Mutations in Isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer1–5. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert alpha-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation6–10. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models (GEMMs) expressing mutant IDH in the adult liver show aberrant response to hepatic injury, characterized by HNF4α silencing, impaired hepatocyte differentiation and markedly elevated levels of cell proliferation. Moreover, mutant IDH and activated Kras, genetic alterations that co-exist in a subset of human IHCCs4,5, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel GEMM of IDH-driven malignancy.

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.

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...

Hepatocyte nuclear factor 4 alpha (HNF4α) is an orphan nuclear receptor commonly known as the master regulator of hepatic differentiation owing to the large number of hepatocyte-specific genes it regulates. Whereas the role of HNF4α in hepatocyte differentiation is well recognized and extensively studied, its role in regulation of cell proliferation is relatively less known. Recent studies have revealed that HNF4α inhibits proliferation not only of hepatocytes but also cells in colon and kidney. Further, a growing number of studies have demonstrated that inhibition or loss of HNF4α promotes tumorigenesis in the liver and colon, and re-expression of HNF4α results in decreased cancer growth. Studies using tissue-specific conditional knockout mice, knock-in studies, and combinatorial bioinformatics of RNA/ChIP-sequencing data indicate that the mechanisms of HNF4α-mediated inhibition of cell proliferation are multifold involving epigenetic repression of pro-mitogenic genes, significant crosstalk with other cell cycle regulators including c-Myc and Cyclin D1, and regulation of miRNAs. Furthermore, studies indicate that post-translational modifications of HNF4α may change its activity and may be at the core of its dual role as a differentiation factor and repressor of proliferation. This review summarizes recent findings on the role of HNF4α in cell proliferation and highlights the newly understood function of this old receptor.