Cancer Research Oncogenic Potential of Retinoic Acid...

Published OnlineFirst March 2, 2010; DOI: 10.1158/0008-5472.CAN-09-2968

Molecular and Cellular Pathobiology
Cancer
Research

Oncogenic Potential of Retinoic Acid Receptor-γin Hepatocellular Carcinoma

Ting-Dong Yan1, Hua Wu1, Hai-Ping Zhang2, Na Lu1, Ping Ye3, Feng-Hai Yu3, Hu Zhou4, Wen-Gang Li2,Xihua Cao4, Ya-Yu Lin1, Jia-You He1, Wei-Wei Gao1, Yi Zhao1, Lei Xie1, Jie-bo Chen4,Xiao-kun Zhang1,4, and Jin-Zhang Zeng1

Abstract

Authors'UniversityHepatobilInstitute for

Note: T-D.

CorresponBiomedical86-592-218Xiao-kun Z10901 Nor3141; Fax:

doi: 10.115

©2010 Am

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Retinoic acid receptors (RAR; α, β, and γ), members of the nuclear receptor superfamily, mediate the pleio-tropic effects of the vitamin A metabolite retinoic acid (RA) and derivatives (retinoids) in normal and cancercells. Abnormal expression and function of RARs are often involved in the growth and development of cancer.However, the underlying molecular mechanisms remain largely elusive. Here, we report that levels of RARγwere significantly elevated in tumor tissues from a majority of human hepatocellular carcinoma (HCC) andin HCC cell lines. Overexpression of RARγ promoted colony formation by HCC cells in vitro and the growthof HCC xenografts in animals. In HepG2 cells, transfection of RARγ enhanced, whereas downregulation ofRARγ expression by siRNA approach impaired, the effect of RA on inducing the expression of α-fetoprotein,a protein marker of hepatocarcinogenesis. In studying the possible mechanism by which overexpression ofRARγ contributed to liver cancer cell growth and transformation, we observed that RARγ resided mainly inthe cytoplasm of HCC cells, interacting with the p85α regulatory subunit of phosphatidylinositol 3-kinase(PI3K). The interaction between RARγ and p85α resulted in activation of Akt and NF-κB, critical regulatorsof the growth and survival of cancer cells. Together, our results show that overexpression of RARγ plays arole in the growth of HCC cells through nongenomic activation of the PI3K/Akt and NF-κB signaling path-ways. Cancer Res; 70(6); 2285–95. ©2010 AACR.

Introduction

Retinoic acid receptors (RAR) and retinoid X receptors(RXR) are members of the nuclear receptor superfamily,which mediate the pleiotropic effects of vitamin A metaboliteretinoic acid (RA) and its natural and synthetic derivatives(retinoids; refs. 1, 2). Both RAR and RXR are encoded by threedistinct genes, α, β, and γ, and they regulate a variety ofimportant cellular processes in normal and cancer cells(1, 2). The distinct spatial and temporal expression patternsof retinoid receptors during development and in adult tis-sues suggest that each of the subtypes has discrete func-tions (1, 2). Altered expression and function of retinoidreceptors are associated with the development of certainmalignancies (3–5). Integration of HBV gene at FRA3A

Affiliations: 1Institute for Biomedical Research, Xiamen; 2First Hospital of Xiamen, Xiamen, China; 3Easterniary Surgery Hospital, Shanghai, China; and 4BurnhamMedical Research, La Jolla, California

Yan, H. Wu, and H-P. Zhang contributed equally to this work.

ding Authors: Jin-Zhang Zeng, Cancer Center, Institute forResearch, Xiamen University, Xiamen 361005, China. Phone:7221; Fax: 86-592-2181879; E-mail: [email protected] orhang, Cancer Center, Burnham Institute for Medical Research,th Torrey Pines Road, La Jolla, CA 92037. Phone: 858-646-858-646-3195; E-mail: [email protected].

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within the RARβ gene (6, 7), altered phosphorylation ofRXRα (8), and limited proteolysis of the RXRα protein (9)have been implicated in the development and progressionof hepatocellular carcinoma (HCC).RARs, like other nuclear receptors, are known to act as

transcriptional factors to regulate gene expression by bindingto cis-acting response elements (retinoic acid response ele-ments, RARE) in the promoter/enhancer region of targetgenes as heterodimer with RXRs (1, 10). However, only a lim-ited number of their target genes are described (11). A con-siderable number of studies have now shown that RARs cannongenomically regulate some rapid biological responses toretinoids (11, 12). Thus, a membrane-associated RARα canmediate the effect of RA on spine formation of hippocampalneurons through phosphorylation of extracellular signal-regulated kinase 1/2 and p70s6k (13), whereas RARγ can bindto and catalytically activate c-Src kinase during neuritogen-esis (14). RARs can also interact with the p85α regulatorysubunit of phosphoinositide 3-kinase (PI3K), leading to itsactivation in various cell types (15–17). Consistently, retinoidreceptors are found to reside in the cytoplasm at certainstages during development (18, 19) and in response to differ-entiation (14, 20), apoptosis (21), and inflammation (22–26).Altered subcellular localization of retinoid receptors likely re-flects changes in the cellular environment but may also playan active role in the regulation of cellular activities. In a re-cent study, we showed that RARγ is unique among three RARsubtypes in that it often resides in the cytoplasm of cancer

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cells (9, 27). Whether and how the cytoplasmic RARγ regu-lates the growth of cancer cells remained undefined.In the present study, we showed that RARγ was over-

expressed in a majority of tumor tissues from HCC patientsand in HCC cell lines. By transfection and siRNA approaches,we showed that RARγ played a critical role in promoting col-ony formation of HCC cells in vitro and the growth of HCCxenografts in animals and in mediating the effect of RA oninducing the expression of α-fetoprotein (AFP), a proteinmarker of hepatocarcinogenesis (28, 29). We also found thatRARγ predominantly resided in the cytoplasm where it inter-acted with p85α, leading to activation of Akt and NF-κB inHCC cells. Together, our results show that RARγ overexpres-sion contributes to the growth and development of HCCthrough its interaction with p85α, which activates both Aktand NF-κB pathways.

Materials and Methods

Reagents. Lipofectamine 2000 and Trizol LS (Invitrogen);enhanced chemilumienescence (ECL) reagents, goat anti-rabbitand anti-mouse secondary antibodies conjugated to horse-radish peroxidase (Thermo); polyclonal antibodies againstRARγ, RARα (C-20), RARβ (C-19), IκBα, Akt1/2/3, pAkt1/2/3,p65, OctA-probe (Flag, D-8), and proliferating cell nuclear an-tigen (PCNA; FL-261) and monoclonal antibodies against pIκ-Bα, Myc (9E10), Hsp60 (H-1), p85α (B-9), AFP (1B10), andFITC-labeled antirabbit IgG (Santa Cruz Biotechnology);monoclonal anti-poly(ADP-ribose) polymerase (PARP) antibody(556494; BD Biosciences); antimouse IgG conjugated withCy3 (Chemicon international); monoclonal antibodies againstβ-actin, α-tubulin, and glyceraldehyde-3-phosphate dehydro-genase (GAPDH) and chemicals including all-trans-RA, cyclo-phosphamide, aflatoxin B1, tumor necrosis factor α (TNFα),MG132, LY-294002, and BMS-345541 (Sigma); protein A beads(GEHealthcare); polyvinylidene difluoride (PVDF)membranes(Millipore); and cocktail of proteinase inhibitors (80-6501-23;Amersham) were used in this study.Tissue samples. Seventeen Chinese patients with primary

HCC without prior chemotherapy were recruited. Tumor andthe adjacent nontumorous tissues were collected after sur-gical resection.Construction of lentivirus-based siRNA expression vector.

RARγ siRNA oligonucleotide (5′-gctaccaagtgcatcatca-3′) ornonsense siRNA oligonucleotide (5′-ttctccgaacgtgtcacgt-3′)with hairpin-containing sequence created as described (30)was ligated into pLL3.7 vector via HpaI/XhoI site. Lentiviruspackaging and infection were performed as described (30).Colony formation. HepG2, QGY-7703, and QSG-7701 cells

(31) and stable clones expressing Myc-RARγ (HepG2/γ andQSG-7701/γ), empty vector (HepG2/v and QSG-7701/v),RARγ siRNA lentiviral vector (HepG2/γi and QGY-7703/γi),and control siRNA (HepG2/ci and QGY-7703/ci) were used. Cellscultured in six-well plates for 14 d were fixed and stained withGiemsa. The number of foci containing >50 cells was scored.Xenografts. Nude mice (BALB/c, SPF grade, 16–18 g, 4–5

weeks old) were injected s.c. with 200 μL of wild-type HepG2

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or stable clone (HepG2/γ, HepG2/γi, HepG2/v, or HepG2/ci)cells (5 × 106 per mouse). Mice with HepG2/wt xenograftswere treated i.p. at day 8 of posttransplantation with saline,cyclophosphamide (CP, 40 mg/kg), RA (10 mg/kg), or aflatox-in B1 (AFB1, 20 μg/kg) once every other day. Tumor size wasmeasured every 4 or 5 d. Three weeks later, mice were sacri-ficed and tumors were removed for assessments (32).Cell fractionation. Total proteins were prepared with a

modified radioimmunoprecipitation assay buffer (33). Cyto-plasmic proteins were purified with Buffer A [10 mmol/LHEPES-KOH (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl,and 0.5 mmol/L DTT], whereas nuclear proteins were pre-pared by resuspending the pellets in high-salt Buffer C[20 mmol/L HEPES-KOH (pH 7.9), 25% glycerol, 420 mmol/LNaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, and 0.5 mmol/LDTT; ref. 33].Immunoprecipitations. Cells were lysed in a buffer con-

taining 2 mmol/L Tris-HCl (pH 7.4), 10 mmol/L EDTA,100 mmol/L NaCl, and 1% IGEPAL. After preclearing withprotein A beads, lysates were incubated with 8 μL of primaryantibody and 40 μL of protein A beads at 4°C. The beads werewashed thrice with 1 mL of cold lysis buffer with exception ofthe NaCl concentration (150 mmol/L).Western blotting. A cocktail of proteinase inhibitors was

included in all protein purification. Equal amount of pro-teins was electrophoresed on an 8% SDS-PAGE gel andtransferred onto PVDF membranes, which were then incu-bated with primary and secondary antibodies and detectedusing ECL system.Immunohistochemistry. Tumor sections were immuno-

stained with antibody against RARγ (1:500) or PCNA(1:500) and detected with corresponding secondary antibody.The slides were counterstained with hematoxylin. The prolif-eration indices were calculated by counting PCNA-positivecells versus total cells from three randomly selected regions.For fluorescent confocal microscopy, HepG2 cells were trans-fected with Myc-RARγ alone or in combination of Flag-p85αand stained with anti-Myc (1:100) and anti-Flag (1:100)antibodies. The slides were then stained with anti-Cy3and anti-FITC antibodies. Cells were also costained with4′,6-diamidino-2-phenylindole to visualize nuclei.Reverse transcription–PCR analysis. Total RNAs were

isolated by Trizol LS. The first strand was synthesized withRevertAid First Strand cDNA synthesis kits (Fermentas; ref.33). The primers for PCR reactions are as follows: for humanAFP, sense: 5′-tcagtgaggacaaactattgg-3′ and antisense: 5′-ctcttcagcaaagcagacttc-3′; for β-actin, sense: 5′-ctggagaagagc-tacgag-3′ and antisense: 5′-tgatggagttgaaggtag-3′.Luciferase reporter assay. Expression of βRARE-lucifer-

ase reporter gene was determined in HepG2 and Hep3B cellsafter treatment with vehicle or 1 μmol/L RA for 20 h. NF-κBluciferase activity was assayed in HepG2 cells treated with10 ng/mL TNFα, 1 μmol/L RA, or vehicle for 6 h. Each trans-fection also included β-galactosidase for normalization ofthe luciferase activities.Statistical analysis. Data were expressed as mean ± SD.

Each assay was repeated in triplicate in three independentexperiments. Statistics was analyzed using Student's t test

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Overexpression of RARγ in HCC


or ANOVA analysis. Values of P < 0.05 were consideredsignificant.

Results

Overexpression of RARγ in HCC tissues and cells. We re-cently reported that RARγ often resides in the cytoplasm ofcancer cells depending on cell culture conditions (27). In thisstudy, we wanted to explore RARγ expression and its cyto-plasmic action in HCC cells. Immunoblotting studies showedthat RARγ was readily detectable in all tumor and surround-ing liver tissues prepared from HCC surgical specimensthough at various levels (Fig. 1A). However, levels of RARγwere significantly higher in tumor tissues than the non-

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tumorous tissues from a majority of patients (82%). Immu-nostaining studies showed that overexpression of RARγ inHCC tissues was predominantly cytoplasmic (Fig. 1B, left),which was consistent with our previous observation thatoverexpressed RARγ could translocate to the cytoplasm(27). The cytoplasmic localization of RARγ in HCC tissueswere further confirmed by subcellular fractionation analysis,whereas in the surrounding liver tissues RARγ was mainlyexpressed in the nucleus (Fig. 1B, right). Thus, our resultsclearly show that RARγ is abnormally overexpressed in thecytoplasm of HCC cells.Our results also showed that RARγ was highly expressed

in the aggressive QGY-7703 HCC cells (Fig. 1C); however,it was only slightly expressed in QSG-7701 hepatocytes, a

Figure 1. Overexpression of RARγin HCC. A, RARγ expression intumor tissues (T) and surroundingliver tissues (S) prepared from HCCsurgical specimens. B, cytoplasmiclocalization of RARγ in HCC.Left, RARγ immunostaining.Right, subcellular fractionationanalysis of RARγ expression.Immunoblottings of Hsp60 andPARP were served as controls forthe purity of cytoplasmic (C) andnuclear (N) fractions, respectively.C, RARγ expression in various HCCcells and QSG-7701 hepatocytes.D, left, distinct growth regulatoryresponses of RA in HepG2 andHep3B cells. Cells were treatedwith increasing concentrations ofRA for 48 h and subjected to MTTassays. Middle, status of RARsubtypes in HepG2 and Hep3Bcells. Right, βRARE-luciferasereporter assays. HepG2 andHep3B cells were treated withincreasing concentrations of RA for8 h. The relative luciferase activitywas normalized to β-galactosidaseactivity.

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precancerous cell line derived from paracancerous tissueof a HCC patient (31). The expression levels of RARγ inQGY-7703 and QSG-7701 cells were comparable with thosein HCC tissues and the paired nontumorous tissues. Com-pared with QSG-7701 hepatocytes, HepG2 and SMMC7721HCC cells also expressed elevated levels of RARγ. How-ever, no detectable level of RARγ was seen in Hep3B cells(Fig. 1C).



RARγ confers growth stimulatory effect of RA. We nextstudied the effect of RA on the growth of RARγ-positiveHepG2 and RARγ-negative Hep3B cells. Treatment of Hep3Bcells with RA resulted in growth inhibition in a dose-dependentmanner (Fig. 1D, left). In contrast, RA treatment enhanced thegrowth of HepG2 cells. To determine whether RARγ expres-sion levels were responsible for the distinct growth responsesof RA, HepG2/γi cell line was then treated with RA. Our result

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Figure 2. Oncogenic activity ofRARγ. A, colony formation assayswere performed in wild-typecells (wt) and in stable clonesexpressing Myc-RARγ (γ), RARγsiRNA (γi), empty vector (v), orcontrol siRNA (ci). The meannumber of foci formed by HepG2/wt cells was normalized to 100%,whereas all other colonies werecompared. B, tumor growth curve.Tumor volume was measuredevery 4 or 5 d after implantation(left). Tumors were removed atthe 21st day of postimplantationand their sizes (middle) andweights (right) were evaluated.C, PCNA immunostaining.Proliferative index was calculatedas percentage of PCNA-positivecells. D, cyclophosphamide (CP),which had beneficial anticanceractivity, and AFB1, which was akey factor for the developmentof HCC, were investigated fortheir regulation of RARγ expressionin HepG2 xenografts. RARγexpression was determined byWestern blotting and its relativelevels were normalized to theGAPDH controls.

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showed that inhibition of RARγ by siRNA was able to renderHepG2 cells sensitive to growth inhibition by RA (Fig. 1D, left),thus showing that RARγ mediates the growth stimulatory ef-fect of RA in HepG2 cells. This was consistent with our obser-vation that RARα and RARβ were similarly expressed inHepG2 and Hep3B cells (Fig. 1D, middle). Interestingly, whenthe effect of RA on activating βRARE reporter gene was exam-ined in HepG2 and Hep3B cells, we observed that RA inducedsimilar luciferase activity in both cell lines (Fig. 1D, right). Thisresult suggests that transcriptional regulation of RA targetgenes is unlikely responsible for the different effects of RAon the growth of HepG2 and Hep3B cells.RARγ enhances colony formation of HCC cells. Colony

formation assays were then used to investigate the role ofRARγ in cell transformation. Figure 2A showed that the abil-ity of HepG2 cells in forming foci was significantly enhancedwhen the cells were transfected with RARγ expression vector.Conversely, inhibition of RARγ by siRNA strikingly impairedHepG2 cells to form colonies. When colony formation wascompared between high-RARγ expressing QGY-7703 HCCcells and low-RARγ expressing QSG-7701 hepatocytes, we ob-served a significant number of foci formed by QGY-7703. Incontrast, QSG-7701 cells seldom formed foci (Fig. 2A). WhenRARγ siRNA was introduced into QGY-7703 cells, it potentlyinhibited their colony formation, whereas overexpression of



RARγ in QSG-7701 cells greatly increased their ability to formcolonies (Fig. 2A). Together, these results convincingly showthat RARγ plays an active role in promoting the clonogenicsurvival of HCC cells.RARγ promotes the growth of HCC xenografts in animals.

We next studied the effect of RARγ on the growth ofHepG2 cells in nude mice. Figure 2B showed that RARγtransfection greatly enhanced the growth of HepG2 xeno-grafts in mice, whereas inhibition of RARγ by siRNAsignificantly inhibited their growth. Consistent with ourin vitro observation showing that RA was growth stimula-tory in HepG2 cells, treatment of mice with RA stimulatedrather inhibited the growth of HepG2 xenografts (112% ofcontrol, data not shown). When PCNA expression, an in-dex of cell proliferation, were examined, we observed thatectopic overexpression of RARγ and RA treatment couldstrongly enhance immunoreactivity of PCNA in HepG2xenograft (Fig. 2C). Interestingly, treatment of mice withcyclophosphamide, a potent cancer chemotherapeuticagent, greatly inhibited the growth of HepG2 xenografts(43% of control, data not shown), which was accompaniedwith reduction of RARγ expression, whereas AFB1, a keyfactor for promoting the development of HCC, increasedRARγ expression (Fig. 2D). These results further showthe role of RARγ in regulating the growth of HCC cells

Figure 3. Induction of AFP by RARγ. A, RT-PCRanalysis of AFP mRNA expression in HepG2cells. Untransfected and RARγ-transfectedHepG2 cells were treated with vehicle or 1μmol/L RA for 6 h. B, effect of RARγ siRNA onAFP mRNA expression. RARγ siRNA-mediatedRARγ silencing was indicated. Cells weretreated with vehicle or 1 μmol/L RA for 6 h.C, regulation of AFP expression in vivo.HepG2 xenografts were treated with saline,cyclophosphamide (CP), or RA, as described inMaterials and Methods. The relative AFPprotein levels by immunoblotting werenormalized to the GAPDH controls.

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and suggest that it may also serve as a target of antican-cer drugs.Induction of AFP by RARγ. As AFP expression is a sensitive

biomarker for hepatocarcinogenesis (28, 29), we then examinedthe role of RARγ in regulation of AFP expression in HepG2 cells.Figure 3A showed that RARγ transfection could strongly induceAFP transcription, which was further enhanced upon RA treat-ment. In contrast, inhibition of RARγ expression by siRNA abol-ished the effect of RA on inducing AFP expression (Fig. 3B). InHepG2 xenografts, AFP levels were reduced by cyclophospha-mide but significantly increased by RA treatment (Fig. 3C). To-gether, these results suggest that abnormally increasedexpression of RARγ may play a role in hepatocarcinogenesis.



RARγ activates NF-κB in HCC cells. Because HCC is path-ologically associated with chronic hepatitis and activation ofNF-κB is a crucial for HCC growth and progression (34, 35),we next studied whether RARγ expression was involved inthe regulation of NF-κB signaling pathway. We first deter-mined the effect of RA on expression of IκBα, a key inhibitorof NF-κB nuclear translocation (36), in HepG2 and Hep3Bcells. Figure 4A showed that RA treatment could strongly re-duce IκBα expression in HepG2 cells, but it was ineffective inHep3B cells. Both cell lines were sensitive to TNFα treat-ment. In the next studies, we showed that transfection ofRARγ into HepG2 cells could dose-dependently reduce IκBαexpression, which was further potentiated by RA treatment


Figure 4. Association of RARγwith IκBα turnover in response toRA. A, IκBα levels in HepG2 andHep3B cells. Cells were treatedwith vehicle, 10 ng/mL TNFα, or1 μmol/L RA for 30 min. B, effectof RARγ transfection on IκBαturnover. Left, HepG2 cells weretransfected with increasingamounts of RARγ plasmid andtreated with vehicle or 1 μmol/LRA for 30 min. Right, HepG2 cellswere cotreated with 1 μmol/L RAand 10 μmol/L MG132 for 30 min.Various controls were indicated.C, RARγ-mediated nucleartranslocation of p65 in HepG2cells. Left, cells were treatedwith vehicle, 10 ng/mL TNFα,or 1 μmol/L RA for 30 min. Totalp65 protein and its nuclear fractionwere analyzed by immunoblotting.Right, cells were immunostainedwith anti-p65 antibody after30-min treatment of vehicleor 1 μmol/L RA. D, RARγoverexpression stimulated NF-κBactivity in HepG2 cells. Cellswere transfected with NF-κBluciferase reporter gene alone or incombination of RARγ expressionvector and treated with vehicle,10 ng/mL TNFα, or 1 μmol/L RAfor 6 h. Each transfection alsoincluded β-galactosidaseconstruct for normalization ofthe luciferase activity.

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(Fig. 4B, left). Our results showed that RA/RARγ-inducedIκBα reduction was due to activation of proteasome degra-dative pathway, as pretreatment of MG132, a potent inhibitorof the 26S proteasome, strongly inhibited this process(Fig. 4B, right). The ability of RARγ to activate the NF-κBpathway was also illustrated by our observation that RAtreatment and RARγ transfection significantly induced nu-clear translocation of p65 (Fig. 4C) and stimulated NF-κB lu-ciferase activity (Fig. 4D). Together, our results show thatRARγ can activate NF-κB pathway in HepG2 cells.Interaction of RARγ with p85α and the activation of

PI3K/Akt in HCC cells. As one of the major upstream activa-



tors of NF-κB is Akt (36, 37), we then examined whetherRARγ activated Akt in HCC cells. Treatment of HepG2 cellswith RA for 20 minutes increased levels of phosphorylatedAkt protein in a dose-dependent manner (Fig. 5A). Such aneffect of RA was not due to its enhancement of total levels ofAkt protein, suggesting that RA could rapidly activate Akt.Transfection of HepG2 cells with RARγ also resulted in Aktactivation, which was further enhanced when cells were trea-ted with RA (Fig. 5A). Thus, RARγmight mediate the effect ofRA on inducing Akt activation in HCC cells.Recent studies showed that certain retinoid receptors

could activate the PI3K/Akt pathway through interaction

Figure 5. RARγ interacts with p85αand mediates the effect of RA on Aktactivation. A, RARγ transfectionenhanced the effect of RA on Aktphosphorylation. Untransfected andRARγ-transfected HepG2 cells weretreated with vehicle or with increasingconcentrations of RA for 20 min.B, interaction of endogenous RARγand p85α. Immunoprecipitation wasconducted by using anti-RARγ (left)and anti-p85α (right) antibodies.C, interaction of Myc-RARγ andFlag-p85α in cotransfection assay.Lysates were immunoprecipitated witheither anti-Flag (left) or anti-Myc (9E10;right) antibodies. Cells were treatedwith vehicle or 1 μmol/L RA for 30 min.IgG was served as a control in thecoimmunoprecipitation. D, subcellularlocalization of RARγ. HepG2 cells weretransfected with Myc-RARγ alone or incombination with Flag-p85α. The cellswere immunostained with anti-Myc(9E10) antibody or costained withanti-Flag antibody.

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with the p85α regulatory subunit of PI3K (15, 17). We there-fore studied the interaction of RARγ with p85α in HepG2cells. Coimmunoprecipitation assays showed that immuno-precipitation of endogenous RARγ protein in HepG2 cellsby anti-RARγ antibody but not by control IgG resulted incoimmunoprecipitation of a significant amount of p85α pro-tein (Fig. 5B). The coimmunoprecipitation of p85α protein byanti-RARγ antibody was enhanced when cells were treated



with RA. Similarly, RARγ was coimmunoprecitated by anti-p85α antibody in HepG2 cells. Furthermore, when HepG2cells were cotransfected with Myc-RARγ and Flag-p85α, im-munoprecipitation of Flag-p85α by anti-Flag antibody butnot by control IgG resulted in coimmunoprecipitation ofMyc-RARγ (Fig. 5C). Similarly, immunoprecipitation ofMyc-RARγ by anti-Myc antibody led to coimmunoprecipita-tion of Flag-p85α. The interaction between Myc-RARγ and


Figure 6. RA induces Akt and IκBαphosphorylation. A, time courseanalysis of Akt activation by RA.HepG2 cells were treated withvehicle or 1 μmol/L RA in thepresence or absence of PI3Kinhibitor LY294002 (10 μmol/L).Phosphorylated and total levels ofboth Akt and IκBα were analyzed.B, left, RA-inducing IκBα turnoverwas reversed by dn-Akt. HepG2cells were transfected withincreasing concentrations ofdn-Akt plasmid. IκBα levels wereanalyzed after the cells weretreated with vehicle or 1 μmol/L RAfor 30 min. Right, effect of dn-Akton NF-κB activity. Luciferaseactivity was determined at 6 hposttreatment of 1 μmol/L RA andnormalized to β-galactosidaseactivity. C, RA-induced Akt/IκBαphosphorylation and IκBα turnoverwere abrogated by RARγ siRNA.HepG2 cells were treated withvehicle or 1 μmol/L RA for 30 min.D, involvement of PI3K/NF-κBsignaling in AFP induction. HepG2cells were treated with 1 μmol/LRA for 30 min with or withoutpreincubation of either PI3Kinhibitor LY294002 (10 μmol/L)or IKK inhibitor BMS-345541(10 μmol/L).

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Flag-p85α occurred in the absence of RA, and the interac-tion could be enhanced by RA treatment. The interaction ofMyc-RARγ with Flag-p85α was also illustrated by theircolocalization in HepG2 cells (Fig. 5D). In the absence ofFlag-p85α, Myc-RARγ was diffusely distributed in both thenucleus and cytoplasm of cells. When cotransfected withFlag-p85α that was primarily cytoplasmic, Myc-RARγ wasmainly found in the cytoplasm, displaying distributionpatterns that were extensively overlapped with those ofFlag-p85α (Fig. 5D). Together, these results show that RARγcan interact with p85α, which may lead to activation ofPI3K/Akt pathway.RARγ activation of Akt precedes NF-κB activation. We

next studied whether activation of Akt by RARγ was requiredfor its activation of NF-κB. Time course analysis of the effectof RA on activation of Akt and NF-κB in HepG2 cells showedthat Akt activation occurred 10 minutes after cells were trea-ted with RA, with the optimal effect at 20 minutes after RAtreatment (Fig. 6A). Such RA-induced Akt activation was ac-companied with IκBα phosphorylation and its degradation.When the cells were pretreated with LY294002, a potentPI3K kinase inhibitor, RA-induced Akt phosphorylation wasreduced. Consequently, IκBα phosphorylation and its degra-dation induced by RA were inhibited (Fig. 6A). Furthermore,transfection of dn-Akt, a dominant-negative Akt mutant (38),could dose-dependently reverse the effect of RA on IκBαdegradation (Fig. 6B, left) and abrogate the effect of RARγon stimulating NF-κB activity in HepG2 cells (Fig. 6B, right).These findings suggest that RARγ-induced IκBα degrada-tion and NF-κB transactivation are mediated by its activa-tion of Akt.The role of RARγ in activating Akt and NF-κB pathways

was further shown by our observation that inhibition ofRARγ in HepG2 cells by siRNA reduced Akt activation andimpaired the effect of RA on IκBα degradation (Fig. 6C). In-terestingly, pretreatment of HepG2 cells with PI3K inhibitorLY294002 or IKK inhibitor BMS-345541 could strongly inhibitthe effect of RA on inducing AFP expression (Fig. 6D), sug-gesting that RA induces AFP expression through its activa-tion of PI3K/Akt and NF-κB pathways.

Discussion

Retinoids and retinoid receptors are critical regulators ofthe growth, differentiation, and apoptosis of normal and ma-lignant cells. Here, we report that RARγ was overexpressed inHCC tissues and cells and that overexpression of RARγ facil-itated the growth of HCC cells through a RARγ/PI3K/Akt/NF-κB signaling axis.Our results showed that RARγ was overexpressed in a ma-

jority of HCC tissues (Fig. 1A) and in several HCC cell lines(Fig. 1C). We showed that elevated RARγ expression was as-sociated with the resistance of HCC cells to the growth in-hibitory effect of RA in in vitro (Fig. 1D) and in animal,which might explain a previous clinical observation showingthat RA treatment of some HCC patients resulted in the de-velopment of more aggressive phenotypes (39). Our resultsfurther showed that RARγ played an active role in promoting



the growth of HCC cells as overexpression of RARγ greatlyenhanced the colony formation of HCC cells (Fig. 2A) andstimulated tumor growth in mice (Fig. 2B). In addition, over-expression of RARγ strongly increased PCNA expression(Fig. 2C), an index of proliferation, and reactivated AFP tran-scription (Fig. 3), a sensitive marker for evaluating tumor ac-tivity during heptocarcinogenesis (28, 29). Consistently, theoncogenic activity of RARγ has been suggested in other tis-sues. In skin, high level of RARγ was believed to mediate theeffect of RA on inducing hyperplasia (40). Overexpression ofRARγ could significantly inhibit RA-induced expression ofRARβ, a tumor suppressor frequently downregulated inmany malignant diseases (41). The role of RARγ in maintain-ing the growth and survival of HCC cells was also illustratedby our observation that inhibition of the growth of HepG2xenografts by cyclophosphamide was associated with a de-crease of RARγ expression (Fig. 2D). Although how cyclo-phosphamide inhibited RARγ expression remains to bedetermined, such an observation suggests that RARγ maybe targeted for drug development.Data presented here show that overexpression of RARγ in

HCC cells plays a critical role in the activation of PI3K/Aktpathway. We recently reported that unlike other retinoid re-ceptors, RARγ often resided in the cytoplasm of cancer cells(27). Consistently, our present study revealed a strong cyto-plasmic accumulation of RARγ in HCC cells but not in thesurrounding liver cells (Fig. 1B). Altered subcellular localiza-tion of retinoid receptors likely reflects changes in the cellu-lar environment, such as abnormal survival signalings, due tothe effects of growth factors during carcinogenesis. Alterna-tively, the cytoplasmic expression of retinoid receptors mayplay an active role in the modulation of signal pathwaysthrough their direct interaction with protein kinase cascades(11, 12). These actions often occur rapidly and are recognizedas nongenomic effects contrasting to their classic genomicRARE-based transcriptional regulation (1, 11, 12). In thisstudy, we showed that RARγ could interact with p85α, whichwas further enhanced by RA treatment (Fig. 5B and C). Asp85α is often highly expressed in human cancers includingHCC (42, 43), p85α may serve to retain RARγ in the cyto-plasm through their physical interaction in HCC. Indeed,transfection of Myc-RARγ alone showed a diffused distribu-tion of Myc-RARγ in both nucleus and cytoplasm. Howevercotransfection of Flag-p85α and Myc-RARγ resulted in in-creased cytoplasmic localization of Myc-RARγ, displaying adistribution pattern that was extensively colocalized withFlag-p85α (Fig. 5D). The interaction of RARγ with p85αmay lead to constitutive activation of Akt often detected inHCC (43, 44) and may also be responsible for RA-induced ac-tivation of Akt in RARγ overexpressing HCC cells (Fig. 5A).We showed that RARγ was essential for Akt activation astransfection of RARγ strongly enhanced the effect of RA onAkt activation (Fig. 5A), whereas downregulation of RARγby siRNA impaired its effect (Fig. 6C). Because variousRAR subtypes are able to activate PI3K/Akt pathway (15,17), this pathway seems to be a general downstream targetof RARs. However, its biological outcomes seem to be differ-ent, depending on different cell types. RARα-dependent

Cancer Res; 70(6) March 15, 2010 2293



Yan et al.

2294


PI3K/Akt signaling was shown to be required for RA-induceddifferentiation in SH-SY5Y neuroblastoma (15), whereasRARβ-dependent PI3K/Akt signaling in human breast can-cer facilitated iodide uptake (17). Our results showed thatRARγ-dependent activation of PI3K/Akt signaling ledto NF-κB activation (Fig. 4C and D) and AFP induction(Figs. 3A and 6D), which was associated with the growthof HCC cells. The PI3K/Akt pathway is one of the majorsurvival pathways in cancer cells (42), whereas activationof NF-κB provides an additional mechanism for cell growthand survival (44). The regulation of NF-κB activity by RARγthrough PI3K/Akt pathway described here is distinct fromthe established mutually antagonistic mode between NF-κBand retinoid acid receptors, which mainly occurs within thenucleus (45).In summary, we report here that RARγ is often over-

expressed in HCC and in liver cancer cells, conferring theirsurvival in vitro and in vivo. RARγ predominantly resides inthe cytoplasm of HCC cells and is able to interact with p85α,leading to activation of the PI3K/Akt and IκBα/NFκB path-ways and induction of AFP. Our characterization of thismechanism may provide a basis for developing novel strate-



gies and agents for treatment of HCC by targeting the RARγ/PI3K/NF-κB signaling axis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Xin Zhang from Chinese National Human Genome Center atShanghai for providing Hep3B cells.

Grant Support

863 Program grant 2007AA09Z404, Natural Science Foundation of Chinagrant 30971445, NSFC/RGC Joint Research Project grant 30931100431, KeyScience and Technology Planning Project grants 2007I0023 and 2008Y0062,Natural Science Foundation grant 2009J01198 of Fujian Province, and 985Project (J-Z. Zeng) and NIH grant CA109345 and U.S. Army Medical Researchand Material Command grant PCRPW81XWH-08-1-0478 (X-k. Zhang).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 08/11/2009; revised 12/04/2009; accepted 01/14/2010; publishedOnlineFirst 03/02/2010.

References
1. Kastner P, Mark M, Chambon P. Nonsteroid nuclear receptors: what
are genetic studies telling us about their role in real life? Cell 1995;83:859–69.

2. Germain P, Chambon P, Eichele G, et al. International Union of Phar-macology: LX. Retinoic acid receptors. Pharmacol Rev 2006;58:712–25.

3. Altucci L, Leibowitz MD, Ogilvie KM, de Lera AR, Gronemeyer H.RAR and RXR modulation in cancer and metabolic disease. NatRev Drug Discov 2007;6:793–810.

4. Khetchoumian K, Teletin M, Tisserand J, et al. Loss of Trim24 (Tif1α)gene function confers oncogenic activity to retinoic acid receptor α.Nat Genet 2007;39:1500–6.

5. Sano K, Takayama T, Murakami K, Saiki I, Makuuchi M. Overexpres-sion of retinoic acid receptor α in hepatocellular carcinoma. Clin Can-cer Res 2003;9:3679–83.

6. Dejean A, de The H. Hepatitis B virus as an insertional mutagene in ahuman hepatocellular carcinoma. Mol Biol Med 1990;7:213–22.

7. Feitelson MA, Lee J. Hepatitis B virus integration, fragile sites, andhepatocarcinogenesis. Cancer Lett 2007;252:157–70.

8. Matsushima-Nishiwaki R, Okuno M, Adachi S, et al. Phosphorylationof retinoid X receptor α at serine 260 impairs its metabolism andfunction in human hepatocellular carcinoma. Cancer Res 2001;61:7675–82.

9. Nomura Y, Nagaya T, Yamaguchi S, Katunuma N, Seo H. Cleavageof RXRα by a lysosomal enzyme, cathepsin L-type protease. Bio-chem Biophys Res Commun 1999;254:388–94.

10. Germain P, Chambon P, Eichele G, et al. International Union ofPharmacology: LXIII. Retinoid X receptors. Pharmacol Rev 2006;58:760–72.

11. Zeng JZ, Zhang XK. Nongenomic actions of retinoids: role of Nur77and RXR in the regulation of apoptosis and inflammation. Anti-inflamm Antiallergy Agents Med Chem 2007;6:315–31.

12. Gronemeyer H, Gustafsson JA, Laudet V. Principles for modulationof the nuclear receptor superfamily. Nat Rev Drug Discov 2004;3:950–64.

13. Chen N, Napoli JL. All-trans-retinoic acid stimulates translation andinduces spine formation in hippocampal neurons through a mem-brane-associated RARα. FASEB J 2008;22:236–45.

14. Dey N, De PK, Wang M, et al. CSK controls retinoic acid receptor

(RAR) signaling: a RAR-c-SRC signaling axis is required for neurito-genic differentiation. Mol Cell Biol 2007;27:4179–97.

15. Masia S, Alvarez S, de Lera AR, Barettino D. Rapid, nongenomic ac-tions of retinoic acid on phosphatidylinositol-3-kinase signaling path-way mediated by the retinoic acid receptor. Mol Endocrinol 2007;21:2391–402.

16. Lopez-Carballo G, Moreno L, Masia S, Perez P, Barettino D. Activa-tion of the phosphatidylinositol 3-kinase/Akt signaling pathway byretinoic acid is required for neural differentiation of SH-SY5Y humanneuroblastoma cells. J Biol Chem 2002;277:25297–304.

17. Ohashi E, Kogai T, Kagechika H, Brent GA. Activation of the PI3kinase pathway by retinoic acid mediates sodium/iodide sympor-ter induction and iodide transport in MCF-7 breast cancer cells.Cancer Res 2009;69:3443–50.

18. Dufour JM, Kim KH. Cellular and subcellular localization of six ret-inoid receptors in rat testis during postnatal development: identifi-cation of potential heterodimeric receptors. Biol Reprod 1999;61:1300–8.

19. Fukunaka K, Saito T, Wataba K, Ashihara K, Ito E, Kudo R. Changesin expression and subcellular localization of nuclear retinoic acid re-ceptors in human endometrial epithelium during the menstrual cycle.Mol Hum Reprod 2001;7:437–46.

20. Katagiri Y, Takeda K, Yu ZX, Ferrans VJ, Ozato K, Guroff G. Modu-lation of retinoid signalling through NGF-induced nuclear export ofNGFI-B. Nat Cell Biol 2000;2:435–40.

21. Cao X, Liu W, Lin F, et al. Retinoid X receptor regulates Nur77/TR3-dependent apoptosis by modulating its nuclear export and mito-chondrial targeting. Mol Cell Biol 2004;24:9705–25.

22. Ghose R, Mulder J, von Furstenberg RJ, Thevananther S, Kuipers F,Karpen SJ. Rosiglitazone attenuates suppression of RXRα-depen-dent gene expression in inflamed liver. J Hepatol 2007;46:115–23.

23. Yu X, Mertz JE. Critical roles of nuclear receptor response elementsin replication of hepatitis B virus. J Virol 2001;75:11354–64.

24. Wang K, Wan YJ. Nuclear receptors and inflammatory diseases. ExpBiol Med (Maywood) 2008;233:496–506.

25. Gyamfi MA, He L, French SW, Damjanov I, Wan YJ. Hepatocyte ret-inoid X receptor α-dependent regulation of lipid homeostasis and in-flammatory cytokine expression contributes to alcohol-induced liverinjury. J Pharmacol Exp Ther 2008;324:443–53.

Cancer Research





26. Ghose R, Zimmerman TL, Thevananther S, Karpen SJ. Endotoxinleads to rapid subcellular re-localization of hepatic RXRα: a novelmechanism for reduced hepatic gene expression in inflammation.Nucl Recept 2004;2:4.

27. Han YH, Zhou H, Kim JH, et al. A unique cytoplasmic localization ofRARγ and its regulations. J Biol Chem 2009;284:18503–14.

28. McIntire KR, Vogel CL, Princler GL, Patel IR. Serum α-fetoprotein asa biochemical marker for hepatocellular carcinoma. Cancer Res1972;32:1941–6.

29. Marrero JA, Feng Z, Wang Y, et al. α-Fetoprotein, des-γ carboxy-prothrombin, and lectin-bound α-fetoprotein in early hepatocellularcarcinoma. Gastroenterology 2009;137:110–8.

30. Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-basedsystem to functionally silence genes in primary mammalian cells,stem cells and transgenic mice by RNA interference. Nat Genet2003;33:401–6.

31. Pan XB, Wei L, Chen HS, Liu F, Gao Y. Liver-derived cell lines QSG-7701 and HepG2 support different HBV replication patterns. ArchVirol 2007;152:1159–73.

32. Hou J, Wang D, Zhang R, Wang H. Experimental therapy of hepato-ma with artemisinin and its derivatives: in vitro and in vivo activity,chemosensitization, and mechanisms of action. Clin Cancer Res2008;14:5519–30.

33. Liu J, Zhou W, Li SS, et al. Modulation of orphan nuclear receptorNur77-mediated apoptotic pathway by acetylshikonin and analogs.Cancer Res 2008;68:8871–80.

34. Maeda S, Kamata H, Luo JL, Leffert H, Karin M. IKKβ couples hepa-tocyte death to cytokine-driven compensatory proliferation that pro-motes chemical hepatocarcinogenesis. Cell 2005;121:977–90.

35. Karin M, Greten FR. NF-κB: linking inflammation and immunity to



cancer development and progression. Nat Rev Immunol 2005;5:749–59.

36. Tak PP, Firestein GS. NF-κB: a key role in inflammatory diseases.J Clin Invest 2001;107:7–11.

37. Roberts LR, Gores GJ. Hepatocellular carcinoma: molecular path-ways and new therapeutic targets. Semin Liver Dis 2005;25:212–25.

38. Han YH, Cao X, Lin B, et al. Regulation of Nur77 nuclear export byc-Jun N-terminal kinase and Akt. Oncogene 2006;25:2974–86.

39. Meyskens FL, Jakobson J, Nguyen B, Weiss GR, Gandara DR,MacDonald JS. Phase II trial of oral β-all trans-retinoic acid in hepato-cellular carcinoma (SWOG 9157). Invest New Drugs 1998;16:171–3.

40. Kang S, Duell EA, Fisher GJ, et al. Application of retinol to humanskin in vivo induces epidermal hyperplasia and cellular retinoid bind-ing proteins characteristic of retinoic acid but without measurableretinoic acid levels or irritation. J Invest Dermatol 1995;105:549–56.

41. Ferrari N, Pfahl M, Levi G. Retinoic acid receptor γ1 (RARγ1) levelscontrol RARβ2 expression in SK-N-BE2(c) neuroblastoma cells andregulate a differentiation-apoptosis switch. Mol Cell Biol 1998;18:6482–92.

42. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT path-way in human cancer. Nat Rev Cancer 2002;2:489–501.

43. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiologyand molecular carcinogenesis. Gastroenterology 2007;132:2557–76.

44. Factor V, Oliver AL, Panta GR, Thorgeirsson SS, Sonenshein GE,Arsura M. Roles of Akt/PKB and IKK complex in constitutive induc-tion of NF-κB in hepatocellular carcinomas of transforming growthfactor α/c-myc transgenic mice. Hepatology 2001;34:32–41.

45. De Bosscher K, Vanden Berghe W, Haegeman G. Cross-talk be-tween nuclear receptors and nuclear factor κB. Oncogene 2006;25:6868–86.

Cancer Res; 70(6) March 15, 2010 2295



2010;70:2285-2295. Published OnlineFirst March 2, 2010.Cancer Res Ting-Dong Yan, Hua Wu, Hai-Ping Zhang, et al. Hepatocellular Carcinoma

inγOncogenic Potential of Retinoic Acid Receptor-

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