The FASEB Journal?Research Communication
Signal transducer and activator of transcription3is a transcriptional factor regulating the gene expression of SALL4
J.Dien Bard,*,1P.Gelebart,*,1H.M.Amin,?L.C.Young,*Y.Ma,?and https://www.doczj.com/doc/2515424994.html,i*,2
*Department of Laboratory Medicine and Pathology,University of Alberta,Edmonton,Alberta,
Canada;?Department of Hematopathology,University of Texas M.D.Anderson Cancer Center,
Houston,Texas,USA;and?Department of Hematopathology,Nevada Cancer Center,Las Vegas,
Nevada,USA
ABSTRACT Both signal transducer and activator of transcription3(STAT3)and SALL4are important in maintaining the pluripotent and self-renewal state of embryonic stem cells.We hypothesized that STAT3,a latent transcriptional factor,may regulate the gene expression of SALL4.In support of this hypothesis, DNA sequence analysis of the SALL4gene promoter revealed four putative STAT3-binding https://www.doczj.com/doc/2515424994.html,ing a SALL4-luciferase reporter gene assay,we found that modulation of the STAT3activity signi?cantly up-regu-lated the luciferase activity.By chromatin immunopre-cipitation,the segment of the SALL4promoter showing the highest af?nity to STAT3was localized to?366to ?163,in which there was only one putative STAT3 binding site starting at?199.Site-directed mutagenesis of all four putative STAT3-binding sites in the SALL4 promoter signi?cantly reduced its responsiveness to STAT3,although the most dramatic effect was seen at the binding site starting at?199.We further tested the functional relationship between STAT3and SALL4us-ing MDA-MB-231,a breast cell line carrying constitutive SALL4expression and STAT3activity.Down-regulation of the STAT3activity using a dominant-negative con-struct resulted in a signi?cant decrease in the expres-sion of SALL4.To conclude,our data suggest that STAT3and SALL4probably cooperate in both physio-logical and pathological states.—Dien Bard,J.,Gele-bart,P.,Amin,H.M.,Young,L.C.,Ma,Y.,Lai,R. Signal transducer and activator of transcription3is a transcriptional factor regulating the gene expression of SALL4.FASEB J.23,1405–1414(2009)
Key Words:cell signaling?luciferase assay?chromatin immu-noprecipitation
Signal transducers and activators of transcription (STATs)consist of7latent cytoplasmic transcription factors that are involved in cell cycle progression,differ-entiation,and survival in response to various cytokines and growth factors(1,2).In contrast to normal cells in which phosphorylation and activation of STAT3is a transient process,cancer cells often have constitutively active STAT3;aberrant STAT3activation has been re-ported in several types of human cancers,including breast cancer(3),pancreatic cancer(4),and lymphomas(5). With the use of a variety of experimental models,it has been shown that constitutive activation of STAT3directly contributes to oncogenesis(6,7).Aside from its roles in oncogenesis,STAT3has been implicated as a major player in murine embryonic development,as targeted disruption of the STAT3gene resulted in embryonic lethality(8).STAT3also has been strongly implicated in the maintenance of the pluripotent and self-renewal state in mouse embryonic stem(ES)cells,because blockade of STAT3signaling using a dominant-negative construct results in a loss in pluripotency and a transition into cell differentiation in these cells(9-11).The importance of STAT3signaling in these cells is probably related to the observation that the self-renewal property of ES cells depends on leukemia inhibitory factor,a cytokine known to trigger the activation of the Janus kinase/STAT3sig-naling pathway(15,16).
In addition to STAT3,the fundamental role of several other transcription factors in the self-renewal process of ES cells also has been established(17-20).One of these proteins is SALL4,a zinc?nger transcriptional factor that is one of four members of the SALL gene family originally cloned on the basis of the DNA sequence homologue to Drosophila spalt(21,22).Mutations of SALL4in humans has been reported in the Okihiro/Duane-radial ray syn-drome,an autosomal dominant disorder that is charac-terized by radial ray defects and associated with defects in multiple organs(21).Recent studies have shown that SALL4plays an essential role in maintaining the pluripo-tency of murine ES cells,because decreasing SALL4 expression by small interfering RNA(siRNA)results in the differentiation of ES cells to the trophoblast stage (23).Similar to STAT3,SALL4has been implicated in tumorigenesis in humans;for instance,its direct role in 1These authors contributed equally to this work.
2Correspondence:Dept.of Laboratory Medicine and Pa-thology,University of Alberta,11560University Ave.,Edmon-ton,AB,Canada T6G1Z2.E-mail:raymondl@cancerboard.ab.ca doi:10.1096/fj.08-117721
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0892-6638/09/0023-1405?FASEB
the pathogenesis of acute myeloid leukemia was recently documented(21,24,25).In hematological neoplasms, SALL4expression was found in acute myeloid leukemia and precursor B-cell lymphoblastic leukemia/lymphoma, but not in those carrying a mature phenotype(25).This result correlates well with the normal expression pattern of SALL4,which is restricted to stem cells.Interestingly, expression of SALL4mRNA also has been found in human epithelial ovarian cancer,a tumor type known to have constitutively active STAT3(26,27).
Because STAT3and SALL4share a similar role in maintaining the pluripotent state of the ES cells,these two proteins may be functionally linked to each other.In this study,we speci?cally tested the hypothesis that STAT3is a transcriptional factor regulating the gene expression of SALL4.We provide evidence that STAT3binds to the promoter of the SALL4gene and signi?cantly modulates the transcriptional activity of SALL4.Our data suggest that SALL4and STAT3may cooperate with each other in regulating the pluripotency of ES cells and promoting tumorigenesis.
MATERIALS AND METHODS
Construction of STAT3C tet-off
The STAT3C construct was a generous gift from Dr.J.F. Bromberg(Memorial Sloan-Kettering Cancer Center,New York, NY,USA),and its properties have been well characterized and described(7).Details of the tetracycline-off STAT3C(STAT3C tet-off)expression system have been described previously(28).
Cell lines and culture
Human breast cancer cell lines MCF-7,MDA-MB-231,MDA-MB-468,and HL-60were obtained from the American Type Culture Collection(Manassas,VA,USA).Mino cells,a mantle cell lymphoma cell line of mature B-cell immunophenotype, were described previously(29).All cell lines were grown at 37°C in5%CO2and maintained in Dulbecco’s modi?ed Eagle’s medium or RPMI1640(Sigma-Aldrich,Oakville,ON, Canada).Culture media were enriched with10%FBS(Life Technologies,Inc.,Grand Island,NY,USA)and antibiotics (10,000U/mL penicillin G and10000?g/mL streptomycin; Life Technologies,Inc.).MCF-7cells permanently trans-fected with the tetracycline-controlled transactivator and TRE-STAT3C plasmids were selected and maintained by the addition of800?g/ml geneticin(Life Technologies,Inc.)to the culture media(28).
SALL4promoter construct
The5?-?anking region of SALL4was ampli?ed using the following primer set:5?-GGGGTACCGCTCAATCAATTAT-TATTATTAC and3?-CCCAAGCTTGCGAGCATCG GGGCG-CCGGGAGAG to generate a fragment of2000bp upstream of the?rst ATG carrying the Kpn and Hind II restriction enzyme site at each end,respectively.Human genomic DNA isolated from the kidney was used to amplify this5?-?anking region of SALL4.After Kpn and Hind II digestion,this fragment was cloned into the promoterless pGL3-Basic luciferase reporter plasmid(Promega,Madison,WI,USA)to generate the SALL4/luciferase reporter.Gene transfection
Transient gene transfection of cell lines with various vectors was performed using the Lipofectamine2000transfection reagent(Invitrogen Life Technologies,Carlsbad,CA,USA) according to the manufacturer’s protocol.Brie?y,breast cancer cell lines were grown in6-well culture plates without antibiotics.When cells reached?90%con?uency,the culture medium was replaced with serum-free Opti-MEM I(Life Technologies,Inc.),and cells were transfected with the DNA:lipofectamine complex.For all of the in vitro experi-ments,STAT3C tet-off MCF-7cells were subjected to transient transfection with2?g of SALL4/luciferase and1?g of ?-galactosidase(?-GAL)plasmid.To regulate the expression of STAT3C in these cells,various concentrations of tetracy-cline(Invitrogen)were added to the cell culture.
Western blot analysis and antibodies
Western blot analysis was performed using standard tech-niques described in detail previously(29).Antibodies used included anti-FLAG(1:3000,Sigma-Aldrich),anti-STAT3(1: 500,Santa Cruz Biotechnology,Inc.,Santa Cruz,CA,USA), anti-pSTAT3(1:500,Santa Cruz Biotechnology,Inc.),anti-SALL4,anti-?-tubulin(1:1000,Santa Cruz Biotechnology, Inc.),and anti-?-actin(1:3000,Sigma-Aldrich).The genera-tion of the SALL4antibody was described previously(25).In brief,to prepare an antipeptide antibody,the peptide MSRRKQAKPQHIN of human SALL4was chosen,and the antibody was produced in rabbits in collaboration with Lam-pire Biological Laboratories(Piperville,PA,USA).
Nuclear extraction
Nuclear extraction of MDA-MB-231,MCF-7,SU-DHL-1,and Mino cells was performed using a nuclear extraction kit according to the manufacturer’s protocol(Panomics,Fre-mont,CA,USA).Brie?y,buffer A was added to1?106cells, and the solution was rocked on ice for10min at200rpm and centrifuged at14,000g for3min at4°C.After the supernatant was removed(the cytoplasmic portion),buffer B was added to the cell pellets,which were rocked on ice for2h at200rpm. Samples were then centrifuged at14,000g for3min at4°C, and the supernatant(the nuclear portion)was transferred to a new microcentrifuge tube for Western blot analysis.
Reverse transcriptase-polymerase chain reaction(RT-PCR)
Total cellular RNA was extracted from MCF-7,MDA-MB-231, MDA-MB-468,Mino,and HL-60cells and10primary breast tumors using TRIzol extraction according to the manufacturer’s protocol(Life Technologies,Inc.).Reverse transcription was performed using500ng of total RNA in a?rst-strand cDNA synthesis reaction with SuperScript reverse transcriptase as rec-ommended by the manufacturer(Invitrogen).Glyceraldehyde-3-phosphate dehydrogenase was included as an internal control. The SALL4primer sequences were obtained from a previously published article(30).The primer sequences for SALL4are forward5?-CATGATGGCTTCCT TAGATGCCCCAG-3?and re-verse5?CCGTGTGTCATGTAGTGA ACCTTTAAG-3?,with an expected product size of500bp.The primer sequences for GAPDH are forward5?-AAGGTCATCCCTGAGCTGA-3?and re-verse5?-CCCTGTTGCTGTAGCCAAAT-3?,with an expected product size of316bp.PCR was performed by adding1?l of RT product into a25-?l volume reaction containing1?buffer,a 200?M concentration of each dNTP,oligonucleotide primer, and0.2U of AmpliTaq polymerase.For DNA ampli?cation, cDNA was denatured at94°C for1min and subjected to primer
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annealing at60°C for1min,followed by DNA extension at72°C for1min for40cycles in a thermal cycler(Applied Biosystems, Foster City,CA,USA).Ampli?ed products were analyzed by DNA gel electrophoresis in1%agarose and visualized by the Alpha Imager3400(Alpha Innotech,San Leandro,CA,USA). Sequencing of the SALL4PCR products was performed using a TOPO TA Cloning Kit(Invitrogen)and a3130XL Genetics Analyzer(Applied Biosystems).
Luciferase assay
STAT3C tet-off MCF-7cells were subjected to double transfection with the SALL4/luciferase reporter construct and?-GAL.Ly-sates were prepared24h after transfection on ice using freshly prepared lysis buffer(1M Tris-HCl,pH7.8;10%Nonidet P-40; and1M dithiothreitol),incubated on ice for15min,and plated onto96-well plates(Corning Life Sciences,Lowell,MA,USA). Samples were analyzed using the FLUOstar Optima microplate reader(BMG Labtech,Offenburg,Germany).A portion of the lysate was used to measure?-GAL,an internal control for transfection ef?ciency.The?-GAL activity was measured colori-metrically at405nm using a microplate reader(Bio-Rad, Mississauga,ON,Canada).The luciferase activity was normal-ized against the?-GAL activity to minimize experimental vari-abilities owing to differences in cell viability or transfection ef?ciency.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation was performed using a Magna ChIP Assay Kit according to the manufacturer’s pro-tocol(Upstate,Charlottesville,VA,USA).In brief,histones were cross-linked to DNA by adding formaldehyde to a?nal concentration of1%and incubated for10min at room temperature.Sonication of chromatin to an average size of ?500bp was performed,followed by immunoprecipitation with protein A magnetic beads and either a rabbit anti-human STAT3antibody or normal IgG antibody overnight at4°C. The protein A magnetic beads were then separated using a magnetic separator(Upstate)and incubated with anti-rabbit secondary antibody for1h at4°C.DNA was recovered using the DNA puri?cation spin columns provided and then used for PCR.Primer pairs were designed by Primer3Input0.4.0to detect the SALL4promoter region containing putative STAT3-binding sites.The primer sequences are as follows: primer1(expected product size,209bp)forward5?-GCCCA-GAGCAGTTATGGAAA-3?and reverse5?-ATTGA CACAT-GATGCCTGGA-3?;and primer2(expected product size,220 bp)forward5?-GATAGCTGGAGCAA GGATGG-3?and re-verse5?-ATGAGCCCTGACAGCTGATT-3?.PCR was per-formed by adding500ng of DNA product into a25-?l reaction containing1?buffer,a200?M concentration of each dNTP,oligonucleotide primer,and0.2U of AmpliTaq polymerase.
Site-speci?c mutagenesis of the SALL4promoter
The human SALL4promoter consists of?2.0kb of genomic DNA.Potential STAT3-binding sites in the SALL4promoter were determined by searching the2.0-kb region for consensus STAT-binding sites,(TTN(4–6)AA),and consensus STAT3-bind-ing sites,TTMXXXDAA(D:A,G,or T;M:A or C)(31).Four sites at positions?199,?1229,?1270,or?1316upstream of the initiation site were selected and speci?cally mutated using the GeneEditor site-directed mutagenesis system according to the manufacturer’s protocol(Promega).Oligonucleotide se-quences are listed below,with mutations underscored and in lowercase:?1316from ATGCCCAGAGCAGTTATGGAAAGAC-CAATC to ATGCCCAGAGCAGgcgtGGA AAGACCAATC,?1270from to GATGATTTCATCAgcgtTTTAACATTTATTG,?1229from TT-GTGTAAAGCAGT TAGCCAAATTAAGTATAC to TTGTGTA-AAGCAGgcgtCCAAATTAAGTATAC,and?199from GAT-CAATGAGGGCTTATTTAAATGATCTC to GATCAATGAGG GCgcgTTTAAATGATCTC.The SALL4/luciferase reporter construct was the template used for the mutagenesis study. Mutants were screened by direct sequencing using the3130XL Genetics Analyzer.Plasmids were ampli?ed using a Maxi-prep kit(Qiagen,Mississauga,ON,Canada).
STAT3dominant-negative vector and SALL4knockdown
The production and characteristics of the adenoviral vector carrying a STAT3dominant-negative construct(Ad-STAT3DN) have been described previously(32).In brief,the adenoviral vector was deleted at the E1A region.With use of the site-directed mutagenesis technique,the STAT3cDNA was modi?ed such that the Tyr705residue was replaced by phenylalanine.The expression of STAT3was driven by the rabbit?-actin promoter and cytomegalovirus enhancer.The construct was epitope-tagged with FLAG(DYKDDDDK)(Kodak,Rochester,NY,USA) at the N terminus.An adenoviral vector expressing green ?uorescence protein(GFP)was used as the negative control. For SALL4knockdown,we used three short-hairpin RNA (shRNA)-expressing retroviral vectors,with one being a neg-ative control(pRS)(Origen,Rockville,MD,USA)and two (nos.7410and7412;Origen)being SALL4speci?c.The production and transfection of these retroviral vectors have been described previously(33).
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt(MTS)assay
To determine the effect of SALL4on cell viability,MDA-MB-231cells were infected with either SALL4shRNA or control shRNA retrovirus,and the MTS assay(Promega)was per-formed after48h.After1000cells were seeded in96-well culture plates,cell viability was measured daily using a color-imetric microplate reader(Bio-Rad)at450nm,and absor-bance values were normalized using Microplate Manager 5.2.1(Bio-Rad).
SALL4immunohistochemistry
Production of the anti-SALL4antibody and details of antigen retrieval and immunohistochemistry have been described previously(30).
RESULTS
SALL4gene promoter contains four STAT3consensus binding sites
As summarized in Tables1and2,DNA sequence analysis of the?2-kb SALL4promoter region in both humans and mice revealed27consensus sequences for STAT protein binding,characterized by TTN(4-6)AA(34).Four se-quences in human SALL4and10in mouse SALL4con-tain TTMXXXDAA(D:A,G,or T;M:A or C),the consensus sequence of STAT3(31,35).In humans,the four STAT3potential binding sites begin at positions ?199,?1229,?1270,and?1316,upstream of the ATG transcription initiation site.
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STAT3regulates SALL4gene expression
To establish the functional relationship between the STAT3activity and SALL4gene expression,we used an in vitro model previously established in our laboratory,in which the expression of STAT3C(hence the STAT3 activity)in MCF-7(a breast cancer cell line)can be down-regulated with the addition of different concentra-tions of tetracycline to the cell culture(28).For simplicity, these cells are labeled STAT3C tet-off MCF-7.As shown in Fig.1A,the FLAG epitope that has been tagged to the STAT3C construct was strongly expressed in the absence of tetracycline.With the addition of increasing doses of tetracycline to the cell culture(20and60?g/ml),there was a gradual decrease in the expression of the FLAG epitope.Accordingly,the total STAT3level,which in-cluded both the endogenous STAT3and exogenous STAT3C,was also decreased.?-Actin,the loading control, was unchanged in all three lanes.
We then examined the protein expression of SALL4in STAT3tet-off cells under different concentrations of tetra-cycline.The blots shown in Fig.1B was derived from the same Western blots used for Fig.1A.Because SALL4B was previously shown to induce acute myeloid leukemia in a transgenic mice model(25),we primarily evaluated the expression of SALL4B(at95kDa)in this study.The protein expression of SALL4was signi?cantly decreased with the addition of tetracycline;it was undetectable at60?g/ml tetracycline.To con?rm these results,RT-PCR was performed and consistent results were obtained.As shown in Fig.2,STAT3C tet-off MCF-7cells expressed SALL4 mRNA at a much higher level in the absence of tetracy-cline treatment,compared with cells treated with tetracy-cline,at20or60?g/ml.HL-60cells(an acute myeloid leukemia cell line)served as a positive control,whereas Mino cells(a mantle cell lymphoma cell line)served as a negative control for SALL4.The sequences of the RT-PCR amplicons for SALL4were con?rmed(results not shown). To further establish the functional relationship be-tween the STAT3activity and SALL4gene expression, STAT3C tet-off MCF-7cells were transfected with a vector containing the human SALL4promoter/pGL3-Basic lu-ciferase reporter.All of the transfection experiments performed included a?-GAL-expressing vector as a con-trol for the transfection ef?ciency.Relatively robust lucif-erase activity was detectable with a high level of STAT3C expression(i.e.,no tetracycline added).As shown in Fig.3, addition of tetracycline resulted in a signi?cant decrease in the SALL4promoter luciferase activity.The relative luciferase activity was calculated by normalizing the lucif-erase activity to the?-GAL activity.
STAT3binds to the SALL4promoter shown by chromatin immunoprecipitation
To determine whether there is direct binding of STAT3to the STAT3-binding consensus sequences on the SALL4 TABLE2.Putative STAT3-binding sites in the mouse SALL4 gene promoter
Site number
Location relative
to ATG Consensus sites TTN(4?6)AA 1?203TTATTTAAA a
2?410TTCAATAAA a
3?439TTCTAGAA
4?482TTACTACAA
5?614TTCCGTAAA a
6?683TTAGGTAA
7?775TTACAACAA
8?794TTCCGAAAA a
9?856TTCTAGCAA
10?869TTCTGGAA
11?942TTAAGTACAA
12?955TTCTAGAAA a
13?983TTCTGTAA
14?1024TTCCCGAA
15?1041TTAGGAAA
16?1076TTGAGAAAA a
17?1100TTAGCCAA
18?1111TTGCAAGGAA
19?1137TTCTTCAA
20?1147TTAGTTGAA a
21?1447TTTCTGTAA
22?1478TTGCAGAA
23?1692TTCCCAGAA a
24?1774TTCCCTTAA a
25?1785TTACACAA
26?1808TTAGATAAA a
27?1919TTATGAAA
a Speci?c STAT3-binding site.
TABLE1.Putative STAT3-binding sites in the human SALL4
gene promoter
Site Location relative to ATG Consensus sites TTN(4?6)AA
1?73TTTCCCAA
2?124TTGATAATAA
3?199TTATTTAAA a
4?255TTTGTCACAA
5?433TTCTACGTAA
6?442TTCCAAAA
7?472TTTGGAGCAA
8?576TTGGGGGAA
9?618TTAATCAAAA
10?722TTTTGGAA
11?799TTTGTGAA
12?847TTGAAGTTAA
13?1046TTAGTAACAA
14?1071TTGCACAA
15?1092TTTTCTGTAA
16?1229TTAGCCAAA a
17?1242TTGTGTAAA
18?1270TTCATTTAA a
19?1316TTATGGAAA a
20?1360TTACAATGAA
21?1512TTGTCCAA
22?1582TTCAACAA
23?1622TTATACATAA
24?1634TTGCTTTAAA
25?1655TTGGCTATAA
26?1691TTATTAAA
27?1879TTTTCCTTAA
a Speci?c STAT3-binding site.
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promoter,chromatin immunoprecipitation was per-formed using MDA-MB-231,a breast cancer cell line expressing constitutively active STAT3.After extraction,chromatin was immunoprecipitated using anti-STAT3an-tibody.Immunoprecipitation with normal rabbit IgG an-tibody was used as the negative control.Figure 4A is a
schematic representation of the primer sets that were designed to include the four putative STAT3-binding sites on the human SALL4promoter,with primer 1spanning three STAT3sites (?1316,?1270,and ?1229)and primer 2spanning one STAT3site (?199).The use of both primer 1and primer 2yielded ampli?able products of the SALL4promoter (Fig.4B ).No ampli?cations were observed in the immunoprecipitation reactions that con-tained normal IgG antibody.The input lane was included as a control for PCR effectiveness.PCR without the addition of DNA templates was used as a negative control.Con?rmation of the STAT3-binding sites using site-directed mutagenesis
To con?rm the functionality of these STAT3-binding sites on the SALL4promoter,site-directed mutagenesis was used to mutate all of the four putative STAT3-binding sites.Mutations were con?rmed by DNA sequencing and are described as follows:?1316from TTATGGAAA to gcgtGGAAA,?1270from TTCATTTAA to gcgtTTTAA,?1229from TTAGCCAAA to gcgtCCAAA,and ?199from TTATTTAAA to gcgTTTAAA.STAT3C tet-off MCF-7cells were transfected with ?-GAL and with either the wild-type or mutated constructs.Subsequently,a lucif-erase assay was performed.After normalization with ?-GAL,the relative luciferase activity for each mutated constructs was compared with that of the wild-type con-struct.The wild-type luciferase promoter demonstrated the highest level of luciferase activity.In contrast,muta-tions at the promoter region at ?199(i.e .,mutant 4)resulted in the most signi?cant down-regulation of SALL4promoter activity (Fig.5).The luciferase activity was also signi?cantly reduced in the other mutants.These results correlated with those found in the chromatin immuno-precipitation assay,supporting the concept that there is more than one functional STAT3-binding site on the SALL4promoter.Nevertheless,the STAT3-binding site
at
Figure 1.SALL4expression in STAT3C tet-off MCF-7cells.STAT3C tet-off MCF-7cells were treated with 0,20,and 60?g/ml tetracycline,and the protein level of SALL4was assessed by Western blots.Results shown are representative of 3separate experiments.A )FLAG epitope tagged to STAT3C was detected using an anti-FLAG antibody.Total cellular STAT3was detected using an anti-STAT3antibody.Addition of increasing concentrations of tetracycline resulted in a substantial decrease in both FLAG and STAT3.B )SALL4was detected using an anti-SALL4antibody.Addition of tetracy-cline at 60?g/ml was suf?cient in reducing the SALL4expression to an undetectable level.MW,molecular
weight.
Figure 2.SALL4mRNA expression in STAT3C tet-off MCF-7cells.RT-PCR studies using STAT3C tet-off MCF-7cells treated with 0,20,and 60?g/ml tetracycline demonstrated a dose-dependent down-regulation of SALL4,with SALL4mRNA expression being absent at 60?g/ml tetracycline.HL-60cells were included as the positive control,and Mino cells were included as the negative control for SALL4.GAPDH was included as an internal control.Results shown are represen-tative of 3separate
experiments.
Figure 3.STAT3-regulated gene expression of SALL4.Lucif-erase assay was performed using STAT3C tet-off MCF-7cells treated with 0and 20?g/ml tetracycline.Cells were tran-siently transfected with the SALL4/luciferase reported con-struct and ?-GAL.Luciferase activity was read using a lumi-nometer,and levels were normalized with ?-GAL activity.Luciferase activity was signi?cantly higher in transfected cells with no tetracycline treatment compared with cells treated with 20?g/ml tetracycline.Triplicate experiments were performed.Error bars ?sd .
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?199appears to be most effective in modulating the gene expression of SALL4.
STAT3regulates the endogenous level of SALL4in human cell lines
Because much of our data presented thus far in this study were based on the use of the SALL4/luciferase reporter gene construct,we tested whether the SALL4expression level can be regulated by modulating STAT3activity in a native human cell line.Although the expression of SALL4is highly restricted in human cells (30),our previous studies had allowed us to identify a variable expression level of SALL4in several breast cancer cell lines,including MCF-7(parental clone),MDA-MB-231,and MDA-MB-468,as shown in Fig.6A .HL-60cells were included as the positive control,and Mino cells were included as the
negative control.Of note,in contrast with the STAT3tet-off cells (a derivative of MCF-7),we were able to detect a low level of SALL4in the parental MCF-7cells.To con?rm SALL4expression in these cell lines,RT-PCR was per-formed and SALL4mRNA was detected in all three breast cancer cell lines (Fig.6B ).The ampli?able PCR products were sequenced,and the products were con?rmed to be full-length SALL4(data not shown).Because SALL4is known to be a transcriptional factor itself,it is expected to be localized to the nucleus (25).Thus,we performed nuclear cytoplasmic fractionation.As shown in Fig.6C ,we con?rmed that SALL4is localized to the nucleus in MDA-MB-231and MCF-7cells.Interestingly,the SALL4nuclear protein level was higher in MDA-MB-231cells,which correlated with a substantially higher pSTAT3level in the nuclei of MB-231cells.The same membrane was reprobed with an antibody reactive with ?-tubulin,a protein largely restricted to the cytoplasm;the absence of ?-tubulin in the nuclear fraction indicates that our frac-tionation method was suf?cient.
Using MDA-MB-231,which expressed both SALL4and constitutively active STAT3,we determined whether STAT3also regulates SALL4gene expression.We inhib-ited STAT3activation with the use of an adenoviral STAT3dominant-negative construct (Ad-STAT3DN),which had been described previously in detail (36).As shown in Fig.7,MDA-MB-231cells infected with Ad-STAT3DN for 24h showed expression of the FLAG epitope that was tagged to the STAT3DN construct.SALL4expression was substantially down-regulated in these cells.These results were in contrast with cells treated with an adenoviral vector expressing GFP (Ad-GFP).Fur-thermore,a signi?cant decrease in viable cells was ob-served in cells infected with Ad-STAT3DN compared with those transfected with Ad-GFP,with no apoptotic activity detectable (see Supplemental Figs.S1and S2).Effects of endogenous SALL4on human cancer cell lines
To assess whether STAT3-regulated expression of SALL4is biologically meaningful,SALL4expression was down-regulated in MDA-MB-231cells using shRNA-mediated knockdown.MDA-MB-231cells were infected with
either
speci?c for the SALL4promoter used in this study.Primer 1consisted of three putative
STAT3-binding sites;primer 2consisted of one putative STAT3-binding site.B )Chromatin immunoprecipitation was performed using MDA-MB-231and an antibody against STAT3.Normal rabbit IgG antibody instead of anti-STAT3served as the negative control.PCR with both primer 1and primer 2revealed amplicons.In contrast,no amplicons were observed in negative control samples.Results shown are representative of 3separate
experiments.
Figure 5.Site-directed mutagenesis of the SALL4gene pro-moter.Site-directed mutagenesis was performed using the SALL4/luciferase reporter construct,with introduction of mutations of 3-4bp in each of the four putative STAT3-binding sites:?1316(Mut 1),?1270(Mut 2),?1229(Mut 3),and ?199(Mut 4).Luciferase activity was determined for each of the four mutants and for the unmutated construct.One of these constructs and ?-GAL were transfected into STAT3C tet-off MCF-7cells,and a luciferase assay was per-formed 24h later.Luciferase activity was normalized against ?-GAL activity.Our results showed that the luciferase activity of all mutants was signi?cantly decreased compared with that of the unmutated construct,with the most signi?cant de-crease found in Mut 4(?199).This experiment was per-formed in triplicate.Error bars ?sd .
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SALL4siRNA retrovirus or a control shRNA retrovirus for 48h.A trypan blue exclusion assay was used to count the number of viable cells and to assess cell morphology.Similar to the results with cells treated with Ad-STAT3DN,there was a signi?cant decrease in the number of viable cells in MDA-MB-231cells treated with SALL4shRNA,but no appreciable apoptosis was detectable.Western blots were performed to con?rm that SALL4expression was down-regulated with the shRNA treatment.As illustrated
in Fig.8A ,SALL4expression was down-regulated by ?50%.To further assess the effects of SALL4on cell growth,an MTS assay was performed 48h after retroviral infection.The cell growth index was measured daily for 4days,and a signi?cant decrease in cell growth was ob-served in the SALL4shRNA-infected cells compared with cells infected with the control shRNA (Fig.8B ).On day 4,there was an ?50%decrease in cell growth in the SALL4shRNA-treated samples compared with the control sam-ples.
Expression of SALL4in primary breast tumors Twenty randomly chosen,formalin-?xed/paraf?n-em-bedded primary breast carcinomas were assessed for https://www.doczj.com/doc/2515424994.html,ing immunohistochemical staining,we found SALL4expression in ?ve cases.In contrast,normal breast epithelial cells are negative for this marker (Fig.9).As reported previously (28),pSTAT3was detect-able only in a subset of breast cancer but not in benign breast epithelium.
DISCUSSION
In view of the common roles of STAT3and SALL4in maintaining the pluripotency of ES cells and their impor-tance in oncogenesis,we investigated whether there is a
Figure 6.SALL4expression in human breast can-cer cell lines.A )Western blot analysis con?rmed the expression of SALL4in breast cancer cell lines,MDA-MB-468,MDA-MB-231,and MCF-7.HL-60cells were included as the positive control,and Mino cells were included as the negative control for SALL4.Results shown are representative of 3separate experiments.B )RT-PCR studies using MDA-MB-468,MDA-MB-231,MCF-7,HL-60,and Mino cells demonstrated the presence of SALL4in all breast cancer cell lines.HL-60cells were in-cluded as the positive control,and Mino cells were
used as the negative control for SALL4.GAPDH was included as an internal control.Results shown are representative of 3separate experiments.C )Nuclear extraction was performed on MDA-MB-231,MCF-7,and Mino cells.Western blot analysis demonstrated the presence of nuclear SALL4in both breast cancer cell lines.?-Tubulin,a cytoplasmic protein,was not detectable.Levels of nuclear SALL4expression seem to correlate with those of STAT3levels in the two breast cancer cell lines.Mino cells were included as the negative control for both SALL4and pSTAT3.Results shown are representative of 3separate experiments.MW,molecular
weight.
Figure 7.Blockade of STAT3signaling down-regulated SALL4.MDA-MB-231cells were treated with Ad-STAT3DN or Ad-GFP for 24h.In the Ad-STAT3DN-treated cells,FLAG was detectable,and total STAT3level was increased.Correlating with these changes,SALL4level was decreased.Cells infected with Ad-GFP served as the negative control.Results shown are representative of 3separate experiments.
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functional link between STAT3and SALL4.Speci?cally,we tested whether STAT3is a transcriptional factor for SALL4.Our results support this concept.First,we have shown that the SALL4promoter carries four STAT3binding consensus sequences.Second,our data support the fact that STAT3indeed binds to these four sites and regulates the gene expression of SALL4,with the binding region starting at ?199being the most in?uential.Third,site-directed mutations at all four STAT3-binding sites signi?cantly reduced the luciferase activity in our SALL4
/
Figure 8.Down-regulation of SALL4inhibited cell growth.A )MDA-MB-231cells were treated with either SALL4shRNA retrovirus or control shRNA retrovirus,and the cell lysates obtained were subjected to Western blots.SALL4levels were decreased in shRNA-treated cells compared with cells treated with control shRNA.Densitom-etry was performed to quantify SALL4expression levels (bottom)and demonstrated ?50%down-regulation of SALL4expression.Results shown are representative of 3separate experiments.Error bars ?sd .B )MTS assay was performed comparing cell growth in MDA-MD-231cells treated with SALL4shRNA retrovirus or control shRNA.After 48h,treatment of MDA-MB-231with SALL4shRNA retrovirus resulted in a signi?cant decrease in cell growth compared with cells treated with control shRNA.On day 4,cell growth index in cells treated with SALL4shRNA was ?50%of that of cells treated with control shRNA.This experiment was performed in triplicate.Error bars ?sd
.
B
D
Figure 9.Immunohistochemical staining to detect SALL4expres-sion in breast cancer.A )Benign breast epithelial cells showed no de?nitive nuclear SALL4stain-ing.B ,C )Two cases of breast cancer also showed no de?nitive SALL4nuclear staining.D )One tumor showed strong and de?n-itive SALL4nuclear staining.Nonspeci?c cytoplasmic stain-ing was detectable in all panels .
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luciferase reporter gene assay,and the reduction was most dramatic with mutations introduced at the?199site. Fourth,using a STAT3dominant-negative construct,we showed that inhibition of STAT3activity signi?cantly decreased the expression of SALL4in a native human breast cancer cell line,MDA-MB-231,which expresses both pSTAT3and SALL4.
Although the focus of this article was to document that STAT3is a transcriptional factor for SALL4,we also had the opportunity to examine whether SALL4is biologically important in breast cancer cells.In this regard,for the ?rst time,we demonstrated that breast cancer cell lines express SALL4at a variable level.SALL4expression is likely to be functional in these cells,because it is localized to the nucleus.Interestingly,in a comparison of the results with MDA-MB-231and MCF-7(Fig.6C),the nu-clear SALL4protein level appears to correlate with that of pSTAT3,although a more comprehensive study is re-quired to con?rm this impression.Our results further support the fact that SALL4expression is of biological importance in breast cancer cell lines,because shRNA-mediated down-regulation of SALL4expression in MDA-MB-231led to a signi?cant decrease in cell growth.In keeping with a previously published study(37),we also found that breast cancer cells treated with a STAT3 dominant-negative construct also showed cell cycle arrest. On the basis of the?ndings described in this study,the suppression of cell growth in breast cancer cell lines induced by STAT3inhibition may be mediated via SALL4.Of note,in contrast with leukemic cells(33), SALL4knockdown in MDA-MD-231cells did not result in detectable apoptosis.We are not certain about this differ-ence,but the apoptotic effects of SALL4knockdown may be cell type-speci?c.
The?nding of relatively consistent expression of SALL4in various breast cancer cell lines was initially surprising to us,considering that SALL4is believed to be largely a stem cell marker(23,38).Nevertheless,it is not uncommon for stem cell markers to be expressed in carcinoma cells.For instance,Oct4,one of the key regu-lators of ES cells,was detectable in a variety of human cancers including cancers of the breast,lung,pancreas, colon,and ovary(39).In addition to Oct4,a relatively recent study by Ezeh et al.(40)identi?ed expression of Nanog,another regulator of ES cells,in breast tumors and cell lines but not in normal breast tissues.Although the biological role of Nanog and Oct4in cancer is largely unknown,the expression of these two markers may cor-relate with tumor aggressiveness.Speci?cally,a recent study reported that expression of Nanog and Oct4is associated with a high-grade histology(41).Similar to SALL4,the interactions between STAT3and both Oct4 and Nanog have been reported to be essential for the maintenance of murine ES cells in a pluripotent state(42) and for oncogenesis(43).For instance,Oct4is reported to provide antiapoptotic effects in murine ES cells,and this effect may be mediated by the STAT3signaling pathway(44).Coexpression of STAT3,Oct4,and Nanog has been reported in bone sarcoma cells,although whether STAT3is responsible for the expression of Oct4and Nanog in these cells was not addressed(43).In light of our?ndings in this study,it is tempting to speculate that STAT3may directly contribute to the aberrant ex-pression of these stem cell markers in various forms of human cancer.
The oncogenic potential of SALL4has been recently revealed by multiple studies.The SALL4B transgenic mice exhibit myelodysplastic-like features including ineffective hematopoiesis and an increased proportion of immature cells(25).The SALL4level was found to be consistently increased with progression to leukemia.SALL4is known to directly target the BMI-1gene,a marker that is impor-tant in predicting the disease progression of acute my-eloid leukemia(24).The functional categories of SALL4 target genes in leukemic cells revealed that SALL4fre-quently binds to target genes involving various apoptotic pathways,including p53,BCL2,TNF,and PTEN(44). Interestingly,SALL4has also been reported to directly bind to?-catenin and activate the Wnt canonical pathway, another pathway implicated in oncogenesis(25).Thus,it is highly likely that SALL4cooperates with a number of different physiological pathways,including the STAT3 signaling pathway,to promote tumorigenesis in various human malignancies.
To conclude,we describe a novel functional link be-tween STAT3and SALL4in which SALL4is a downstream target gene of STAT3.We have located the speci?c STAT3-binding regions in the SALL4gene promoter.Our data suggest that STAT3and SALL4probably cooperate in both physiological and pathological
states.
We thank Dr.Hanan Armanious for her technical assis-tance.This study was supported by research grants from the Canadian Institute of Health Research and the Canadian Cancer Society awarded to R.L.P.G.is a recipient of a Lymphoma Research Foundation of Canada Fellowship Award.
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Received for publication July22,2008.
Accepted for publication December4,2008.
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