Progression of HCC in mice is associated with a downregulation in the expression of hepatocyte nuclear factors

Abstract

Hepatocyte nuclear factors (HNF) play a critical role in development of the liver. Their roles during liver tumorigenesis and progression of hepatocellular carcinomas (HCC) are, however, poorly understood. To address the role of HNFs in tumor progression, we generated a new experimental model in which a highly differentiated slow-growing transplantable mouse HCC (sgHCC) rapidly gives rise in vivo to a highly invasive fast-growing dedifferentiated variant (fgHCC). This in vivo model has allowed us to investigate the fundamental mechanisms underlying HCC progression. A complete loss of cell polarity, a decrease in cell-cell and cell-extracellular matrix (ECM) adhesion, elevation of telomerase activity, and extinction of liver-specific gene expression accompanies tumor progression. Moreover, cells isolated from fgHCCs acquired the ability to proliferate rapidly in culture. These alterations were coupled with a reduced expression of several liver transcription factors including HNF4, a factor essential for hepatocyte differentiation. Forced re-expression of HNF4␣1 in cultured fgHCC cells reversed the progressive phenotype and induced fgHCC cells to reestablish an epithelium and reform cell-ECM contacts. Moreover, fgHCC cells that expressed HNF4␣1 also re-established expression of the profile of liver transcription factors and hepatic genes that are associated with a differentiated hepatocyte phenotype. Importantly, re-expression of HNF4␣1 in fgHCC reduced the proliferation rate in vitro and diminished tumor formation in congenic recipient mice. In conclusion, loss of HNF4 expression is an important determinant of HCC progression. Forced expression of this factor can promote reversion of tumors toward a less invasive highly differentiated slow-growing phenotype. (HEPATOLOGY 2004;39:1038 -1047 H epatocellular carcinoma (HCC) is one of the world's most common cancers, with chronic hepatitis B and C infection and prolonged exposure to hepatocarcinogens being the major risk factors. 1 The development of a malignant phenotype is considered a multi-step process that results from the accumulation of genetic alterations. The consequence of these alterations is the evolution of a tumor towards a more aggressive phenotype. HCC progression from a well differentiated to a less differentiated form is accompanied by striking changes in the morphological and genetic properties of the cells and is one of the most critical steps of liver tumorigenesis. 2,3 Hepatic tumor progression is defined by a decrease in differentiation, an extinction of tissue-specific gene expression, acceleration of cell proliferation, loss of epithelial morphology, increased invasiveness, and ultimately metastasis. While a variety of inter-and intracellular signaling pathways essential for the control of liver function and proliferation have been described, the mo-

An epistatic mini-circuitry between the transcription factors Snail and HNF4α controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs

“…A 1:20 dilution of starting chromatin DNA was used as PCR template for input normalization. hepatocytes, 18,19 re-establishes a differentiated phenotype to invasive hepatocellular carcinoma 19 and, as recently we demonstrated, controls the hepatic epithelial phenotype by direct repression of master EMT regulators including Snail. 2 MiRs-200 induce epithelial differentiation of cancer stem cells by direct targeting the transcriptional repressors of E-cadherin ZEB1 and ZEB2, and other stemness genes.…”

“…Our data are in line with previous evidences indicating these miRs as central factors of the regulation of epithelial phenotype. 19 Our results suggest the epistatic relationship depicted in Figure 6 in which transcriptional and post-transcriptional regulators are involved. The proposed mechanism implies that the execution of a stemness program requires the active repression of a differentiation program, whereas the maintenance of the hepatocyte one requires the active repression of stemness traits.…”

Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines

“…We showed expression of HNF4a to be well correlated with that of TCF2 and to be upregulated by inducing TCF2 expression using a methyltransferase inhibitor, which suggests alterations in the hepatocyte nuclear factor network can be reversed by inducing TCF2 through demethylation of the gene. Consistent with that idea, Lazarevich et al (2004) reported that expression of HNF4a is downregulated in hepatocellular carcinoma and its restoration suppresses tumour growth. It has also been reported that HNF4a is downregulated in other types of tumours (Lemm et al, 1999) and that it induces expression of endothelial Fas ligand (FasL), which prevents cancer cell transmigration (Osanai et al, 2002).…”

Embryonic development of the liver†

“…Expression of HNF4␣ is lost in a mouse model of hepatocellular carcinoma progression in which slow-growing tumors lose their epithelial characteristics and transform to a fast-growing invasive form. 144 Moreover, when expression of HNF4␣ is forced in the fast-growing invasive hepatocellular carcinoma cells, proliferation is reduced and tumor formation is suppressed. 144 In addition to controlling hepatocyte differentiation during embryogenesis, HNF4␣ is also required to maintain a differentiated hepatocyte phenotype, since disruption of Hnf4 in adult livers also dramatically affects hepatic gene expression and severely disrupts liver function.…”

“…144 Moreover, when expression of HNF4␣ is forced in the fast-growing invasive hepatocellular carcinoma cells, proliferation is reduced and tumor formation is suppressed. 144 In addition to controlling hepatocyte differentiation during embryogenesis, HNF4␣ is also required to maintain a differentiated hepatocyte phenotype, since disruption of Hnf4 in adult livers also dramatically affects hepatic gene expression and severely disrupts liver function. 135 Although the data still need to be validated by genetic analyses, when chromatin immunoprecipitation was used to probe human promoter microarrays for HNF4␣ DNA elements within HepG2 cells, HNF4␣ was shown to occupy a surprising 12% (1,575 genes) of genes represented on the Hu13K microarray.…”