2010;70:6516-6526. Published OnlineFirst August 3, 2010.Cancer Res
Yingduan Cheng, Hua Geng, Suk Hang Cheng, et al.
Commonly Silenced in Multiple Carcinomas
Suppressor That Represses Multiple Oncogenes and Is KRAB Zinc Finger Protein ZNF382 Is a Proapoptotic Tumor
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Molecular and Cellular Pathobiology
KRAB Zinc Finger Protein ZNF382Is a Proapoptotic Tumor Suppressor That Represses Multiple Oncogenes and Is Commonly Silenced in Multiple Carcinomas
Yingduan Cheng 1,Hua Geng 1,Suk Hang Cheng 2,Pei Liang 2,Yan Bai 3,Jisheng
Li 1,Gopesh Srivastava 4,Margaret H.L.Ng 2,Tatsuo Fukagawa 5,Xiushan Wu 3,Anthony T.C.Chan 1,and Qian Tao 1
Abstract
Zinc finger transcription factors are involved broadly in development and tumorigenesis.Here,we report that the little studied zinc finger transcription factor ZNF382functions as a tumor suppressor in multiple carcinomas.Although broadly expressed in normal tissues,ZNF382expression was attenuated in multiple carcinoma cell lines due to promoter CpG methylation.ZNF382was also frequently methylated in multiple primary tumors (nasopharyngeal,esophageal,colon,gastric,and breast).Ectopic expression of ZNF382in silenced tumor cells significantly inhibited their clonogenicity and proliferation and induced apo-ptosis.We further found that ZNF382inhibited NF-κB and AP-1signaling and downregulated the expression of multiple oncogenes including MYC,MITF,HMGA2,and CDK6,as well as the NF-κB upstream factors STAT3,STAT5B,ID1,and IKBKE ,most likely through heterochromatin silencing.ZNF382could suppress tumorigen-esis through heterochromatin-mediated silencing,as ZNF382was colocalized and interacted with heterochro-matin protein HP1and further changed the chromatin modifications of ZNF382target oncogenes.Our data show that ZNF382is a functional tumor suppressor frequently methylated in multiple carcinomas.Cancer Res;
70(16);6516–26.?2010AACR.
Introduction
Cancer is caused by aberrant gene regulation,including in-activation of negative regulators of cell proliferation [includ-ing tumor suppressor genes (TSG)]and activation of positive regulators (such as oncogenes;ref.1).TSGs function through the following mechanisms:protecting the genome from mu-tagenic events,fine tuning the cell cycle regulation,inducing apoptosis in cells that escape normal cell cycle control,and inhibiting cellular migration and metastasis (2).While genet-ic alteration is a hallmark of human cancers,aberrant epi-genetic modification of tumor cells is also important in initiating carcinogenesis (3).Promoter CpG methylation causes the loss of TSG functions,which occurs frequently
during tumor development and progression (4).Currently,it is well accepted that epigenetic alterations even precede genetic changes during tumorigenesis (4).Clinically,TSG methylation can be used as an epigenetic biomarker for tu-mor diagnosis and prognosis prediction.
Zinc finger protein (ZFP)is the largest family of transcrip-tional factors with their zinc fingers binding to promoters to activate or repress gene expression (5).ZFPs also interact with other proteins to function in various signaling pathways and sometimes even bind to dsRNA (6).About one third of ZFPs contain a Krüppel-associated box (KRAB)domain.KRAB-con-taining ZFPs (KRAB-ZFP)are involved in cell differentiation,proliferation,apoptosis,and neoplastic transformation (7).For instance,ATM and p53–associated KZNF protein (APAK)can interact with p53to suppress p53-mediated apoptosis (8);ZNF23has growth-inhibitory potential (9);and ZBRK1,a BRCA1-dependent transcriptional repressor,plays a critical role in the control of cell growth and survival (10).
KRAB-ZFPs are also involved in heterochromatin forma-tion,which in turn leads to epigenetic silencing.KRAB-ZFPs bind to specific DNA sequences and recruit KRAB-associated protein 1(KAP1),which forms heterochromatin with HP1,SETDB1,and histone deacetylase (HDAC)inhibitor to silence target gene expression (7).The active form of KRAB-ZFPs is required to initiate heterochromatin formation;thus,irregu-lar KRAB-ZFPs result in failed formation of heterochromatin and uncontrollable gene expression leading to tumorigenesis.A large number of ZFPs are located in several gene clusters at the long arm of chromosome 19.Deletion of 19q is frequent in multiple cancers including cervical cancer,esophageal
Authors'Affiliations:1State Key Laboratory in Oncology in South China/Cancer Epigenetics Laboratory,Sir YK Pao Center for Cancer,Department of Clinical Oncology,Hong Kong Cancer Institute and Li Ka Shing Institute of Health Sciences,and 2Department of Anatomical and Cellular Pathology,Chinese University of Hong Kong,Shatin,Hong Kong;3The Center for Heart Development,Key Lab of MOE for Development Biology and Protein Chemistry,College of Life Sciences,Hunan Normal University,Changsha,China;4Department of Pathology,University of Hong Kong,Hong Kong;and 5National Institute of Genetics and The Graduate University for Advanced Studies,Mishima,Shizuoka,Japan
Note:Supplementary data for this article are available at Cancer Research Online (https://www.doczj.com/doc/6815590883.html,/).
Corresponding Author:Qian Tao,Room 315,Cancer Center,PWH,Chinese University of Hong Kong,Shatin,Hong Kong.Phone:852-2632-1340;Fax:852-2648-8842;E-mail:qtao@https://www.doczj.com/doc/6815590883.html,.hk.doi:10.1158/0008-5472.CAN-09-4566
?2010American Association for Cancer Research.Cancer Research
6516
squamous cell carcinoma(ESCC),and nasopharyngeal carci-noma(NPC;refs.11–13).Here,we investigated the potential tumor suppressor function of ZNF382,a novel19q13.12gene that we identified previously(14).We found that ZNF382 was epigenetically inactivated in common carcinomas in-cluding NPC and ESCC.We further showed that ectopic expression of ZNF382suppressed clonogenicity and prolifer-ation and induced apoptosis in tumor cells lacking endoge-nous ZNF382expression.ZNF382also repressed NF-κB and AP-1signaling and inhibited the expression of multiple oncogenes including the NF-κB upstream factors STAT3, STAT5B,ID1,and IKBKE.
Materials and Methods
Cell lines,tumors,and normal tissue samples
A series of tumor cell lines were used(15).Immortalized normal epithelial cell lines(NP69,NE1,NE3,NE083,Het-1A, HMEC,and HMEpC)were also included.HCT116cell lines with genetic knockout(KO)of DNA methyltransferases (DNMT):HCT116DNMT1?/?(1KO),HCT116DNMT3B?/?(3BKO),and HCT116DNMT1?/?DNMT3B?/?(DKO)cells (gifts of Bert Vogelstein,Johns Hopkins University,Baltimore, MD),were grown with either0.4mg/mL geneticin or 0.05mg/mL hygromycin or both.These cell lines were obtained either from the American Type Culture Collection or from our collaborators with no authentication performed. Human normal adult and fetal tissue RNA samples were purchased commercially(Stratagene or Millipore Chemicon; ref.16).DNA samples of normal nasopharyngeal and esoph-ageal tissues from healthy individuals were extracted as described previously(16).Samples of Asian Chinese primary NPC,nude mouse–passaged NPC tumors originated from North Africans,paired Hong Kong Chinese esophageal carci-nomas(T)and the matching surgical marginal normal tis-sues(N),and other carcinomas were used for methylation study(15–18).
Semiquantitative reverse transcription-PCR and
real-time PCR
The expression of ZNF382in cell lines and tissues was examined by reverse transcription-PCR(RT-PCR),with GAPDH as an internal control.Primers used are listed in Supplementary Table S1.RT-PCR was performed for32cy-cles with hot-start Go-Taq(Promega;ref.16).The signals were quantified by densitometry using the ImageJ software (https://www.doczj.com/doc/6815590883.html,/ij/)developed by Wayne Rasband (Research Services Branch,National Institute of Mental Health,Bethesda,Maryland).Real-time PCR was performed according to the manufacturer's protocol(HT7900system, Applied Biosystems),with the expression level of ZNF382 in normal tissues(larynx and esophagus)set as baseline. To screen for ZNF382target genes,we examined a panel of oncogenes(primers listed in Supplementary Table S2). For chromatin immunoprecipitation(ChIP)-quantitative PCR,the primers for c-MYC and STAT3promoters are listed in Supplementary Table S1.Methylation-specific PCR and bisulfite genomic sequencing
DNA bisulfite treatment,methylation-specific PCR(MSP), and bisulfite genomic sequencing(BGS)were performed as previously described(19).Several pairs of MSP primers tar-geting the methylated or unmethylated alleles of the promot-er region were designed and tested,and only the optimal pairs with best amplification efficiency and specificity were used(ZNF382m3/ZNF382m5and ZNF382u3/ZNF382u5;Sup-plementary Table S1).These primer pairs were also tested for not amplifying any unbisulfited DNA and thus were specific to bisulfite-converted DNA.MSP was performed for35cycles using AmpliTaq-Gold(Applied Biosystems;ref.19).BGS pri-mers were ZNF382BGS3and ZNF382BGS2(Supplementary Table S1).Amplified BGS products were TA-cloned,and five to six randomly chosen colonies were sequenced.
5-Aza-2′-deoxycytidine and trichostatin A treatment Tumor cells(1×105/mL)were allowed to grow over-night.The culture medium was then replaced with fresh medium containing5-aza-2′-deoxycytidine(Aza)at a final concentration of5to10μmol/L(Sigma-Aldrich;ref.20). Cells were allowed to grow for72hours with change of Aza-containing medium every24hours;some were further treated with the HDAC inhibitor trichostatin A(TSA)for an additional24hours.Cells were then harvested for DNA and RNA extractions.
Stress treatment
Heat shock was performed as previously described(15).In brief,the cells were incubated at42°C for1hour,followed by recovery in5%CO2incubator at37°C for2hours.Then,the cells were harvested for RNA extraction.
Gene cloning and plasmid construction
The full-length open reading frame(ORF)of ZNF382was cloned from adult testis cDNA library by nested-PCR. ZNF382CF2/ZNF382CR2was used for the first-round PCR and ZNF382CF3/ZNF382CR3was subsequently applied for the second-round PCR.All PCR reactions were performed with Pfu polymerase(Stratagene).The PCR product was then digested with Bam HI and ligated into pcDNA3.1(+)with the sequence and orientation confirmed.The same ORF segment was also subcloned into pIRES-ZsGreen1for flow cytometry analysis.
Subcellular localization
HCT116or COS7cells(5×104)were seeded on coverslips in a six-well plate.Cells were transfected with pcDNA3.1(+)-Flag-ZNF382using Fugene6(Roche).In COS7cells,ZNF382 was also cotransfected with RFP-HP1γ(21).Twenty-four hours after transfection,cells were fixed and stained with anti-Flag M2monoclonal antibody(F3165,Sigma),then incu-bated with FITC-conjugated rabbit anti-mouse IgG F(ab)2 antibody(F0313,DAKO).Subsequently,cell nuclei were stained with4′,6-diamidino-2-phenylindole(DAPI)and im-aged using an inverted fluorescence microscope(Olympus, Japan).
Methylation of ZNF382in Multiple Carcinomas
Colony formation assay
Monolayer culture was performed.HONE1,KYSE510,and HCT116cells(1×105per well)were seeded in a12-well plate and transfected with pcDNA3.1(+)-Flag-ZNF382plas-mid or the control vector(0.8μg each),using Fugene6. Forty-eight hours after transfection,cells were collected and plated at appropriate density in a six-well plate and sub-jected to G418(0.4mg/mL)selection for10to12days,with selective medium refreshed every3days.Surviving colonies (>50cells per colony)were counted after staining with Gen-tian Violet(ICM Pharma).Total RNA from the transfected cells was extracted,treated with DNase I,and analyzed by RT-PCR to confirm the ectopic expression of ZNF382.All the experiments were performed in triplicate wells three times.
Cell proliferation assay
Cell proliferation was measured by flow cytometry(BD Biosciences)with7-hydroxy-9H(1,3-dichloro-9,9-dimethyla-cridin-2-one succinimidyl ester(DDAO-SE;Invitrogen) staining.The cells were transiently transfected with pIRES-ZsGreen1-ZNF382or control vector.Twenty-four hours after transfection,1×105cells were replated in a 12-well plate.After another24hours,cells were washed with PBS and incubated with5μmol/L DDAO-SE(Molecu-lar Probes)in PBS for15minutes at37°C.The reaction was stopped by replacing the staining solution with fresh,pre-warmed medium,after which the cells were incubated for an additional30minutes at37°C.Cells were then harvested at the starting time point and at48hours,and the fluores-cence intensity was measured by flow cytometry for green fluorescent protein(GFP)–positive cells.The experiment was repeated three times independently.
Apoptosis assay
Apoptosis was assessed using the Annexin V-phycoerythrin (PE)Apoptosis Detection Kit I(BD Biosciences)by flow cy-tometry.Cell apoptosis and viability were measured by Annexin V-PE and7-amino-actinomycin(7-AAD)staining. Cell populations were counted as viable(Annexin V?, 7-AAD?),early apoptotic(Annexin V+,7-AAD?),necrotic (Annexin V?,7-AAD+),and late apoptotic cells(Annexin V+,7-AAD+).HCT116cells transfected with pIRES-ZsGreen1or pIRES-ZsGreen1-ZNF382were harvested at 48hours after transfection.GFP-positive cells were sorted by flow cytometry and the apoptotic status was analyzed after staining with Annexin V-PE and7-AAD.Both late and early apoptotic cells were counted together for relative apoptotic changes.All the experiments were performed three times.
Apoptosis was also determined by DAPI staining.Forty-eight hours after transfection of pIRES-ZsGreen1-ZNF382 or control vector,cells were fixed with pre-cold methanol for10minutes at?20°C.Subsequently,cell nuclei were stained with DAPI(0.5μg/mL)for10minutes at room temperature and examined under a fluorescence micro-scope.Condensed or fragmented nuclei indicated apoptotic changes.Coimmunoprecipitation and Western blot
For coimmunoprecipitation,1×106HEK293cells were cotransfected with2μg of pcDNA3.1(+)-Flag-ZNF382and pEGFP-C1-HP1β(mouse HP1β;a gift from Alain Verreault, Institute for Research in Immunology and Cancer,Mon-treal,Canada)using Fugene6.Forty-eight hours after transfection,cells were lysed in200μL of lysis buffer [50mmol/L NaCl,20mmol/L Tris(pH7.6),1%NP40,1×protease inhibitor mixture]for1hour on ice.Cell lysates were centrifuged at10,000×g at4°C for10minutes.A total of200μg of protein were used for coimmunoprecipi-tation reaction.First,total protein lysates were precleared by20μL of protein G-agarose with incubation at4°C for 30minutes.After a brief centrifugation,the supernatant was transferred to a new1.5-mL tube.The immunoprecip-itation reaction was performed with2μg of the indicated antibody and50μL of protein-G slurry for overnight incubation at4°C.The precipitated materials were separat-ed by SDS-PAGE,transferred onto a polyvinylidene difluor-ide(PVDF)membrane,and immunoblotted as described previously(18).
For Western blot analysis,48hours after transfection,cells were harvested and lysed in lysis buffer[10mmol/L Tris-HCl (pH7.4),1%SDS,10%glycerol,5mmol/L MgCl2,1mmol/L phenylmethylsulfonyl fluoride,1mmol/L sodium orthovan-date,5μg/mL leupeptin,and21μg/mL aprotinin].A total of 30μg of protein lysates were separated by SDS-PAGE and transferred onto a PVDF membrane.The dilution of primary antibodies was according to the company's recommendation. Proteins were visualized using the enhanced chemilumines-cence detection system.
Chromatin immunoprecipitation
ChIP assays were performed according to the protocol of the kit manufacturer(Upstate).Specific H3K4me3anti-body was used(ab8580,Abcam).The same amount of non-specific IgG was used as antibody control.Both input DNA and immunoprecipitated DNA were purified with Qiagen DNA mini kit.All samples were analyzed by real-time PCR.The fold enrichment of target sequence was deter-mined using the following formula:fold enrichment= 2(ΔCT of input?ΔCT of IP'ed DNA).The experiment was repeat-ed three times independently.
Luciferase reporter assay
To screen for signaling pathways modulated by ZNF382, several signaling pathway luciferase reporters were evaluated in ZNF382-transfected HCT116and HEK293cells,including NF-κB-luc,p53-luc,AP-1-luc,SRE-luc(Stratagene),and TOP-FLASH[kindly provided by Prof.Christof Niehrs,German Cancer Research Center(DKFZ),Heidelberg,Germany]. Luciferase assay was done as previously described(22).All the experiments were performed in triplicates.
Statistical analysis
Data were presented as mean±SD.Statistical analysis was carried out with Student's t test.P<0.05was considered as statistically significant difference.
Cheng et al.
Results
Expression profiling of ZNF382in normal tissues and tumor cell lines
We previously identified a novel KRAB-ZFP,ZNF382,at 19q13.12(14).By semiquantitative RT-PCR,which is more sen-sitive than the previous Northern blot,we found that ZNF382 was expressed in all normal adult tissues and fetal tissues ex-amined,as well as in normal peripheral blood mononuclear cells,but with varying expression levels(Fig.1A).In contrast,ZNF382expression was frequently silenced or reduced in mul-tiple carcinoma cell lines of nasopharyngeal,lung,esophageal, colon,stomach,breast,and cervical cancers,but infrequently in liver and prostate cancers(Fig.1B).This expression pattern was further confirmed by real-time PCR in some representative cell lines(Fig.1C).ZNF382was also highly expressed in seven immortalized normal epithelial cell lines examined(NP69,NE1, NE3,NE083,Het-1A,HMEC,and HMEpC).These results sug-gest that ZNF382is a broadly expressed gene but is frequently disrupted in multiple carcinoma cell
lines.
Figure1.A,top,the ZNF382CGI.Transcription start site is indicated by a curved arrow.The CGI,MSP,and BGS regions analyzed are indicated. Bottom,expression profile of ZNF382in human normal adult and fetal tissues by semiquantitative RT-PCR with GAPDH as a control.B,representative analyses of ZNF382expression and promoter methylation in tumor cell lines and immortalized epithelial cells.M,methylated;U,unmethylated.
C,real-time PCR analysis of ZNF382expression in several representative cell lines.The expression level of ZNF382in NPC cell lines was normalized to that in normal larynx,whereas the expression in esophageal carcinoma cell lines was compared with that in normal esophagus.D,high-resolution methylation analysis of the ZNF382promoter by BGS in tumor cell lines and immortalized normal cell line NP69.
Methylation of ZNF382in Multiple Carcinomas
Frequent inactivation of ZNF382by promoter
CpG methylation
Sequence analysis revealed that the ZNF382promoter con-tains a CpG island(CGI;Fig.1A),indicating CpG methylation may be a major mechanism silencing its expression in cancer cells(20).MSP analysis showed that the ZNF382promoter was methylated in6of6NPC,1of1hypopharyngeal,3of 5(60%)of lung,16of18(89%)esophageal,4of4colon,15 of15(100%)gastric,4of13(30%)hepatocellular,5of10 (50%)breast,and3of4cervical cancer cell lines and with no methylation detected in3prostate cancer cell lines (Fig.1B).In contrast,obvious ZNF382methylation was not detected in7immortalized normal epithelial cell lines (Fig.1B),suggesting that the methylation of ZNF382is tumor specific.A good correlation between downregulation and methylation of ZNF382was observed in these carcinoma cell lines.We further examined ZNF382methylation in detail by high-resolution BGS analysis.The results confirmed those of the MSP analysis(Fig.1D).
Pharmacologic and genetic demethylation restores ZNF382expression
To determine whether CpG methylation directly mediates ZNF382downregulation,several carcinoma cell lines,C666-1, HK1,HNE1,HONE1,HCT116,EC109,KYSE510,KYSE520, MB231,and MB435,were treated with the DNA methyltrans-ferase inhibitor Aza alone or combined with the HDAC inhib-itor TSA.The treatment restored ZNF382expression, accompanied by a decrease of methylated promoter alleles and an increase of unmethylated alleles(Fig.2A).ZNF382 expression could also be activated by genetic demethylation through double KO of both DNMT1and DNMT3B(DKO cell line),but not through single KO of DNMT1or DNMT3B(1KO or3BKO cell line),in a colorectal cancer cell line model(Fig. 2B),indicating that the maintenance of ZNF382methylation was mediated by DNMT1and DNMT3B together,similar to the other TSGs we have examined(15,16).Detailed BGS analysis confirmed the demethylation of ZNF382(Fig.2C). These results indicate that promoter methylation directly mediates the transcriptional silencing of ZNF382. Promoter methylation disrupts the stress
response of ZNF382
Three heat shock factor binding sites were predicted in the ZNF382promoter[one localizes within the CGI whereas the other two are adjacent to the CGI(MatInspector;http:// www.genomatix.de)],indicating that ZNF382might be a stress-responsive gene(Fig.2D).We thus examined the re-sponse of ZNF382to heat shock stimulation in several cell lines.ZNF382expression was significantly increased on heat shock treatment in HK1,CNE1(weakly unmethylated),and CNE2(hemi-unmethylated)cell lines.This response was, however,abolished in completely methylated HCT116cells. In contrast,heat shock–activated ZNF382expression was rescued in pharmacologically demethylated HCT116cells (Fig.2D).Taken together,these results show that ZNF382is indeed stress responsive,and promoter methylation disrupts its cellular protective response to environmental stresses.Frequent ZNF382methylation in primary tumors
We further examined ZNF382methylation in multiple pri-mary tumors and the corresponding normal tissues.ZNF382 was not methylated in normal nasopharyngeal(0of10)and esophageal epithelial tissues(0of7),but was frequently methylated in88.9%(48of54)of Asian Chinese primary NPC,2of3nude mice-passaged undifferentiated NPC tu-mors from North Africans(C15,C17,and C18),and71.4% (20of28)of esophageal,72.7%(8of11)of colon,63.6% (7of11)of gastric,and18.2%(2of11)of breast tumors (Fig.3A and B).Aberrant methylation was also detected in 10.7%(3of28)paired surgical marginal esophageal tissues from esophageal carcinoma patients,which could be due to the presence of premalignant lesions or infiltrating tumor cells.The transcript level of ZNF382was reduced in19of21 methylated primary tumors but not in unmethylated primary tumors as examined by semiquantitative RT-PCR(Fig.3C), indicating a good correlation between ZNF382promoter methylation and its transcriptional silencing in primary tu-mors.It should be noted that primary tumors,if not micro-dissected,would also have background expression of ZNF382transcripts due to infiltrating normal cells(17), which could explain the situation of the two not downregu-lated,methylated cases(#62and#73).Nevertheless,these results show that ZNF382methylation is a common event in multiple tumorigenesis.
ZNF382is a nuclear protein inhibiting the NF-κB and AP-1signaling pathways
KS1,the rat homologue of human ZNF382,is a nuclear protein that functions as a transcription repressor(23,24). We examined the subcellular localization of ZNF382.Indirect immunofluorescent staining showed that Flag-tagged ZNF382was also a nuclear protein(Fig.4A).Furthermore, by screening several signaling pathways with luciferase reporter assays,we found that ZNF382significantly sup-pressed NF-κB and AP-1reporter activities in HCT116tumor cells;however,in an immortalized normal epithelial cell line, HEK293,it only significantly inhibited AP-1activity(Fig.4B). We further confirmed the inhibitory effect of ZNF382on NF-κB activity in two other tumor cell lines,KYSE150and HNE1(Fig.4C).Both NF-κB and AP-1signaling are impor-tant in cell proliferation,survival,apoptosis,and malignant transformation(25,26).Thus,ZNF382could induce apopto-sis and inhibit cell proliferation by suppressing both the NF-κB and the AP-1signaling pathways.
ZNF382inhibits clonogenicity and proliferation and induces apoptosis of tumor cells
To assess the functions of ZNF382in tumor cells,Flag-ZNF382–expressing plasmid was transfected into HONE1, KYSE510,and HCT116cells with completely methylated/ silenced ZNF382.Ectopic expression of ZNF382dramatically reduced the colony formation efficiency of these cells(down to10.2–42.9%;P<0.01;Fig.5A).The ZNF382inhibition of cell proliferation was further determined by flow cytometry with DDAO-SE staining.Forty-eight hours after transfection,the fluorescence intensity in ZNF382-transfected HCT116cells
Cheng et al.
was higher than that in control vector –transfected cells after GFP sorting,which provided direct evidence that ZNF382could inhibit tumor cell proliferation (Fig.5B).
To explore the mechanism of tumor suppression by ZNF382,we performed an apoptosis assay using flow cyto-metry with Annexin V-PE and 7-AAD double staining.pIRES-ZsGreen1,which could translate the gene of interest and ZsGreen1from a single bicistronic mRNA,was used to sort transfected positive cells.We determined the percentage of Annexin V(+/?)and 7-AAD(+/?)cells for GFP-positive cells.In HCT116cells,ectopic ZNF382expression resulted in a significant increase of apoptotic cells as compared with control (Fig.5C).Apoptotic induction was further confirmed by DAPI staining at the individual-cell level.pIRES-ZsGreen1-ZNF382–transfected cells (GFP positive)showed obvious condensed or fragmented nuclei,a remarkable cell apoptotic feature,which was not observed in vector control –transfected cells (Fig.5D).Evidence of apoptosis was also detected by Western blot.Ectopic expression of ZNF382could signifi-cantly increase the cleavage of poly(ADP-ribose)polymerase (PARP)in HCT116and HNE1cells as compared with control (Fig.5C).Taken together,these results support that ZNF382functions as a tumor suppressor by inducing apoptosis and inhibiting the proliferation of tumor
cells.
Figure 2.Restoration of ZNF382by demethylation.A,pharmacologic demethylation with Aza alone or Aza combined with TSA (A +T)activates ZNF382expression in tumor cell lines,accompanied by demethylation of the promoter.B,genetic demethylation through double KO of DNMT1and DNMT3B in HCT116cells restores ZNF382expression,but not with single KO of DNMT1(1KO)or DNMT3B (3BKO).C,detailed BGS analysis confirms the
demethylation of ZNF382promoter in HCT116-DKO cell line.D,epigenetic silencing of ZNF382results in the disruption of its response to heat shock stress.Top,locations of heat shock factor (HSF)binding sites and the ZNF382promoter CGI are indicated;bottom,ZNF382expression is upregulated in
weakly unmethylated (HK1and CNE1)or hemi-unmethylated (CNE2)cell lines but not in the fully methylated cell line (HCT116)upon heat shock treatment,and pharmacologic demethylation of HCT116is able to restore this heat shock response of ZNF382.
Methylation of ZNF382in Multiple Carcinomas
ZNF382represses the expression of multiple oncogenes including NF-κB pathway upstream effectors through heterochromatin silencing
Because ZNF382(KS1)is a repressor,we screened for its potential target genes by examining the expression changes of34major oncogenes that have been reported to be in-volved in proliferation/transformation/apoptosis inhibition and signaling pathways,after ectopic ZNF382expression in HCT116.Real-time PCR analysis revealed that,as compared with vector control–transfected cells,ZNF382expression sig-nificantly downregulated the expression of multiple onco-genes at the mRNA level,including STAT3,MYC,ID1, IKBKE,STAT5B,MITF,HMGA2,and CDK6,whereas the ex-pression of other oncogenes such as ID2and CCND1was not affected(Fig.6A).Downregulation of STAT3and STAT5B was further confirmed by Western blot(Fig.6A).Some of these oncogenes were known to be NF-κB pathway upstream effectors,such as STAT3,STAT5B,IKBKE,and ID1(27–30). Thus,ZNF382could inhibit the NF-κB pathway by suppres-sing its upstream effectors.
To explore the possible mechanism of transcriptional re-pression mediated by ZNF382,we checked the involvement of heterochromatin silencing,as previous study revealed that KS1is located in the nucleus and interacts with KAP1(23,24) whereas KAP1is known to form a complex with heterochro-matin proteins(HP)to exert gene silencing(31).Indirect immunofluorescence staining for Flag-tagged ZNF382in HCT116and COS7cells clearly showed that ZNF382is a nu-clear protein(Fig.4A)and also colocalizes with HP1(Fig.6B). The interaction between ZNF382and HP1βwas further con-firmed by coimmunoprecipitation assay(Fig.6B).It is well known that H3K4me3(trimethylation of histone H3at the lysine4residue)is a marker of transcriptionally active chro-matin,whereas H3K9me3modification is a marker of inac-tive chromatin(32).Our ChIP assays further showed that ectopic expression of ZNF382could significantly decrease the levels of H3K4me3on the promoters of its target onco-genes,STAT3and c-MYC(Fig.6C).Taken together,our re-sults suggest that ZNF382is functionally involved in heterochromatin-mediated gene silencing,by the suppres-sion of multiple oncogenes.
Discussion
In this report,we found that ZNF382,a KRAB-ZFP we iden-tified previously,is frequently downregulated by
promoter Figure3.Representative analyses of ZNF382methylation in normal tissues and primary tumors by MSP.A,normal epithelial tissues.B,primary NPC, esophageal carcinomas(T),and their surgical marginal normal tissues(N)and other carcinomas.C,semiquantitative RT-PCR analysis of ZNF382 expression in primary NPC relative to GAPDH expression levels.The expression levels of ZNF382in NPC tumors were normalized to that in the normal control cell line NP69.
Cheng et al.
CpG methylation in multiple tumors.We further found that ectopic expression of ZNF382in silenced carcinoma cells dra-matically inhibits their colony formation by inhibiting cell proliferation and inducing apoptosis.We also showed that ZNF382inhibits NF-κB and AP-1signaling and suppresses the expression of multiple oncogenes likely through hetero-chromatin silencing.Thus,ZNF382functions as a broad tu-mor suppressor in human cancers.
KRAB-ZFPs play important roles in various epigenetic regulations.During early mouse development,KRAB domain triggers de novo promoter methylation (33).Zfp57maintains both maternal and paternal imprints in mice (34).The KRAB domain binds to KAP1,which further recruits HP1,SETDB1(H3K9methyltransferase),and HDAC complexes,whereas the zinc finger domain specifically binds to nucleation sites,forming heterochromatin for gene silencing (35).Defects in chromatin-related activities,such as chromatin assembly and remodeling,may trigger tumorigenesis (36).The disrup-tion of DNA binding proteins (KRAB-ZFPs)may thus cause failure in heterochromatin formation and contribute to tu-morigenesis.We found that ZNF382colocalizes and interacts with heterochromatin protein HP1,together with the previ-ous report of the colocalization of the ZNF382rat homolo-gene KS1with KAP1(24),indicating that ZNF382is indeed involved in heterochromatin formation and silencing.Thus,ZNF382might directly bind to target gene promoters as a repressor (24)or form heterochromatin on target gene pro-moters together with KAP1and HP1,leading to transcrip-tional repression of target genes.The epigenetic disruption of ZNF382would cause the aberrant expression of normally repressed genes mediated by heterochromatin.
Indeed,we found that ZNF382could significantly repress the RNA levels of multiple oncogenes involved in neoplastic transformation,apoptosis,cell cycle,and proliferation,in-cluding MYC,STAT3,ID1,IKBKE,STAT5B,MITF,HMGA2,and CDK6.We further verified that the downregulation of STAT3and MYC by ZNF382was mainly mediated through heterochromatin silencing.Among these targets,MYC,MITF ,and CDK6are involved in cell cycle regulation and apoptosis (37–39).STAT3,MITF ,and HMGA2are involved in
neoplastic
Figure 4.A,nuclear localization of ZNF382by indirect
immunofluorescence in HCT116and COS7cell lines.Left,FITC green fluorescence of Flag-tagged ZNF382;middle,DAPI-stained cell nuclei;right,merged images.
B,the effects of ZNF382on several signaling pathways in HCT116and HEK293cells were assessed by dual-luciferase reporter assays.*,P <0.05;**,P <0.01.C,ZNF382significantly inhibits NF-κB
activity in KYSE150and HNE1cell lines.*,P <0.05;**,P <0.01.
Methylation of ZNF382in Multiple Carcinomas
transformation(40–42).Some targets are NF-κB upstream regulatory factors,such as STAT3,STAT5B,ID1,and IKBKE. STAT3and STAT5B are required for maintaining NF-κB ac-tivity(27,28).ID1promotes cell survival by regulating NF-κB activity in prostate and breast cancers(30,43);IKBKE is an oncogene controlling the activity of NF-κB in cell prolifera-tion and malignant transformation(29).In agreement, ZNF382inhibits the oncogenic NF-κB signaling pathway as well as the AP-1signaling pathway.
NF-κB is a dimeric transcriptional factor,involved in the regulation of cell proliferation,apoptosis,angiogenesis,and cell invasion(25).NF-κB is highly activated in many cancers, accompanied by the upregulation of its downstream onco-proteins.Thus,the NF-κB pathway is considered as a cell survival and antiapoptotic signaling pathway(25).Some TSGs exert their tumor-suppressive functions by inhibiting the NF-κB pathway,such as CYLD and CHFR(44,45). NF-κB also cross-talks with the AP-1signaling pathway (46).AP-1is another dimeric factor regulating multiple cel-lular processes including proliferation,apoptosis,and differ-entiation.Activated AP-1not only induces apoptosis for certain tumors or specific stages of tumorigenesis but also promotes cell survival for other tumor types(26).Several TSGs can inhibit the proliferation of colon cancer by inhi-biting AP-1activity,such as PDCD4and HINT1(47,48).KS1, the rat homologue of human ZNF382,antagonizes Ras-, Gα12-,or Gα13-induced neoplastic transformation,but does not induce apoptosis(23).Since NF-κB and AP-1are both downstream targets of Ras,Gα12and Gα13(25,26,49, 50),ZNF382could antagonize Ras oncogene–induced trans-formation by inhibiting downstream events such as NF-κB and AP-1
signaling.
Figure5.ZNF382is a functional TSG.A,left,representative colony formation assay with monolayer culture.Right,ectopic ZNF382expression in tumor cells was confirmed by RT-PCR.Bottom,quantitative analysis of colony formation ability in ZNF382-transfected cells.The number of G418-resistant colonies(>50cells)in each vector-transfected cells was set to100.**,P<0.01.B,ZNF382inhibits cell proliferation in HCT116cells by DDAO-SE assay.The fluorescence intensities of ZNF382-and control vector–transfected HCT116cells at different time points were indicated by different colors. C,left,relative proportions of apoptotic cells in ZNF382-and control vector–transfected HCT116cells as analyzed by flow cytometry after staining with Annexin V and7-AAD.Right,Western blot of apoptotic indicator,cleaved PARP,in ZNF382-transfected HCT116and HNE1cells.D,ZNF382induced the apoptosis-associated cell nuclear changes.Cells were stained with DAPI and photographed under a fluorescence microscope48h after transfection. Red arrows,transfection-positive cells.
Cheng et al.
In conclusion,we found that ZNF382is a functional TSG,inducing apoptosis,inhibiting cell proliferation,and suppres-sing multiple oncogenes.The antitumorigenic effect of ZNF382may act through suppressing both the NF-κB and the AP-1signaling pathways (Fig.6D),whereas epigenetic si-lencing of ZNF382would activate these cancer signaling path-ways during tumorigenesis.In addition,the high frequency and tumor-specific methylation of ZNF382in NPC and esophageal carcinomas indicate that it could be used as a potential epigenetic biomarker for their molecular diagnosis.Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Bert Vogelstein,George Tsao,Michael Obster (Dolly Huang),Kai-Tai Yao,and Gui-yuan Li for some of the cell lines;DSMZ (German Collection of Microorganisms and Cell Cultures)for the KYSE cell lines (Shimada et al.,Cancer 69:277-284;1992);Jianming Ying for normal nasopharyngeal DNA samples;and Ying Ying for critical reading of the manuscript.
Grant Support
Hong Kong RGC grant 474407,Chinese University of Hong Kong Scheme C,and a Chinese University of Hong Kong direct grant.
The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section 1734solely to indicate this fact.
Received 12/16/2009;revised 06/09/2010;accepted 06/15/2010;published OnlineFirst 08/03/2010.
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