Hepatocellular adenomas (HCAs) are rare benign

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1 Loss of Hepatocyte Nuclear Factor 1 Function in Human Hepatocellular Adenomas Leads to Aberrant Activation of Signaling Pathways Involved in Tumorigenesis Laura Pelletier, 1,2 * Sandra Rebouissou, 1,2 * Alain Paris, 3 Estelle Rathahao-Paris, 3 Elisabeth Perdu, 3 Paulette Bioulac-Sage, 4,5 Sandrine Imbeaud, 6 and Jessica Zucman-Rossi 1,2 Hepatocellular adenomas (HCAs) are benign liver tumors that usually develop in women who are taking oral contraceptives. Among these tumors, biallelic inactivating mutations of the hepatocyte nuclear factor 1 (HNF1A) transcription factor have been frequently identified and in rare cases of hepatocellular carcinomas developed in noncirrhotic liver. Because HNF1A meets the genetic criteria of a tumor suppressor gene, we aimed to elucidate the tumorigenic mechanisms related to HNF1 inactivation in hepatocytes. We searched for signaling pathways aberrantly activated in human HNF1A-mutated HCA (H-HCA) using a genome-wide transcriptome analysis comparing five H-HCA with four normal livers. We validated the main pathways by quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting in a large series of samples. Then, we assessed the role of HNF1 in the observed deregulations in hepatocellular cell models (HepG2 and Hep3B) by silencing its endogenous expression using small interfering RNA. Along with the previously described induction of glycolysis and lipogenesis, H-HCA also displayed overexpression of several genes encoding growth factor receptors, components of the translation machinery, cell cycle, and angiogenesis regulators, with, in particular, activation of the mammalian target of rapamycin (mtor) pathway. Moreover, estradiol detoxification activities were shut down, suggesting a hypersensitivity of H-HCA to estrogenic stimulation. In the cell model, inhibition of HNF1 recapitulated most of these identified transcriptional deregulations, demonstrating that they were related to HNF1 inhibition. Conclusion: H-HCA showed a combination of alterations related to HNF1 inactivation that may cooperate to promote tumor development. Interestingly, mtor appears as a potential new attractive therapeutic target for treatment of this group of HCAs. (HEPATOLOGY 2010;51: ) Abbreviations: ACL, adenosine triphosphate citrate lyase; FAS, fatty acid synthase; HCA, hepatocellular adenomas; HCC, hepatocellular carcinoma; H-HCA, human hepatocyte nuclear factor 1 -mutated hepatocellular adenoma; HNF1, hepatocyte nuclear factor 1 ; mtor, mammalian target of rapamycin; RT-PCR, reverse transcription polymerase chain reaction; sirna, small interfering RNA. From 1 Institut National de la Sante et de la Recherche Medicale (INSERM), U674, Génomique fonctionnelle des tumeurs solides, Paris, France; 2 Université Paris Descartes, Paris, France; 3 INRA UMR 1089, Xénobiotiques, Toulouse, France; 4 INSERM, U889, Université Bordeaux 2, IFR66, Bordeaux, France; 5 CHU Bordeaux, Hôpital Pellegrin, Bordeaux, France; 6 Array s/image, Genexpress, Functional Genomics and Systems Biology for Health UMR 7091, CNRS, Université Paris, 6 Pierre et Marie Curie, Villejuif, France. *These authors contributed equally to this work. Received June 12, 2009; accepted September 8, Address reprint requests to: Jessica Zucman-Rossi, INSERM, U674, Functional Genomic of Solid Tumors, 27 rue Juliette Dodu, Paris, France. zucman@cephb.fr. Copyright 2009 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Supported by the INSERM (Réseau de Recherche clinique et en santé des populations) and SNFGE. L.P. and S.R. are supported by a doctoral fellowship from the Ministère de la Recherche and the Ligue Nationale Contre le Cancer, respectively. Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article. 557

2 558 PELLETIER, REBOUISSOU, ET AL. HEPATOLOGY, February 2010 Hepatocellular adenomas (HCAs) are rare benign liver tumors usually occurring in young women taking oral contraceptives and occasionally associated with the use of anabolic corticosteroid or with type I glycogen storage disease. 1 3 Recently, HCA has been described as a heterogeneous disease including at least three main subtypes of tumors in which histological phenotypes are closely related to specific genetic alterations and clinical features. 4 6 The most frequent group of HCAs harbor biallelic inactivating mutations of hepatocyte nuclear factor 1 (HNF1A) gene and are phenotypically characterized by marked steatosis. 4,6,7 In 90% of the cases, HNF1 -mutated HCA (H-HCA) are sporadic lesions displaying somatic mutations. However, in rare families with an inherited mutation in one allele of HNF1A, maturity-onset diabetes of the young type 3 patients are predisposed to develop familial liver adenomatosis that is defined by the presence of more than 10 HCA nodules in the liver, which is clinically difficult to manage Thus, HNF1A meets the genetic criteria of a tumor suppressor gene. 7 However, liver adenomatosis is a rare complication of maturity-onset diabetes of the young type 3, and its development may be promoted by heterozygous germline mutation inactivating CYP1B1, a key metabolism enzyme responsible for the formation of hydroxylated and genotoxic metabolites of estrogen. 11 HNF1 is an atypical homeodomain-containing protein that was originally identified as a hepatocyte-specific transcriptional regulator. 12 In vivo and in vitro models of HNF1 inactivation demonstrated that this transcription factor plays an important role in hepatocyte differentiation and is also crucial for metabolic regulation and liver function Interestingly, and consistent with the identified tumor suppressor function of HNF1 in humans, hnf1 -deficient mice develop a dramatic liver enlargement that has been associated with increased hepatocyte proliferation, sometimes accompanied by dysplasia. 14,16,17 However, the role of HNF1 in the control of cell proliferation or survival in hepatocytes is still poorly understood. To gain insight into the tumorigenic mechanisms related to HNF1 inactivation, we searched for tumorigenic pathways aberrantly activated in human H-HCA. The role of HNF1 in the observed deregulations was subsequently assessed in vitro by inhibiting its endogenous expression in human liver cancer cell lines by using small interfering RNA. Patients and Methods Patients and Samples. Liver tissues were collected in nine French surgery departments from 1992 to They were immediately frozen in liquid nitrogen and stored at 80 C until used for molecular studies. The whole series of HCA used for the different molecular analyses included 23 H-HCA previously described 4,6 and 17 normal livers taken from patients resected with primary liver tumors developed in the absence of cirrhosis (Supporting Table 1). All of the patients were recruited in accordance with French law and institutional ethical guidelines. The study was approved by the ethical committee of Hôpital Saint-Louis, Paris, France. Microarray Analysis. Transcriptional profiling of five H-HCA and four normal liver tissues were performed using Affymetrix oligonucleotide GeneChips HG- U133A (GEO accession number GSE7473), as previously described. 5,18 Cell Lines and Small Interfering RNA Transfection. HepG2 and Hep3B cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in Dulbecco s modified eagle medium with high glucose (Invitrogen) supplemented with 10% fetal calf serum, penicillin 100 IU/mL, and streptomycin 100 g/ml. Small interfering RNA (sirna) transfections were performed according to the manufacturer s protocol, in six-well plates using the lipofectamine RNAiMax reagent (Invitrogen) with two different sequences of sirna duplexes targeting HNF1 (NM_000545) (Ambion) as follows: GGUCUUCACCUCAGACACUtt (exon ) or GGCAGAAGAACCCUAGCAAtt (ex3 3450). Block-iT Alexa Fluor Red Fluorescent Oligo sirna (Invitrogen) was used as a double-stranded RNAnegative control. In most experiments, 10 nm of each sirna was transfected in triplicate, except for dose effect studies, in which several sirna concentrations were tested (0, 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 5, 10, and 50 nm) to obtain different levels of HNF1 expression. Cells were prepared for quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting analyses either 3 or 7 days after transfection. The absence of cross-reaction of the HNF1 sirna duplexes with the HNF1 sequence was checked by comparing the expression level of HNF1 transcript in cells transfected with sirna targeting HNF1 with the control sirnatransfected cells (data not shown). Quantitative RT-PCR. Quantitative RT-PCR was performed in duplicate as previously described 19 by using predesigned primers and probe sets from Applied Biosystems (Supporting Table 2). Ribosomal 18S (R18S) was used for the normalization of expression data, and the 2 CT method was applied. The final results were expressed as the fold differences in target gene expression in tested samples compared with the mean expression value

3 HEPATOLOGY, Vol. 51, No. 2, 2010 PELLETIER, REBOUISSOU, ET AL. 559 of normal tissues (for tumor analysis) or with the control sirna in cell lines. Western Blotting. Western blot analyses were performed as described, 18 using the primary antibodies specific for ErbB2 (V-ERB-B2 avian erythroblastic leukemia viral oncogene homolog 2), mammalian target of rapamycin (mtor), phospho-mtor ser2448, 4E-BP1, phospho 4E-BP1 (eukaryotic translation initiation factor 4Ebinding protein 1) thr37/46 (Cell Signaling Technology, diluted 1:500, except for phospho-mtor that was used at 1:200 dilution); eif4g3 (eukaryotic translation initiatin factor 4-gamma, 3) (a gift of Dr. N. Sonenberg, 1:500), eef1a2 (eukaryotic translation elongation factor 1, alpha-2) (Abnova, 1:500); HNF1 and cyclin D1 (Santa Cruz Biotechnology, 1:100) and L-FABP (Liver fatty acid binding protein) (a gift of Dr. J. Gordon, 1:2000). Polyclonal rabbit anti-actin (1:3000, Sigma) was used as loading control. Lipid Staining. HepG2 cells were grown on slides for 7 days and fixed with 4% formaldehyde in phosphatebuffered saline 1X. After washing, cells were stained with Oil Red O (Sigma) dissolved in 60% isopropyl alcohol (20 ), and counterstaining was performed with 50% hematoxylin (Harris, Gill II, PAP1, Labonord). Estrogen Metabolism. Human liver S9 fractions were prepared from 100 to 135 mg frozen tissue. The tissues were mechanically disrupted in an Elvehjem potter in 0.1 M phosphate buffer (ph 7.4), and the homogenates were subsequently subjected to centrifugation at 600g for 10 minutes. The S9 fraction was obtained from the supernatant after centrifugation at 9000g for 20 minutes, and the upper solid fatty material was discarded at the same time. Then, S9 fractions were stored at 80 C until analyses. Estradiol-17 ( 2 ) 2-hydroxylase and 4-hydroxylase activities were determined from 1 mg S9 protein by quantifying 2- and 4-o-methyl-ether derivatives of E 2, using radio high-pressure liquid chromatography as previously described in Rizzati et al esterification activity was determined from 2 mg S9 protein, by measuring 2-17-fatty acid esters formation, as previously described in Paris and Rao. 21 Detailed procedures are provided in the Supporting Methods. Statistical Analysis. All of the values reported are means standard deviation. Statistical analyses were performed using GraphPad Prism version 4 software, and significance was determined using either the nonparametric Mann-Whitney test for unpaired data or the twotailed t test. Difference was considered significant at P In all graphs, *, **, and *** indicate differences between groups at P 0.05, P 0.01, and P 0.001, respectively. Results Gene Expression Profiles in HNF1 -Mutated HCA. To study the role of HNF1 inactivation in human liver tumorigenesis, we used oligonucleotide microarrays to compare expression profiles between H-HCA and control normal liver tissues. Statistical analysis of gene expression identified 338 and 212 genes that were, respectively significantly underexpressed and overexpressed in H-HCA compared with normal liver samples (Supporting Table 3). Several classes of genes related to normal hepatocyte function were altered in this subtype of adenoma, including amino acid, glucose, lipid, xenobiotic, and steroid hormone metabolism (Supporting Table 4). A large fraction of differentially expressed genes also were involved in transcriptional regulation, and few genes encoded receptors, growth factors, or proteins implicated in translation and cell proliferation (Supporting Table 4). On examination of these data, we selected a set of deregulated genes that may be relevant for tumorigenesis, and we further validated their expression using quantitative RT-PCR and western blotting in a large series of H-HCA and in hepatocellular cell lines inactivated for HNF1 by sirna. HNF1 Inhibition in Human Liver Cancer Cell Lines. HepG2 cells were transfected with a control sirna and two sirna targeting exon 3 or exon 8-9 junction, leading to 87% and 98% HNF1A messenger RNA (mrna) extinction, respectively (Fig. 1A). To modulate the HNF1 expression and to study dose-dependent response of candidate genes putatively regulated by HNF1, we used a range of different concentrations of HNF1 sirna (Fig. 1B). Indeed, there was a close correlation between the expression of HNF1A and different genes known to be directly targeted by HNF1, as shown for FGB in Fig. 1B. Using sirna ex8-9, we observed an extinction of HNF1 protein as soon as 24 hours, with maximal inhibition of HNF1 and its transactivated gene FABP1 72 hours after transfection (Fig. 1C) that was maintained for at least 7 days (Fig. 2). In all the following experiments, we used the most efficient sirna (ex8-9). However, to verify that the observed deregulations were related to HNF1 inactivation, we confirmed the results using the ex3 sirna in HepG2 cells and ex8-9 sirna in a second hepatocellular cell line (Hep3B), reaching 91% of HNF1A mrna inhibition (Supporting Fig. 1 and other Supporting data). Activation of Lipogenesis. Presence of a severe steatosis is the most striking phenotype constantly observed in H-HCA 4,6 (Fig. 2D). Accordingly, we previously demonstrated that H-HCA displayed a strong alteration in glucose and lipid metabolism gene expression profiles,

4 560 PELLETIER, REBOUISSOU, ET AL. HEPATOLOGY, February 2010 Fig. 1. Inhibition of HNF1 expression in HepG2 cells. (A) HepG2 cells were transfected independently with two different sequences of sirna directed against HNF1A (HNF1 ex8-9 and HNF1 ex3) (sih), or with a control sirna (sic). Inhibition efficiencies were assessed 3 days after transfection by measuring the expression level of HNF1A and two of its transactivated genes (FABP1 and FGB) by quantitative RT-PCR (two-tailed t test). (B) Correlation between HNF1A and FGB mrna expression 3 days after transfection of HepG2 cells with different concentrations of HNF1 ex8-9 sirna (Spearman s rank correlation test). (C) Time course analysis of HNF1 and LFABP protein expression after transfection with control (sic) and HNF1 ex8-9 (sih) sirna using western blotting. Fig. 2. Activation of lipogenesis. (A, B) Expression of glycolysis and lipogenesis genes in H-HCA and HepG2 cells transfected with HNF1 ex8-9 sirna after 3 (A) or 7 (B) days. mrna levels were analyzed by quantitative RT-PCR and are expressed as an n-fold difference in gene expression relative to the mean expression value of normal liver tissues (NL, n 10, mean 1 dotted line) for H-HCA (T, n 16) (twotailed Mann-Whitney test), or relative to cells transfected with control sirna (sic) (mean 1 dotted line) for HepG2 cells transfected with HNF1 sirna (sih) (twotailed t test). NE: Not expressed in the cell line. (C) Overexpression of ACL and FAS proteins in HepG2 cells transfected with HNF1 ex8-9 sirna after 7 days compared with cells transfected with control sirna analyzed by western blotting. (D) Typical aspect of a H-HCA with marked lipid overload. (E) HepG2 cells transfected with HNF1 ex8-9 sirna showing marked lipid overload compared with HepG2 cells transfected with control sirna, after Oil Red O staining and hematoxylin counterstain.

5 HEPATOLOGY, Vol. 51, No. 2, 2010 PELLETIER, REBOUISSOU, ET AL. 561 Fig. 3. Overexpression of receptors, growth factors, and translational and cell cycle regulators in H-HCA. (A) Quantitative RT-PCR validation of gene array expression data comparing H-HCA (T; n 16) with normal liver tissues (NL; n 10) (twotailed Mann-Whitney test). (B) Western blotting validation of quantitative RT-PCR results comparing H-HCA (n 6) with normal livers (n 4). leading to an aberrant induction of glycolytic and lipogenic genes expression with a repression of gluconeogenesis that predict elevated rates of lipogenesis 18 (Supporting Table 4; Fig. 2A, B). Moreover, we found that most of these gene deregulations were related to HNF1 inactivation in our cell model. First, the four genes (PCK1, PCK2, FBP1, and G6PT1) involved in the gluconeogenesis pathway that were underexpressed in H-HCA were also significantly repressed after HNF1 extinction in HepG2 cells (Fig. 2A). Among the five glycolytic genes altered in H-HCA, three of them (GCKR, PKLR, and PKM2) showed the same profile of deregulation in HNF1 -inhibited HepG2 cells. In contrast, glucose-6-phosphate isomerase (GPI) displayed an opposite regulation in HepG2 cells, and glucokinase was not expressed (Fig. 2A). All of the transcriptional modifications observed in both gluconeogenesis and glycolysis pathways occurred immediately after HNF1 extinction and were maintained 7 days later, suggesting that their expression is directly controlled by HNF1 (Supporting Fig. 2B and Supporting Table 4). Second, in the lipogenic pathway, 11 genes were significantly overexpressed in H-HCA compared with normal livers; six of them were also significantly up-regulated in HNF1 -inhibited HepG2 cells compared with control sirna-transfected cells (Fig. 2B). This included three crucial genes involved in the first steps of fatty acid synthesis: ACLY, ACACA, and FASN, encoding, respectively, the adenosine triphosphate citrate lyase (ACL), acetyl- CoA carboxylase, and fatty acid synthase (FAS) enzymes. ACLY and FASN overexpression was confirmed at the protein level (Fig. 2C). Notably, these transcriptional changes were late events, because they were observed only 7 days after HNF1 inhibition except for the two elongases (ELOVL1 and ELOVL5) that were already overexpressed after 3 days (Supporting Fig. 2C and Supporting Table 4). Furthermore, the overexpression of lipogenic genes was correlated with lipid overload in HNF1 -inhibited cells, which showed more and larger lipid vacuoles than control sirna-transfected cells (Fig. 2E). These results suggested that several of these lipogenic genes are indirectly regulated by HNF1. Finally, in HNF1 -inactivated HepG2 as in H-HCA, we did not find overexpression of several transcription factors known to regulate lipogenic genes expression; in particular, SREBP1 was not overexpressed (Fig. 2B and Supporting Fig. 2A). Almost all of the results found after 3 days of HNF1 extinction were confirmed in Hep3B cells and in HepG2 cells using ex3 sirna (Supporting Fig. 2B and 2C and Supporting Table 4). However, lipogenesis could not be studied at 7 days in Hep3B and in HepG2 cells using ex3 sirna, because in these conditions, HNF1 suppression was not maintained. Overexpression of Receptors, Growth Factors, and Translational and Cell Cycle Regulators. In H-HCA samples, our microarray analysis identified an overexpression of several genes encoding critical proteins that are known to be involved in a variety of tumors. Among them, ERBB2 transcript, which encodes a tyrosine kinase receptor, was fivefold increased in H-HCA compared with normal livers. This overexpression was correlated with a strong accumulation of the corresponding protein in tumors (Fig. 3A, B). This transcriptional overexpression was not related to gene amplification, because no ERBB2 gene amplification was found in seven H-HCA cases using comparative genomic hybridization based on single nucleotide polymorphism genotyping (data not shown). Both PDGFA and PDGFB mrna, encoding two growth factors implicated in angiogenesis and cell proliferation, were also significantly increased in H-HCA compared with normal livers (Fig. 3A). Moreover, we identified up-regulation of several members of the trans-

6 562 PELLETIER, REBOUISSOU, ET AL. HEPATOLOGY, February 2010 Fig. 4. Overexpression of receptors, growth factors, and translational and cell cycle regulators in HNF1 -inhibited HepG2 cells. (A) Messenger RNA expression levels were compared between HepG2 cells transfected with HNF1 ex8-9 sirna (sih) and with control sirna (sic) (two-tailed t test). (B) Correlations between expression of HNF1A mrna, and the five genes significantly overexpressed in HNF1 inhibited HEPG2 cells were analyzed using a range of sirna concentrations, and significance was assessed by Spearman s rank correlation test. All graphs plot quantitative RT-PCR results relative to cells transfected with control sirna. (C) Cyclin D1 and eif-4g3 expression was analyzed using western blotting after transfection with different concentrations of HNF1 ex8-9 sirna. All analyses were performed 3 days after transfection. lational machinery. In particular, mtor, which is known to play a pivotal role in the regulation of translation, was overexpressed in H-HCA at both transcriptional (4.5-fold) and protein level (Fig. 3A, B). Accordingly, compared with normal livers, phospho-mtor was significantly elevated in H-HCA, as well as 4E-BP1 phosphorylation, which is a well-known downstream target phosphorylated by mtor (Fig. 3B). These results indicated that mtor pathway was aberrantly activated in H-HCA. In addition, these tumors displayed an elevation of eif-4g3 and eef1a2 at mrna and protein levels, that are involved in protein translation initiation and elongation, respectively (Fig. 3A, B). Finally, H-HCA showed overexpression of CCND1 mrna (3.6-fold), which encodes cyclin D1, a key cell cycle regulatory protein (Fig. 3A). This result was confirmed by western blotting analysis, demonstrating a constant accumulation of cyclin D1 in H-HCA (Fig. 3B). Except for mtor and ERBB2, significant overexpression of five other aforementioned genes (EIF4G3, EEF1A2, PDGFA, PDGFB, and CCND1) was also found in HepG2 cells 3 and 7 days after HNF1 inhibition compared with control-sirna-transfected cells (Fig. 4A and Supporting Fig. 3A). These data display a similar pattern of deregulation using HNF1 sirna targeting exon 3 or in Hep3B cells (Supporting Fig. 3B-D). Remarkably, using the sirna concentration range, we identified a strong negative correlation between the expression of HNF1 mrna and mrna expression of these five genes (Fig. 4B). Moreover, we confirmed that cyclin D1 and eif-4g3 were closely correlated to HNF1 expression even at the protein level (Fig. 4C). Alteration of Estrogen Metabolism. Interestingly, the pattern of deregulation observed in genes encoding xenobiotic and steroid hormone-metabolizing enzymes, suggested that H-HCA may have impaired ability to inactivate the active form of estradiol (Supporting Table 4). Indeed, in these tumors, we confirmed the dramatic decrease in mrna level of CYP1A1, CYP1A2, and CYP3A4, which encodes the three principal cytochrome P450 isoforms catalyzing E 2 oxidation in human liver (Fig. 5A). In addition,

7 HEPATOLOGY, Vol. 51, No. 2, 2010 PELLETIER, REBOUISSOU, ET AL. 563 Fig. 5. Impaired estradiol metabolism. (A, B) Expression of estradiol detoxification-related genes was validated using quantitative RT-PCR. (A) H-HCA (T; n 16) were compared with normal liver tissues (NL; n 10) (two-tailed Mann-Whitney test) and (B) HepG2 cells transfected with sirna HNF1 ex8-9 (sih) were compared with cells transfected with control sirna (sic) 3 days after transfection (two-tailed t test). (C) Activities of E 2-2 hydroxylation, E 2-4-hydroxylation, and E 2-17-acylation were compared between H-HCA (n 3) and normal liver tissues (n 3) (one-tailed t test). compared with normal livers, H-HCA showed a twofold decrease in HSD17B2 transcript (Fig. 5A) encoding the 17 -hydroxysteroid dehydrogenase, which is responsible for the interconversion of active E 2 to the less potent E 1. CYP1A1 and HSD17B2 were also downregulated by HNF1 inactivation in HepG2 and Hep3B cells (Fig. 5B and Supporting Fig. 4), whereas CYP1A2 and CYP3A4 were not expressed in these cell lines at baseline. According to the observed strong down-regulation of cytochrome P450 isoforms, activities of 2-hydroxylation and 4-hydroxylation of E 2 were suppressed in H-HCA samples as compared with normal livers (Fig. 5C). Furthermore, in these tumors, a significant threefold increase in activity of E acylation to long chain fatty acid was found compared with normal livers, predicting elevated concentrations of estrogen fatty acid esters which are known to be highly potent and long-lived estrogens 22 (Fig. 5C). Collectively, these results support the hypothesis that stimulation of growth by estrogen would be enhanced in H-HCA tissue. Discussion Biallelic inactivating mutations of HNF1A have been identified in 35% to 50% of HCA and in rare cases of hepatocellular carcinomas (HCC) developed in the absence of cirrhosis. 4,6,7 Hence, this genetic event appears to be an important driver of HCA development and may represent an early step in malignant transformation of hepatocytes. Using a genome-wide transcriptome analysis, we identified altered expression in several genes that may contribute to the pathogenesis of H-HCA (Fig. 6). Moreover, we demonstrated that a number of these transcriptional changes were related to the loss of HNF1 activity in hepatocellular cells. A role for HNF1 in the control of liver metabolism has been well documented. Mice deficient in hnf1 develop severe liver steatosis that is very similar to that observed in H-HCA. As previously shown, human adenomas display coordinated aberrant overexpression of glycolytic and lipogenic genes and inhibition of gluconeogenesis, which may be responsible for elevated rates of lipid synthesis and hepatocyte fat overload. 18 Interestingly, our cellular model of HNF1 inactivation recapitulates most of the glycolysis and lipogenesis gene deregulations and the fat overload observed in H-HCA. In the gluconeogenic and glycolytic pathways, almost all of the down-regulated genes were previously shown as targets or putative targets positively regulated by HNF1, and the expression was suppressed immediately after HNF1 extinction. In contrast, most of the significant overexpressions observed in the lipogenic pathway arose later, suggesting that they are an indirect consequence of HNF1 inhibition. These observations suggest a repressor function of HNF1 that remains to be demonstrated on the corresponding promoters. Surprisingly, as shown in H-HCA, none of the well-known transcription factors involved in the control of lipogenesis was activated in our cell model. Our results showed an obvious role of HNF1 in the negative control of lipogenesis that may involve a complex regulatory network whose understanding needs further investigation. An emerging view suggests that metabolic reprogramming commonly observed in tumor cells is required for survival and to satisfy increased energetic and anabolic needs. Particularly, high glycolysis and lipogenesis rates, as shown in H-HCA, are a hallmark of many human tumors, and recent findings demonstrated that these metabolic adaptations function downstream from oncogenic signalings. 23 Moreover, several works showed that inhibi-

8 564 PELLETIER, REBOUISSOU, ET AL. HEPATOLOGY, February 2010 Overexpression Underexpression/Inactivation ATP Nucleotids Amino acids Membrane synthesis Lipid modification of proteins ErbB2 ErbB2 PI3K PTEN PDGFA PDGFB PDK1 Angiogenesis Estrogen metabolism E2-fatty acid Acylation Activates Inhibits estrone (E1) HSD17B2 Glycolysis Metformin Lipogenesis AMPK AKT TSC2 TSC1 Rheb Amino Acids ++ E2 Proliferation 17 -estradiol (E2) CYP3A4 CYP1A1 CYP1A2 2/4-OH-estradiol Cell division LKB1 G2 M Cell cycle G1 S Cyclin D1 mtor 4E-BP1 eif4g3 S6K eif4e eif4b Translation initiation Rapamycin CCI779 RAD001 AP23573 eef1a2 Synthesis of proteins involved in cell growth and survival Translation elongation Fig. 6. Scheme of the different pathways altered in human H-HCA and hypothetical consequences. In adenoma, loss of HNF1 activity leads to many alterations that may shift cellular activities toward a proliferative state. Indeed, in cooperation with loss of cell cycle control, metabolic reprogramming resulting from elevated levels of glycolysis, and lipogenesis may fuel cell proliferation by providing energy and precursors required for the synthesis of macromolecules. Furthermore, activation of the mtor pathway and the translational machinery may promote the accumulation of critical proteins necessary for cell growth and survival. Overexpression of proangiogenic factors also may contribute to tumor development by stimulating neovascularization. Finally, suppression of estradiol detoxification activities may lead to estradiol accumulation and growth advantage. Some of these pathways can be targeted by specific pharmacological inhibitors (indicated in italic bold font), such as metformin, which inhibits both lipogenesis and mtor pathways, or rapamycin and its derivatives that target mtor. tion of lipogenesis is able to reduce cell proliferation and tumor growth in vitro and in human tumor xenograft in nude mice Importantly, we also identified an aberrant activation of the mtor signaling in H-HCA. mtor has been recognized as a key regulator of cell proliferation and growth, in particular through its ability to stimulate protein translation by phosphorylating S6K and 4E-BP1 proteins, leading to selective enhancement in synthesis of a subset of key growth-promoting proteins. 27 The abnormal activation of the mtor pathway has been implicated in a wide variety of tumors. 28 Interestingly, in the welldifferentiated hepatocellular cell line, HepaRG, increased mtor activity was demonstrated to confer a neoplastic phenotype to the hepatocytes by altering the translation of genes necessary for moderating hepatocellular growth and establishing normal hepatic energy homeostasis. 29 Moreover, several recent studies identified activation of the mtor pathway in 15% to 50% of HCC, and showed antineoplastic activity of mtor inhibitors in experimental models of HCC Collectively, these findings pinpoint a major role for mtor signaling in the control of hepatocyte growth and survival. We showed herein that mtor pathway activation in H-HCA may occur through elevated mrna and protein levels of mtor as well as the strong accumulation of the ErbB2 receptor, as previously demonstrated in breast cancer overexpressing ErbB2. 34 In addition, the identified up-regulation of components of the translational machinery working downstream of mtor, such as the initiation factor eif- 4G3 and the elongation factor eef1a2, also may contribute to maintaining high levels of protein synthesis in H-HCA, thereby promoting the accumulation of critical factors necessary for cell growth and survival. Remarkably, eef1a2 has been shown to have transforming activity. 35 Recently, frequent EEF1A2 overexpression has been identified in HCC, and gene silencing was shown to reduce cell proliferation and increase apoptosis rates in HepG2 and Hep3B cell lines. 36,37 In addition, a loss of critical cell cycle checkpoint through the abnormal elevated levels of cyclin D1 was observed in H-HCA and the increased levels of PDGFA and particularly PDGFB may promote tumor neovascularization. 38 Although mtor and ERBB2 overexpression were recurrently observed in H-HCA, these deregulations were not found after in vitro silencing of HNF1. This result may reflect a more complex regulation of these two genes involving cooperation of HNF1

9 HEPATOLOGY, Vol. 51, No. 2, 2010 PELLETIER, REBOUISSOU, ET AL. 565 with other factors that may be altered in our cellular models. In contrast, the strong negative correlation between the expression level of HNF1 and the other aforementioned genes suggests that HNF1 plays an important role in directing their basal promoter activity and may directly repress their expression in physiological conditions. As for other tumor suppressor genes involved in human benign tumorigenesis, understanding why HNF1 inactivation promotes formation of benign tumors rather than malignant tumors remains a difficult challenge. Indeed, it is surprising that many molecular deregulations identified in the benign H-HCA are also commonly found in malignancies. The effect of any mutation and its importance in tumorigenesis are likely to vary, depending on when and where it occurs and has its effects. One can speculate that in HNF1 -inactivated hepatocytes, feedback controls of the cell population are still functional and may add stability to the system, rendering exponential expansion and therefore malignant transformation impossible unless these regulatory mechanisms are altered during progression. The existence of specific pathways involved in the development of benign tumors is not clear. However, it is interesting to note that, as identified in H-HCA, aberrant activation of the mtor pathway resulting from the inactivation of many tumor-suppressor genes has been commonly found in several other benign lesions characterized by cell growth abnormalities such as hamartomas syndromes, as well as cardiac hypertrophy. 28 The last point of the current study was to investigate the link between estrogen and H-HCA development. Several lines of evidence coming from both animal models and human epidemiological studies indicated that estrogens may act as liver tumor promoters. 1,39,40 In humans, the relationship between HCA and oral contraceptives has been well established, and several reports have described tumor regression after the withdrawal of hormonal agents. 41,42 In addition, some cases of HCA enlargement have been reported during pregnancy, presumably as a result of estrogen stimulation. 43 Despite some discrepancy between the studies, overall, the weight of evidence suggested that estrogen receptors are present in HCA. 44 In our series of H-HCA, we also found detectable levels of ER receptors using western blotting, suggesting that these tumors may respond to estrogen stimulation (data not shown). Our findings suggested that H-HCA have a severely impaired ability to eliminate the active form of estradiol and may present an increased capacity to generate E 2-17-fatty acid esters that are potent long-acting estrogens. The ability to increase E acylation may be a direct consequence of fatty acids accumulation in these adenomas. Hence, estrogens may confer growth advantage to the HNF1 -inactivated hepatocytes, as a result of an abnormal prolonged time of hormonal exposure. In conclusion, this study provides new insights into the tumorigenic mechanisms related to HNF1 inactivation in the liver. In H-HCA, we identified alterations that may cooperate to reorganize cellular activities supporting bioenergetics, macromolecular synthesis, and ultimately cell division (Fig. 6). Although the exact role of HNF1 in the control of these processes remains to be more accurately studied, our results suggest that this transcription factor may act as a transcriptional repressor. 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