaging. CHIP was performed to identify the binding sites of FoxO1 at the regulatory region of the autophagy genes. Three different liver fibrosis mice models were used to verify the role of NCX1 in liver fibrogenesis in vivo. Results: The protein expression of NCX1 in human liver fibrogenesis tissue was markedly increased, and the expression level was positively correlated with the degree of liver fibrogenesis. The numbers of autophagosomes formation and autophagy genes expression were markedly upregulated under TGF-β treatment in HSCs cell lines. Moreover, TGF- β induced the increase of [Ca 2+ ]i in HSC cells, and NCX1 shRNA and inhibitor KB-R7943 and [Ca 2+ ]i chelator BAPTA-AM attenuated the TGF- β induced [Ca 2+ ]i increase, autophagy genes expression, and HSCs activation. Mechanism researches have confirmed that increased [Ca 2+ ]i significantly decreased FoxO1 phosphorylation at Ser256 via CaMKK- β/PI3K/AKT signal pathway in HSCs cell lines, FOXO1 dephosphorylation and nuclear translocation further promoted the transcriptional activity of autophagy genes and HSCs activation. In vivo, NCX1 inhibitor KB-R7943 effectively inhibited the liver fibrosis and autophagy gene expression in three different liver fibrosis mice models. Conclusions: NCX1-mediated calcium signal plays an important role in autophagy of HSC activation, implying that targeting the novel CaMKK- β/PI3K/FOXO1 pathway might serve as therapeutic strategies for the liver fibrogenesis.