HNF1α Inactivation Promotes Lipogenesis in Human Hepatocellular Adenoma Independently of SREBP-1 and Carbohydrate-response Element-binding Protein (ChREBP) Activation

Biallelic inactivating mutations of the transcription factor 1 gene (TCF1), encoding hepatocyte nuclear factor 1α (HNF1α) were identified in 50% of hepatocellular adenomas (HCA) phenotypically characterized by a striking steatosis. To understand the molecular basis of this aberrant lipid storage, we performed a microarray transcriptome analysis validated by quantitative reverse transcription-PCR, Western blotting, and lipid profiling. In mutated HCA, we showed a repression of gluconeogenesis coordinated with an activation of glycolysis, citrate shuttle, and fatty acid synthesis predicting elevated rates of lipogenesis. Moreover, the strong down-regulation of liver fatty acid-binding protein suggests that impaired fatty acid trafficking may also contribute to the fatty phenotype. In addition, transcriptional profile analysis of the observed deregulated genes in non-HNF1α-mutated HCA as well as in non-tumor livers allowed us to define a specific signature of the HNF1α-mutated HCA. In these tumors, lipid composition was dramatically modified according to the transcriptional deregulations identified in the fatty acid synthetic pathway. Surprisingly, lipogenesis activation did not operate through sterol regulatory element-binding protein-1 (SREBP-1) and carbohydrate-response element-binding protein (ChREBP) that were repressed. We conclude that steatosis in HNF1α-mutated HCA results mainly from an aberrant promotion of lipogenesis that is linked to HNF1α inactivation and that is independent of both SREBP-1 and ChREBP activation. Finally, our findings have potential clinical implications since lipogenesis can be efficiently inhibited by targeted therapies. Biallelic inactivating mutations of the transcription factor 1 gene (TCF1), encoding hepatocyte nuclear factor 1α (HNF1α) were identified in 50% of hepatocellular adenomas (HCA) phenotypically characterized by a striking steatosis. To understand the molecular basis of this aberrant lipid storage, we performed a microarray transcriptome analysis validated by quantitative reverse transcription-PCR, Western blotting, and lipid profiling. In mutated HCA, we showed a repression of gluconeogenesis coordinated with an activation of glycolysis, citrate shuttle, and fatty acid synthesis predicting elevated rates of lipogenesis. Moreover, the strong down-regulation of liver fatty acid-binding protein suggests that impaired fatty acid trafficking may also contribute to the fatty phenotype. In addition, transcriptional profile analysis of the observed deregulated genes in non-HNF1α-mutated HCA as well as in non-tumor livers allowed us to define a specific signature of the HNF1α-mutated HCA. In these tumors, lipid composition was dramatically modified according to the transcriptional deregulations identified in the fatty acid synthetic pathway. Surprisingly, lipogenesis activation did not operate through sterol regulatory element-binding protein-1 (SREBP-1) and carbohydrate-response element-binding protein (ChREBP) that were repressed. We conclude that steatosis in HNF1α-mutated HCA results mainly from an aberrant promotion of lipogenesis that is linked to HNF1α inactivation and that is independent of both SREBP-1 and ChREBP activation. Finally, our findings have potential clinical implications since lipogenesis can be efficiently inhibited by targeted therapies. Hepatocyte nuclear factor 1-α (HNF1α) 3The abbreviations used are: HNF1α, hepatocyte nuclear factor 1-α; HNF4α, hepatocyte nuclear factor 4-α; ACL, ATP citrate lyase; ChREBP, carbohydrate-response element-binding protein; SREBP-1, sterol regulatory element-binding protein-1; FA, fatty acid; FAS, fatty acid synthase; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; UFA, unsaturated fatty acids; L-FABP, liver fatty acid-binding protein; G6P, glucose-6-phosphate; GK, glucokinase; HCA, hepatocellular adenomas; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PPARγ, peroxisome proliferator-activated receptor-γ; PS, phosphatidylserine; PI, phosphatidylinositol; RT, reverse transcription. is a transcription factor that controls the expression of liver-specific genes, such as β-fibrinogen, α1-antitrypsin, and albumin (1Courtois G. Morgan J.G. Campbell L.A. Fourel G. Crabtree G.R. Science. 1987; 238: 688-692Crossref PubMed Scopus (282) Google Scholar). Heterozygous germline mutations in the gene encoding HNF1α (TCF1 (transcription factor 1)) are responsible for an autosomal dominant form of non-insulin-dependent diabetes mellitus called maturity onset diabetes of the young type 3 (MODY3), in which subjects usually develop hyperglycemia before 25 years of age (2Yamagata K. Oda N. Kaisaki P.J. Menzel S. Furuta H. Vaxillaire M. Southam L. Cox R.D. Lathrop G.M. Boriraj V.V. Chen X. Cox N.J. Oda Y. Yano H. Le Beau M.M. Yamada S. Nishigori H. Takeda J. Fajans S.S. Hattersley A.T. Iwasaki N. Hansen T. Pedersen O. Polonsky K.S. Turner R.C. Velho G. Chèvre J.C. Froguel P. Bell G.I. Nature. 1996; 384: 455-458Crossref PubMed Scopus (1056) Google Scholar). More recently, we identified HNF1α as a tumor suppressor gene involved in human liver tumorigenesis since we found biallelic inactivating mutations of this gene in 50% of hepatocellular adenomas (HCA) and in rare cases of well differentiated hepatocellular carcinomas developed in the absence of cirrhosis (3Bluteau O. Jeannot E. Bioulac-Sage P. Marques J.M. Blanc J.F. Bui H. Beaudoin J.C. Franco D. Balabaud C. Laurent-Puig P. Zucman-Rossi J. Nat. Genet. 2002; 32: 312-315Crossref PubMed Scopus (288) Google Scholar). HCA are rare benign primary liver tumors closely related to oral contraceptive intake (4Edmondson H.A. Henderson B. Benton B. N. Engl. J. Med. 1976; 294: 470-472Crossref PubMed Scopus (449) Google Scholar). Recently, in a comprehensive analysis of genotype-phenotype correlations in a large series of 96 HCA, we showed that HNF1α mutations define a homogeneous group of tumors phenotypically characterized by the recurrent presence of a marked steatosis (5Zucman-Rossi J. Jeannot E. Nhieu J.T. Scoazec J.Y. Guettier C. Rebouissou S. Bacq Y. Leteurtre E. Paradis V. Michalak S. Wendum D. Chiche L. Fabre M. Mellottee L. Laurent C. Partensky C. Castaing D. Zafrani E.S. Laurent-Puig P. Balabaud C. Bioulac-Sage P. Hepatology. 2006; 43: 515-524Crossref PubMed Scopus (632) Google Scholar). To get insight into the underlying molecular mechanisms that drive the fatty phenotype in human HNF1α-mutated HCA, we performed a transcriptome analysis using cDNA and Affymetrix microarrays. Gene expression profiles were compared between non-tumor livers and HNF1α-mutated HCA. Among the differentially expressed genes, we focused our analysis on genes involved in lipid homeostasis, and we searched for a possible alteration in fat transport, degradation, and synthesis processes. Transcriptomic deregulations were further validated in an additional series of tumors, but also at the protein level, and we analyzed the precise composition of accumulated lipids. Patients and Samples—A series of 40 HCA, 25 non-steatotic non-tumor livers, and 11 steatotic non-tumor livers was collected in nine French surgery departments from 1992 to 2004. Liver tissues were immediately frozen in liquid nitrogen and stored at -80 °C until used for molecular studies. Among the 38 patients with an HCA, the sex ratio (male: female) was 1:9, and the mean age was 37 (median = 37 years ranging from 14 to 56). All the patients were recruited in accordance with French law and institutional ethical guidelines. The study was approved by the ethical committee of the Hôpital Saint-Louis, Paris, France. All HCA were screened for HNF1α and β-catenin mutation, pathological slides were reviewed, and all HCA were classified as described previously (5Zucman-Rossi J. Jeannot E. Nhieu J.T. Scoazec J.Y. Guettier C. Rebouissou S. Bacq Y. Leteurtre E. Paradis V. Michalak S. Wendum D. Chiche L. Fabre M. Mellottee L. Laurent C. Partensky C. Castaing D. Zafrani E.S. Laurent-Puig P. Balabaud C. Bioulac-Sage P. Hepatology. 2006; 43: 515-524Crossref PubMed Scopus (632) Google Scholar). Twenty-five HCA from 23 patients were HNF1α-mutated, 11 demonstrated steatosis in more than two-thirds of hepatocytes, eight demonstrated steatosis in one-third to two-thirds of hepatocytes, three demonstrated steatosis in less than one-third of hepatocytes, and three were not steatotic. Fifteen HCA had no mutations in HNF1α, four of them were β-catenin-mutated, five were inflammatory, and nine demonstrated steatosis. Non-tumor liver tissues were taken from patients resected with primary liver tumors developed in the absence of cirrhosis. Steatotic non-tumor livers demonstrated lipid storage either in more than two-thirds of hepatocytes (four cases) or in one-third to two-thirds of hepatocytes (seven cases), and were of various etiologies: hepatitis C virus infection (one case) dysmetabolic syndrome (six cases), alcohol (three cases), and unknown etiology (one case). Microarray Analysis—Total RNA was extracted from frozen tissues using Qiagen RNeasy kits (Qiagen) according to the manufacturer’s instructions. RNA integrity was assessed using RNA 6000 nano chips and the Agilent 2100 bioanalyzer. RNA quality control was performed as described previously (6Imbeaud S. Graudens E. Boulanger V. Barlet X. Zaborski P. Eveno E. Mueller O. Schroeder A. Auffray C. Nucleic Acids Res. 2005; 33: e56Crossref PubMed Scopus (421) Google Scholar). Transcriptional profiling of HNF1α-mutated HCA and non-tumor liver tissues was performed using two different microarray approaches. Minimum Information about a Microarray Experiment (MIAME)-compliant data (7Brazma A. Hingamp P. Quackenbush J. Sherlock G. Spellman P. Stoeckert C. Aach J. Ansorge W. Ball C.A. Causton H.C. Gaasterland T. Glenisson P. Holstege F.C. Kim I.F. Markowitz V. Matese J.C. Parkinson H. Robinson A. Sarkans U. Schulze-Kremer S. Stewart J. Taylor R. Vilo J. Vingron M. Nat. Genet. 2001; 29: 365-371Crossref PubMed Scopus (3337) Google Scholar) have been deposited in Gene Expression Omnibus (GEO) at NCBI and are accessible through GEO Series accession number GSE7473. The detailed procedure of analysis is provided in the supplemental experimental procedures. Quantitative RT-PCR—Quantitative RT-PCR was performed as described previously (8Rebouissou S. Vasiliu V. Thomas C. Bellanne-Chantelot C. Bui H. Chretien Y. Timsit J. Rosty C. Laurent-Puig P. Chauveau D. Zucman-Rossi J. Hum. Mol. Genet. 2005; 14: 603-614Crossref PubMed Scopus (100) Google Scholar) using predesigned primers and probe sets from Applied Biosystems for the detection of R18S, FABP1 (liver fatty acid-binding protein), PCK1 (phosphoenolpyruvate carboxykinase 1, soluble), PCK2 (phosphoenolpyruvate carboxykinase 2, mitochondrial), FBP1 (fructose-1,6-bisphosphatase), G6PT1 (glucose-6-phosphate transporter 1), GCK (glucokinase), GCKR (glucokinase regulatory protein), GPI (glucose-6-phosphate isomerase), ME1 (malic enzyme 1), MDH1 (NADPH malate dehydrogenase, soluble), ACLY (ATP citrate lyase), PKLR (pyruvate kinase, liver and red blood cell), PKM2 ((pyruvate kinase muscle 2) detection of both PKM1 and PKM2 transcripts), ACACA (acetyl-CoA carboxylase-α), FASN (fatty acid synthase), ELOVL1 (elongation of very long chain fatty acids protein 1), ELOVL2 (elongation of very long chain fatty acids protein 2), ELOVL5 (ELOVL family member 5, elongation of very long chain fatty acids), SCD (stearoyl-CoA desaturase), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), SREBP-1 (detection of both SREBP-1a and SREBP-1c isoforms), SREBP-1a, CHREBP, PPARα (peroxisome proliferator-activated receptor-α), PPARγ, LXRα (liver X receptor α), and HNF4α. Ribosomal 18 S (R18S) was used for the normalization of expression data. The relative amount of measured mRNA in samples was determined using the 2-ΔΔCT method where ΔΔCT = (CTtarget - CTR18S)sample - (CTtarget - CTR18S)calibrator. Final results were expressed as the n-fold differences in target gene expression in tested samples when compared with the mean expression value of non-tumor tissues. Western Blotting—Total protein extracts were obtained after homogenization in radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology). Nuclear extracts were prepared using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce), and protein concentration was determined using a Pierce BCA protein assay kit. Primary antibodies were used at the following dilutions: rabbit polyclonal anti-L-FABP 1:2000 (a gift of Dr. J Gordon), mouse monoclonals anti-fatty acid synthase (anti-FAS) and anti-SREBP-1 (detecting both the SREBP-1a and the SREBP-1c isoforms) 1:500 (Pharmingen), polyclonals rabbit anti-glucokinase (anti-GK) (Santa Cruz Biotechnology) and anti-ACL (Cell Signaling Technology) 1:200, and polyclonal rabbit anti-ChREBP 1:1000 (Novus Biologicals). We used polyclonal rabbit anti-actin (1:3000, Sigma) and polyclonal rabbit anti-lamin A/C (1:500, Cell Signaling Technology) as loading controls to normalize the signal obtained for total and nuclear protein extracts, respectively. Detection of signals was performed using the ECL SuperSignal West Pico chemiluminescent substrate (Pierce) with either anti-mouse (1:4000, Amersham Biosciences) or anti-rabbit (1:2000, Santa Cruz Biotechnology) horseradish peroxidase-conjugated IgG as second antibodies. Glucose-6-phosphate Measurement—Glucose-6-phosphate (G6P) concentrations were determined enzymatically in normal and tumor samples by exploiting the selective and quantitative conversion of G6P to 6-phosphogluconic acid in the presence of NADP+ and glucose-6-phosphate dehydrogenase, as described previously (9Bandsma R.H. Wiegman C.H. Herling A.W. Burger H.J. ter Harmsel A. Meijer A.J. Romijn J.A. Reijngoud D.J. Kuipers F. Diabetes. 2001; 50: 2591-2597Crossref PubMed Scopus (57) Google Scholar). Histochemical Analysis of Glycogen Storage—For this analysis, we used liver tissues frozen through immersion in isopentane cooled in liquid nitrogen. Periodic acid Schiff histochemical staining was performed on frozen section from five HNF1α-mutated HCA and five non-mutated HCA as well as their matched non-tumor livers. Lipid Profiling—Detailed protocols used for lipid profiling are provided in the supplemental experimental procedures. Statistical Analysis—All the values reported are mean ± S.D. Statistical analysis was performed using GraphPad Prism version 4 software, and significance was determined using the non-parametric Mann-Whitney test for unpaired data. Difference was considered significant at p < 0.05. Gene Expression Profiles in HNF1α-mutated HCA—Our cDNA and Affymetrix microarray experiments analyzed the expression of a total of 15,000 different genes comparing, respectively, eight HNF1α-mutated HCA with their corresponding non-tumor liver and five HNF1α-mutated HCA with four non-tumor livers. Computational analysis of both experiments identified 375 and 222 genes, respectively, significantly down- and up-regulated in HNF1α-mutated HCA. Among these genes, a large fraction was related to the normal hepatocyte function including carbohydrate and lipid metabolism, detoxification, and synthesis of secreted proteins such as complement and coagulation factors (supplemental Table 1). Among differentially expressed genes, promoter regions of 33 were previously identified to bound HNF1α in primary human hepatocytes using chromatin immunoprecipitation-on-chip (ChIP-on-chip) technology (10Odom D.T. Zizlsperger N. Gordon D.B. Bell G.W. Rinaldi N.J. Murray H.L. Volkert T.L. Schreiber J. Rolfe P.A. Gifford D.K. Fraenkel E. Bell G.I. Young R.A. Science. 2004; 303: 1378-1381Crossref PubMed Scopus (1093) Google Scholar). All but four of these genes were down-regulated in HNF1α-mutated HCA (supplemental Table 1), which is in accordance with the well known transactivating function of HNF1α. In addition, we identified 82 genes showing a common pattern of expression with liver from hnf1α-null mice (11Shih D.Q. Bussen M. Sehayek E. Ananthanarayanan M. Shneider B.L. Suchy F.J. Shefer S. Bollileni J.S. Gonzalez F.J. Breslow J.L. Stoffel M. Nat. Genet. 2001; 27: 375-382Crossref PubMed Scopus (367) Google Scholar) (supplemental Table 1). Interestingly, 19 of these common deregulated genes were involved in glucido-lipidic metabolism and particularly in bile acid metabolism, cholesterol, and fatty acid synthesis (supplemental Table 1). Fatty Acid Transport and Oxidation—In HNF1α-mutated HCA, we identified an important modification in the expression profiles of apolipoprotein genes. Particularly, components of the high density lipoprotein particles encoded by APOM, LPAL2, APOF, LPA, and APOA4 were strongly down-regulated, whereas APOL3 was up-regulated (supplemental Table 1). In contrast, the expression of critical genes for hepatocyte very low density lipoprotein assembly and secretion such as APOB and MTP was not affected. We also found a dramatic decrease in the mRNA of FABP1 encoding the liver fatty acid-binding protein (L-FABP). This result was confirmed by quantitative RT-PCR (-68-fold change, Fig. 1A) and by Western blotting analysis that showed an absence of L-FABP in mutated HCA (Fig. 1B). In contrast, we did not find any change in the expression of genes encoding mitochondrial and peroxisomal β-oxidation enzymes. The expression level of genes encoding plasma membrane transporters such as CD36 was also normal in mutated HCA as well as the LDLR gene. However, we identified a number of deregulated genes related to the lipogenesis pathway (Figs. 2A and 3A).FIGURE 2Gluconeogenesis repression, glycolysis and citrate shuttle activation in HNF1α-mutated HCA. A, schematic representation of the gluconeogenic pathway (gray arrows), glycolytic pathway, and citrate shuttle (black arrows). Dashed arrows indicate several reaction steps. Genes whose expression was deregulated in HNF1α-mutated HCA are in solid boxes. The up arrows and down arrows indicate transcripts that are up-regulated and down-regulated, respectively, when compared with non-tumor livers. The asterisk indicates a significant increase in the product quantity. OAA, oxaloacetate. B, quantitative RT-PCR validation of gene array expression data. Results are expressed as the n-fold difference in gene expression relative to the mean expression value of non-tumor livers. Data are mean ± S.D. *, **, and ***, difference between groups at p < 0.05, 0.01, and 0.001, respectively. C, protein level of GK (52 kDa) and ACL (125 kDa) was compared by Western blotting analysis between two HNF1α-mutated HCA (T1, T2) and their respective corresponding non-tumor liver (N1, N2). β-Actin was used as loading control. D, G6P content measurement. Values are mean ± S.D. **, difference between groups at p < 0.01. E, frozen sections of a non-HNF1α-mutated HCA (panel a) and an HNF1α-mutated HCA (panel b). Glycogen storage in cytoplasm of hepatocytes (red color with periodic acid Schiff staining, here in dark) is increased in HNF1α-mutated HCA (panel b) when compared with non-mutated HCA (panel a). F, mRNA level of PKLR and PKM2 genes was assessed using quantitative RT-PCR. Results are expressed as the n-fold difference in gene expression relative to the mean expression value of non-tumor livers. Data are mean ± S.D. ***, difference between groups at p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Activation of the fatty acid synthetic pathway in HNF1α-mutated HCA. A, schematic representation of the fatty acid synthetic pathway. Dashed arrows indicate several reaction steps that occur only in plants. Genes whose expression was deregulated in HNF1α-mutated HCA are in solid boxes. The up arrow indicates transcript up-regulation when compared with non-tumor livers. The asterisk indicates fatty acid species significantly raised in HNF1α-mutated HCA when compared with non-tumor livers. B, quantitative RT-PCR validation of gene array expression data. Results are expressed as the n-fold difference in gene expression relative to the mean expression value of non-tumor livers. Data are mean ± S.D. **, ***, difference between groups at p < 0.01 and 0.001, respectively. C, protein level of FAS (265 kDa) was compared by Western blotting analysis between two HNF1α-mutated HCA (T1, T2) and their respective corresponding non-tumor liver (N1, N2). β-Actin was used as loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gluconeogenesis Repression and Glycolysis Activation—The expression of four genes (FBP1, encoding the fructose-1,6-bisphosphatase; PCK1 and PCK2, encoding the cytosolic and the mitochondrial forms, respectively, of the phosphoenolpyruvate carboxykinase (PEPCK) enzyme; and G6PT1 encoding the glucose-6-phosphate transporter 1) critical to promote hepatic glucose production was significantly decreased in HNF1α-mutated HCA (Fig. 2B), indicating a repression of the gluconeogenesis pathway in these tumors (Fig. 2A). In contrast, glycolysis was activated in HNF1α-mutated HCA since we found a strong up-regulation of glucokinase at the mRNA (GCK, 11-fold) and protein level (GK, Fig. 2, B and C). In addition, we found a 3-fold decrease in the expression of the GCKR transcript whose product acts as a negative regulator of glucokinase (Fig. 2B). According to a predicted increased glucose phosphorylation rate, we showed a significantly higher level of glucose-6-phosphate in HNF1α-mutated HCA (Fig. 2D). Moreover, periodic acid Schiff staining revealed a glycogen overload in these tumors consistent with G6P increase (Fig. 2E). We also found an up-regulation of GPI mRNA (4-fold, Fig. 2B), which encodes the glucose phosphate isomerase, a second glycolytic enzyme. In contrast, PKLR mRNA, which encodes the liver-specific pyruvate kinase that catalyzes the last irreversible step of glycolysis, was 3-fold decreased in HNF1α-mutated HCA (Fig. 2F). However, PKLR down-regulation may be balanced by a 2-fold increase of PKM2 gene that encodes the muscle-specific pyruvate kinase (Fig. 2F). Activation of the Citrate Shuttle—Besides the observed gluconeogenesis inhibition and glycolysis activation that predict an overproduction of pyruvate, a substrate for the mitochondrial synthesis of acetyl-CoA, the citrate shuttle system that exports acetyl-CoA into the cytosol was also activated in HNF1α-mutated HCA (Fig. 2A). Indeed, we found a 12-fold overexpression of ME1 mRNA, which encodes the malic enzyme, and a moderate 2-fold increase of MDH1 transcript, and ACLY mRNA encoding the ATP citrate lyase (ACL) was 3.7-fold overexpressed in HNF1α-mutated HCA (Fig. 2B). As ATP citrate lyase is crucial to provide the unique cytoplasmic source of the acetyl-CoA lipogenic precursor, we confirmed by Western blotting the ACL protein overexpression (Fig. 2C). Stimulation of the Fatty Acid Synthetic Pathway—As shown in Fig. 3A, the fatty acid synthetic pathway was also stimulated in HNF1α-mutated HCA. We found an increase of the acetyl-CoA carboxylase transcript (ACACA, 2.6-fold) and a strong overexpression of fatty acid synthase at both the transcriptional (FASN, 7-fold) and the protein level (FAS, Fig. 3, B and C). In the downstream steps of fatty acid elongation and desaturation, we also identified an increase in the mRNA level of three genes encoding elongases including ELOVL1 (3.4-fold), ELOVL2 (2.2-fold), and ELOVL5 (2.3-fold) and of three genes encoding desaturases such as SCD (6-fold), FADS1 (3-fold), and FADS2 (4.7-fold, Fig. 3B). Transcriptional Alteration of Glucido-lipidic Genes Defines an Expression Pattern That Is Specific of HNF1α-mutated HCA—To search whether the pattern of alteration in the expression of the glucido-lipidic genes was specific of HNF1α inactivation, we compared the expression profiles of the 19 aforementioned genes between HNF1α-mutated HCA and a group of non-mutated HCA containing or not containing steatosis, using quantitative RT-PCR. In this analysis, we also included steatotic non-tumor livers from different etiologies and non-steatotic non-tumor livers. We then performed a non-supervised analysis of the results using a hierarchical clustering algorithm to group the genes as well as the samples on the basis of similarity in their expression pattern. This analysis accurately classified all samples in two major clusters according to their HNF1α status (Fig. 4). Steatotic non-tumor livers and non-steatotic non-tumor livers were gathered with the non-HNF1α-mutated HCA. Gene cluster analysis identified two main groups corresponding to down-regulated (cluster A) and up-regulated (cluster B) genes in HNF1α-mutated HCA (Fig. 4). As expected, four out of five genes identified in cluster A had been previously characterized as HNF1α transactivated targets. Interestingly, all up-regulated genes have never been previously found regulated by HNF1α. Within cluster B, three subgroups of genes (subgroups 1, 2, and 3) showed a highly correlated overexpression in HNF1α-mutated HCA (supplemental Fig. 1). For example, ACLY level of expression was correlated to ACACA overexpression in 16 HNF1α-mutated HCA (Spearman r = 0.72, p = 0.001) (supplemental Fig. 1). In non-HNF1α-mutated liver tissues, only few genes demonstrated a significant transcriptional deregulation (supplemental Fig. 2). In steatotic non-tumor livers, expression of only two genes, PCK2 and ME1, followed an expression pattern as seen in HNF1α-mutated HCA (supplemental Fig. 2). In non-HNF1α-mutated steatotic HCA, nine genes involved in gluconeogenesis, glycolysis, and citrate shuttle were significantly deregulated when compared with the non-steatotic non-tumor livers (supplemental Fig. 2). One of these, GCK, showed an inversed pattern of deregulation when compared with the HNF1α-mutated HCA. However, except for a modest elevation in ELOVL1 transcript, expression of genes related to the fatty acid synthetic pathway were unchanged in this group of steatotic adenomas. Finally, in the group of non-HNF1α-mutated non-steatotic HCA, only four genes showed a significant change in expression pattern (FBP1, GPI, ACLY, ELOVL1). These results indicated that the patterns of expression of the 19 analyzed genes were specific to HNF1α inactivation. Down-regulations could be directly attributed to the loss of the classical HNF1α transactivation activity. In contrast, overexpressed genes demonstrated a coordinated activation, but none are known to date to be directly controlled by HNF1α. Activation of Lipogenesis in HNF1α-mutated HCA Is Independent of SREBP-1 and ChREBP Activation—Among the lipogenic genes whose expression was increased in HNF1α-mutated HCA, 8 out of 13 were previously described as SREBP-1c targets. However, using quantitative RT-PCR, we did not find any significant variation in the mRNA level of total SREBP-1 in HNF1α-mutated HCA when compared with non-tumor livers nor in SREBP-1a (Fig. 5A). Moreover, surprisingly, Western blotting studies revealed a decrease in the nuclear active form of SREBP-1 in the tumors when compared with their corresponding non-tumor liver (Fig. 5B), and the microarray study also found an increase in INSIG1 (insulin-induced gene 1) mRNA (2-fold, supplemental Table 1) whose product is known to inhibit SREBP-1 cleavage processing. We then looked for a potential alteration of ChREBP expression, another key transcriptional activator of fatty acid synthesis in the liver. RT-PCR results showed a significant decrease in the mRNA of CHREBP in HNF1α-mutated HCA (-1.8-fold, Fig. 5A), and Western blotting studies revealed a decrease of the protein level in the nucleus (Fig. 5B). Using the same samples where we demonstrated a repression of both SREBP-1 and ChREBP, we found an obvious overexpression of the FAS and GK proteins, which are known to be transactivated by these two transcription factors (Fig. 5B). Although unexpected, these results suggest that increased lipogenic gene expression found in HNF1α-mutated HCA is not related to SREBP-1 and ChREBP activation. FAS and GK overexpression were confirmed in three additional cases of HNF1α-mutated HCA (supplemental Fig. 3). In two of these three cases, ChREBP was decreased, and the nuclear active form of SREBP-1 was highly reduced or unchanged. In the remaining case, we found a mild elevation of ChREBP and nuclear SREBP-1 when compared with the non-tumoral counterpart. On the whole, we observed an increased expression of FAS and GK together with a clear repression of SREBP-1 and ChREBP in four out of six HNF1α-mutated HCA analyzed. Interestingly, no similar pattern of expression was found in the non-HNF1α-mutated HCA tested (supplemental Fig. 3). We also checked for the transcriptional level of other transcription factors that have been implicated in the regulation of glucido-lipidic metabolism (Fig. 5A). Except for a 1.9-fold elevation of PPARγ transcript, the mRNA level of FOXO1A (forkhead box O1A), PPARα, LXRα, and HNF4α was not significantly different in HNF1α-mutated HCA when compared with non-tumor livers (Fig. 5A). Finally, the RXR isotypes genes were not differentially expressed in the microarray analysis. Total Lipid Composition Is Modified in HNF1α-mutated HCA—We compared lipid profiles of four non-steatotic non-tumor livers with that of five HNF1α-mutated HCA. Total fatty acid content was 5.8-fold increased in HNF1α-mutated HCA (Fig. 6A), and the fatty acid profile was modified. Saturated (SFA) and monounsaturated (MUFA) fatty acids were 7- and 8-fold increased, respectively (Fig. 6A), as a consequence of the accumulation of all SFA and MUFA