Mammalian Target of Rapamycin Activation Impairs Hepatocytic

Research Article

Mammalian Target of Rapamycin Activation Impairs Hepatocytic Differentiation and Targets Genes Moderating Lipid Homeostasis and Hepatocellular Growth Romain Parent, Deepak Kolippakkam, Garrett Booth, and Laura Beretta

Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington

Abstract Wnt/h-catenin, phosphatase and tensin homologue (PTEN)/ The mammalian target of rapamycin (mTOR) pathway, a phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of major regulator of translation, is frequently activated in rapamycin (mTOR), and the telomerase immortalization enzyme, hepatocellular carcinomas. We investigated the effects of are frequently dysregulated in HCC (2, 3). The pathway of mTOR activation in the human HepaRGcells, which possess relevance to this study is the PTEN/PI3K/Akt/mTOR pathway. potent hepatocytic differentiation capability. Differentiation In mice, hepatocyte-specific PTEN deficiency results in HCCs (4). of HepaRGcells into functional and polarized hepatocyte-like In other organs, tumor formation resulting from PTEN ablation cells correlated with a decrease in mTOR and Akt activities. is suppressed by treatment of the mice with the rapamycin Stable cell lines expressing an activated mutant of mTOR were derivative CCI-779, a specific inhibitor of the mTOR kinase (5, 6). generated. Sustained activation of mTOR impaired the The ability of rapamycin to suppress tumor formation in PTEN- deficient mice has been attributed to the mTOR dependence of hepatocytic differentiation capability of these cells as shown by impaired formation of bile canaliculi, absence of polarity, PI3K signaling. mTOR stimulates protein translation through and reduced secretion of A1-antitrypsin. An inhibitor of phosphorylation of 4E-BP1 and p70 S6 kinase (7, 8). Over- mTOR, rapamycin, was able to revert this phenotype. Further- expression of the activated phosphorylated form of mTOR was more, increased mTOR activity in HepaRGcells resulted in observed in 15% of human HCCs and increased expression of their resistance to the antiproliferative effects of transforming p70 S6 kinase was found in 45% of cases and positively correlated growth factor-B1. Profiling of polysome-bound transcripts with tumor size (9). The aim of this study was to examine the role indicated that activated mTOR specifically targeted genes of activated mTOR in mediating the behavior of hepatocytic cells posttranscriptionally regulated on hepatocytic differentiation. during neoplastic transformation. Three major biological networks targeted by activated mTOR The HepaRG cell line has been established from the nontumoral were identified: (a) cell death associated with tumor necrosis region of a resected HCV-associated HCC. These cells display factor superfamily members, IFNs and caspases; (b) lipid bipotent differentiation-inducible properties and share some homeostasis associated with the transcription factors features with liver progenitor cells (10). Throughout differentiation, PPARA,PPARD, and retinoid X receptor B; and (c) liver HepaRG cells evolve from a homogeneous, dedifferentiated, development associated with CCAAT/enhancer binding pro- depolarized, epithelial phenotype showing no specific organization tein A and hepatic mitogens. In conclusion, increased mTOR to a well structurally defined and polarized monolayer closely activity conferred a preneoplastic phenotype to the HepaRG resembling those formed in primary human hepatocyte culture, cells by altering the translation of genes vital for establishing with bright canaliculi-like structures (10). At the hepatocytic normal hepatic energy homeostasis and moderating hepato- differentiated state, hepatocytic polarization markers such as ZO-1 cellular growth. [Cancer Res 2007;67(9):4337–45] 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 Introduction a) are expressed at levels similar to those found in normal liver Hepatocellular carcinoma (HCC) is the fifth most common cause biopsies (10, 11). Finally, iron storage and metabolism, typical of cancer, and its incidence is increasing worldwide because of the features of mature hepatocytes, never observed in HCC, remain dissemination of hepatitis B and C virus infections. The overall intact in HepaRG cells (12). Although HepaRG cells do bear prognosis for patients with HCC is dismal with few effective chromosomal aberrations (11) and cannot be considered normal treatment options (1). Understanding the pathways involved in liver parenchymal cells with differentiation-inducible properties, hepatocarcinogenesis is of particular interest because these factors they constitute a powerful model for studying the role of specific may represent novel therapeutic targets for the treatment of pathways on hepatocytic differentiation and for evaluating the patients with chronic liver diseases that predispose them to consequences of their dysregulation on hepatocarcinogenesis. development of HCC. Specific signaling pathways, such as p53, Materials and Methods Cell culture and generation of #TOR and control HepaRGclones. Note: Supplementary data for this article are available at Cancer Research Online The HepaRG cell line was cultured in William’s E medium (Invitrogen) (http://cancerres.aacrjournals.org/). supplemented with 10% FCS (Cellgro), 100 units/mL penicillin, 100 Ag/mL À Requests for reprints: Laura Beretta, Public Health Sciences Division, Fred streptomycin (Invitrogen), 5 Ag/mL insulin (Sigma), and 5 Â 10 5 mol/L Hutchinson Cancer Research Center, Seattle, WA 98109. Phone: 206-667-7080; Fax: 206- hydrocortisone hemisuccinate (Sigma). To generate DTOR and control 667-2537; E-mail: [email protected]. Â 6 A I2007 American Association for Cancer Research. HepaRG clones, 2 10 cells were transfected with 2 g of pCDNA3 plasmid doi:10.1158/0008-5472.CAN-06-3640 (Invitrogen), bearing or not the DTOR insert (provided by Dr. Edinger), www.aacrjournals.org 4337 Cancer Res 2007; 67: (9). May 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research using Lipofectamine 2000 (Invitrogen). After 24 h, 600 Ag/mL G418 RNeasy mini-kit clean-up protocol (Qiagen). First-strand cDNA, double- (Invitrogen) was added to the culture medium, allowing selection of stranded cDNA, and cRNA were synthesized, and cRNA was fragmented G418-resistant clones. using Affymetrix kits and guidelines.1 All cRNA final products were tested in To induce differentiation, a two-step procedure was used as previously terms of amount and integrity by Bioanalyzer (Agilent) before microarray described (10). Cells were seeded at a density of 4 Â 104/cm2 and hybridization. cRNA samples were processed on Affymetrix HGU133A maintained for 2 weeks in the growth medium. Then, the culture medium arrays with strict adherence to the labeling, hybridization, and staining was supplemented with 1% DMSO (Sigma) and 20 ng/mL epidermal growth protocols provided by Affymetrix. GeneChip image analysis was done using factor (Peprotech) for 2 additional weeks. For reversion experiments, GCOS v1.4 (Affymetrix). Probe-level analysis, preprocessing, and normali- rapamycin (2 ng/mL) was added from day 3 postseeding until the end of the zation steps were carried out using GeneTraffic 3.2.-11 (Iobion Stratagene differentiation process. For transforming growth factor-h (TGF-h) treat- Microarray Analysis Software). ment, cells were seeded at a density of 104/cm2. After 24 h, TGF-h Data mining. The Ingenuity Pathway Analysis2 was used to analyze (Peprotech) was added at a concentration of 2.5 ng/mL in medium selected probe sets obtained from the microarray data. Each gene identifier supplemented with 0.5% FCS. After 72 h, cells were analyzed with a was mapped to its corresponding gene object in the Ingenuity Pathways FACScan analyzer equipped with the Cell Quest software (Becton Knowledge Base. The application uses a right-sided Fisher’s exact test to Dickinson). Cell culture pictures were taken using a phase-contrast identify networks that had higher odds ratio of containing significant genes. microscope (Nikon) equipped with the Metamorph software. Differentiation These genes, called Focus Genes, were then overlaid onto a global molecular was evaluated morphologically by counting bile canaliculi (refringent area) network. Networks of these Focus Genes were then algorithmically at the intersection of two or three hepatocyte-like cells (10). generated. For the selected probe sets listed in Supplementary Tables S1 Western blotting and ELISA. Cells were lysed in 50 mmol/L Tris-HCl and S2, the corresponding data from the Total RNA data sets were (pH 8), 150 mmol/L NaCl, 0.1% SDS, 1% NP40, supplemented with protease extracted. Scatter plots were then drawn and the corresponding correlation inhibitors (Complete, Roche). Twenty micrograms of proteins were resolved coefficients were calculated. Microarray data have been deposited into the on 5%, 10%, or 15% SDS-polyacrylamide gels and electrotransferred onto Array Express repository3 under the accession no. E-MEXP-958. nitrocellulose membrane (Amersham Biosciences). Equal loadings and homogeneous blotting were confirmed by Ponceau red staining. Membranes Results were blocked with 5% nonfat milk in TBS and incubated with primary antibodies overnight. The following antibodies were used: anti–phospho- Down-regulation of the Akt-mTOR pathway on hepatocytic Akt, anti-Akt, anti–phospho-mTOR, anti-mTOR, anti–4E-BP1, anti–p70 S6 differentiation. We investigated the Akt-mTOR pathway activity kinase (Cell Signaling Technologies; dilution, 1/2,000); anti-AU1 tag (Babco; in the human HepaRG cells on their differentiation into dilution, 1/2,000); anti-p21(WAF1/CIP1) (PharMingen; dilution, 1/2,000). hepatocyte-like cells. The amount of phosphorylated Akt (Ser473 Horseradish peroxidase–conjugated immunoglobulins (Dako) were used residue) decreased on differentiation, whereas total Akt amounts as secondary antibodies and proteins were visualized with enhanced remained unchanged, indicative of a reduction in Akt activity on chemiluminescence reagent (Amersham Biosciences). a1-Antitrypsin quan- differentiation. Amounts of total and Ser2448-phosphorylated titation in the supernatant was done using an ELISA with an a1-antitrypsin mTOR also decreased throughout the differentiation process capture antibody (Antibody Shop), a rabbit anti–a1-antitrypsin detection antibody (Dako), and a donkey anti-rabbit immunoglobulin-horseradish (Fig. 1A). Reduced phosphorylation of the mTOR substrates, 4E- peroxidase conjugate (Jackson ImmunoResearch). BP1 and p70 S6 kinase, was also observed on differentiation Reverse transcription-PCR. One microgram of DNase I–treated (Fig. 1B). In conclusion, the activity of the Akt-mTOR pathway (Promega) total RNA was reverse transcribed using Moloney murine decreases during differentiation of HepaRG cells into hepatocyte- leukemia virus reverse transcriptase and random hexamers (Invitrogen) like cells. for 50 min at 42jC. Primers for cyclin D1 and actin were 5¶-GGATGCTGG- Generation of HepaRG cells expressing a constitutively AGGTCTGCGA-3¶ and 5¶-AGAGGCCACGAACATGCAAG-3¶,5¶-TGGACTTC- activated mTOR mutant and initial characterization. To ¶ ¶ ¶ GAGCAAGAGATGG-3 and 5 -GGAAGGAAGGCTGGAAGAGTG-3 , respec- determine the role of mTOR in hepatocytic differentiation and tively. PCR cycle numbers for cyclin D1 and actin were 35 and 23 cycles, the consequence of its dysregulation, we generated HepaRG clones respectively. expressing a constitutively activated mTOR mutant (DTOR). mTOR Polysome-bound RNA preparation. Before harvest, cycloheximide (100 Ag/mL) was added to the medium for 3 min. The medium was then contains a kinase domain and a repressor domain. The kinase removed and the cells were washed with ice-cold PBS containing 100 Ag/mL activity of mTOR increases by 3.5- to 10-fold when the repressor cycloheximide. The cells were then scraped, centrifuged at 800 Â g for domain is deleted (14). DTOR- and backbone vector–bearing cells 5 min at 4jC, and cytoplasmic RNA was obtained by lysing the cell pellet were generated and screened for the expression of the DTOR-AU1 in 1 mL of polysome buffer containing 10 mmol/L Tris-HCl (pH 8.0), tag fusion protein and of total mTOR (Fig. 2A). To confirm the 140 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.5% NP40, and a RNase inhibitor, functional activity of the transgene, 4E-BP1 phosphorylation was RNasin (500 units/mL; Promega). After the removal of nuclei, the examined in proliferative and differentiated clones. Consistent with cytosolic supernatant was supplemented with 100 Ag/mL cycloheximide, the induced enzymatic activity of DTOR, 4E-BP1 phosphorylation A 665 g/mL heparin, 20 mmol/L DTT, and 1 mmol/L phenylmethane- was higher in DTOR-bearing cells than in control cells at both the sulfonyl fluoride. Mitochondria and membrane debris were removed by proliferative and differentiated states. In particular, 4E-BP1 centrifugation, and postmitochondrial supernatant was overlaid onto a A remained in majority phosphorylated on differentiation in the 15% to 40% sucrose gradient (13). Fractions (750 L) were collected from F the bottom of each gradient and deproteinated with 100 Ag of proteinase DTOR-expressing cells (65 3%) whereas 4E-BP1 was only K in the presence of 1% SDS and 10 mmol/L EDTA. After acid phenol partially phosphorylated at the differentiated stage in control cells extraction, RNA integrity was controlled by electrophoresis analysis on (39 F 2% for vector-bearing cells and 42 F 1% for HepaRG cells; 1.2% agarose gel. Densitometry (GelDoc, Bio-Rad) was used to determine Fig. 2B). 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. 1 http://www.affymetrix.com/support/technical/technotesmain.affx Double-stranded cDNA and cRNA synthesis and microarray 2 https://analysis.ingenuity.com hybridization. Total RNA and polysomal RNAs were purified using the 3 http://www.ebi.ac.uk/arrayexpress

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Increased cell size is associated with tumorigenesis. The size of containing HepaRG cells and in control cells. Similar amounts of DTOR-expressing cells was significantly higher at days 5 and 7 this cytokine (<70 pg/mL/d) were detected in the supernatant of postseeding, compared with vector-containing cells (day 5, P = all cell lines, independently of DTOR expression (data not shown). 0.017; day 7, P = 0.035) and parental HepaRG cells (day 5, P = 0.018; In comparison, conventional doses of TGF-h used in in vitro day 7, P = 0.029; Fig. 2C). At the seeding density used in this study, assays are between 2,000 and 5,000 pg/mL. Therefore, the confluence is reached after 4 days. The cell size of DTOR-expressing capability of DTOR to modulate the antiproliferative effects of cells was not significantly changed until day 7 and, therefore, DTOR TGF-h1 was tested. No apoptosis was observed in any cell line on cells, unlike control cells, did not seem to be sensitive to TGF-h1 treatment (data not shown). Phase-contrast microscopy confluency. Cyclin D1 promotes mitogen-independent cell cycle analysis indicated that DTOR-expressing HepaRG cells were less progression in primary hepatocytes. To evaluate the capacity of susceptible to the acquisition of a spindle-shaped morphology, DTOR-expressing cells to proliferate even at the end of the typical of TGF-h1–induced response (Fig. 5A). In addition, DTOR- differentiation process, cyclin D1 mRNA expression levels were expressing cells were resistant to the antiproliferative effect of measured by semiquantitative reverse transcription-PCR (Fig. 2D). TGF-h1 compared with control cells with a proliferation Cyclin D1 mRNA levels were significantly higher in DTOR- inhibition rate of 9 F 2% in DTOR-expressing cells, 51 F 9% in expressing cells compared with controls. Taken together, these vector-containing cells (P = 0.043), and 38 F 15% in HepaRG cells data suggest that increased mTOR activity results in a proliferative (P = 0.003; Fig. 5B). Finally, expression of the cell cycle inhibitor advantage in HepaRG cells on differentiation. p21(WAF1/CIP1) was induced on TGF-h1 treatment in control cell Impaired hepatocytic differentiation in #TOR-expressing lines but not in DTOR-expressing cells (Fig. 5C). Taken together, HepaRGcells and its reversion by rapamycin. The differenti- these results suggest that increased mTOR activity leads to loss of ation capability along the hepatocytic lineage was analyzed in TGF-h responsiveness in hepatocytes. DTOR-expressing cells. Whereas no morphologic differences were Comparative polysome-bound RNA profiling of #TOR- noticed between proliferative cell lines, striking differences were expressing HepaRGcells and control cell lines on hepatocytic observed at the end of the differentiation process (Fig. 3A). differentiation and data mining. To identify the mechanisms by Differentiated hepatocytes display refractile cellular borders, dark which activated mTOR impairs hepatocytic differentiation and cytosol, clearly delineated nuclei, and tridimensional polarization generates a preneoplastic phenotype in the HepaRG cells, profiling with the appearance of refringent circular canaliculi vertically. of total RNAs and polysome-bound RNAs was done in DTOR- DTOR-expressing cells did not establish any structured monolayer expressing cells and in control cells (vector-bearing and parental in contrast to control cell lines, and none of the morphologic HepaRG) at the proliferative stage and at the end of the criteria described above was observed in DTOR cells in contrast differentiation protocol using Affymetrix microarrays. mTOR is a 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/cm2 in DTOR cells, 492 F 124/cm2 in vector- containing cells (P = 0.021), and 342 F 51/cm2 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 vector- bearing cells was 101 F 3% of the amount secreted by HepaRG cells (P < 0.001; Fig. 3C). To further confirm that impaired differentiation of DTOR- bearing HepaRG cells was indeed due to increased mTOR kinase activity and to exclude any clonal effect, differentiation of DTOR- expressing 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/cm2 in rapamycin-treated DTOR cells and 50 F 10/ cm2 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. B # Figure 1. Down-regulation of the Akt/mTOR pathway on hepatocytic Loss of TGF- responsiveness in TOR-expressing HepaRG differentiation. Differentiation of HepaRG cells was induced as described in cells. TGF-h plays a critical role in the transition of stem cells or Materials and Methods. Expression levels of phospho-Akt (P-Akt), Akt, progenitor cells to a fully differentiated phenotype in the liver phospho-mTOR (P-mTOR), and mTOR (A) and of phospho-4E-BP1 (P-4E-BP1), 4E-BP1, phospho-p70 S6 kinase (P-p70S6k), and p70 S6 kinase parenchyma and controls cell growth and apoptosis in the liver. (B) were assessed by immunoblotting. P, proliferative stage; D, differentiated Active TGF-h1 secretion levels were assessed in the DTOR- stage. Representative of three independent experiments. www.aacrjournals.org 4339 Cancer Res 2007; 67: (9). May 1, 2007

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. regulator of translation and, therefore, we used polysome-bound The contents of the Supplementary Tables S1 and S2 were RNA profiling for our analysis to detect changes occurring at both analyzed using the Ingenuity Systems Pathways Knowledge Base4 transcriptional and translational levels. We selected polysome- (March 2006 release). This database enables search for gene bound transcripts significantly modified in control cells by at least product interactions and annotations coming from curated data 2-fold on differentiation in three independent experiments but not from publications and peer-reviewed resources. The Ingenuity in DTOR cells (P V 0.05). These include 590 up-regulated pathway analysis identified two molecular networks generated (Supplementary Table S1) and 49 down-regulated (Supplementary from the up-regulated transcripts displaying 100% overlay Table S2) transcripts. between the selected regulated genes found in our study and To investigate whether activation of mTOR was affecting the software-preselected members. The first network includes 35 transcripts regulated at the transcriptional level or at posttran- genes associated with the proapoptotic tumor necrosis factor scriptional levels on differentiation of control cells, total RNA fold (TNF) superfamily– and caspase-related pathways and with the changes in control cells were plotted against polysome-bound IFN system (Fig. 6C). It includes two members of the TNF RNA fold changes in control cells for these selected transcripts. superfamily, TNFSF10 [TNF-related apoptosis-inducing ligand For the up-regulated transcripts, the slope of the regression curve (TRAIL)] and TNFSF13 (APRIL), and six members of the TNF calculated from all experimental values was 0.6222 (Fig. 6A). The receptor superfamily, TNFRSF1A, TNFRSF10C (TRAILR3), slope of this curve is greater than zero, indicative of a positive TNFRSF25 (death receptor 3, Apo3), TNFRSF5 (CD40), TNFRSF6B correlation between polysome-bound RNA and total RNA fold (decoy receptor 3), and TNFRSF11B. The other main components change values. However, the correlation coefficient for this of this network are caspase-8 (CASP8) and caspase-9 (CASP9), regression curve was 0.3798, showing a poor correlation between Januskinase2 (JAK2), and signal transducer and activator of changes in the polysome-bound fractions and changes in total transcription 1 (STAT1). Other proteins associated with increased RNA on differentiation of HepaRG cells. Similarly, a poor susceptibility to apoptosis include CRADD, FADD, TRADD, MADD, correlation between changes in the polysome-bound fractions IHPK2, sphingomyelin phosphodiesterase 1, and programmed cell and changes in total RNA on differentiation of HepaRG cells was death 8. Finally, two members of this network are IFN-inducible observed for the down-regulated genes, with a correlation genes: 2¶,5¶-oligoadenylate synthetase-1 (OAS1) and IFI16. IFI16 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. 4 http://www.ingenuity.com

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. mTOR and Hepatocytic Differentiation 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

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.

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 apolipo- protein 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 Figure 3. Impaired hepatocytic differentiation of HepaRG cells expressing posttranscriptional regulation of genes vital for modulating A, an activated mutant of mTOR. DTOR, control vector, and HepaRG parental sensitivity to apoptosis, lipid homeostasis, and hepatocellular cells were induced to differentiate as described in Materials and Methods. Pictures (magnification, Â40) were taken in three randomly selected fields at growth. 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 Discussion quantified by ELISA in the supernatant of each cell line at the differentiated stage and normalized by cell count. Columns, mean of three independent In this study, we used the human HepaRG cell line to increase experiments; bars, SD. our knowledge about the possible involvement of mTOR activation www.aacrjournals.org 4341 Cancer Res 2007; 67: (9). May 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research in the development of HCC. HepaRG cells display potent mTOR is a critical component of translational control (7). To hepatocytic differentiation–inducible properties and share some identify the altered events in DTOR-expressing HepaRG cells features with liver progenitor cells (10). The number of human liver leading to their preneoplastic phenotype, we analyzed polysome- progenitor cells correlates with the severity of liver diseases such bound RNAs by microarray and selected changes occurring on as chronic viral hepatitis, alcoholic and nonalcoholic fatty liver differentiation in control cells but not in DTOR cells. Comparing diseases, which predispose patients to HCC. Activation of changes of the selected transcripts at both the total RNA and progenitor cells in these diseases suggests that they form a polysome-bound RNA levels indicated that mTOR specifically potential target cell population, precursors of HCC, and, indeed, a targeted genes posttranscriptionally regulated on differentiation subtype of human HCCs express one or more markers of of HepaRG cells. mTOR impaired the up-regulation of a large progenitor cells (15, 16). number of members of the TNF/caspase transduction pathway. We showed that the activity of mTOR decreases on hepatocytic These include TNFSF10 (TRAIL) and caspase-8, known to play a differentiation of the HepaRG cells and that sustained mTOR crucial role in inducing apoptosis in human hepatocytes and activation impairs their differentiation and their polarization HCCs (20). Caspase-8, a key mediator of death receptor–induced status. DTOR-expressing cells were unable to settle tridimensional apoptosis, has previously been reported to be frequently organization and polarization, mandatory for normal endocrine inactivated by epigenetic silencing in many tumors. This network and exocrine functionality of the hepatocyte. In agreement with our also shows that mTOR impaired the up-regulation of JAK2 and observation that control HepaRG cells but not DTOR-expressing STAT1 and of IFN-induced proteins such as OAS1 or IFI16 (>50- cells reached a mature hepatocytic functional state, induction of fold). There is currently no information on IFI16 in the liver but radixin and catenin-y1, genes involved in epithelium polarization IFI16 is an essential mediator of growth inhibition in medullary and organization (17), was impaired by mTOR activation. Loss of thyroid carcinoma cells (21) and of p53 and p21(WAF1/CIP1) hepatocytic and trabecular organization in the lobule is a hallmark functions (22). IFI16 expression is increased by TRAIL in breast of many HCCs and HCC grades have been defined according to carcinoma cells (23). Noteworthy, both up-regulated caspases these features (18). Another hallmark of many HCCs is their loss of identified (caspase-8 and caspase-9) belong to the initiator responsiveness to TGF-h1. TGF-h1 has antiproliferative and caspases family, whereas none of the members of the effector proapoptotic properties in normal hepatocytes and in rat partial caspase family (caspase-3, caspase-6, and caspase-7; ref. 24) was hepatectomy models. In our study, DTOR expression conferred affected, supporting the fact that control cells did not undergo resistance to TGF-h1–induced inhibition of proliferation. Recent apoptosis in culture. Numerous additional transcripts coding for studies suggested that mTOR abolishes TGF-h/ALK5–mediated antiproliferative, neoplastic transformation inhibitors and proa- Smad3 activation (19), in agreement with our observed antagonism poptotic proteins were identified to be up-regulated in control between the Akt/mTOR pathway and TGF-h1 antitumorigenic cells on differentiation but not in DTOR-expressing cells. Normal function. hepatocytes are highly sensitive to cell death upon, for example,

Figure 5. Loss of TGF-h responsiveness in DTOR-expressing HepaRG cells. Cells were incubated with TGF-h1for3d. 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.

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Figure 6. Correlation between total RNA and polysomal RNA fold changes and Ingenuity pathway analysis. Scatter plots were drawn for the selected 615 up-regulated probe sets (A) and the selected 49 down-regulated probe sets (B) between the log-transformed polysome-bound RNA fold changes and the corresponding total RNA fold changes. Dottedline, total/polysome-bound RNA ratio of 1 (slope = 1). Solidline, regression curve calculated from all plots. C and D, the identified networks are represented as nodes displayed using various shapes that represent the functional class of the gene product and lines/arrows displayed with various labels that describe the specific relationship between the nodes. Gene abbreviation is located within the symbol. The significance of the shapes of the symbols, and the letters mentioned between nodes can be found in the Ingenuity website (http://www.ingenuity.com). Solidlines, direct interaction. Dottedlines, indirect interaction. An asterisk appears next to any gene for which the input file contained more than one identifier. C, a total of 35 differentially expressed genes were revealed in this network with a highly significant score of 50. Gene symbols found in this network: ADM, adrenomedullin; CASP8, caspase-8, apoptosis-related cysteine protease; CASP9, caspase-9, apoptosis-related cysteine protease; TNFRSF5, TNF receptor superfamily, member 5 or CD40; CRADD, CASP2 and RIPK1 domain containing adaptor with death domain; CTNND1, catenin (cadherin-associated protein), y1; DHFR, dihydrofolate reductase; ENO1, enolase 1, (a); FADD, Fas (TNFRSF6)-associated via death domain; FAF1, Fas (TNFRSF6)-associated factor 1; IFI16, IFN, g-inducible protein 16; IHPK2, inositol hexaphosphate kinase 2; JAK2, Janus kinase 2; MADD, mitogen-activated protein kinase activating death domain; OAS1, 2¶,5¶-oligoadenylate synthetase 1; PDCD8, programmed cell death 8; PDE4A, phosphodiesterase 4A, cyclic AMP-specific; PECAM1, platelet/endothelial cell adhesion molecule; PIAS1, protein inhibitor of activated STAT, 1; PIP5A1C, phosphatidylinositol-4-phosphate 5-kinase, type I, g; PTBP1, polypyrimidine tract-binding protein 1; PTPN6, protein tyrosine phosphatase, nonreceptor type 6; RBM5, RNA-binding motif protein 5; SMPD1, sphingomyelin phosphodiesterase 1, acid lysosomal; SRPX, sushi-repeat-containing protein, X-linked; STAT1, signal transducer and activator of transcription 1; TCEB2, transcription elongation factor B (SIII), polypeptide 2; TNFRSF25, TNF receptor superfamily, member 25; TNFRSF10C, TNF receptor superfamily, member 10c, decoy; TNFRSF11B, TNF receptor superfamily, member 11b; TNFRSF1A, TNF receptor superfamily, member 1A; TNFRSF6B, TNF receptor superfamily, member 6b, decoy; TNFSF10, TNF (ligand) superfamily, member 10 (TRAIL); TNFSF13, TNF (ligand) superfamily, member 13 (APRIL); TRADD, TNFRSF1A-associated via death domain. D, a total of 35 differentially expressed genes were revealed in this network with a highly significant score of 50. Gene symbols found in this network: ACOX1, acyl-CoA oxidase 1; AKAP13, akinase(PRKA)anchorprotein13;ARNT, aryl hydrocarbon receptor nuclear translocator; C19ORF29, chromosome 19 open reading frame 29; CEBPD, CCAAT/enhancer binding protein, y; CITED2, CREB binding protein/p300-interacting transactivator, 2; CPT2, carnitine palmitoyltransferase II; DDIT4, DNA-damage-inducible transcript 4; EFNA1, ephrin-A1; FABP4, fatty acid–binding protein 4; FGB, fibrinogen h chain; FKBP1A, FK506 binding protein 1A; FN1, fibronectin 1; FRAP1, FK506 binding protein 12-rapamycin associated protein 1; GLUD1, glutamate dehydrogenase 1; GPHN, gephyrin; HADHA, hydroxyacyl-CoA dehydrogenase/3- ketoacyl-CoA thiolase/enoyl-CoA hydratase, a subunit; HADHB, hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, h subunit; IL1R1, interleukin-1 receptor, type I; MIA, melanoma inhibitory activity; MLYCD, malonyl-CoA decarboxylase; NAB2, nuclear polyadenylated RNA-binding protein; PDHA1, pyruvate dehydrogenase (lipoamide) a1; PDK2, pyruvate dehydrogenase kinase, isoenzyme 2; PDK3, pyruvate dehydrogenase kinase, isoenzyme 3; PDK4, pyruvate dehydrogenase kinase, isoenzyme 4; PPARA, peroxisome proliferator–activated receptor, a; PPARD, peroxisome proliferator–activated receptor, y; PRKAR1A, protein kinase, cyclic AMP-dependent, regulatory, type I, a; PRKAR2A, protein kinase, cyclic AMP-dependent, regulatory, type II, a; RHEB, Ras homologue enriched in brain; RXRB, retinoid X receptor h; ST6GAL1, ST6 h-galactosamide a-2,6-sialyltranferase 1; TCF4, transcription factor 4; TSC2, tuberous sclerosis 2. www.aacrjournals.org 4343 Cancer Res 2007; 67: (9). May 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research drug-induced liver toxicity, and three-dimensional polarization show lowered plasma levels of free fatty acid, triglyceride, and sensitizes hepatocytes to Fas apoptotic signaling (25). Therefore, cholesterol as well as marked changes in PPARa and apoliproteins sustained activation of mTOR may contribute to neoplastic cell (43). Therefore, by impairing induction of C/EBPa on hepatocytic expansion by altering receptor-induced apoptosis sensitivity. Our differentiation, mTOR is affecting numerous vital liver-specific data also suggest an important molecular cross-talk between the functions. TRAIL and IFN pathways in liver progenitor cells. mTOR may Dysregulation of pleiotropic growth factors and their receptors induce resistance to TRAIL- and/or IFN-induced apoptosis. represent a central protumorigenic principle in human hepato- Defects in IFN signaling that result in loss of expression of IFN- carcinogenesis. Especially the IGF, HGF, and TGF-h pathways, all inducible proteins are associated with cellular immortalization, an found affected by activated mTOR in the HepaRG cells, contribute important early event in the development of human cancer. to proliferation, antiapoptosis, and invasive behavior of tumor cells. mTOR activation impaired the induction of the transcription HGF is the primary agent promoting the proliferation and factors PPARa,PPARy, and RXRh and their target genes. The apoptosis resistance of mature hepatocytes. Serum HGF levels lipid-lowering function of PPARa occurs across a number of are strongly associated with liver diseases including insulin mammalian species, thus showing the essential role of this resistance and nonalcoholic steatohepatitis. Blockage of HGF nuclear receptor in lipid homeostasis and normal liver function. suppresses HCC in mice by inhibiting tumor cell motility and Mice deficient in PPARa lack hepatic peroxisomal proliferation, angiogenesis (44). FGF2, VEGF, and PDGFB are potent mitogenic have an impaired expression of several hepatic target genes, and and angiogenic factors and stimulate tumor growth (45). show a massive accumulation of lipids in their livers (26). Ethanol Expression of VEGF and FGF2 is altered in patients with HCC impairs fatty acid catabolism in liver by blocking PPARa- and, interestingly, VEGF and FGF2 concentrations are elevated mediated responses contributing to the development of alcoholic before the emergence of HCC (46). By maintaining high levels of fatty liver, which can be overcome by PPARa agonists (27). HCV expression of these factors on hepatocytic differentiation, mTOR infection is also associated with altered expression and function may accelerate abnormal proliferation and angiogenesis. of PPARa (28). PPARy also plays a role in lipid metabolism In conclusion, we have shown that an enhanced activity of including cholesterol efflux and fatty acid oxidation (29, 30); mTOR, as found in clinical HCC samples, is capable of preventing activates fat metabolism to prevent obesity (31); and regulates hepatocytic differentiation not only through inhibition of induction fatty acid synthesis, glucose metabolism, and insulin sensitivity of C/EBPa but also by preventing hepatocytic polarization. In (32). Interestingly, it has been reported that PPARy attenuates addition, sustained mTOR activity may lead to reduced suscepti- colon carcinogenesis (33). RXR heterodimers serve as key bility to TGF-h antiproliferative effects and to TRAIL/TNF– and regulators in cholesterol homeostasis by governing reverse IFN-induced apoptosis. Sustained mTOR activity may also lead to cholesterol transport from peripheral tissues, bile acid synthesis abnormal expression of genes modulating lipid homeostasis, in liver, and cholesterol absorption in intestine (34). Liver-specific hepatocellular growth, and angiogenesis. These effects, taken loss of function of retinoic acid leads to steatohepatitis and liver together, could contribute to the neoplastic transformation of tumors in vivo (35). Therefore, sustained mTOR activity may these cells. Our study also suggests that combination therapy contribute to the development of steatosis observed in most strategies aimed at overcoming TRAIL and IFN resistance and y HCCs by impairing lipid homeostasis. PPARa and PPAR defects may be effective for treatment of HCC Another major target of DTOR is C/EBPa. This transcription with activated mTOR or PTEN deletion. factor regulates two aspects of hepatic terminal differentiation: induction of differentiation-specific genes and repression of Acknowledgments mitogenesis (36–38). C/EBPa reduces HCC susceptibility in mice Received 10/2/2006; revised 1/5/2007; accepted 2/9/2007. (39) and down-regulation of C/EBPa in HCC correlates with tumor Grant support: NIH/National Institute of Diabetes and Digestive and Kidney size and progression (40). C/EBPa-deficient mice present with Diseases grant DK066840 (Fred Hutchinson Cancer Research Center). The costs of publication of this article were defrayed in part by the payment of page severely disturbed liver architecture with acinar formation in a charges. This article must therefore be hereby marked advertisement in accordance pattern suggestive of either regenerating liver or HCC, abnormally with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. A. Edinger (University of California, Los Angeles, CA) for the gift of active hepatocytic proliferation, impaired hepatic glycogen storage, the DTOR constructs and Drs. C. Tre´poand M-A. Petit (Institut National de la Sante´et and accumulation of lipids in the liver (41, 42). These mice also de la Recherche Me´dicale Unit 271, Lyon, France) for the gift of HepaRG cells.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Mammalian Target of Rapamycin Activation Impairs Hepatocytic Differentiation and Targets Genes Moderating Lipid Homeostasis and Hepatocellular Growth

Romain Parent, Deepak Kolippakkam, Garrett Booth, et al.

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