Acetaminophen (APAP) overdose results in acute liver failure and has limited treatment options. Previous studies show that stimulating liver regeneration is critical for survival after APAP overdose, but the mechanisms remain unclear. In this study, we identified major signaling pathways involved in liver regeneration after APAP-induced acute liver injury using a novel incremental dose model. Liver injury and regeneration were studied in C57BL/6 mice treated with either 300 mg/kg (APAP300) or 600 mg/kg (APAP600) APAP. Mice treated with APAP300 developed extensive liver injury and robust liver regeneration. In contrast, APAP600-treated mice exhibited significant liver injury but substantial inhibition of liver regeneration, resulting in sustained injury and decreased survival. The inhibition of liver regeneration in the APAP600 group was associated with cell cycle arrest and decreased cyclin D1 expression. Several known regenerative pathways, including the IL-6/STAT-3 and epidermal growth factor receptor/c-Met/mitogen-activated protein kinase pathways, were activated, even at APAP600, where regeneration was inhibited. However, canonical Wnt/β-catenin and NF-κB pathways were activated only in APAP300-treated mice, where liver regeneration was stimulated. Furthermore, overexpression of a stable form of β-catenin, where serine 45 is mutated to aspartic acid, in mice resulted in improved liver regeneration after APAP overdose. Taken together, our incremental dose model has identified a differential role of several signaling pathways in liver regeneration after APAP overdose and highlighted canonical Wnt signaling as a potential target for regenerative therapies for APAP-induced acute liver failure.

HNF4α, the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation via unknown mechanisms. We investigated the mechanisms of HNF4α-induced inhibition of hepatocyte proliferation using a novel TAM-inducible, hepatocyte specific HNF4α knockdown mouse model. Hepatocyte specific deletion of HNF4α in adult mice resulted in increased hepatocyte proliferation with a significant increase in liver to body weight ratio. We determined global gene expression changes using Illumina HiSeq-based RNA sequencing, which revealed that, a significant number of up-regulated genes following deletion of HNF4α were associated with cancer pathogenesis, cell cycle control, and cell proliferation. The pathway analysis further revealed that c-Myc-regulated gene expression network was highly activated following HNF4α deletion. To determine whether deletion of HNF4α affects cancer pathogenesis, HNF4α knockdown was induced in mice treated with the known hepatic carcinogen diethylnitrosamine (DEN). Deletion of HNF4α significantly increased the number and size of DEN-induced hepatic tumors. Pathological analysis revealed that tumors in HNF4α deleted mice were well-differentiated hepatocellular carcinoma (HCC) and mixed HCC-cholangiocarcinoma. Analysis of tumors and surrounding normal liver tissue in DEN-treated HNF4α knockout mice showed significant induction in c-Myc expression. Taken together, deletion of HNF4α in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN-induced hepatic tumors secondary to aberrant c-Myc activation.

Farnesoid X receptor (FXR), the primary bile acid-sensing nuclear receptor, also is known for its anticancer properties. It is known that FXR deficiency in mice results in spontaneous hepatocellular carcinoma (HCC), but the mechanisms are not completely understood. We report that sustained activation of the Wnt/␤-catenin pathway is associated with spontaneous HCC in FXR-knockout (KO) mice. HCC development was studied in FXR-KO mice at 3, 8, and 14 months of age. No tumors were observed at either 3 or 8 months, but the presence of HCC was observed in 100% of the FXR-KO mice at the age of 14 months. Further analysis revealed no change in ␤-catenin activation in the livers of 3-month-old FXR-KO mice, but a moderate increase was observed in 8-month-old FXR-KO mice. ␤-Catenin activation further increased significantly in 14-month-old tumor-bearing mice. Further analysis revealed that two independent mechanisms might be involved in ␤-catenin activation in the livers of FXR-KO mice. Activation of canonical Wnt signaling was evident as indicated by increased Wnt4 and dishevelled expression along with glycogen synthase kinase-3␤ inactivation. We also observed decreased expression of E-cadherin, a known regulator of ␤-catenin, in FXR-KO mice. The decrease in E-cadherin expression was accompanied by increased expression of its transcriptional repressor, Snail. Consistent with the increased HCC in FXR-KO mice, we observed a significant decrease in FXR expression and activity in human HCC samples. Taken together, these data indicate that a temporal increase in the activation of Wnt/␤-catenin is observed during spontaneous HCC development in FXR-KO mice and is potentially critical for tumor development.

Hepatocyte nuclear factor-4␣ (HNF4␣) is known as the master regulator of hepatocyte differentiation. Recent studies indicate that HNF4␣ may inhibit hepatocyte proliferation via mechanisms that have yet to be identified. Using a HNF4␣ knockdown mouse model based on delivery of inducible Cre recombinase via an adeno-associated virus 8 viral vector, we investigated the role of HNF4␣ in the regulation of hepatocyte proliferation. Hepatocyte-specific deletion of HNF4␣ resulted in increased hepatocyte proliferation. Global gene expression analysis showed that a majority of the downregulated genes were previously known HNF4␣ target genes involved in hepatic differentiation. Interestingly, Ն500 upregulated genes were associated with cell proliferation and cancer. Furthermore, we identified potential negative target genes of HNF4␣, many of which are involved in the stimulation of proliferation. Using chromatin immunoprecipitation analysis, we confirmed binding of HNF4␣ at three of these genes. Furthermore, overexpression of HNF4␣ in mouse hepatocellular carcinoma cells resulted in a decrease in promitogenic gene expression and cell cycle arrest. Taken together, these data indicate that, apart from its role in hepatocyte differentiation, HNF4␣ actively inhibits hepatocyte proliferation by repression of specific promitogenic genes. adeno-associated virus; Ect2; gene repression; hepatocyte proliferation; liver regeneration THE LIVER IS KNOWN for its exceptional capability to regenerate in response to surgical removal, as well as following druginduced liver injury. Liver regeneration is critical in maintaining liver health, because the liver is the major site of xenobiotic detoxification and is exposed to a variety of drugs and chemicals. The mechanisms of initiation and progression of liver regeneration are well studied, but the mechanisms of termination of regeneration are not completely understood (27). It is important to understand the mechanisms of termination of liver regeneration, because many of the pathways involved in stimulation of liver regeneration can be carcinogenic if left unchecked. It is known that the mechanisms of termination of regeneration are essential to maintain controlled liver growth and inhibit progression to hepatocellular carcinoma (HCC). However, the detailed mechanisms of termination of regeneration and their link to prevention of HCC are not completely clear.Hepatocyte nuclear factor-4␣ (HNF4␣, representative public ID NR2A1) is considered the master regulator of hepatocyte differentiation. It plays an important role in the regulation of many hepatocyte-specific genes, including those involved in glycolysis, gluconeogenesis, ureagenesis, fatty acid metabolism, bile acid synthesis, drug metabolism, apolipoprotein synthesis, and blood coagulation (11, 15, 18 -21). Recent studies suggest a novel role of HNF4␣ in the regulation of cell proliferation (8,9,11,12,15,23,26,29,36). Data indicate that HNF4␣ may have antiproliferative activity in human embryonic kidney cells and rat pancreatic beta cell...

Background/Aims: Hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and hepatoblastoma (HB) are the main hepatic malignancies with limited treatment options and high mortality. Recent studies have implicated Hippo Kinase pathway in cancer development but detailed analysis of Hippo Kinase signaling in human hepatic malignancies, especially CC and HB, is lacking. Methods: We investigated Hippo Kinase signaling in HCC, CC and HB using cells and patient samples. Results: Increased expression of yes-associated protein (Yap), the downstream effector of the Hippo Kinase pathway, was observed in HCC cells and siRNA-mediated knockdown of Yap resulted in decreased survival of HCC cells. The density-dependent activation of Hippo Kinase pathway characteristic of normal cells was not observed in HCC cells and CCLP cells, a cholangiocarcinoma cell line. Immunohistochemistry of Yap in HCC, CC and HB tissues indicated extensive nuclear localization of Yap in majority of tissues. Western blot analysis performed using total cell extracts from patient samples and normal livers showed extensive activation of Yap. Marked induction of glypican-3, CTGF and Survivin, the three Yap target genes was observed in the tumor samples. Further analysis revealed significant decrease in expression and activity of Lats kinase, the main upstream regulator of Yap. However, no change in activation of Mst-2 kinase, the upstream regulator of Lats kinase was observed. Conclusions: These data show that Yap induction mediated by inactivation of Lats is observed in hepatic malignancies. These studies highlight Hippo Kinase pathway as a novel therapeutic target for hepatic malignancies.