Mammalian Target of Rapamycin Activation Impairs Hepatocytic Differentiation and Targets Genes Moderating Lipid Homeostasis and Hepatocellular Growth

The mammalian target of rapamycin (mTOR) pathway, a major regulator of translation, is frequently activated in hepatocellular carcinomas. We investigated the effects of mTOR activation in the human HepaRG cells, which possess potent hepatocytic differentiation capability. Differentiation of HepaRG cells into functional and polarized hepatocyte-like cells correlated with a decrease in mTOR and Akt activities. Stable cell lines expressing an activated mutant of mTOR were generated. Sustained activation of mTOR impaired the hepatocytic differentiation capability of these cells as shown by impaired formation of bile canaliculi, absence of polarity, and reduced secretion of A1-antitrypsin. An inhibitor of mTOR, rapamycin, was able to revert this phenotype. Furthermore , increased mTOR activity in HepaRG cells resulted in their resistance to the antiproliferative effects of transforming growth factor-B1. Profiling of polysome-bound transcripts indicated that activated mTOR specifically targeted genes posttranscriptionally regulated on hepatocytic differentiation. Three major biological networks targeted by activated mTOR were identified: (a) cell death associated with tumor necrosis factor superfamily members, IFNs and caspases; (b) lipid homeostasis associated with the transcription factors PPARA, PPARD, and retinoid X receptor B; and (c) liver development associated with CCAAT/enhancer binding protein A and hepatic mitogens. In conclusion, increased mTOR activity conferred a preneoplastic phenotype to the HepaRG cells by altering the translation of genes vital for establishing normal hepatic energy homeostasis and moderating hepato-cellular growth.


Introduction
Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer, and its incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infections.The overall prognosis for patients with HCC is dismal with few effective treatment options (1).Understanding the pathways involved in hepatocarcinogenesis is of particular interest because these factors may represent novel therapeutic targets for the treatment of patients with chronic liver diseases that predispose them to development of HCC.Specific signaling pathways, such as p53, Wnt/h-catenin, phosphatase and tensin homologue (PTEN)/ phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and the telomerase immortalization enzyme, are frequently dysregulated in HCC (2,3).The pathway of relevance to this study is the PTEN/PI3K/Akt/mTOR pathway.In mice, hepatocyte-specific PTEN deficiency results in HCCs (4).In other organs, tumor formation resulting from PTEN ablation is suppressed by treatment of the mice with the rapamycin derivative CCI-779, a specific inhibitor of the mTOR kinase (5,6).The ability of rapamycin to suppress tumor formation in PTENdeficient mice has been attributed to the mTOR dependence of PI3K signaling.mTOR stimulates protein translation through phosphorylation of 4E-BP1 and p70 S6 kinase (7,8).Overexpression of the activated phosphorylated form of mTOR was observed in 15% of human HCCs and increased expression of p70 S6 kinase was found in 45% of cases and positively correlated with tumor size (9).The aim of this study was to examine the role of activated mTOR in mediating the behavior of hepatocytic cells during neoplastic transformation.
The HepaRG cell line has been established from the nontumoral region of a resected HCV-associated HCC.These cells display bipotent differentiation-inducible properties and share some features with liver progenitor cells (10).Throughout differentiation, HepaRG cells evolve from a homogeneous, dedifferentiated, depolarized, epithelial phenotype showing no specific organization to a well structurally defined and polarized monolayer closely resembling those formed in primary human hepatocyte culture, with bright canaliculi-like structures (10).At the hepatocytic differentiated state, hepatocytic polarization markers such as ZO-1 and CD26 and liver-specific proteins such as albumin and transferrin, the glycolytic enzyme aldolase B, and enzymes involved in detoxification (CYP2E1, CYP3A4, and glutathione S-transferase a) are expressed at levels similar to those found in normal liver biopsies (10,11).Finally, iron storage and metabolism, typical features of mature hepatocytes, never observed in HCC, remain intact in HepaRG cells (12).Although HepaRG cells do bear chromosomal aberrations (11) and cannot be considered normal liver parenchymal cells with differentiation-inducible properties, they constitute a powerful model for studying the role of specific pathways on hepatocytic differentiation and for evaluating the consequences of their dysregulation on hepatocarcinogenesis.
To induce differentiation, a two-step procedure was used as previously described (10).Cells were seeded at a density of 4 Â 10 4 /cm2 and maintained for 2 weeks in the growth medium.Then, the culture medium was supplemented with 1% DMSO (Sigma) and 20 ng/mL epidermal growth factor (Peprotech) for 2 additional weeks.For reversion experiments, rapamycin (2 ng/mL) was added from day 3 postseeding until the end of the differentiation process.For transforming growth factor-h (TGF-h) treatment, cells were seeded at a density of 10 4 /cm 2 .After 24 h, TGF-h (Peprotech) was added at a concentration of 2.5 ng/mL in medium supplemented with 0.5% FCS.After 72 h, cells were analyzed with a FACScan analyzer equipped with the Cell Quest software (Becton Dickinson).Cell culture pictures were taken using a phase-contrast microscope (Nikon) equipped with the Metamorph software.Differentiation was evaluated morphologically by counting bile canaliculi (refringent area) at the intersection of two or three hepatocyte-like cells (10).
Polysome-bound RNA preparation.Before harvest, cycloheximide (100 Ag/mL) was added to the medium for 3 min.The medium was then removed and the cells were washed with ice-cold PBS containing 100 Ag/mL cycloheximide.The cells were then scraped, centrifuged at 800 Â g for 5 min at 4jC, and cytoplasmic RNA was obtained by lysing the cell pellet in 1 mL of polysome buffer containing 10 mmol/L Tris-HCl (pH 8.0), 140 mmol/L NaCl, 1.5 mmol/L MgCl 2 , 0.5% NP40, and a RNase inhibitor, RNasin (500 units/mL; Promega).After the removal of nuclei, the cytosolic supernatant was supplemented with 100 Ag/mL cycloheximide, 665 Ag/mL heparin, 20 mmol/L DTT, and 1 mmol/L phenylmethanesulfonyl fluoride.Mitochondria and membrane debris were removed by centrifugation, and postmitochondrial supernatant was overlaid onto a 15% to 40% sucrose gradient (13).Fractions (750 AL) were collected from the bottom of each gradient and deproteinated with 100 Ag of proteinase K in the presence of 1% SDS and 10 mmol/L EDTA.After acid phenol extraction, RNA integrity was controlled by electrophoresis analysis on 1.2% agarose gel.Densitometry (GelDoc, Bio-Rad) was used to determine fractions in which the 28S/18S ratio equals 2 (i.e., fractions corresponding to polysome-bound RNA).The polysome-bound RNA-containing fractions were pooled from each sucrose gradient according to the distribution profile.
Double-stranded cDNA and cRNA synthesis and microarray hybridization.Total RNA and polysomal RNAs were purified using the RNeasy mini-kit clean-up protocol (Qiagen).First-strand cDNA, doublestranded cDNA, and cRNA were synthesized, and cRNA was fragmented using Affymetrix kits and guidelines. 1 All cRNA final products were tested in terms of amount and integrity by Bioanalyzer (Agilent) before microarray hybridization.cRNA samples were processed on Affymetrix HGU133A arrays with strict adherence to the labeling, hybridization, and staining protocols provided by Affymetrix.GeneChip image analysis was done using GCOS v1.4 (Affymetrix).Probe-level analysis, preprocessing, and normalization steps were carried out using GeneTraffic 3.2.-11(Iobion Stratagene Microarray Analysis Software).
Data mining.The Ingenuity Pathway Analysis 2 was used to analyze selected probe sets obtained from the microarray data.Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base.The application uses a right-sided Fisher's exact test to identify networks that had higher odds ratio of containing significant genes.These genes, called Focus Genes, were then overlaid onto a global molecular network.Networks of these Focus Genes were then algorithmically generated.For the selected probe sets listed in Supplementary Tables S1  and S2, the corresponding data from the Total RNA data sets were extracted.Scatter plots were then drawn and the corresponding correlation coefficients were calculated.Microarray data have been deposited into the Array Express repository 3 under the accession no.E-MEXP-958.

Results
Down-regulation of the Akt-mTOR pathway on hepatocytic differentiation.We investigated the Akt-mTOR pathway activity in the human HepaRG cells on their differentiation into hepatocyte-like cells.The amount of phosphorylated Akt (Ser 473 residue) decreased on differentiation, whereas total Akt amounts remained unchanged, indicative of a reduction in Akt activity on differentiation.Amounts of total and Ser 2448 -phosphorylated mTOR also decreased throughout the differentiation process (Fig. 1A).Reduced phosphorylation of the mTOR substrates, 4E-BP1 and p70 S6 kinase, was also observed on differentiation (Fig. 1B).In conclusion, the activity of the Akt-mTOR pathway decreases during differentiation of HepaRG cells into hepatocytelike cells.
Generation of HepaRG cells expressing a constitutively activated mTOR mutant and initial characterization.To determine the role of mTOR in hepatocytic differentiation and the consequence of its dysregulation, we generated HepaRG clones expressing a constitutively activated mTOR mutant (DTOR).mTOR contains a kinase domain and a repressor domain.The kinase activity of mTOR increases by 3.5-to 10-fold when the repressor domain is deleted (14).DTORand backbone vector-bearing cells were generated and screened for the expression of the DTOR-AU1 tag fusion protein and of total mTOR (Fig. 2A).To confirm the functional activity of the transgene, 4E-BP1 phosphorylation was examined in proliferative and differentiated clones.Consistent with the induced enzymatic activity of DTOR, 4E-BP1 phosphorylation was higher in DTOR-bearing cells than in control cells at both the proliferative and differentiated states.In particular, 4E-BP1 remained in majority phosphorylated on differentiation in the DTOR-expressing cells (65 F 3%) whereas 4E-BP1 was only partially phosphorylated at the differentiated stage in control cells (39 F 2% for vector-bearing cells and 42 F 1% for HepaRG cells; Fig. 2B).
Increased cell size is associated with tumorigenesis.The size of DTOR-expressing cells was significantly higher at days 5 and 7 postseeding, compared with vector-containing cells (day 5, P = 0.017; day 7, P = 0.035) and parental HepaRG cells (day 5, P = 0.018; day 7, P = 0.029; Fig. 2C).At the seeding density used in this study, confluence is reached after 4 days.The cell size of DTOR-expressing cells was not significantly changed until day 7 and, therefore, DTOR cells, unlike control cells, did not seem to be sensitive to confluency.Cyclin D1 promotes mitogen-independent cell cycle progression in primary hepatocytes.To evaluate the capacity of DTOR-expressing cells to proliferate even at the end of the differentiation process, cyclin D1 mRNA expression levels were measured by semiquantitative reverse transcription-PCR (Fig. 2D).Cyclin D1 mRNA levels were significantly higher in DTORexpressing cells compared with controls.Taken together, these data suggest that increased mTOR activity results in a proliferative advantage in HepaRG cells on differentiation.
Impaired hepatocytic differentiation in #TOR-expressing HepaRG cells and its reversion by rapamycin.The differentiation capability along the hepatocytic lineage was analyzed in DTOR-expressing cells.Whereas no morphologic differences were noticed between proliferative cell lines, striking differences were observed at the end of the differentiation process (Fig. 3A).Differentiated hepatocytes display refractile cellular borders, dark cytosol, clearly delineated nuclei, and tridimensional polarization with the appearance of refringent circular canaliculi vertically.DTOR-expressing cells did not establish any structured monolayer in contrast to control cell lines, and none of the morphologic criteria described above was observed in DTOR cells in contrast to the two control cell lines.Bile canaliculi were counted in the three cell lines at the end of the differentiation process, indicating a quasi-absence of tridimensional polarization in DTOR-expressing cells [25 F 25/cm 2 in DTOR cells, 492 F 124/cm 2 in vectorcontaining cells (P = 0.021), and 342 F 51/cm 2 in HepaRG cells (P = 0.004); Fig. 3B].The capacity of hepatocytes to secrete plasmatic proteins in appropriate amounts correlates with their differentiation status.Levels of secreted a1-antitrypsin were measured in the three cell lines at the end of the differentiation protocol.The amount of a1-antitrypsin released in the supernatant by DTOR-expressing cells was 40.75 F 8% of the amount secreted by HepaRG cells, whereas the amount released by vectorbearing cells was 101 F 3% of the amount secreted by HepaRG cells (P < 0.001; Fig. 3C).
To further confirm that impaired differentiation of DTORbearing HepaRG cells was indeed due to increased mTOR kinase activity and to exclude any clonal effect, differentiation of DTORexpressing HepaRG cells was carried out in the presence of an inhibitor of mTOR, rapamycin, in two independent experiments.Morphologic evaluation showed an organized and polarized phenotype in rapamycin-treated DTOR cells (Fig. 4A), with a bile canaliculi density comparable to levels found in control cell lines [693 F 233/cm 2 in rapamycin-treated DTOR cells and 50 F 10/ cm 2 in vehicle-treated DTOR cells (P = 0.05); Fig. 4B; see also Fig. 3B].In conclusion, increased mTOR activity impairs the morphologic and biochemical hepatocytic differentiation of the HepaRG cells.
Loss of TGF-B responsiveness in #TOR-expressing HepaRG cells.TGF-h plays a critical role in the transition of stem cells or progenitor cells to a fully differentiated phenotype in the liver parenchyma and controls cell growth and apoptosis in the liver.Active TGF-h1 secretion levels were assessed in the DTOR-containing HepaRG cells and in control cells.Similar amounts of this cytokine (<70 pg/mL/d) were detected in the supernatant of all cell lines, independently of DTOR expression (data not shown).In comparison, conventional doses of TGF-h used in in vitro assays are between 2,000 and 5,000 pg/mL.Therefore, the capability of DTOR to modulate the antiproliferative effects of TGF-h1 was tested.No apoptosis was observed in any cell line on TGF-h1 treatment (data not shown).Phase-contrast microscopy analysis indicated that DTOR-expressing HepaRG cells were less susceptible to the acquisition of a spindle-shaped morphology, typical of TGF-h1-induced response (Fig. 5A).In addition, DTORexpressing cells were resistant to the antiproliferative effect of TGF-h1 compared with control cells with a proliferation inhibition rate of 9 F 2% in DTOR-expressing cells, 51 F 9% in vector-containing cells (P = 0.043), and 38 F 15% in HepaRG cells (P = 0.003; Fig. 5B).Finally, expression of the cell cycle inhibitor p21 (WAF1/CIP1) was induced on TGF-h1 treatment in control cell lines but not in DTOR-expressing cells (Fig. 5C).Taken together, these results suggest that increased mTOR activity leads to loss of TGF-h responsiveness in hepatocytes.
Comparative polysome-bound RNA profiling of #TORexpressing HepaRG cells and control cell lines on hepatocytic differentiation and data mining.To identify the mechanisms by which activated mTOR impairs hepatocytic differentiation and generates a preneoplastic phenotype in the HepaRG cells, profiling of total RNAs and polysome-bound RNAs was done in DTORexpressing cells and in control cells (vector-bearing and parental HepaRG) at the proliferative stage and at the end of the differentiation protocol using Affymetrix microarrays.mTOR is a regulator of translation and, therefore, we used polysome-bound RNA profiling for our analysis to detect changes occurring at both transcriptional and translational levels.We selected polysomebound transcripts significantly modified in control cells by at least 2-fold on differentiation in three independent experiments but not in DTOR cells (P V 0.05).These include 590 up-regulated (Supplementary Table S1) and 49 down-regulated (Supplementary Table S2) transcripts.
To investigate whether activation of mTOR was affecting transcripts regulated at the transcriptional level or at posttranscriptional levels on differentiation of control cells, total RNA fold changes in control cells were plotted against polysome-bound RNA fold changes in control cells for these selected transcripts.For the up-regulated transcripts, the slope of the regression curve calculated from all experimental values was 0.6222 (Fig. 6A).The slope of this curve is greater than zero, indicative of a positive correlation between polysome-bound RNA and total RNA fold change values.However, the correlation coefficient for this regression curve was 0.3798, showing a poor correlation between changes in the polysome-bound fractions and changes in total RNA on differentiation of HepaRG cells.Similarly, a poor correlation between changes in the polysome-bound fractions and changes in total RNA on differentiation of HepaRG cells was observed for the down-regulated genes, with a correlation coefficient of 0.0791 (Fig. 6B).These results suggest that mTOR activity is specifically targeting transcripts modified at a posttranscriptional level on hepatocytic differentiation of HepaRG cells.
The contents of the Supplementary Tables S1 and S2 were analyzed using the Ingenuity Systems Pathways Knowledge Base4 (March 2006 release).This database enables search for gene product interactions and annotations coming from curated data from publications and peer-reviewed resources.The Ingenuity pathway analysis identified two molecular networks generated from the up-regulated transcripts displaying 100% overlay between the selected regulated genes found in our study and the software-preselected members.The first network includes 35 genes associated with the proapoptotic tumor necrosis factor (TNF) superfamily-and caspase-related pathways and with the IFN system (Fig. 6C).It includes two members of the TNF superfamily, TNFSF10 [TNF-related apoptosis-inducing ligand (TRAIL)] and TNFSF13 (APRIL), and six members of the TNF receptor superfamily, TNFRSF1A, TNFRSF10C (TRAILR3 ), TNFRSF25 (death receptor 3, Apo3), TNFRSF5 (CD40), TNFRSF6B (decoy receptor 3), and TNFRSF11B.The other main components of this network are caspase-8 (CASP8) and caspase-9 (CASP9), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 1 (STAT1).Other proteins associated with increased susceptibility to apoptosis include CRADD, FADD, TRADD, MADD, IHPK2, sphingomyelin phosphodiesterase 1, and programmed cell death 8. Finally, two members of this network are IFN-inducible genes: 2 ¶,5 ¶-oligoadenylate synthetase-1 (OAS1) and IFI16.IFI16 showed the largest fold change (54.1-fold) and is also associated with proapoptotic functions, in addition to negative regulator of cell growth.In addition to the transcripts depicted in this network, several transcripts also involved in susceptibility to apoptosis included cell death activator CIDE-3, insulin-like growth factor (IGF)-binding protein 1, tuberous sclerosis complex protein 2, growth arrest-specific 2, and programmed cell death 4 (Table S1).
The second network is associated with lipid and carbohydrate metabolisms and includes 35 genes (Fig. 6D).These include the peroxisome proliferator-activated receptors a and y (PPARa and PPARy) and the retinoid X receptor h (RXRh), a modulator of PPARa activity.PPARs and RXRs induce the expression of proteins and enzymes involved in pyruvate metabolism, such as PDK2, PDK3, PDK4, and PDHA1, and in lipid metabolism such as HADHA, HADHB, C/EBPy, ACOX1, and FABP4.In addition to the transcripts depicted in this network, transcripts involved in lipid transport and/or metabolism included CCAAT/enhancer binding protein (C/EBP)-a, solute carrier family 27-member 3, sterol regulatory element-binding transcription factor 2, lipoic acid synthetase, low-density lipoprotein receptor-related protein 1, and apolipoprotein M (Table S1).
Remarkably, down-regulation of the growth factors vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), fibroblast growth factors 2 and 5 (FGF2 and FGF5), and PDGFB upon differentiation of the HepaRG cells was inhibited by DTOR (Table S2).
Taken together, these results indicate that DTOR affects the posttranscriptional regulation of genes vital for modulating sensitivity to apoptosis, lipid homeostasis, and hepatocellular growth.

Discussion
In this study, we used the human HepaRG cell line to increase our knowledge about the possible involvement of mTOR activation  in the development of HCC.HepaRG cells display potent hepatocytic differentiation-inducible properties and share some features with liver progenitor cells (10).The number of human liver progenitor cells correlates with the severity of liver diseases such as chronic viral hepatitis, alcoholic and nonalcoholic fatty liver diseases, which predispose patients to HCC.Activation of progenitor cells in these diseases suggests that they form a potential target cell population, precursors of HCC, and, indeed, a subtype of human HCCs express one or more markers of progenitor cells (15,16).
We showed that the activity of mTOR decreases on hepatocytic differentiation of the HepaRG cells and that sustained mTOR activation impairs their differentiation and their polarization status.DTOR-expressing cells were unable to settle tridimensional organization and polarization, mandatory for normal endocrine and exocrine functionality of the hepatocyte.In agreement with our observation that control HepaRG cells but not DTOR-expressing cells reached a mature hepatocytic functional state, induction of radixin and catenin-y1, genes involved in epithelium polarization and organization (17), was impaired by mTOR activation.Loss of hepatocytic and trabecular organization in the lobule is a hallmark of many HCCs and HCC grades have been defined according to these features (18).Another hallmark of many HCCs is their loss of responsiveness to TGF-h1.TGF-h1 has antiproliferative and proapoptotic properties in normal hepatocytes and in rat partial hepatectomy models.In our study, DTOR expression conferred resistance to TGF-h1-induced inhibition of proliferation.Recent studies suggested that mTOR abolishes TGF-h/ALK5-mediated Smad3 activation (19), in agreement with our observed antagonism between the Akt/mTOR pathway and TGF-h1 antitumorigenic function.
mTOR is a critical component of translational control (7).To identify the altered events in DTOR-expressing HepaRG cells leading to their preneoplastic phenotype, we analyzed polysomebound RNAs by microarray and selected changes occurring on differentiation in control cells but not in DTOR cells.Comparing changes of the selected transcripts at both the total RNA and polysome-bound RNA levels indicated that mTOR specifically targeted genes posttranscriptionally regulated on differentiation of HepaRG cells.mTOR impaired the up-regulation of a large number of members of the TNF/caspase transduction pathway.These include TNFSF10 (TRAIL) and caspase-8, known to play a crucial role in inducing apoptosis in human hepatocytes and HCCs (20).Caspase-8, a key mediator of death receptor-induced apoptosis, has previously been reported to be frequently inactivated by epigenetic silencing in many tumors.This network also shows that mTOR impaired the up-regulation of JAK2 and STAT1 and of IFN-induced proteins such as OAS1 or IFI16 (>50fold).There is currently no information on IFI16 in the liver but IFI16 is an essential mediator of growth inhibition in medullary thyroid carcinoma cells ( 21) and of p53 and p21 (WAF1/CIP1) functions (22).IFI16 expression is increased by TRAIL in breast carcinoma cells (23).Noteworthy, both up-regulated caspases identified (caspase-8 and caspase-9) belong to the initiator caspases family, whereas none of the members of the effector caspase family (caspase-3, caspase-6, and caspase-7; ref. 24) was affected, supporting the fact that control cells did not undergo apoptosis in culture.Numerous additional transcripts coding for antiproliferative, neoplastic transformation inhibitors and proapoptotic proteins were identified to be up-regulated in control cells on differentiation but not in DTOR-expressing cells.Normal hepatocytes are highly sensitive to cell death upon, for example,
drug-induced liver toxicity, and three-dimensional polarization sensitizes hepatocytes to Fas apoptotic signaling (25).Therefore, sustained activation of mTOR may contribute to neoplastic cell expansion by altering receptor-induced apoptosis sensitivity.Our data also suggest an important molecular cross-talk between the TRAIL and IFN pathways in liver progenitor cells.mTOR may induce resistance to TRAIL-and/or IFN-induced apoptosis.Defects in IFN signaling that result in loss of expression of IFNinducible proteins are associated with cellular immortalization, an important early event in the development of human cancer.mTOR activation impaired the induction of the transcription factors PPARa, PPARy, and RXRh and their target genes.The lipid-lowering function of PPARa occurs across a number of mammalian species, thus showing the essential role of this nuclear receptor in lipid homeostasis and normal liver function.Mice deficient in PPARa lack hepatic peroxisomal proliferation, have an impaired expression of several hepatic target genes, and show a massive accumulation of lipids in their livers (26).Ethanol impairs fatty acid catabolism in liver by blocking PPARamediated responses contributing to the development of alcoholic fatty liver, which can be overcome by PPARa agonists (27).HCV infection is also associated with altered expression and function of PPARa (28).PPARy also plays a role in lipid metabolism including cholesterol efflux and fatty acid oxidation (29,30); activates fat metabolism to prevent obesity (31); and regulates fatty acid synthesis, glucose metabolism, and insulin sensitivity (32).Interestingly, it has been reported that PPARy attenuates colon carcinogenesis (33).RXR heterodimers serve as key regulators in cholesterol homeostasis by governing reverse cholesterol transport from peripheral tissues, bile acid synthesis in liver, and cholesterol absorption in intestine (34).Liver-specific loss of function of retinoic acid leads to steatohepatitis and liver tumors in vivo (35).Therefore, sustained mTOR activity may contribute to the development of steatosis observed in most HCCs by impairing lipid homeostasis.
Another major target of DTOR is C/EBPa.This transcription factor regulates two aspects of hepatic terminal differentiation: induction of differentiation-specific genes and repression of mitogenesis (36)(37)(38).C/EBPa reduces HCC susceptibility in mice (39) and down-regulation of C/EBPa in HCC correlates with tumor size and progression (40).C/EBPa-deficient mice present with severely disturbed liver architecture with acinar formation in a pattern suggestive of either regenerating liver or HCC, abnormally active hepatocytic proliferation, impaired hepatic glycogen storage, and accumulation of lipids in the liver (41,42).These mice also show lowered plasma levels of free fatty acid, triglyceride, and cholesterol as well as marked changes in PPARa and apoliproteins (43).Therefore, by impairing induction of C/EBPa on hepatocytic differentiation, mTOR is affecting numerous vital liver-specific functions.
Dysregulation of pleiotropic growth factors and their receptors represent a central protumorigenic principle in human hepatocarcinogenesis. Especially the IGF, HGF, and TGF-h pathways, all found affected by activated mTOR in the HepaRG cells, contribute to proliferation, antiapoptosis, and invasive behavior of tumor cells.HGF is the primary agent promoting the proliferation and apoptosis resistance of mature hepatocytes.Serum HGF levels are strongly associated with liver diseases including insulin resistance and nonalcoholic steatohepatitis.Blockage of HGF suppresses HCC in mice by inhibiting tumor cell motility and angiogenesis (44).FGF2, VEGF, and PDGFB are potent mitogenic and angiogenic factors and stimulate tumor growth (45).Expression of VEGF and FGF2 is altered in patients with HCC and, interestingly, VEGF and FGF2 concentrations are elevated before the emergence of HCC (46).By maintaining high levels of expression of these factors on hepatocytic differentiation, mTOR may accelerate abnormal proliferation and angiogenesis.
In conclusion, we have shown that an enhanced activity of mTOR, as found in clinical HCC samples, is capable of preventing hepatocytic differentiation not only through inhibition of induction of C/EBPa but also by preventing hepatocytic polarization.In addition, sustained mTOR activity may lead to reduced susceptibility to TGF-h antiproliferative effects and to TRAIL/TNF-and IFN-induced apoptosis.Sustained mTOR activity may also lead to abnormal expression of genes modulating lipid homeostasis, hepatocellular growth, and angiogenesis.These effects, taken together, could contribute to the neoplastic transformation of these cells.Our study also suggests that combination therapy strategies aimed at overcoming TRAIL and IFN resistance and PPARa and PPARy defects may be effective for treatment of HCC with activated mTOR or PTEN deletion.

Figure 2 .
Figure 2. Generation of DTOR-expressing HepaRG cells and initial characterization.A, stable clones of HepaRG cells expressing the empty vector or DTOR were generated.The mTOR transgene expression was examined by Western blotting with antibodies against the AU1 tag or mTOR.B, phosphorylation status of 4E-BP1 in proliferative (P) and differentiated (D ) DTOR, vector, and HepaRG cells by Western blot with an antibody against 4E-BP1.Representative of three independent experiments.C, cell sizes for DTOR, vector, and HepaRG cells were measured by flow cytometry.Columns, mean of three independent experiments; bars, SD.D, cyclin D1 and h-actin transcripts at the differentiated state were amplified by PCR and the PCR products were resolved on a 2% agarose gel.

Figure 4 .
Figure 4. Reversion by rapamycin of the altered phenotype.DTOR-bearing cells were treated throughout the differentiation protocol with rapamycin (2 ng/mL) or vehicle alone.A, morphology.B, columns, mean bile canaliculi densities from three randomly selected fields and from two independent experiments; bars, SE.

Figure 3 .
Figure 3. Impaired hepatocytic differentiation of HepaRG cells expressing an activated mutant of mTOR.A, DTOR, control vector, and HepaRG parental cells were induced to differentiate as described in Materials and Methods.Pictures (magnification, Â40) were taken in three randomly selected fields at the proliferative (P ) and differentiated (D) stages.Representative of three independent experiments.B, bile canaliculi were counted from three randomly selected fields in three independent experiments.C, a1-antitrypsin levels were quantified by ELISA in the supernatant of each cell line at the differentiated stage and normalized by cell count.Columns, mean of three independent experiments; bars, SD.

Figure 5 .
Figure 5. Loss of TGF-h responsiveness in DTOR-expressing HepaRG cells.Cells were incubated with TGF-h1 for 3 d.A, pictures were taken from three randomly selected fields.B, cells were analyzed by fluorescence-activated cell sorting using the cell count function of the device.Columns, mean of three experiments; bars, SD.C, p21 (WAF1/CIP1) expression levels were assessed by immunoblotting.