Thiazolidinediones inhibit hepatocarcinogenesis in hepatitis B virus-transgenic mice by peroxisome proliferator-activated receptor γ-independent regulation of nucleophosmin

Thiazolidinediones Inhibit Hepatocarcinogenesis inHepatitis B Virus–Transgenic Mice by PeroxisomeProliferator-Activated Receptor c–Independent

Regulation of NucleophosminAndrea Galli,1,2 Elisabetta Ceni,1,2 Tommaso Mello,1,2 Simone Polvani,1,4 Mirko Tarocchi,1,2

Francesca Buccoliero,1 Francesca Lisi,1 Laura Cioni,1 Barbara Ottanelli,1 Valeria Foresta,1

Guido Mastrobuoni,3 Gloriano Moneti,3 Giuseppe Pieraccini,3 Calogero Surrenti,1 and Stefano Milani1,2

Antidiabetic thiazolidinediones (TZD) have in vitro antiproliferative effect in epithelialcancers, including hepatocellular carcinoma (HCC). The effective anticancer propertiesand the underlying molecular mechanisms of these drugs in vivo remain unclear. In addi-tion, the primary biological target of TZD, the ligand-dependent transcription factor per-oxisome proliferator-activated receptor c (PPARc), is up-regulated in HCC and seems toprovide tumor-promoting responses. The aim of our study was to evaluate whetherchronic administration of TZD may affect hepatic carcinogenesis in vivo in relation toPPARc expression and activity. The effect of TZD oral administration for 26 weeks wastested on tumor formation in PPARc-expressing and PPARc-deficient mouse models of he-patic carcinogenesis. Proteomic analysis was performed in freshly isolated hepatocytes bydifferential in gel electrophoresis and mass spectrometry analysis. Identified TZD targetswere confirmed in cultured PPARc-deficient hepatocytes. TZD administration in hepatitisB virus (HBV)–transgenic mice (TgN[Alb1HBV]44Bri) reduced tumor incidence in theliver, inhibiting hepatocyte proliferation and increasing apoptosis. PPARc deletion in he-patocytes of HBV-transgenic mice (Tg[HBV]CreKOc) did not modify hepatic carcinogen-esis but increased the TZD antitumorigenic effect. Proteomic analysis identifiednucleophosmin (NPM) as a TZD target in PPARc-deficient hepatocytes. TZD inhibitedNPM expression at protein and messenger RNA levels and decreased NPM promoter activ-ity. TZD inhibition of NPM was associated with the induction of p53 phosphorylationand p21 expression. Conclusion: These findings suggest that chronic administrationof TZD has anticancer activity in the liver via inhibition of NPM expression andindicate that these drugs might be useful for HCC chemoprevention and treatment.(HEPATOLOGY 2010;52:493-505)

Abbreviations: DIGE, difference gel electrophoresis; EMSA, electrophoretic mobility shift assays; HCC, hepatocellular carcinoma; NPM, nucleophosmin; PCNA,proliferating cell nuclear antigen; PGZ, pioglitazone; PPARc, peroxisome proliferator-activated receptor c; PPRE, peroxisome proliferator response element; RGZ,rosiglitazone; TZD, thiazolidinedione.From the 1Gastroenterology Unit, Department of Clinical Pathophysiology; 2Center of Excellence for Research, Transfer and High Education, DENOthe; and 3Mass

Spectrometry Center, University of Florence, Florence, Italy; 4FiorGen Farmacogenomic Foundation, Sesto Fiorentino, Florence, Italy.Received October 22, 2009; accepted March 8, 2010.Supported by This work was supported by grants from Ministero dell’ Istruzione, dell’ Universita, e della Ricerca (MIUR), Regione Toscana (TRESOR project),

Cassa di Risparmio di Firenze (CRF), and FiorGen Foundation.Address reprint requests to: Andrea Galli, M.D., Ph.D., Gastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Viale Pieraccini

6, 50139 Florence, Italy. E-mail: [email protected]; fax: þ39 (055) 4222.409.Copyright VC 2010 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.23669Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article.

493

Hepatocellular carcinoma (HCC) is the mostfrequent solid tumor of the liver. Half a mil-lion cases occur annually, making it the fifth

most common malignancy in men and the ninth mostcommon in women.1

Hepatocarcinogenesis is a multistep process involv-ing genetic and epigenetic events that accumulate dur-ing chronic liver diseases. The extent of hepatic dys-function limits therapeutic options for HCC andsurvival of patients with this tumor remains dismal, asthe average survival from time of diagnosis of unresect-able HCC is measured in months.2 In this scenarioHCC is therefore an attractive target for identificationof potential chemopreventive drugs.Thiazolidinediones (TZD) are a class of antidiabetic

drugs which attenuate insulin resistance and impairedglucose tolerance in humans as well as in several animalmodels of non–insulin-dependent diabetes mellitus.3 Themechanisms of TZD action are still being investigatedbut it has been clearly demonstrated that some of theireffects are mediated through activation of the peroxisomeproliferator-activated receptor-c (PPARc), a member ofthe nuclear receptor superfamily of ligand-dependenttranscription factors predominantly expressed in adipo-cytes but also in other normal and transformed cells.4

Beyond the metabolic actions, several studies indi-cate that TZD may have also anticancer properties ina variety of different epithelial malignancies. IndeedTZD treatment of cancer cells cultured in vitro orimplanted in nude mice causes reduction of growthrate, cell differentiation and apoptosis.5

Despite the suggestions that TZD might favor can-cer remission, there are conflicting data on whetherPPARc activation promote or suppress tumorigenesiswhen applied in animal model of cancer.6 Studies incolon and breast carcinogenesis have shown that TZD-dependent activation of PPARc leads to an increase oftumor formation.7,8 In addition, PPARc is overex-pressed in many epithelial tumor cells and regulatesthe production of hepatocyte growth factor which canfavor tumor growth, suggesting that this nuclear recep-tor might represent a prosurvival factor.9

It has been previously shown that in human HCC,cancer cells express PPARc and treatment with trogli-tazone, the first TZD initially approved for clinicaluse, induces a dose dependent reduction of cell prolif-eration, and a significant increase of apoptosis by amechanism involving the induction of the cell cycle in-hibitor p27.10 Conversely, recent results indicate thatspecific PPARc inhibitors prevent adhesion to extracel-lular matrix and induce anoikis, causing a more effec-tive cell death than TZD.11

Given these apparently discrepant observations onwhether PPARc activation could be growth-inhibitoryor tumor-promoting in hepatic cancer cells, this studywas designed to analyze the potential in vivo anticancereffect of chronic oral administration of TZD in a HBV-related model of hepatocarcinogenesis and to define thecorrelation between TZD actions and PPARc expres-sion and transcriptional activity in hepatocytes. Here weshow that TZD treatment inhibits tumor formation inHBV transgenic mice with a significant reduction of he-patocyte proliferation and increased apoptosis inde-pendently of PPARc expression. Proteomic analysisidentifies nucleophosmin (NPM), a nucleolar proteininitially characterized in the process of ribosomal RNAassembly and transport, as master coordinator of TZDantineoplastic action in hepatocytes.

Materials and Methods

Transgenic Animals and Experimental Design. Toachieve a selective elimination of PPARc in the liver ofTgN(Alb1HBV)44Bri mice,12 we realized a tripletransgenic animal where the liver-specific Cre expres-sion, obtained by placing Cre DNA under the controlof albumin promoter, deletes PPARc in hepatocytes.Parental transgenic mice were obtained from The Jack-son Laboratories (Bar Harbor, ME). Breeding detailsand histopathological diagnoses are specified in theSupporting Information. Nine-month-old male trans-genic mice were treated for 26 weeks with daily gavageadministration of TZD (3.0 mg/kg/day) (rosiglitazone[RGZ] or pioglitazone [PGZ]) or with the non-TZDPPARc ligand GW1929 (5.0 mg/kg/day). Control ani-mals were treated with vehicle alone.Immunohistochemistry. The proliferation of hepatic

cells was estimated by immunostaining for PCNA andCyclin D1 whereas apoptosis was detected by stainingfor activated caspase-3 and caspase-7. PCNA, CyclinD1, and apoptotic labeling indexes (LI), were semi-quantitatively evaluated by counting the percentage ofimmunoreactive hepatocytes in at least 10 randomlyselected fields using the image processing and analysissoftware Image J.13 (W.S. Rasband, ImageJ, U.S. NIH,Bethesda, MD, http://rsb.info.nih.gov/ij/, 1997-2009.)Hepatocyte Isolation and Proliferation Assay. He-

patocytes were isolated by a two-step collagenase perfu-sion of the liver through the inferior cava vein.14

Hepatocytes were plated at a density of 0.3 � 106

per 35-mm dish in DMEM/F12 medium. After 4hours attachment, cells were starved in serum-freemedia. DNA synthesis in primary hepatocyte cultures

494 GALLI ET AL. HEPATOLOGY, August 2010

was measured by [3H]thymidine incorporation. Addi-tionally, apoptosis was assessed morphologically byHoechst 33342 staining (Sigma Chemical, Germany)and fluorescence microscopy (Carl Zeiss, Germany).Isolation of Nuclear Protein Extract and DNA

Binding Assay. Nuclear proteins were extracted fromisolated hepatocytes based on a micropreparationmethod.15 Electrophoretic mobility shift assays(EMSA) were performed by radiolabeling double-stranded oligonucleotides corresponding to the PPARResponse Element (PPRE) ARE7 (50-TGCACATTTCACCCAGAGAGAAGGGATTGA-30).Transfection of Cultured Hepatocytes. For transfec-

tion, the Amaxa nucleofection technology (Lonza AG,Belgium) was employed. Hepatocytes were transfectedfollowing the manufacturer’s instructions. Briefly, 100lL of 2 � 106 cell suspension were mixed with 2.5 lgof (ARE7)3-tk-luciferase reporter plasmid or with 2.5lg NPM promoter construct. pSV2CAT (2 lg) wasused as internal control for transfection efficiency. Themixture was nucleofected with the T-028 nucleofectorprogram. After transfection, hepatocytes were trans-ferred into six-well plates and culture medium wasreplaced 4 hours later. After 48 hours, the cells wereharvested for luciferase and CAT assays.15

Western Blot. Protein extracts were fractionated by12% sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis. Proteins were detected incubating primaryantibodies overnight at 4�C, followed by the appropri-ate secondary antibody conjugated with horseradishperoxidase (1:1000) 2 hours at room temperature.Blots were developed with the enhanced chemilumi-nescence detection system ECL plus kit (PharmaciaBiosciences, Piscataway, NJ).Proteomic Analysis. Proteomic analysis was per-

formed by difference gel electrophoresis (DIGE). Sam-ples preparation for two-dimensional DIGE and massspectrometry identification are described in the Sup-porting Information.Statistical Analysis. Results are expressed as mean

6 standard deviation (SD) or standard error (SE).Multiple comparisons were performed by one-wayanalysis of variance with Bonferroni’s correction. AP value less than 0.05 was considered statisticallysignificant.

Results

TZD Inhibits Tumor Formation in HBVTransgenic Mice. We chose to examine the effect ofTZD chronic administration on a mouse model of

HBV-related hepatocarcinogenesis. Hepatocytes oftransgenic mice TgN(Alb1HBV)44Bri express andaccumulate the large HBsAg protein, resulting insevere chronic hepatocellular injury. This condition isconstantly followed by the development of dysplastichepatic lesions that progress after the ninth month oflife to hepatocellular adenomas and carcinomas.12

TZD (RGZ or PGZ), or a non-TZD n-aryl tyrosineactivator of PPARc (GW1929) or vehicle alone(CTRL) were administered daily by oral gavage toHBV transgenic mice for 26 weeks starting from theninth month of life. Four vehicle-treated, one RGZ-treated, three PGZ-treated, and four GW1929-treatedanimals died during the study and were not included inthe effective numbers: the observed deaths were notcaused by the treatments but are caused by natural andtechnical reasons (i.e., the protracted TZD administra-tion by gavage as demonstrated by necroscopy examina-tion). In the control group, 96% of mice developed he-patocellular adenomas and in 42% of them, we foundhepatocellular carcinomas after sacrifice (Table 1, Fig.2A).TZD oral administration markedly suppressed the

tumorigenic process in treated mice. Of the 56 TZD-treated mice, only three mice had evident hepatocellu-lar nodules larger than 2 mm, and 12 mice were com-pletely devoid of macroscopically visible formations(Fig. 1A). The smaller number and size of neoplasticfoci in TZD-treated mice correlated with the smallerliver mass reflecting an apparent difference in thegrowth rate of preneoplastic and neoplastic lesions ascompared with controls. On the contrary treatmentwith GW1929 exerted no effect on tumor formationin HBV transgenic mice.TZD Inhibits Hepatocyte Proliferation and

Induces Apoptosis in HBV Transgenic Mice. The in-hibition of tumor formation by TZD correlated withthe reduction of the proliferative activity and increasedfrequency of apoptosis of liver cells measured by PCNA(Figs. 1B, 2D), cyclin D1 (Figs. 1B, 2C) and activatedcaspase-3 and caspase-7 immunostaining (Figs. 1B, 2B).At the microscopic level, TZD-treated livers exhibitedonly mild dysplasia with considerably less cellular andnuclear enlargement and without advanced nuclearatypia when compared with control littermates (Fig.1A). Although nodular regeneration was significantlyreduced in TZD-treated animals, no differences in de-generative alterations, chronic inflammation and livercell necrosis were documented (Supporting InformationTable 1). Serum concentration of alanine aminotrans-ferase (ALT) was not modified by TZD treatment (Fig.2E) whereas a-fetoprotein, a marker of hepatocellular

HEPATOLOGY, Vol. 52, No. 2, 2010 GALLI ET AL. 495

regeneration and transformation, was drasticallyreduced in TZD-treated but not in GW1929-treatedtransgenic mice (Fig. 2F).PPARc Transcriptional Activity Is Not Involved in

TZD Inhibition of Hepatocyte Proliferation. Todetermine the effect of TZD and GW1929 on PPARctranscriptional activity in HBV transgenic mice, theability of nuclear proteins extracted from isolated he-patocytes to bind a PPRE probe (ARE-7), that haspreviously been shown to bind preferentially PPARcover other PPAR isoforms,15 was tested by EMSA.Nuclear extracts from hepatocytes isolated from con-trol transgenic mice contained proteins that retardedthe ARE-7 oligonucleotide (Fig. 3A). PPRE bindingwas increased in extracts from hepatocytes isolated byTZD and GW1929-treated animals suggesting aligand activation of PPARc (Fig. 3A, lanes 2-4). Thespecificity of this band was confirmed by super-shift inextracts incubated with antibody against PPARc (Fig.3A, lanes 5-8). The ability of these drugs to modulatePPARc activation was confirmed by the inducedexpression of GLUT-2, a PPARc target gene,16 in he-patocytes isolated from both TZD-treated andGW1929-treated mice (Fig. 3B, lanes 1-4).In cultured HBV-derived mouse hepatocytes, TZD

and GW1929 similarly induced PPARc transactivationas monitored by the activity of transfected (ARE-7)3-tk-luc reporter (Fig. 3D) but only TZD were able to inducea dose-dependent inhibition of DNA synthesis (Fig.3C), thus confirming the direct effect of TZD on hepa-tocytes proliferation. The inhibition of DNA synthesisby TZD was not modified by transfection of dominantnegative PPARc (DN-PPARc) (Fig. 3E) that, on thecontrary, abolished the ligand-induced reporter activity(Fig. 3F) and GLUT-2 expression (Fig. 3B, lanes 5-10).Taken together, these results show that PPARc acti-

vation by TZD is not correlated with the ability ofthese drugs to inhibit hepatocyte DNA synthesis.Genetic Deficiency of PPARc Does Not Alter Car-

cinogenesis in HBV Transgenic Mice and Does NotInfluence the Antitumor Effect of TZD. In considera-

tion that HCC arise from clonal expansion of hepato-cytes in TgN(Alb1HBV)44Bri mice,17 we generated astrain of HBV transgenic mice with specific deletionof PPARc gene in hepatocytes (Supporting Informa-tion Fig. 1A-C) in order to clarify the role of PPARcsignaling in tumor development in HBV transgenicmice and evaluate whether the anticancer effects ofTZD were mediated by this nuclear receptor in vivo.No differences in tumor incidence, latency, size, histo-

pathology, and disease progression were observed in ani-mals carrying the PPARc deletion (Tg[HBV]CreKOccompared to parental HBV transgenic mice and controlPpargf/f/Tg[HBV]Bri44 mice (Supporting InformationFig. 1D,E).Five vehicle-treated animals, two RGZ-treated, five

PGZ-treated, and two GW1929-treated animals diedbefore the end of the study and were not included inthe effective numbers. The effect of TZD administra-tion on incidences, multiplicities, histological features,and size distribution of tumors are summarized inSupporting Information Table 2.Administration of TZD almost halved the number

of hepatic tumors in Tg(HBV)CreKOc (Fig. 4) and itcorrelated with a significant increase of apoptosis (Sup-porting Information Fig. 2) suggesting that the anti-cancer effect of these drugs is independent of PPARcexpression in hepatocytes.Protein Expression Profiling in Hepatocytes Iso-

lated from PPARc-Deficient HBV Transgenic MiceAfter TZD Administration. To identify novel proteintargets that are differentially regulated under chronicoral administration of TZD independently by PPARc,we performed two-dimensional gel electrophoresis andmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry in primaryhepatocytes isolated from Tg(HBV)CreKOc mice.We used samples from 10 different vehicle-treated

and RGZ-treated animals detecting an average of 3527spots (Fig. 5A). MALDI-TOF peptide fingerprint anal-ysis characterized 26 proteins that were significantlydifferential expressed; these proteins are listed in

Table 1. TZD Oral Administration Inhibits Hepatocarcinogenesis in HBV Transgenic Mice

Incidence %Multiplicity Maximum tumor size Liver Weight

Treatments Adenomas Carcinomas Mean 6 SE Mean 6 SE (mm) Mean 6 SE (g)

Vehicle 96.15 (25/26) 42.30 (11/26) 6.23 6 0.70 8.7 6 0.87 3.73 6 0.56

RGZ 58.62 (17/29) 27.58 (8/29) 2.58 6 0.53* 1.4 6 0.12* 1.85 6 0.29*

PGZ 59.25 (16/27) 25.92 (7/27) 3.11 6 0.67* 1.8 6 0.17* 1.92 6 0.26*

GW1929 92.30 (24/26) 53.84 (14/26) 5.53 6 0.62 9.3 6 0.79 3.86 6 0.45

The values reported were calculated as described in the Supporting Information Materials and Methods. The number of mice with adenomas/carcinomas per

total number of mice is given in parentheses.

*P< 0.05 versus vehicle-treated animals.


Supporting Information Table 3, with their corre-sponding molecular weight, isoelectric point (pI), andrecognized function according to the Swiss-Prot data-base. The majority of them belong to cytoskeleton,

chaperones, and stress/redox regulatory systems. Wechose to further investigate nucleophosmin (NPM)because this nucleolar protein, involved in cell growthand transformation,18 was consistently down-regulated

Fig. 1. TZD suppresses hepatic tumorigenesis in HBV transgenic mice. (A) Spontaneous liver tumor development is inhibited by 6 months oraladministration of RGZ and PGZ, but not of GW1929. Macroscopic and microscopic (original magnification: �100) of representative livers areshown. (B) Representative immunostaining for activated caspase-3 and caspase-7, PCNA, and cyclin D1 of HBV transgenic mouse liver at theend of treatments. Scale bar ¼ 50 lm.


at protein (Fig. 5B,D) and messenger RNA (mRNA)levels (Fig. 5C) in hepatocytes of TZD-treated mice,but it was unaffected by GW1929. Moreover, the roleof NPM in the development of liver tumors is com-pletely unknown. A dose-dependent reduction on NPMprotein and mRNA expression was confirmed by west-ern blot and RT-PCR analysis in PPARc-deficient hepa-tocytes cultured in vitro and treated with TZD (Sup-porting Information Fig. 3A,B). TZD affected NPMexpression in hepatocytes at the transcriptional level asdemonstrated by the TZD inhibition of NPM pro-moter activity in transient transfection experiments(Supporting Information Fig. 3C). This effect was notinfluenced by cotransfection with wtPPARc or withDN-PPARc (Supporting Information Fig. 3D).The effect of RGZ on NPM expression was also con-

firmed both in hepatocytes isolated from TgN(Alb1HBV)44Bri mice cultured in vitro and in human and micehepatoma cell lines (HuH7 and Hepa 1-6) (SupportingInformation Fig. 4). Analysis of the cellular localization

of NPM in TZD-treated hepatoma cells showed a sig-nificant nucleolar reduction of both NPM and its phos-phorylated form (pThr199-NPM) (Supporting Infor-mation Fig. 5). Moreover, TZD treatment localizedpThr199-NPM in nuclear speckles (Supporting Infor-mation Fig. 5, insets), possibly reflecting a reduction inmessenger RNA processing.18

AMPK Activation Is Involved in TZD Inhibitionof Hepatocyte Proliferation and NPM Expression. -Recently, it has been demonstrated that TZD suppressgrowth factors tumor-promoting activity via AMPKactivation.20 Inhibition of AMPK activity by the spe-cific AMPK inhibitor, compound C, or the dominantnegative AMPKa2(D157A), completely prevented thegrowth arrest induced by TZD treatment in PPARc-deficient hepatocytes (Fig. 6A,B). Furthermore, TZDtreatment induced phosphorylation of AMPK both invivo, as documented in freshly-isolated hepatocytesfrom PPARc-deficient mice (Fig. 6C) and in vitro, incultured hepatocytes (Fig. 6D). Consistent with our

Fig. 2. TZD inhibit proliferationand induce apoptosis in hepato-cytes of HBV transgenic mice. (A)Graphic representation showing thepercentage of RGZ (n ¼ 29), PGZ(n ¼ 27), GW1929 (n ¼ 26), andcontrol (CTRL; n ¼ 26) treatedmice with liver tumors. *P < 0.02,**P < 0.01. (B) Histograms showthat apoptotic index is significantlyhigher in TZD than in CTRL andGW1929-treated mice. (C,D) Histo-grams show a significant inhibitionof labeling index of cyclin D1 andPCNA labeling indexes in RGZ-treated and PGZ-treated mice. Val-ues are expressed as mean per-centage 6 SE (n ¼ 15 in eachgroup of mice) *P < 0.05. (E, F)serum alanine aminotransferaseand a-fetoprotein levels weredetermined in venous blood sam-ples after 6 months of oral drugadministration. Data are expressedas mean 6 SD. *P < 0.01 versusCTRL or GW1929 treatment.


results, expression of the dominant-negative AMPKreverted the TZD-mediated inhibition of NPM expres-sion (Fig. 6E). These results strongly suggest that TZD in-hibit hepatocytes proliferation through AMPK activation.Nucleophosmin/p53 Interaction Mediates the

PPARc-Independent Inhibition of Hepatocyte Prolif-eration by TZD Treatment. In consideration thatNPM is involved in cell death and proliferation interactingwith the tumor suppressor p53,18 we tested whether NPMoverexpression could antagonize TZD effect via p53.Cultured hepatocytes isolated from Tg(HBV)CreKOc

mice were transfected with vector expressing FLAG-tagged NPM under CMV promoter (WT-NPM) or amutant variant with a deletion of the 120 c-terminal

amino acids of NPM (NPMDC) required for the bind-ing to p53. High levels of FLAG-tagged NPM or NPMmutant proteins were achieved in the transfected cells,whereas no FLAG-tagged proteins were detected in sam-ples transfected with control vector (Fig. 7A, inset).Increased expression of WT-NPM completely abrogatedthe growth inhibitory effect of TZD but it was not asso-ciated with an increase of either thymidine incorporationor incidence of apoptosis in control cultured hepatocytes.On the contrary, expression of the mutant NPMDC didnot modify the antiproliferative and proapoptotic effectsof TZD (Fig. 7A,B) suggesting that these antidiabeticdrugs induce cell growth arrest by inhibiting NPMexpression and consequently its interaction with p53.

Fig. 3. PPARc transactivation is not required for TZD inhibition of hepatocyte proliferation. (A) PPRE-binding activity was evaluated in nuclearextract from hepatocytes isolated from vehicle or TZD-treated TgN(Alb1HBV)44Bri mice. EMSA was performed with a probe containing a copy ofa PPARc response element (ARE-7) as described in supplemental materials and methods. Where indicated, antibodies against PPARc (anti-PPARc) were used to confirm PPARc presence in the PPRE binding complexes. (B, lane 1-4) TZD effect on the PPARc target GLUT-2 was evaluateby western blot analysis of protein extracts from hepatocytes isolated from vehicle and TZD-treated (or GW1929-treated) animals and in culturedhepatocytes transfected with the DN-PPARc or with the pcmx empty vector (B, lane 5-10). Representative data from three independent experi-ments, each of which was performed in triplicate, are shown. (C) DNA synthesis was measured by [H3]thymidine incorporation into DNA of cul-tured hepatocytes treated with increased concentration of TZD or GW1929 and in hepatocytes transfected with the PPARcDN or with the pcmxempty vector (E). (D, F) PPARc transcriptional activity was monitored in hepatocytes treated with the same experimental condition describedabove and cotransfected with ARE-73-TK-Luc reporter and pSV2-CAT as an internal control for transfection efficiency. Results are expressed asmean 6 SD from three experiments done in sextuplicate. *P < 0.05 versus respective controls. **P < 0.02 versus pcmx-transfected cells.


It has been shown that NPM interacts with p53 andregulates p53 phosphorylation at the Serine-15 residuewhich is crucial for p53 transactivation and subsequentapoptotic signals transduction.21 We thus determined

whether TZD-inhibited NPM expression may affectp53 activity in PPARc-deficient hepatocytes. Asshown in Fig. 7C, TZD induced both P-p53Ser-15 andits target gene cyclin-dependent kinase inhibitorp21WAF1/CIP1. Strikingly, over expression of NPMsignificantly reduced the TZD-induced P-p53Ser-15 andp21 expression, whereas overexpression of the mutantNPMDC failed to oppose TZD effect on p53 activa-tion (Fig. 7D). In parallel, induction of P-p53Ser-15

was also demonstrated in freshly isolated hepatocytesfrom TZD-treated Tg(HBV)CreKOc mice comparedto vehicle-treated animals (Fig. 7E). Taken togetherthese results indicate that the PPARc-independentantiproliferative effect of TZD is mediated by NMPthrough a mechanism involving p53.

Discussion

Our study shows that chronic administration of twodifferent TZD, significantly inhibit tumor formationin a HBV-related mouse model of hepatocarcinogene-sis. This effect was correlated by in vivo and in vitro

Fig. 4. TZD inhibit tumor formation independently of PPARc expres-sion. Tumors were enumerated in mice livers and the multiplicity quan-tified as described in the Supporting Information Materials andMethods section. *P < 0.05 versus respective controls; #P < 0.03versus RGZ-treated TgN(Alb1HBV)44Bri mice; P < 0,05 versus PGZ-treated TgN(Alb1HBV)44Bri.

Fig. 5. Proteome analysis in hepatocytes from Tg(HBV)CreKOc mice. (A) Two-dimensional DIGE was carried out with 50 lg proteins of hepato-cytes isolated from CTRL and RGZ-treated Tg(HBV)CreKOc mice, and labeled with Cy5 (red) and Cy3 (green), respectively. Gels from three differ-ent experiments were scanned using Typhoon TRIO scanner. Identified proteins with at least two-fold change in volume are indicated by arrows.(B) Western blot analysis was carried out with 30 lg of total proteins using specific antibodies against NPM and b-actin. (C) Total RNA wasextracted from freshly isolated hepatocytes and reverse transcribed. Real-time PCR was performed with specific primers for NPM and GAPDH(Applied Biosystems Inc., Foster City, CA). (D) Representative immunohistochemistry for NPM of hepatic tissue obtained from vehicle-treated andRGZ-treated Tg(HBV)CreKOc mice (original magnification: �1000).


inhibition of hepatocyte proliferation and induction ofapoptosis with negligible effects on the degenerativeand the inflammatory responses. On the contrary, thenon-TZD PPARc agonist GW1929 had no effect ontumor formation and hepatocyte proliferation althoughthis drug is able to induce PPARc transactivation andtarget gene expression in mouse hepatocytes. This sug-gests that PPARc activation is unlikely involved in theantitumor effect of TZD in mouse liver.Previous in vitro evidences suggest that the antiproli-

ferative effect of TZD is independent of PPARc activa-tion; indeed, troglitazone induced growth arrest by in-hibition of translation initiation in PPARc�/�

embryonic stem cells.22 Similarly, we found that in he-patocytes isolated from HBV transgenic mice, thegrowth inhibitory effect of TZD is dissociated fromthe ability of these drugs to promote PPARc transacti-vation. In fact, ectopic expression of DN-PPARc wasunable to revert the growth inhibitory effect of TZD.

Although PPARc is clearly recognized as master reg-ulator of lineage-specific cell differentiation that differsaccording to the cellular type,23 the correlationbetween PPARc activation and programmed cell deathinduced by TZD is doubted. In pancreatic cancer cells,TZD-induced PPAR-dependent growth arrest is pri-marily mediated by cell differentiation without proa-poptotic effects.24 Conversely, TZD analogues, whichhave a double bond adjoining the terminal thiazolidi-nedione ring that is responsible for the abrogation ofthe PPARc ligand property, retain the ability to induceapoptosis with a potency equal to that of their parentalTZD in cancer cell lines,25 suggesting that mechanismsinvolved in TZD-induced differentiation differ fromthose mediating apoptosis. The dissociation of TZDeffects on apoptosis from their original pharmacologi-cal activity (i.e., PPARc activation), is in line with theobservation that sensitivity of cancer cells to TZD-induced growth inhibition does not correlate with the

Fig. 6. TZD inhibit hepatic carcinogenesis by activating AMPK. (A) DNA synthesis was measured by [3H]thymidine incorporation in cultured he-patocytes isolated from Tg(HBV)CreKOc mice. The effect of compound C was evaluated by preincubating the cells for 30 minutes before TZD(20lM) or vehicle treatment. (B) Inhibition of AMPK activity was obtained by transfection with DN-AMPK which harbors a mutation in catalyticsubunit (dnAMPKa2D157A). Control cells were transfected with empty vector. Data from three independent experiments, each of which was per-formed in triplicate, are shown. Results are expressed as mean 6 SD, *P < 0.01 versus respective controls. Phosphorylation of AMPK wasdetected by western blot in cultured PPARc-deficient hepatocytes after incubation with RGZ (20 lM) at different time points (D) and in hepato-cytes freshly isolated from TZD or GW1929-treated Tg(HBV)CreKOc mice (C). The effect of AMPK inhibition on TZD-modulated expression of NPMwas evaluated by western blot in cultured hepatocytes transfected with either dnAMPKa2D157A or control vector (E).


PPARc expression levels, and there exists a three ordersof magnitude discrepancy between the concentrationrequired to produce antitumor effects and that neededto modify insulin action.26

The PPARc-independent proapoptotic effect ofTZD was confirmed in triple transgenic animalsTg(HBV)CreKOc in which Cre specifically deletesPPARc in hepatocytes. In this experimental model,genetic deficiency of PPARc does not modify the pro-cess of hepatic carcinogenesis and tumor developmentwhen compared to parental HBV transgenic mice.This is in agreement with the demonstration thatPPARc deletion does not alter the development of ex-perimental prostate and breast cancers.27,28 In addi-tion, when mice expressing a constitutively active formof PPARc in mammary glands were bred to transgenicmice prone to mammary gland cancer, the resulting

offspring develop tumors with greatly acceleratedkinetics,8 suggesting a pro-oncogenic role of PPARc.Moreover, in human pancreatic and ovarian cancersexpression profiles indicate a strong overexpression ofPPARc that positively correlate with higher pT stagesand higher tumor grade.29,30

Interestingly, our experiments showed that inTg(HBV)CreKOc mice TZD administration inhibitstumor formation with a potency significantly higherthan in parental and control mice. Moreover, PPARcectopic expression in PPARc-deficient hepatocytesreduced the antiproliferative effect of TZD (Support-ing Information Fig. 6).How PPARc expression limits TZD anticancer

activities remains speculative. We hypothesize that thenet effect of TZD in cancer cells is the result of thebalance of PPARc-mediated (pro-oncogenic) and

Fig. 7. NPM expression antagonizes TZD-induced hepatocytes apoptosis via p53 activation. (A) DNA synthesis was measured by [H3]thymidineincorporation in cultured hepatocytes transfected with the FLAG-tagged wild type (wt NPM) or with the mutant variant (NPMDC) NPM. Controlcells were transfected with empty vector. Data from three independent experiments, each of which was performed in triplicate, are shown. Resultsare expressed as mean 6 SD, *P < 0.02 versus respective controls. Inset, overexpression of wt NPM and NPMDC protein in cultured hepato-cytes: western blot analysis was performed with anti-FLAG antibody. (B) Apoptotic hepatocytes were detected by staining with the DNA bindingfluorescent dye Hoechst 33342. Apoptotic cells were expressed as a percentage of the total number of cell counted. Results are expressed asmean 6 SE of four independent experiments, each of which was performed in duplicate, *P < 0.05 versus respective controls. (C) Effect ofTZD and GW1929 on p53 activation in cultured or freshly isolated (E) hepatocytes. (D) Effect of wtNPM or NPMDC expression on p53 activation.Whole-cell lysates were analyzed by immunoblotting with antibodies specific for phospho-p53Ser-15, total p53, p21, and b-actin.


PPARc-independent (anti-oncogenic) mechanisms thatdepends on different factors including receptor expres-sion levels, phosphorylation status, expression of theheterodimeric partners, and the presence of endoge-nous ligands.31 This might explain the limited thera-peutic efficacy of TZD treatment in oncological trialsexcept for tumor types with reduced levels and possibleloss of function of PPARc such as prostate and thyroidcancers.32,33

In vitro studies have suggested that TZD mediateantiproliferative effects through a complexity ofPPARc-independent mechanisms. Experimental evi-dence indicates that troglitazone and ciglitazone blockBH3 domain-mediated interactions between Bcl-2family members and facilitate the degradation of cyclinD1 through proteasome-mediated proteolysis.34,35 Inour study, we identified a novel molecular target bywhich TZD inhibit hepatocyte proliferation in vivo.Proteomic analysis showed that TZD reduce theexpression of NPM, a nucleolar protein characterizedas a central regulator of ribosomal RNA processingthat has been found to be more abundant in tumorand growing cells than in corresponding normalcells.17

In HCC, NPM overexpression is correlated withclinical parameters, such as serum a-fetoprotein leveland tumor pathological grading, suggesting that NPMmight serve as a potential marker for HCC.36 Inagreement, in our mouse model we found a progres-sive age-related increase of NPM that parallels theincrease of PCNA-LI in hepatocytes (Supporting Infor-mation Fig. 7).TZD inhibited the expression of NPM at protein

and mRNA levels in both isolated hepatocytes andhepatoma cell lines, and significantly repressed NPMpromoter activity independently of the ectopic expres-sion of wild-type PPARc or DN-PPARc. These dataare in agreement with the absence of PPRE in theNPM promoter (A. Galli, E. Ceni, L. Cioni, unpub-lished observation, 2009).The molecular pathway involved in the PPARc-in-

dependent regulation of gene expression by TZD isscarcely known. TZD have been recently shown toactivate AMPK, a cellular sensor of energy status.37

AMPK may suppress tumorigenesis regulating cellgrowth via inhibition of mammalian target of rapamy-cin (mTOR) signaling and p53 activation, and it isindicated as a beneficial target for cancer treatment.38

We showed that TZD induced AMPK activation inPPARc-deficient hepatocytes and that inhibition ofAMPK activity completely prevented the TZD-induced growth arrest and NPM expression. These

results are in agreement with the observation thatTZD specifically inhibit IGF-I tumor-promoting activ-ity in mouse skin through activation of AMPK andsubsequent inhibition of mTOR pathway.20 In addi-tion, AMPK activation was demonstrated to inducep53 phosphorylation and p53-dependent apoptotic celldeath in response to energetic stress.39

Although there is no evidence for a direct involve-ment of NPM in the regulation of the apoptotic ma-chinery, NPM might function as an antiapoptotic pro-tein through indirect mechanisms. Interaction withp53 might be an important step by which NPMinhibits programmed cell death. In fact, NPM overex-pression protects mouse embryonic fibroblast againsthypoxic cell death, but this effect is not observed incell that lacks p53.40

We showed that in cultured PPARc-deficient hepa-tocytes, ectopic expression of wild-type NPM signifi-cantly blocked TZD inhibition whereas a mutant vari-ant lacking the p53-interacting domain did notprevent TZD antiproliferative and proapoptoticactions. Similarly, in malignant haematopoietic cells,the same NPM mutant does not prevent apoptosis inresponse to stress stimuli, unlike the overexpression ofwild-type NPM.41 NPM-p53 interaction inhibits p53phosphorylation at the serine 15, and subsequentlyrepresses p53 target genes expression such as the cellcycle inhibitor p21. However, in hepatic cells TZDmay promote p53 phosphorylation by inhibitingNPM gene expression. Interestingly, NPM has alsobeen shown to interact with p53 in hypoxic cells andto inhibit hypoxia-induced p53 phosphorylation onthe same residue.42 Besides, regulation of p53 expres-sion and activity by TZD has been also demonstratedin human cholangiocarcinoma cells.43 In considerationthat the ability of AMPK to induce cell cycle arrest isdependent on p53 phosphorylation at Ser15,39 itmight be conceivable that TZD modify p53 phospho-rylation status and activity by an AMPK-mediateddown-regulation of NPM.In conclusion, we have shown that chronic adminis-

tration of TZD inhibits hepatic tumor formation inmice with a PPARc-independent mechanism. Further-more, we found that the anticancer activity of thesedrugs in the liver was mediated, at least in part, by in-hibition of NPM expression and p53 activation. Col-lectively, these observations provide new insight intothe molecular mechanisms of hepatic carcinogenesisand emphasize relevant clinical implication. In fact,recent studies have consistently indicated TZD as anti-fibrotic drugs for treatment of different chronic hepaticdiseases,44,45 and moreover, our data suggest that these


molecules or correlated analogues, alone or in combi-nation with other anticancer drugs, may have transla-tional relevance in effective strategies for HCC chemo-prevention and treatment.

Acknowledgment: The authors thank Dr. R. Evansfor PPARc constructs and Dr. Q. Pang for NPM con-structs; Dr. K. Guan for dnAMPKa2D157A plasmidand Professor M. Luconi for many helpful commentsand suggestions. We also thank Dr. D. Lucci for thestatistical support.

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Thiazolidinediones inhibit hepatocarcinogenesis in hepatitis B virus-transgenic mice by peroxisome proliferator-activated receptor γ-independent regulation of nucleophosmin

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