HNF1α is a transcription factor involved in hepatocyte differentiation and is important for normal liver function. Here, we show that HNF1α might also be important for maintenance of epithelial phenotype in hepatocytes. Liver cancer cell lines in which HNF1α expression was inhibited by siRNA underwent an epithelial-mesenchymal transition and lost hepatocyte differentiation and epithelial phenotype. Expression of proteins involved in tight and adherens junctions, like ZO-1 and E-cadherin, was diminished, leading to loss of cell-cell contacts and reorganization of cytoskeleton. Cells transfected with HNF1α siRNA also showed an overexpression of mesenchymal markers and of several key transcription factors involved in EMT development, in particular Snail1 and Snail2.
Under-expression of E-cadherin has previously been described in a mouse model of HNF1α inactivation. In this mouse model in which pancreatic β-cell over expressed a dominant-negative mutant of HNF1α, pancreatic islets showed abnormal architecture with, in particular, a reduced expression of E-cadherin [29
]. It was then suggested that E-cadherin could be a target of HNF1α. A putative HNF1α binding site was found in intron 2 of human E-cadherin gene and HNF1α acts as an enhancer on the chicken E-cadherin gene but further studies are required to understand the regulation of E-cadherin by HNF1α. Our results showed a strong correlation between E-cadherin and HNF1α expression, supporting the hypothesis of a regulation of E-cadherin expression by HNF1α, whether direct or indirect. HNF1α has also been shown to be a positive regulator of other molecules of cellular junctions, tight junction component claudin-2 [30
] and gap junction protein connexin32 [31
The HNF1 homeoprotein family contains another member apart from HNF1α, HNF1β. HNF1α and HNF1β are highly homologous protein that can recognize the same binding site and form heterodimers [32
]. They are both expressed in the polarized epithelium of several tissues (liver, kidney, pancreas and digestive tract), though in a sequential manner, which led to the assumption that they could be involved in epithelial differentiation [33
]. In the liver, HNF1β is expressed earlier during development but in adult hepatocytes HNF1α is predominant, whereas HNF1β is weakly expressed [20
]. HNF1β inactivation has been linked to EMT in ovarian cancer [34
]. Ovarian carcinoma cell lines where HNF1β function was knockdown by siRNA or transfection with a dominant-negative mutant showed reduced E-cadherin expression and underwent epithelial-mesenchymal-like transition, associated with Slug overexpression. HNF1β overexpression lead to down-regulation of Snail and Slug expression. In ovarian tumors, expression of HNF1β was associated with E-cadherin. Altogether, these results support a role of HNF1β in the maintenance of epithelial phenotype. As HNF1α and β have very close activity and can recognize the same genes, HNF1α inactivation in hepatocytes could trigger the same reactions.
Repression of E-cadherin and other epithelial markers by HNF1α could also go through other molecules regulated by HNF1α. In particular, EMT regulators Snail1/2 and ZEB1/2 are able to repress E-cadherin expression through interaction with specific E-boxes of the E-Cadherin promoter [35
]. Snail1 has recently been shown to be repressed by HNF1α in hepatocytes, through binding of HNF1α to a consensus site in Snail1 promoter [37
]. HNF1α can repress Snail1 expression alone or in cooperation with HNF4α, another important regulator of hepatocyte differentiation [37
Hepatocyte differentiation is achieved through a complex network of cross regulation between transcription factors, especially between HNF1α and HNF4α [20
]. There is a regulational hierarchy between those proteins since HNF4α expression precedes that of HNF1α and activates the expression of HNF1α [38
]. On the other hand, HNF1α is also capable of activating HNF4α expression, which defines a regulatory loop assuring the expression of HNF1α and HNF4α in hepatocytes [21
]. Moreover, HNF1α can repress its own expression and the expression of other targets of HNF4α, through interaction with HNF4α [40
]. HNF4α has been involved in epithelium formation and it has been shown to regulate the expression of several epithelial markers and components of cell junctions [41
]. HNF4α has been recently shown to negatively regulate mesenchymal molecules (vimentin, fibronectin and desmin) and EMT master regulator Snail1 [37
]. Moreover, HNF4α inactivation induces EMT in embryonic mouse kidneys [43
]. Interestingly, HNF1α seems to cooperate with HNF4α to suppress mesenchymal markers expression as well as Snail1 [37
]. Since HNF4α was down-regulated in HNF1α-inhibited hepatocytes, the EMT observed in these cells could also go partially through HNF4α inhibition.
Genes involved in cell mobility are also up regulated in HNF1α-inhibited cells, like metalloproteinases, but also PDGFA and B, which have been previously described as over expressed in HNF1α-inactivated tumors and cell lines [17
]. PDGF growth factors are involved in angiogenesis but they are also autocrine factors involved in EMT and are necessary for TGFβ-induced migration and tumor progression in hepatocytes [25
]. Our results show that the EMT induced by HNF1α inhibition is associated with increased cell migration.
To induce EMT, HNF1α could also control directly the expression of growth factors capable of inducing EMT. Among those factors, we showed that TGFβ1 was up-regulated in cells transfected with HNF1α siRNA and that the expression of TGFβ1 was inversely correlated to the expression of HNF1α, suggesting close regulation. Yet it is not clear whether it is this overexpression that trigger the EMT observed in these cells or not. In particular, TGFβ can induce the under expression of HNF4α in rat primary hepatocytes and in immortalized murine hepatocytes [45
]. Therefore, HNF4α down regulation in HNF1α-inhibited cells could also be due to TGFβ1 over-expression. Further studies are necessary to understand the role of TGFβ1 overexpression in the development of EMT induced by HNF1α inhibition.
Interestingly, we also found an overexpression of TGFβ1 in HNF1α-mutated HCA, but neither SMAD7 nor TGFBI up-regulation, nor changes in TGFβ-activation markers. Moreover, an analysis of H-HCA transcriptome failed to identify a TGFβ signature in H-HCA, whether early or late, as defined by Courlouarn et al. [26
] (data not shown). In particular we didn't identify any change in the expression of EMT markers at the transcriptional level in H-HCA. Neither could we analyze the expression of EMT markers at the borders of these tumors by immunostaining because of the important steatosis observed in H-HCA that makes the staining in tumors highly heterogeneous. However, H-HCA present ill-defined borders, that look like local invasions of the adjacent non tumor liver, which is compatible with EMT (Figure ).
The role of TGFβ1 overexpression in these benign tumors remains unclear. TGFβ has a dual effect on tumor development. In early carcinogenesis, TGFβ activation induces cell death and in late carcinogenesis, it is involved in invasion and EMT development [46
]. In tumorous cell lines, cells are at a late stage of carcinogenesis and therefore TGFβ is prone to induce EMT. Whereas in benign tumors, we could think that TGFβ overexpression would induce apoptosis but HNF1α-mutated HCA do not show important necrosis and transcriptomic analysis did not reveal important changes in genes involved in apoptosis or cell cycle arrest [17
]. In the liver, TGFβ has also been involved in hepatic differentiation and fibrosis [47
]. HNF1α-mutated adenomas are developed in normal livers and do not show fibrosis, so this aspect of TGFβ is irrelevant, but HNF1α and TGFβ are both involved in hepatic differentiation. TGFβ pathway is involved in several steps of liver development, in particular in hepatoblast proliferation and differentiation [48
]. Weak TGFβ concentrations are needed for hepatoblast differentiation into hepatocytes. As HNF1α is involved in late hepatocyte differentiation, we suggest that HNF1α negative control of TGFβ1 expression could be associated with establishment/maintenance of hepatocyte differentiation and arrest of proliferation.