3172

HNF4A

Wnt-beta-catenin signaling participates in the epithelial-mesenchymal transition (EMT) in a variety of cancers; however, its involvement in hepatocellular carcinoma (HCC) and downstream molecular events is largely undefined. HNF4alpha is the most prominent and specific factor maintaining the differentiation of hepatic lineage cells and a potential EMT regulator in HCC cells. However, the molecular mechanisms by which HNF4alpha maintains the differentiated liver epithelium and inhibits EMT have not been completely defined. In this study, we systematically explored the relationship between Wnt-beta-catenin signaling and HNF4alpha in the EMT process of HCC cells. Our results indicated that HNF4alpha expression was negatively regulated during Wnt-beta-catenin signaling-induced EMT through Snail and Slug in HCC cells. In contrast, HNF4alpha was found to directly associate with TCF4 to compete with beta-catenin but facilitate transcription co-repressor activities, thus inhibiting expression of EMT-related Wnt-beta-catenin targets. Moreover, HNF4alpha may control the switch between the transcriptional and adhesion functions of beta-catenin. Overexpression of HNF4alpha was found to completely compromise the Wnt-beta-catenin-signaling-induced EMT phenotype. Finally, we determined the regulation pattern between Wnt-beta-catenin signaling and HNF4alpha in rat tumor models. Our studies have identified a double-negative feedback mechanism controlling Wnt-beta-catenin signaling and HNF4alpha expression in vitro and in vivo, which sheds new light on the regulation of EMT in HCC. The modulation of these molecular processes may be a method of inhibiting HCC invasion by blocking Wnt-beta-catenin signaling or restoring HNF4alpha expression to prevent EMT.

epithelial-to-mesenchymal transition (EMT) is a complex multistep process in which phenotype switches are mediated by a network of transcription factors (TFs). Systematic characterization of all dynamic TFs controlling EMT state transitions, especially for the intermediate partial-EMT state, represents a highly relevant yet largely unexplored task. Here, we performed a computational analysis that integrated time-course EMT transcriptomic data with public cistromic data and identified three synergistic master TFs (ETS2, HNF4A and JUNB) that regulate the transition through the partial-EMT state. Overexpression of these regulators predicted a poor clinical outcome, and their elimination readily abolished TGF-beta-induced EMT. Importantly, these factors utilized a clique motif, physically interact and their cumulative binding generally characterized EMT-associated genes. Furthermore, analyses of H3K27ac ChIP-seq data revealed that ETS2, HNF4A and JUNB are associated with super-enhancers and the administration of BRD4 inhibitor readily abolished TGF-beta-induced EMT. These findings have implications for systematic discovery of master EMT regulators and super-enhancers as novel targets for controlling metastasis.CI - (c) The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Gene expression-based classification systems have identified an aggressive colon cancer subtype with mesenchymal features, possibly reflecting epithelial-to-mesenchymal transition (EMT) of tumor cells. However, stromal fibroblasts contribute extensively to the mesenchymal phenotype of aggressive colon tumors, challenging the notion of tumor EMT. To separately study the neoplastic and stromal compartments of colon tumors, we have generated a stroma gene filter (SGF). Comparative analysis of stroma(high) and stroma(low) tumors shows that the neoplastic cells in stroma(high) tumors express specific EMT drivers (ZEB2, TWIST1, TWIST2) and that 98% of differentially expressed genes are strongly correlated with them. Analysis of differential gene expression between mesenchymal and epithelial cancer cell lines revealed that hepatocyte nuclear factor 4alpha (HNF4alpha), a transcriptional activator of intestinal (epithelial) differentiation, and its target genes are highly expressed in epithelial cancer cell lines. However, mesenchymal-type cancer cell lines expressed only part of the mesenchymal genes expressed by tumor-derived neoplastic cells, suggesting that external cues were lacking. We found that collagen-I dominates the extracellular matrix in aggressive colon cancer. Mimicking the tumor microenvironment by replacing laminin-rich Matrigel with collagen-I was sufficient to induce tumor-specific mesenchymal gene expression, suppression of HNF4alpha and its target genes, and collective tumor cell invasion of patient-derived colon tumor organoids. The data connect collagen-rich stroma to mesenchymal gene expression in neoplastic cells and to collective tumor cell invasion. Targeting the tumor-collagen interface may therefore be explored as a novel strategy in the treatment of aggressive colon cancer.

During kidney development, the metanephric mesenchyme (MM) develops into the nephron through mesenchymal-epithelial transition (MET). We have previously reported that knock-down of the expression of hepatocyte nuclear factor 4 alpha (Hnf4a) gene induces failure of cellular organization in the condensed mesenchyme (CM) of cultured embryonic kidneys. To elucidate the details of MET during nephrogenesis, embryonic mouse kidneys were analyzed by electron microscopy, immunohistochemistry, and molecular biology. The findings showed that the intercellular junction, but not the basal lamina, was present in the CM. Additionally, immediately after Hnf4a gene expression, the expression of epithelial genes (Krt8, Tjp1, and Cdh1) increased, and those of mesenchymal genes (Acta1 and Vim) decreased, in the CM compared to the MM. To clarify the relationship between MET and Hnf4alpha, the fibroblast cell line with forced expression of Hnf4alpha protein were analyzed. In this model, it was noted that Hnf4alpha induced increasing epithelial and decreasing mesenchymal gene expression. In these, up-regulation of Pvrl1, -2, and Mllt4 genes which mediate the formation of apico-basal polarity, were found. These results, and those of previous findings, indicate that Hnf4alpha protein is associated with the initiation of MET in early nephrogenesis.

Determining the origin of liver cancer stem cells is important for treating hepatocellular carcinoma. Tg737 deficiency plays an important role in the malignant transformation of liver stem cells, but the underlying mechanism remains unclear. Here we established a chemical-induced mouse hepatoma model and found that Tg737 and hepatocyte nuclear factor 4-alpha (HNF4alpha) expression decreased and epithelial-mesenchymal transition (EMT)-related marker expression increased during liver cancer development. To investigate the underlying mechanism, we knocked down Tg737 in WB-F344 (WB) rat hepatic oval cells. Loss of Tg737 resulted in nuclear beta-catenin accumulation and activation of the Wnt/beta-catenin pathway, which further promoted EMT and the malignant phenotype. XAV939, a beta-catenin inhibitor, attenuated WB cell malignant transformation due to Tg737 knockdown. To clarify the relationships of Tg737, the beta-catenin pathway, and HNF4alpha, we inhibited Snail and overexpressed HNF4alpha after Tg737 knockdown in WB cells and found that Snail and HNF4alpha comprise a negative feedback circuit. Taken together, the results showed that Tg737 regulates a Wnt/beta-catenin/Snail-HNF4alpha negative feedback circuit, thereby blocking EMT and the malignant transformation of liver stem cells to liver cancer stem cells.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.