Proinﬂammatory Homeobox Gene, ISX, Regulates Tumor … · This study assessed a total of 123 patients with HCCs from July 2004 to November 2009 from multiple medical center

Clinical Studies

Proinflammatory Homeobox Gene, ISX, Regulates TumorGrowth and Survival in Hepatocellular Carcinoma

Shih-Hsien Hsu1, Li-Ting Wang1, King-Teh Lee3,4, Yao-Li Chen6,7, Kwei-Yan Liu1, Jau-Ling Suen1,Chee-Yin Chai2, and Shen-Nien Wang1,4,5

AbstractChronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms

linking inflammation and tumor formation remain obscure. In this study, we compared the expression ofinterleukin (IL)-6 and cyclin D1 (CCND1) with the IL-6–induced homeobox gene ISX (intestine-specifichomeobox) in 119 paired specimens of HCCs and adjacent normal tissues and also in paired specimens from11 patients with non-HCCs. In pathologic analysis, ISX exhibited a tumor-specific expression pattern and ahigh correlation to patient survival time, tumor size, tumor number, and progression stage. Enforcedexpression of ISX accelerated cell proliferation and tumorigenic activity in hepatoma cells through CCND1induction. In contrast, short hairpin RNA–mediated attenuation of ISX in hepatoma cells decreased cellproliferation and malignant transformation in vitro and in vivo. A high positive correlation existed in humanhepatoma tumors between ISX and CCND1 expression. Together, our results highlight ISX as an importantregulator in hepatoma progression with significant potential as a prognostic and therapeutic target in HCCs.Cancer Res; 73(2); 508–18. �2012 AACR.

IntroductionHepatocellular carcinoma (HCC), the fifth most commonly

occurring cancer and the third leading cause of cancer-relateddeaths every year worldwide, has a multiple step progressionwith a high evidence of association with chronic inflammationexposure induced by environmental toxin intake and/or viralinfection, such as HBV or HCV (1). The chronic inflammationoften interacts with innate and adaptive immune responses,and the detailed regulatory mechanism leading to HCC tumorformation is unclear to date.

D-type cyclins are the major regulators governing G1

progression to S-phase in response to mitogenic and onco-genic signals and serve as markers for human malignancies(2, 3). Through binding with and activation of their associ-

ated cyclin-dependent kinases, CDK4 and CDK6, cyclin D–CDK complexes phosphorylate the retinoblastoma tumorsuppressor gene products, pRB, and pRB-related proteins,p130 and p107 (4, 5). This phosphorylation aborts growth-inhibitory functions of pRB, which leads to release of theE2F1 transcription factors and allows induction of E2F1target genes that are required for progressing into S-phase(6). The growth-promoting functions and deregulationexpression of D cyclins is a driving force toward increasedtumor proliferation and transforming activity in severalhuman cancers (2, 7). D cyclin overexpression in humancancers is driven by several mechanisms including tran-scriptional activation, genome alteration, posttranscription-al regulation, and posttranslational protein stabilization (2,3). Furthermore, several mouse models have also shown thatcyclin D1 can cooperate with other oncogenes to transformcells (8, 9). The oncogenic characterization of cyclin D1explored in cancer models and human cancers suggeststhat cyclin D1 and its associated CDKs activity may be apotential therapeutic target (3).

Homeobox genes, a superfamily of transcription factorswith homeo domains, play an essential role in controllingcell growth, differentiation, and morphogenesis during earlyembryonic development (10). Deregulation of homeoboxgenes appears to increase cell survival and proliferation andinhibit cell differentiation (11, 12). Recently, many homeo-box genes were found to be aberrantly expressed in a widevariety of human tumor masses (13). Intestine-specifichomeobox (Isx) is a newly identified pair family homeoboxtranscription factor, which shows a intestine-specificexpression pattern in both adult and fetal intestines (14).Phylogenetic analysis showed that the homeo domain of Isx

Authors' Affiliations: 1Graduate Institute of Medicine, 2Department ofPathology, 3Graduate Institute of Healthcare Administration, KaohsiungFaculty of Medicine, College of Medicine, Kaohsiung Medical University;4Division of Hepatobiliary Surgery, Department of Surgery; 5Department ofSurgery, Faculty of Medicine, Kaohsiung Medical University Hospital,Kaohsiung; 6Division of General Surgery, Department of Surgery, Chan-ghua Christian Hospital, Changhua; and 7School of Medicine, Chung ShanMedical University, Taichung, Taiwan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

S.-H. Hsu and L.-T. Wang contributed equally to this work.

Corresponding Authors: Shih-Hsien Hsu, Graduate Institute of Medicine,Kaohsiung Medical University, 807, Kaohsiung, Taiwan, R.O.C. Phone:886-7-3121101, ext 2021; Fax: 886-7-3212062; E-mail:[email protected]; and Shen-Nien Wang; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-2795

�2012 American Association for Cancer Research.

CancerResearch

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on May 12, 2021. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 5, 2012; DOI: 10.1158/0008-5472.CAN-12-2795

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belongs to the paired subfamily and is homologous to Pax3,Pax7, and Prrx1. Targeted disruption of Isx in mice revealedthat Isx was required for intestine-specific regulation of thehigh-density lipoprotein (HDL) receptor and cholesteroltransporter scavenger receptor class B, type 1 (SRB1), forvitamin A metabolism (14).In this study, we identified Isx as a proinflammatory cyto-

kine-induced homeobox gene, which is ectopically expressedin HCCs. By directly binding to the cyclin D1 promoter, Isxregulated cellular cyclin D1 expression, which furtherincreased cell proliferation and transforming activity. Fur-thermore, both Isx and cyclin D1 were upregulated in bothhepatoma tumor cell lines and tumor masses. The cellularfunction and tumor-specific expression pattern suggeststhat Isx is an important activator in proliferation and tumorformation in HCCs.

Materials and MethodsPatientsThis study assessed a total of 123 patients with HCCs from

July 2004 to November 2009 from multiple medical center[Chung Ho Memory Hospital (89 HCCs) and Changhua Chris-tian Hospital (34 HCCs)] with HBV and/or HCV infection and11 patients with non-HCCs (4 infected with HBV/HCV and 7non-infected with HBV or HCV) were enrolled into the Isxstudy, 119 of whom had adequate follow-up data for analysis.Experiments of human samples have been approved by Insti-tutional Review Boards of Kaohsiung Medical University(Kaohsiung, Taiwan; ref. 15).

Plasmids and cell linesFull-length Isx cDNA was PCR-amplified from a human

placenta cDNA library (GIBCO/BRL) and different truncatedIsx cDNAs were subcloned into the pEGFP/C1 vector (Clo-neTech) to express the GFP-tagged Isx protein. PGIPZ wasused for Isx short hairpin RNA interference (shRNAi) con-struction. The following sequences were used to constructIsx shRNAi: shRNAi-1(1031-1051): 50-TGAGCCTGTCCTT-CTCCATTG-30 and shRNAi-2(1367-1387): 50-AGCAGGAGA-AGATTGGCAACC-30. HEK 293, Hep G2, PLC5, HA22T,SK-Hep1, Huh7, and Hep 3B cell lines were subculturedand maintained according to ATCC protocol. Transfectionwas conducted using the Lipofectamine Transfection Kit(GIBCO/BRL).

Western blot, luciferase reporter assays, andimmunohistochemical staining analysisWestern blot, luciferase reporter assays, and immunohisto-

chemical (fluorescence) staining analysis were done as previ-ously described (15).

Quantitative PCR, chromatin immunoprecipitation, andEMSAThe above assays were analyzed as previous described (15).

Tumorigenic assay of Isx in soft agar andnudemice assayCells (104 or 5 � 103) in 1-mL culture medium were mixed

with an equal volume of 0.6% of top agar and plated onto 60-

mm dishes with 0.5% bottom agar (15). More detail processescould be found in Supplementary Methods and Materials.

Statistical analysisThe quantitative variables are presented as means and SDs.

Statistical differences were determined with 2-sample t test.Pearson correlations were used to determine the correlationcoefficient between expression levels of Isx and cyclin D1.Statistical analysis of categorical variables was carried outusing c2 analysis, one-way ANOVA, and Fisher exact analysis.P < 0.05 was considered statistically significant.

ResultsPatient characteristics

Isx is an ectopically expressed homeobox gene found inhepatoma microarray analysis (16); however, the correlationbetween clinical outcome of HCCs and cellular function ofIsx is unknown to date. To characterize the regulatory effectand clinical outcome of Isx in HCCs, 123 patients with HCCsfrom 2 medical centers were enrolled into the Isx cohortstudy from July 2004 to November 2009. Of these, 119 hadadequate follow-up data for analysis. The baseline charac-teristics of patients with HCCs and non-HCCs were com-pared between groups with non-HCCs, low and high Isxexpression, and the results are shown in Table 1. The overallsurvival time of all patients was 48 months. There weresignificant associations in terms of albumin (P ¼ 0.0040),bilirubin (P ¼ 0.0282), ac sugar (P ¼ 0.0281), tumor size (P <0.0001), number of tumors (P < 0.0001), and tumor grade (P <0.0001), but not in age, sex, alanine aminotransferase (ALT),aspartate aminotransferase (AST), alkaline phosphatase,a-fetoprotein, g-glutamyltranspeptidase (g-GT), cholesterol,and triglycerides (Table 1).

The Isx expression pattern was then detected in HCC tumorsamples by immunohistochemistry staining with anti-Isx poly-clonal antibody (Fig. 1A). Isx (brown, yellow arrows) showed atumor-specific expression pattern in HCC tumor mass (left)comparedwith the adjacent normal liver tissue (black star) andwas detected in both cytoplasm (red arrow) and nuclei (blankarrow) in tumor cells (right). IsxmRNA expression in HCC liversamples (81 patients with high Isx expression and 38 patientswith low Isx expression) was significantly upregulated com-pared with patients with non-HCCs analyzed with one-wayANOVA (Fig. 1B, P < 0.001). Analysis of the survival curves ofpatients with HCC found that patients with HCCs with lowerIsx expression had a significantly longer survival time thanthose with higher Isx expression after liver excision surgery(Fig. 1C, P¼ 0.0238). Isx expression seems to correlate with theexpression of cyclin D1, an important cell-cycle regulator.Three HCC tumor species (with HBV and HCV infection) and6 different hepatoma cell lines were found to have high ectopicIsx protein expression than normal liver tissue (with no HBV/HCV infection; Fig. 1D). The Isx and cyclin D1 mRNA expres-sion in HCC tumor specimens from 99 patients with HCCsand 11 non-HCC liver biopsies showed that the Isx mRNAexpression was highly associated with cyclin D1 expressionin all patients (R ¼ 0.7415, P < 0.0001; Fig. 1E). These resultssuggest that Isx plays an important regulatory role in HCC

Isx Regulates Cyclin D1 Expression

www.aacrjournals.org Cancer Res; 73(2) January 15, 2013 509




Table 1. Baseline characteristics of 119 patients with HCCs compared with 11 patients with non-HCCs

GroupNon-HCC(n ¼ 11)

Isx mRNA(�3; n ¼ 38)

Isx mRNA(>3; n ¼ 81) P

Age [mean(SD)] 61.5 (3.8) 57.7 (2.1) 59.0 (1.4) 0.6851Sex 0.4063b

Male 7 31 61Female 4 7 20

ALT, U/L 0.0930b

<40 10 24 3940–<100 1 12 34�100 0 2 8

ALT, U/L 0.1018b

<40 10 20 3740–<100 1 14 35�100 0 4 9

Bilirubin, mg/dL 0.0282b,c

<1.5 11 27 51�1.5 0 11 30

Albumin, mg/dL 0.0040b,c

<4 10 18 31�4 1 20 50

a-Fetoprotein, ng/mL 0.7382b

<2 10 26 552–<5 0 4 7�5 1 8 19

Triglyceridea 0.0664b

<240 11 15 28�240 0 7 5

Ac sugara 0.0281b,c

<100 10 S 12100–<120 0 6 8�120 1 S 13

Size, cm <0.0001b,c

None 11 0 0<2 0 12 152–<5 0 17 40�5 0 9 26

Number of tumors <0.0001b,c

0 11 0 01 0 29 632 0 6 8<2 0 3 10

Modified TNM <0.0001b,c

0 0 0I 15 45II 15 24III (IIIA and IIIB) 8 12

NOTE: P values were calculated by Fisher exact tests. Patients: 123 patients with HCC from 2 medical centers [Chung Ho MemorialHospital (89 HCC) and Changhua Christian Hospital (34 HCC)] with HBV and/or HCV infection and 11 non-HCCs.aNondetectable data of patients from Changhua Christian Hospital.bP values were calculated by Fisher exact test.cP < 0.05.

Hsu et al.

Cancer Res; 73(2) January 15, 2013 Cancer Research510




progression and patients survival. The highly correlatedexpression between Isx and cyclin D1 also suggests that Isxcould be a predictive marker for cyclin D1 expression andhepatoma growth.

Proinflammatory cytokines induced Isx expressionthrough NF-kB signaling pathwayTo explore the association of Isx expression with inflam-

matory cytokines in HCC tumor formation, the proinflam-matory cytokines elevated in serum or liver tissue ofpatients with hepatoma in previous studies (17–19) werefirst used to treat 6 HCC cells (Hep G2, Hep 3B, SK-Hep1,Huh7, PLC/PRF/5, and HA22T) to evaluate the inductionactivity of Isx. Proinflammatory cytokines, such as IL6, IL-1,and TNF-a, were shown to be able to increase the expres-sion of Isx mRNA at both the lower and higher concentra-tions used (5 and 20 ng/mL, respectively) for 8 hours (Fig.2A). Higher, but not lower, doses of IL8, IL1b, and TNF-balso were shown to increase Isx mRNA expression. Tofurther clarify the regulatory signaling of Isx by these

cytokines, the Isx promoter region was subcloned into aluciferase assay system. IL6, an elevated cytokine in serumsample of patients with HCCs (20), showed a dose-depen-dent regulatory effect on the promoter activity of Isx (Fig.2B and C). Also, IL6 (10 ng/mL) was shown to increase Isxprotein and cyclin D1 expression in most of hepatoma celllines except Huh7 as well as NF-kB signaling (p-p65 andIkBa) activation (Fig. 2 D). E2F1 in Isx-transfected cells,such as Hep G2, SK-Hep1, and Huh7, also showed anincrease in protein level. Furthermore, the Hep G2 cellstreated with IL6 (10 ng/mL) were separately coincubatedwith different kinase-specific inhibitors to evaluate thetranscriptional activation and the Isx expression was ana-lyzed with Western blotting. The activated Isx proteinexpression induced by IL6 treatment was aborted dosedependently by an NF-kB–specific inhibitor, BAY 11-7085(5 and 25 mmol/L; Fig. 2E). The expression correlationbetween Isx and IL-6 was also analyzed in patients withHCCs. Isx mRNA showed high correlated expression withIL6 mRNA (R2 ¼ 0.80; Supplementary Fig. S1). These results

Figure 1. Ectopic Isx showed atumor-specific expression patternand significantly decreased survivaltime in HCCs. A, Isx expressionshowed a tumor-specific expressionpattern in HCC tumor species. Isx,brown; HCC, yellow arrows. Blackstar (�), area of normal hepatocytes.Isx (cytoplasm), red arrows; Isx(nuclear), black arrow. B, Isx mRNAsignificantly expressed in HCCspecies. C, high Isx mRNAexpression in HCCs decreasedsurvival time in HCC prognosisanalyzed by Kaplan–Meier survivalanalysis. D, ectopic expression of Isxhighly associated with cyclin D1expression in HCC cell lines andpatients. HCC1, 2, and 3, HCC tumormass. Hep G2, Hep 3B, SK-Hep1,Huh7, PLC/PRF/5, and HA22T, HCCtumor cell lines. E, the mRNAexpression of cyclin D1 in non-HCCand HCC tumor species showedhighly correlated expression with IsxmRNA expression.

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showed that IL6 transcriptionally activated Isx expressionthrough the activation of NF-kB signaling.

The transcriptional regulatory elements induced by IL6 onIsx promoter (Fig. 3A) were then analyzed by promoter assayand chromatin immunoprecipitation (ChIP). IL6 significantlyincreased Isx promoter–driven luciferase activity (3,945� 253)until the promoter sequence had been deleted to a lengthshorter than 220 bp (Fig. 3B). Different segments of oligonu-cleotides between �320 and �220 bp in the Isx promoterregion were then synthesized to determine the NF-kB (p65)binding affinity by electrophoresis mobility shift assay (EMSA)in vitro. The nuclear proteins of IL6-treated cells showed highbinding affinity and were supershifted by the addition of ananti-p65 antibody in the �294 to �318 bp region on the Isxpromoter (Fig. 3C). Deletion of this NF-kB binding elementshowed abortion of the luciferase activity induced by IL6 (Fig.3B). Furthermore, the Isx promoter region (�320 to �220 bp)could be pulled down (3.91-fold) in p65 (Rel A) immunopre-cipitates treated with IL6 compared with vehicle-treated cells(Fig. 3D). This promoter-binding activity of p65 induced by IL-6

was abated when the cells were treated with NF-kB–specificinhibitor, BAY 11-7085 (5 mmol/L), MAPK kinase inhibitor(U0126, 5 mmol/L), and JAK/STAT2 inhibitor (AG490, 20mmol/L). These results suggested that signals (MAPK andJAK/STAT2) activated by IL6 could activate Isx mRNA expres-sion through increasing NF-kB (p65) binding activity onIsx promoter. However, NF-kB signaling activated by IL6-regulated Isx expression at both the transcriptional and trans-lational level.

Moreover, dietary vitamin A intake has been shown toregulate intestinal Isx expression (3), and its metabolites aresuggested to play a role in tumorigenesis. We next examinedthe potential role of vitamin A and its related metabolites onIL6-induced Isx expression. Higher levels of retinoid X receptor(RXR) binding to the Isx promoter were detected in hepatomacells than those noted in normal hepatocytes that showed nodetectable expression of Isx (Fig. 3E). Interestingly, while IL6treatment increased Isx expression in hepatoma cells, thelevels of the binding of RXRa to the Isx promoter weresignificantly reduced (ranging from 52% to 58%) in IL6-treated

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Figure 2. Inflammatory cytokinesinduced Isx expression. A,cytokines and chemokinesinduced Isx mRNA expression inHep G2 cells. ��, P < 0.005;��, P < 0.001. B, IL6 induced Isxpromoter-driven luciferase activityin Hep G2 cells. ��, P < 0.001.C, IL6 showed a dose-dependentactivation on Isx promoter–drivenluciferase activity in Hep G2 cells.��, P < 0.001. D, IL6 activates Isxprotein expression and NF-kBsignaling. E, specific inhibitors forNF-kB signaling pathway abatedthe activation of IsxmRNA (bottom)and protein expression (top)induced by IL6 (10 ng/mL).�,P < 0.001, compared with vehicletreatment; #, P < 0.001, comparingwith IL6. (n ¼ 3, means � SD).

Hsu et al.





hepatocytes, SK-Hep1 cells and, to a lesser degree, in Hep G2cells (18% reduction; Fig. 3F). These results suggested that theIsx expression induced by IL6 was a retinoid-independentregulatory pathway.

Isx expression enhanced cell proliferation in HCC celllinesTo characterize the cellular function of Isx, wild Isx and

different truncated proteins tagged with GFP were trans-fected and characterized in Hep G2 cells. Overexpressed Isxprotein (green) was mainly detected in nuclei (blue), but Isxprotein with the deleted homeobox domain was detected inwhole cells including cytoplasm and nuclei, with loss of itsoriginal nuclear localization (Fig. 4A). Interestingly, the GFPprotein fused with the Isx homeobox domain showed anuclear translocation pattern similar to that of the wild Isxprotein (Fig. 4A). The cell proliferation activity of Isx in HepG2 cells was then determined with cell growth curves and[H3] thymidine incorporation analysis. Hep G2 cells trans-fected with Isx or GFP with Isx homeo domain showed ahigher proliferation rate than that of cells transfected with

mock only or Isx protein without the homeo domain (Fig.4B). Also, overexpressed Isx increased [H3] thymidine incor-poration activity 4.9-fold in comparison to mock-transfectedHep G2 cells after 16 hours incubation (Fig. 4C). The increasein proliferation activity induced by Isx was further observedin cell-cycle analysis with flow cytometry. The Hep G2 cellstransfected with Isx showed an increase in sub-G1 (1.6%–4.2%) and S-phase cell-cycle population (20.3%–30.5%) and adecrease in G1 (42.8%–39.6%) and G2–M (25.7%–18.9%) cell-cycle population compared with mock-transfected only (Fig.4D). These results suggested that Isx increased cell prolif-eration to speed up cell-cycle progression from G1 to S-phasedespite an increase in sub-G1 apoptotic cells.

Isx upregulated cyclin D1 and E2F1 expression in HCCcells

To determine the direct targets regulated by Isx, establishedregulators of the G1–S transition were monitored by Westernblot analysis. As shown in Fig. 5A, overexpression of Isx in HepG2 cells increased D cyclins (1 and 3) and E2F1 proteinexpression and cells transfected with Isx containing a deleted

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Figure 3. Proinflammatory cytokine, IL6, transcriptionally increased Isx expression through NF-kB activation. A, illustration of serious Isx promoter deletionconstructs. B, Isx promoter assay induced by IL6 treatment. ��, P < 0.001. C, IL6 increased NF-kB (p65) binding activity on different oligonucleotidesof Isx promoter. V, nuclear proteins extracted fromvehicle-treated cells. D, NF-kB–specific inhibitor, BAY11-7085(5mmol/L), abated the Isx promoter–bindingactivity of NF-kB (p65) inducedby IL-6. ��,P<0.001. E, hepatoma showedhigher binding activity of RXRson the Isx promoter than hepatocyte. ��,P<0.001.F, IL6 decreased Isx promoter–binding activity of RXRs in hepatocyte and hepatoma. �, P < 0.05. (n ¼ 3, means � SD).






homeobox domain showed reduced activation effect on Dcyclins and E2F1 expression. The activation effects of cyclinD1 and E2F1 by Isx were further confirmed in 5 hepatomacell lines. Cyclin D1 in Isx-transfected cells except Huh7showed an increase in protein expression level (Fig. 5B).E2F1 in Isx-transfected cells, such as Hep G2, SK-Hep 1, andHuh7, also showed an increase in protein level. The regu-latory effect of Isx on these effected G1–S regulators wasfurther addressed by promoter assay. E2F1 (�1,630 to þ30bp) and cyclin D1 (�1,721 to þ21 bp) promoter regionsamplified from human placental genome by PCR were sub-cloned into a luciferase expression system. Hep G2 cellstransfected with Isx protein significantly increased lucifer-ase activity driven by cyclin D1 and E2F1 promoter, respec-tively, as compared with mock-transfected cells (Fig. 5C).This result further confirmed that Isx protein activatedcyclin D1 and E2F1 expression transcriptionally. To clarifythe possible regulatory site of Isx, the cyclin D1 promoterand different truncated promoter regions of cyclin D1 (Fig.5D, top) were subcloned into luciferase reporter constructsand transfected into Hep G2 cells to evaluate the transcrip-tional activation effects of Isx. As shown in Fig. 5D (bottom),Isx upregulated the luciferase activity driven by the cyclin D1promoter and the upregulated luciferase activity induced byIsx expression decreased to the mock-transfected level whenthe cyclin D1 promoter region was shorter than �260 bp.This result suggested that transcriptional regulation of Isxon cyclin D1 promoter was through the binding sequencebetween �260 and �297 bp.

Isx increased cyclin D1 expression through directlybinding to the cyclin D1 promoter in vitro and in vivo

To further delineate the regulatory elements of Isx on thecyclin D1 promoter region, the different regions of oligonu-cleotide between �260 and �297 bp in cyclin D1 promoterwere synthesized for EMSA analysis in vitro. The nuclearproteins extracted from Hep G2 cells transfected with Isxcould specifically bind to the region between �260 and�272 bp, and the binding complex also showed a super shift(arrow) of the anti-Isx antibody in vitro (Fig. 5E). The cyclin D1promoter fragment (�197 to �330 bp) bonded by Isx wasdetected by ChIP assay in vivo and showed an increase up to11.6-fold compared with mock-transfected cells (Fig. 5F).The binding activity of Isx on cyclin D1 promoter abated afterNF-kB inhibitor (BAY 11-7085, 5 mmol/L) treatment (Fig. 5F).Interestingly, MAPK (ERK1/2) inhibitor (PD98059, 50 mmol/L)also showed a decrease to baseline on the cyclin D1 promoterbinding activity of Isx (Fig. 5F). The regulatory effect of Isx oncyclin D1 was also determined in patients with HCCs. Isx(green) showed highly expression localization with cyclin D1(red) and Ki67(pink), a cell proliferation marker; Supplemen-tary Fig. S2). These results showed that Isx directly bound tocyclin D1 promoter and activated cyclin D1 (E2F1) expressiontranscriptionally.

Isx expression was essential for cyclin D1 expression andproliferation activity in HCC cells

To further evaluate the essential role of Isx on cell prolif-eration and tumorigenic activity, 2 sequence-specific shRNAi

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Figure 4. Overexpressed Isxincreased cell-cycle progression inHep G2 cells. A, GFP fused Isxexpression pattern in Hep G2 cells.Top, truncated construct maps ofIsx.Bottom, thecellular localizationof Isx and truncated Isx proteins.Isx, green; F-actin, red; nuclei,nuclei, blue [40,6-diamidino-2-phenylindole (DAPI)]. B, Isxoverexpression increased cellproliferation and cell growth.�, P < 0.001. C, Isx increasedcellular thymidine incorporationactivity. �, P < 0.001. D, cellpopulation analysis in different cell-cycle phases of cells transfectedwith different Isx-truncatedprotein.� and #, P < 0.001, compared withmock-transfected only (n ¼ 3,means � SD).

Hsu et al.





of Isx were transfected into 4 types of hepatoma cells withectopic Isx expression (Hep G2, Hep 3B, SK-Hep1, and HA22T)and the knockdown efficiency in these hepatoma cells wasexamined byWestern blotting (Supplementary Fig. S3 and Fig.6A). Isx protein expression was decreased 85% in Hep G2 cells

but the knockdown was less efficient in Hep 3B cells. Thesehepatoma cells with Isx knockdown were first examined todetermine the cell proliferation activity by [H3] thymidineincorporation assay. The [H3] thymidine incorporation ratein Isx knocked down cells showed a significant decrease of 62%,

Figure 5. Isx increased cyclin D1 expression through direct promoter binding. A, overexpressed Isx increased the expression of cyclin D1 and E2F1.Arrowhead, truncated Isx expression. B, Isx expression increased cyclin D1 and E2F1 expression in hepatoma cells. Arrow, E2F1. C, Isx transcriptionallyactivated cyclin D1 and E2F1 promoter activity. ��, P < 0.001. D, Isx transcriptionally activated cyclin D1 promoter activity. Top, series of deletionconstructs of cyclin D1 promoter. Bottom, relative luciferase activity. E, Isx nuclear extract from Hep G2 cells specifically binds the oligonucleotides of thecyclin D1 promoter region (�260 to �272 bp) in vitro. The DNA-binding activity of Isx nuclear extract was analyzed with EMSA. F, Isx directly boundto the promoter region of cyclin D1 determined with ChIP assay in vivo. Top, agarose gel electrophoresis analysis of ChIP. Arrowhead, cyclin D1 promoterregion (�197 to �330 bp); positive, 10% of input control; negative, no antibody. Bottom, the quantitative statistical analysis of cyclin D1 promoter–bindingactivity of Isx was determined by ChIP assay. �, P < 0.001, compared with Isx-transfected only (n ¼ 3, means � SD).






56%, 54%, and 68%, respectively, in thymidine incorporationactivity compared with those that were mock-transfected (Fig.6B). Furthermore, many G1–S transition regulators, such ascyclin D1 (84%), CDK4 (62%), and E2F1 (86%), were signifi-cantly downregulated in Isx shRNAi cells (Fig. 6C and Supple-mentary Fig. S4). Some apoptotic factors were also shown to beelevated in Isx shRNAi cells (Supplementary Fig. S5).Othertumor suppressors, such as p19 and p21, did not show anyregulatory effect.

Isx expression was critical for hepatoma tumorigenicactivity

Following the determination of cellular proliferation, thetransformational and tumorigenic activity of Isx shRNAicells were then determined with a foci formation on softagar anchorage-independent assay in vitro and tumorgrowth in nude mice in vivo. As shown in Fig. 6D, over-expression of Isx increased the transforming activity (54%)in Isx-transfected cells compared with mock-transfectedHep G2 cells; however, Isx shRNAi cells showed a significant

decrease in transforming activity (84%) in comparison withmock-transfected cells in vitro. Furthermore, Hep G2 (Fig.6E) and Hep 3B cells (Fig. 6F) with overexpressed Isx showedan increase in tumor size and tumor growth activity in nudemice in vivo compared with mock-transfected cells, and IsxshRNAi cells showed a significant decrease in tumor size andtumor growth activity compared with mock-transfectedcells. These results suggest that Isx plays an essential rolein promoting the transforming and tumorigenic activity ofhepatoma cells.

DiscussionIn this study, we identified Isx as a proto-oncogenic homeo-

box gene specifically overexpressed in HCC tumor cells. Isx, anIL6-induced homeobox gene, upregulated cyclin D1 and E2F1expression through the NF-kB signaling pathway. Throughdirect binding to the cyclin D1 promoter, Isx increased cellularcyclin D1 expression through which it regulated the cellproliferation and transforming activity of HCC tumor cellsin vitro and in vivo. The tumor-specific expression pattern,

Figure 6. Isx expression was essential for proliferation and transforming activity in HCC cells. A, Isx expression in Hep G2 cells was knocked down with2 sequence-specific shRNAi constructs. B, hepatoma cells were transfected with Isx shRNAi constructs and showed a decrease in [H3] thymidineincorporation activity. �, P < 0.001. C, the cyclin D1 and E2F1 expression was decreased in Isx shRNAi cells. D, Isx shRNAi cells showed a decreasein tumor-transforming activity in vitro. � and #, P < 0.001 compared with vector group. E and F, Isx shRNAi cells (Hep G2 and Hep 3B) showed adecrease in tumorigenic activity in vivo. ��, P < 0.001 compared with vector-transfected group. A and B, P < 0.001 compared with mock-transfectedgroup (n ¼ 3, means � SD).

Hsu et al.





highly correlated to survival time, tumor progression, andcyclin D1 (IL-6) expression in patients with HCCs, suggestedthat Isx plays an essential role in hepatoma tumor formationand prognosis.HCC is a chronic liver disease that occurs as a multistep

process characterized by progressive accumulation of genet-ic alterations causing aberrant growth, malignant transfor-mation, and metastasis (21). Homeobox genes, essentialregulatory transcription factors for multiple body plandevelopment (10), have been found to be deregulated inmany malignancies and are thought to be potential onco-genes (11). Few homeobox genes have been found to beinvolved in HCC development, excluding Hox, Prox1, andCDX2; however, most of them are expressed both in normaland in tumor cells and are thought to act as tumor sup-pressors in the liver (22–24). In this study, we found Isx, inparticular, ectopically expressed in tumor cells of HCCtumor mass, but not normal cells, and highly correlated tocyclin D1 expression in HCC tumor cells. This tumor-specificpattern and the gene silencing results suggest that Isx is animportant proto-oncogenic protein in HCC development.Isx, a gut-specific transcription factor, was a putative repres-sor for intestinal SRBI and BCMO1 expression that conse-quently repressed vitamin A production and downstreamretinoic acid (RA) signaling (3, 14). Retinoid-induced signal-ing is important in regulating cell growth, differentiation,and development (25) and found to abnormally expressand highly associated with tumor development in manymalignances, including HCCs (26). Deficiency in retinoland RXRa expression was found in tumor cells, but not inthe adjacent normal hepatocytes in patients and animalswith HCCs, which was adverse to Isx expression in patientswith HCCs (27).Also, our study suggested that IL6-induced Isx expression in

hepatoma cells was independent of the retinoid signaling.These results appeared to be at variance with the previousstudies showing that dietary vitamin A intake was able toincrease intestinal Isx expression (3). IL6 might regulate vita-min A metabolism via Isx expression, but this is, at present,uncertain in the case ofHCCs.More studieswould be needed toelucidate the regulatory effect. Furthermore, the regulatoryactivity on vitamin A by ectopic Isx expression is still unclear,although the defects of vitamin A metabolism have beenobserved in Isx knockout mice.In Isx�/� mice, the cell growth regulatory and transform-

ing activity were not observed in tissue development and theoffspring were born in the expected numbers and appearedhealthy for at least 1 year (14). This regulatory effect of Isx onproliferation and the tumor-specific ectopic expression pat-tern in HCC tumor cells suggests a malignant geneticalteration profile induced by chronic inflammation existsin HCC tumor cells. This malignant alteration circumventsactivated Isx expression and a redundant regulatory effect inthe intestine aborted the cellular proliferation effects. Thegenomic alterations at different stages are still unclear. Inthis study, ectopic Isx expression was detected in patientswith HCCs, and some patients with non-HCCs infected withHBV and/or HCV (with no hepatitis) also showed the

decreasing trend in the level of Isx mRNA expression,although the difference did not reach statistical significance.Isx expression in different liver disease stages was stillunclear because the limitation in sample collection and thedetailed regulatory mechanism of Isx in HCC tumorigenesisneeds further investigation.

Chronic inflammation induced by HBV, HCV, and alcoholare always correlated to HCC development and serves as themost common factor involved in HCC progression (28, 29).Many proinflammatory cytokines, such as TNF-a, IL1, and IL6,are found at high serum concentrations in patients with HCCs;however, the pathologic role of these cytokines in HCC devel-opment is unclear to date (18, 20). In this study, we showed thatIsx showed high expression correlation to IL6 in patients withHCCs and was an activated proto-oncogene through inflam-matory cytokines (IL-6 or TNF-a) that then subsequentlyregulate downstream cell-cycle regulators, such as cyclin D1and E2F1, to progress to cell proliferation and transformationin HCCs. This result provides a positive linkage betweeninflammation and HCC development. IL6, one of the proin-flammatory cytokines, has been reported to have high expres-sion levels in patients with HCCs (19, 20) and plays an impor-tant regulatory role in HCC development (18). Many viralproteins fromHBV or HCV, and also alcohol abuse, all increasethe IL6 expression level in liver cells (28, 30). IL6 activatesmanysignaling pathways to transform cellular responses, includingJAK/STATs, MAPK, and AKT/PI3K (17, 31), and, throughactivating JAK/STAT3 signaling, NF-kB and downstreamgenes, such as Isx, are consequently activated to mediate cellsurvival and G1 to S cell-cycle transition. In this study, onepotential IkB-binding site (RNNYYCC) on the Isx promoterregion (�294 to �318 bp) was found to respond to NF-kBregulatory activation in Isx expression. The results of thiselement deletion on the Isx promoter showed that this poten-tial IkB element was essential to NF-kB activation. Also, fromthe mouse model, increased IL6 production has also beenimplicated in the pathogenesis of HCCs (32). Furthermore, inliver regeneration, IL6 regulates cellular proliferation throughthe activation of cyclin D1 expression in hepatocytes, whichprovides a model for cyclin D1 activation by proinflammatorycytokines in HCC development (33).

In summary, in this study, we found that Isx was an impor-tant tumor-specific proto-oncogenic homeobox gene. Throughdirect regulation of cyclin D1 and E2F1 expression, Isx regu-lated the proliferation of tumor cells and their transformingactivity, and it appears worthwhile to further investigate itscellular function in HCCs.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S.-H. Hsu, K.-T. Lee, K.-W. Liu, S.-N. WangDevelopment of methodology: S.-H. Hsu, L.-T. Wang, S.-N. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.-T.Wang, Y.-L. Chen, K.-W. Liu, J.-L. Suen, C.-Y. Chai,S.-N. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.-H. Hsu, L.-T. Wang, K.-W. Liu, S.-N. WangWriting, review, and/or revision of the manuscript: S.-H. Hsu, S.-N. Wang






Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y.-L. Chen, J.-L. SuenStudy supervision: S.-H. Hsu, K.-T. Lee, S.-N. Wang

Grant SupportThis work was supported, in part, by research grant KMUH100-0R20,

KMUH100-0R21, KMUER007-4, and NSC-101-2320-B-037-042 from the NationalScience Council, Taiwan.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received July 24, 2012; revised October 20, 2012; accepted October 26, 2012;published OnlineFirst December 5, 2012.

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2013;73:508-518. Published OnlineFirst December 5, 2012.Cancer Res Shih-Hsien Hsu, Li-Ting Wang, King-Teh Lee, et al. and Survival in Hepatocellular Carcinoma

, Regulates Tumor GrowthISXProinflammatory Homeobox Gene,

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Proinﬂammatory Homeobox Gene, ISX, Regulates Tumor … · This study assessed a total of 123 patients with HCCs from July 2004 to November 2009 from multiple medical center