Early Growth Response 1 Acts As a Tumor Suppressor in Vivo and in Vitro Via Regulation of P53

Research Article

Early Growth Response 1 Acts as a Tumor Suppressor In vivo and In vitro via Regulation of p53

Anja Krones-Herzig,1 Shalu Mittal,1 Kelly Yule,1 Hongyan Liang,2 Chris English,1 Rafael Urcis,1 Tarun Soni,1 Eileen D. Adamson,2 and Dan Mercola1,3

1Sidney Kimmel Cancer Center, San Diego, California and 2The Burnham Institute; 3Cancer Center, University of California at San Diego, La Jolla, California

Abstract human tumor cell lines express little or no Egr1 in contrast to their normal counterparts (9–12). Furthermore, Egr1 is decreased or The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor undetectable in small cell lung tumors, human breast tumors promoter. Egr1-null mouse embryo fibroblasts bypass repli- (11, 13), and human gliomas (12). Reexpression of Egr1 in human tumor cells inhibits transformation. The mechanism of suppression cative senescence and exhibit a loss of DNA damage response h and an apparent immortal growth, suggesting loss of p53 involves the direct induction of TGF- 1 leading to an autocrine- functions. Stringent expression analysis revealed 266 tran- mediated suppression of transformation (8), increased fibronectin, scripts with >2-fold differential expression in Egr1-null mouse and plasminogen activator inhibitor (9). Egr1 also has been embryo fibroblasts, including 143 known genes. Of the 143 implicated in the regulation of p53 in human melanoma cells genes, program-assisted searching revealed 66 informative leading to apoptosis (14–16), and the proapoptotic tumor genes linked to Egr1. All 66 genes could be placed on a single suppressor gene PTEN also is directly regulated by Egr1 (17). Recently, we have identified and established a new role of Egr1 as regulatory network consisting of three branch points of known a ‘‘gatekeeper’’ of p53-dependent growth-regulatory mechanisms in Egr1 target genes: TGFb1, IL6,andIGFI. Moreover, 19 replicative senescence and cell growth (18). This result was additional genes that are known targets of p53 were identified, revealed by examination of primary mouse embryo fibroblasts indicating that p53 is a fourth branch point. Electrophoretic (MEF) isolated from Egr1-null mice developed previously by mobility shift assay as well as chromatin immunoprecipitation Lee et al. (19) and Topilko et al. (20). Egr1-null cells from either confirmed that p53 is a direct target of Egr1. Because deficient strain express no Egr1 protein and much reduced p53 protein. p53 expression causes tumors in mice, we tested the role of These cells completely bypass replicative senescence in culture, Egr1 in a two-step skin carcinogenesis study (144 mice) that thereby appearing to be immortal without passing through a revealed a uniformly accelerated development of skin tumors ‘‘crisis’’ stage. Moreover, these cells fail to arrest following treatment in Egr1-null mice (P < 0.005). These studies reveal a new role with DNA-damaging agents, such as g-irradiation (18) and 12-O- for Egr1 as an in vivo tumor suppressor. (Cancer Res 2005; tetradecanoylphorbol-13-acetate (TPA; see below). Replicative 65(12): 5133-43) senescence and the DNA damage response of the Egr1-null cells may be restored by infection with an Egr1-expressing retrovirus in Introduction contrast to p53-null MEFs. In the Egr1-null cells, p53 is not The transcription factor early growth response 1 (Egr1)isa expressed and the p53 gene remains entirely free of mutation to member of the immediate-early gene family and encodes a 59-kDa high passage numbers. In contrast, the wild-type (WT) cells express phosphoprotein observed at f80 kDa by electrophoresis. Egr1 is normal levels of p53, arrest at low passage in normal tissue culture, involved in the regulation of cell growth and differentiation in and undergo crisis. WT cells that survive crisis inevitably exhibit response to signals, such as mitogens, growth factors, and stress mutations of the p53 gene. We speculated that Egr1 exerts a stimuli (1–4). Egr1 has a COOH-terminal DNA-binding domain gatekeeper function over p53, thereby allowing the null cells to consisting of three zinc fingers that regulates transcription through bypass replicative senescence and avoid the mutation association GC-rich elements. The 9-bp DNA consensus-binding sequence with the survival of crisis. However, the exact mechanism whereby GCG(G/T)GGGCG has been identified in the promoter of several Egr1 functions in the regulation of p53-dependent growth arrest is growth-regulatory genes (5). Besides the highly conserved DNA- unknown, and there is no evidence that Egr1 mediates these roles binding domain, Egr1 also contains a nuclear localization signal, in a whole organism. two activator domains, and one repressor domain (6). In addition, Here, we examined gene expression in the genetically defined Egr1 binds to regulatory proteins called NAB1 and NAB2 (nerve Egr1-deficient MEFs. These cells exhibit at least 266 significant growth factor-I A-binding protein) that repress its transcriptional differences in transcript expression compared with WT cells, many activity (7). of which could be recognized as Egr1 target genes or downstream Analysis of certain human tumor cells and tissues indicate that signal transduction mediators. Indeed, an extended regulatory Egr1 exhibits prominent tumor suppressor function (5, 8–11). Many network based on four ‘‘nodes’’ could be constructed. Three nodes are upstream of numerous recognized Egr1-regulated genes. However, a fourth node, and potential Egr1 target gene, p53,is Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). upstream of at least 19 genes not previously recognized as Egr1- A. Krones-Herzig and S. Mittal contributed equally to this work. regulated genes but are well-recognized mediators of p53 function. Requests for reprints: Dan Mercola, Sidney Kimmel Cancer Center, 10835 Altman Electrophoretic mobility shift assay (EMSA) and chromatin Row, San Diego, CA 92121. Phone: 858-450-5990, ext. 370; Fax: 858-450-3251; E-mail: [email protected]. immunoprecipitation (ChIP) experiments directly confirmed the I2005 American Association for Cancer Research. binding of the p53 promoter by Egr1 in cells both in vitro and www.aacrjournals.org 5133 Cancer Res 2005; 65: (12). June 15, 2005

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Research in vivo. Moreover, TPA-induced skin tumors appeared rapidly in TGFb1, TGFb1I, GRO1, IGFII, THSP2, and procollagen XI. Primers were Egr1-null mice compared with WT and heterozygous littermates. selected for these genes with the aid of Primer Express software (Applied These studies indicate that Egr1 regulates an extensive network of Biosystems) and are available on request. The results were normalized to genes that include direct regulation of p53 at the level of RNA and the relative amounts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). protein expression. Our observations indicate that this mechanism Chromatin immunoprecipitation assay. WT and null MEFs and may function in vitro and in vivo to control DNA damage–induced human prostate cancer DU145 cells were treated with 1% formaldehyde for growth arrest. Loss of this mechanism in vivo may be related to 20 minutes to cross-link protein to binding sites on DNA. Egr1-containing increased skin tumor formation in Egr1-deficient mice. fragments were recovered by immunoprecipitation as described earlier (24). The ChIP-captured DNA was then screened for p53 promoter fragments by PCR analysis using the following p53 specific primers: (mouse) 5V- Materials and Methods GTAGAGTAAGCCCCCGGAAG-3V and 5V-GGTTACCGGGATTCAAAACA-3V Cells and cell culture. MEFs were prepared as described earlier (21) (the amplified fragments correspond to À925 and À424 region of the mouse from 19-day-old embryos from Egr1 WT, Egr1-null, and Egr1 heterozygous p53 promoter from AF287146); (human) 5V-TGGGAGTTGTAGTCTGAA- mice kindly provided by Dr. J. Milbrandt (Washington University Medical CGCTTC-3V and 5V-GAGAAGCTCAAAACTTTTAGCGCC-3V (the amplified School, St. Louis, MO) (19). Cultures of WT and Egr1-null MEFs were fragments correspond to À693 to +89 region of the human p53 promoter established and maintained in parallel for >60 passages. The predicted from X54156). Genomic DNA was used as a control for the amplification genotype and expression properties of the MEFs derived from Egr1-null and efficiency of each primer pair. heterozygous mice were confirmed by PCR-based analysis of DNA and RNA Electrophoretic mobility shift assay. WT and Egr1-null MEFs were and by Western analysis for protein expression as described previously (18). grown in tissue culture to confluence, incubated in serum-free medium for Oligonucleotide microarray analysis. Total cellular RNA was isolated 1 hour, and then supplemented with 20% fetal bovine serum for 1 hour. by using RNeasy kits (Qiagen Inc., Valencia, CA) and quantified. The total Cells were then lysed in hypotonic buffer (10 mmol/L HEPES, 25 mmol/L RNA quality was determined using the standard Affymetrix protocol. For KCl, 1 mmol/L EDTA, 1 mmol/L EGTA) containing 1:100 protease hybridization, total RNA (10 Ag) from WT or Egr1-null MEFs was reverse inhibitor cocktail (Sigma, St. Louis, MO) on ice for 10 minutes. Samples transcribed using an oligo(dT) primer harboring a T7 RNA polymerase were centrifuged at 1,500 rpm for 7 minutes and the pellets were promoter at the 5V end (Genset, San Diego, CA). Following second-strand incubated on ice for 30 minutes in nuclear extract buffer (50 mmol/L Tris- synthesis, biotinylated cRNA probes were produced. The probes were then HCl, 420 mmol/L KCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA) with 1:100 fragmented and hybridized to Affymetrix MGU75Av2 array representing protease inhibitor cocktail. The samples were centrifuged at 5,000 rpm for 12,566 mouse transcripts using the standard Affymetrix protocol (http:// 30 minutes at 4jC and the supernatants were removed for protein www.affymetrix.com). Quantitative analysis of the data from the micro- concentration determination by the BCA method as per manufacturer’s arrays was carried out using the MAS1 suite (http://www.affymetrix.com). instructions (Pierce Biotechnology, Rockford, IL). These extracts were used Two replicated RNA preparations from separate cultures of WT MEFs as nuclear protein in the gel shift assays below. Double-stranded synthetic and two of Egr1-null MEFs were hybridized to four Affymetrix MGU75Av2 oligonucleotides (IDT DNA, Coralville, IA) corresponding to possible Egr1- arrays leading to four different comparisons of data sets. In general, a gene binding sites as determined by transcription element search system was considered to be differentially expressed in Egr1-null MEFs when three were labeled with [g-32P]ATP using T4 polynucleotide kinase or by criteria were met: (a) when ‘‘called present’’ (MAS1 suite), (b) when [a-32P]dATP using the Klenow fragment as per manufacturer’s instruc- exhibiting a z2-fold change in net fluorescence relative to WT MEFs, and tions (Invitrogen, Carlsbad, CA). The five sites tested were probe A 5V- (c) when these criteria were satisfied by all four possible comparisons of the TTTCCCTTTCTCCCCCGCCCTCCCTTCA-3V (nucleotides 886-913 from replicate WT and Egr1-null MEF data sets. The >2-fold change criterion was AF287146), probe B 5V-AAACCGTGGGGTTTGGGGGTGGGGCAGTG-3V, chosen in correspondence to the recommended cutoff for significant probe C 5V-AATGGAAGCTTGGCGGGCGGGATGAACGTT-3V, probe D 5V- change by the manufacturer (22) and because it has been shown that AAATCCTGCGGGGCGGGGTGGCGGGGGGTT-3V, and probe E 5V-CTTTCT- difference of >2-fold change is detected >98% of the time by the Affymetrix CCCCATCTCTCCCCCCTTCTTAA-3V (nucleotides 850-876 from AF287146). assay (23). Gel shift assays were done as described previously (9) with modifications Reconstruction of the early growth response 1 network. The as noted here. Briefly, nuclear protein (20 Ag) and labeled DNA (104-105 significant differentially expressed genes from the expression analysis were cpm) were incubated in 20 AL binding buffer (25 mmol/L HEPES, selected for analysis. An algorithm was developed to convert the Affymetrix 60 mmol/L KCl, 2 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.5 mmol/L DTT, probe IDs to RefSeq IDs (NM numbers) using the LocusLink database. Of 100 Ag/mL spermidine, 10% glycerol, 100 Ag/mL bovine serum albumin) the 266 differentially expressed probes, 143 were identified as annotated for 20 minutes at ambient temperature. Reaction mixtures containing genes in the LocusLink database. These known genes were then further anti-Egr1 (Santa Cruz Biotechnology, Santa Cruz, CA) were preincubated analyzed for their potential connection to Egr1 or Egr1-regulated genes for 20 minutes at ambient temperature before the addition of probe. In by examination of the literature using BiblioSphere (Genomatix Software some cases, an oligonucleotide (10 ng) with the consensus sequences for GmbH, Munich, Germany; http://www.genomatix.de) for uncovering re- the binding of the transcription factor Sp1, 5V-ATTCGATCGGGGCGGGGC- ported regulatory relationships between genes. BiblioSphere is a data GAGC-3V (Santa Cruz Biotechnology), was added to reduce background. mining tool intended to provide gene relationships from literature Reactions were resolved by electrophoreses on ‘‘prerun’’ 4% nondenaturing databases and genome-wide promoter analysis. Over 12 million Pubmed polyacrylamide gels run in 0.5Â Tris-borate EDTA, vacuum dried with abstracts were scanned by this strategy using >500,000 gene names, heat, and exposed to film at À80jC. synonyms, and the Genomatix proprietary semantic relation concepts. Two-stage carcinogenesis. WT Egr1 heterozygous and Egr1-null mice Relationships defined with this tool were confirmed by manual examination of C57BL/6 background were produced from two Egr1 heterozygous in Pubmed, and the confirmed related genes were entered into the breeding pairs [a generous gift from Dr. J. Milbrandt (19)]. A total of 144 Genomatix package to determine secondary relationships. Iteration led to a mice ages 2 to 9 months were divided into 11 treatment groups and used for network relating all informative genes. An algorithm was developed to the two-step skin carcinogenesis assay as described previously (25). The identify the human homologues for these selected genes from the dorsal surface of all mice was shaved over f2 Â 2 cm surface area before LocusLink. the start of the experiment. Two days after shaving, a mutagen and tumor Quantitative real-time PCR. RNA expression levels of selected genes initiator, 7,12-dimethylbenz(a)anthracene (DMBA) in acetone, was applied from the microarray results were quantified by quantitative real-time PCR at a concentration of 7.8 mmol/L (200 AL of 2.0 mg/mL) to the shaved (Q-PCR; Applied Biosystems ABI7900, Foster City, CA) as described area of the mice. After 7 days, the mice were treated thrice weekly with a previously (18). Genes validated by PCR were Bax, Fas, GADD45, p53, p21, tumor promoter TPA in ethanol at a concentration of 1 mmol/L (20 ALof

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0.61 mg/mL) for 18 weeks. Control groups of at least 15 mice each included basis for the additional significant changes observed here, we mice with no treatment, treatments with DMBA alone, TPA alone, or vehicle looked for known relationships. Of the 266 significantly altered alone (acetone-only group, ethanol-only group) for each of the three gene expression values between WT and Egr1-null MEFs, 151 genotypes (WT, heterozygous, and Egr1 null). The onset of the tumors and expression values represent 143 genes identified by their their growth was monitored for the next 18 weeks. After 18 weeks of TPA annotation in the LocusLink database (Supplementary Table S1). treatment, necropsy was done. Samples of tumors and representative organs were fixed in 10% buffered formalin and embedded in paraffin, and The remaining 115 altered expression values represent either 5-Am-thick sections were prepared and stained (H&E) for histologic expressed sequence tags or genes that have not been annotated analysis. yet. Although several genes exhibiting significant change in RNA Statistics. Pearson correlation coefficients with Bartlett’s m2 probabil- expression in MEFs were recognizable as directly regulated by ities, regression coefficients, and t tests (unpaired, multiple variance) were Egr1 (4, 29), the vast majority of genes were of uncertain carried out as implemented by Systat 4.0 (Evanston, IL) and Excel 2000 relationship to Egr1. We carried out alternate rounds of (Microsoft Corp., Redmond, WA). application of the software BiblioSphere followed by direct confirmation in Pubmed/Medline/Entrez to define putative relationships. Two genes that were linked by BiblioSphere were Results considered as related if they also were cited as coregulated or Gene expression profile of normal cycling wild-type and correlated in some fashion without regard to direction or early growth response 1–null mouse embryo fibroblasts mechanism of regulation. Those genes confirmed as correlated reveals differential expression of growth and matrix-related with Egr1 or Egr1 target genes (see Materials and Methods) were genes. To identify genes regulated by Egr1, we compared the gene used as input to BiblioSphere Genomatix software for the expression profiles of WT and Egr1-null MEFs using the mouse successive cycle. Using this iterative process, 66 of the 143 genes Affymetrix GeneChip (MGU74Av2) representing 12,566 genes. Two were confirmed as previously observed in correlation with direct replicated RNA preparations from separate cultures of WT MEFs or indirect Egr1 regulation in one or more experimental studies of and Egr1-null MEFs were hybridized to four Affymetrix MGU75Av2 the mouse (references are given in Supplementary Table S1). The arrays leading to four different potential comparisons of data sets. remaining 77 genes have no reported regulatory associations In general, a gene was considered to be differentially expressed in with Egr1 in the databases consulted. The 66 informative genes Egr1-null MEFs (a) when ‘‘called present,’’ (b) when exhibiting a z2- and associated references are summarized in Table 1 and Fig. 2. fold change in net fluorescence relative to WT MEFs as When direction of induction was reported, this was preserved in recommended (23), and (c) when these criteria were observed for the map. all four possible comparisons of the replicate WT and Egr1-null The use of successive cycles revealed the number of putative MEF data sets. Using these criteria, 266 genes were identified as intermediate regulatory steps relating any gene to Egr1 (Fig. 3). differentially expressed. Of this group, 108 genes were down- Indeed, all 66 genes identified as informative Egr1-associated genes regulated and 158 were up-regulated in Egr1-null MEFs compared could be related by a single-branched regulatory network (Fig. 3). with WT MEFs. Numerous known Egr1 target genes, such as The majority of Egr1-regulated genes are described as under the TGFb1 (5, 26), IGFII (27), and Fas antigen (CD95; ref. 28), were direct transcriptional regulation of Egr1 (target genes) or within readily recognized (for a review, see ref. 4). Other notable genes are one or two downstream regulatory steps of an Egr1 target gene listed in Table 1. Several of the differentially expressed genes are (Fig. 3). Two nodes, TGFb1, a known direct target gene of Egr1 (30), involved in growth differentiation and cell cycle control. In this and IL6, a known direct regulator of Egr1 (31, 32), are upstream regard, TGF-h1 mRNA expression was decreased 7.2-fold in Egr1- regulators of >30 of these genes (Fig. 2). IGFI constitutes a third null MEFs and p21Cip1/Waf1 mRNA expression was decreased 2.5- node with five known subsequent target genes, including Egr1 fold. On the other hand, cyclin E and cyclin A2 mRNA expression itself. Several target genes noted here have been observed to be was increased f2-fold in Egr1-null MEFs. These features suggest regulated in combination consistent with the regulatory pathway. growth deregulation consistent with previous observation of very Moreover, many of the expression changes observed here are also rapid and apparently immortal growth exhibited by primary Egr1- supported by individual experimental observations in the litera- null cells from the time of explant (18). ture, suggesting that some of the regulatory mechanisms governing To confirm the microarray gene expression results, the expression are common among several cells and tissues. Thus, expression level of 12 differentially expressed genes listed in Table virtually all 66 informative genes of the original set of 143 known 1 were also tested by Q-PCR (Fig. 1). The fold induction/repression genes defined as significantly differentially expressed between WT calculated by Q-PCR assays produced a remarkable concordance Egr1 and Egr1-null MEFs for which there is readily accessible with the corresponding ratio determined in the Affymetrix literature may be placed on a single regulatory map consistent with GeneChip analysis. In this regard, stimulatory or inhibitory effects published observations. The consistency between genes observed of Egr1 gene ablation was confirmed in all cases examined. Indeed, by expression analysis to be differentially regulated and the calculation of the Pearson correlation coefficient of the Affymetrix published observations provides support for the accuracy of the results with real-time PCR results yielded a value of 0.94, which is identification of genes whose expression is significantly altered in significant (P V 0.001). Confirmation of target gene expression was Egr1-null fibroblasts. also examined by using an independent RNA preparation from WT Early growth response 1 binds to the promoter of p53 MEFs and Egr1-null MEFs with a different passage number (data in vitro and in vivo. One of the genes exhibiting a significant not shown). Thus, the sum of the results supports the reliability of decrease in expression in Egr1-null fibroblasts is p53. The decrease the microarray gene expression data analysis for selecting genes was confirmed by Q-PCR (Fig. 1). As further indication of the exhibiting significantly changed RNA levels. significance of this change, 18 of the 66 genes that make up the Derivation of network map relating early growth response Egr1-regulatory map have no known direct regulatory link to Egr1 1–regulated genes. To determine whether there is a rational but all are well-recognized direct target genes of p53 (Table 1). www.aacrjournals.org 5135 Cancer Res 2005; 65: (12). June 15, 2005

Table 1. Sixty-six significantly differentially expressed genes between Egr1-null and WT MEFs and related by the Egr1- regulatory pathway

Cluster RefSeq no. Symbol Gene name and type Affymetrix References expression

Cell cycle, growth and maintenance, and apoptosis AJ010108 NM_021515 Ak1 Adenylate kinase 1 À2.075 Up 1, 2 U00937 NM_007836 GADD45 Growth arrest and DNA damage 45 À0.6 Up 3-5 AW048937 NM_007669 Cdkn1a Cyclin-dependent kinase inhibitor 1A (P21) À2.8 Up 6 U09507 NM_007669 Cdkn1a Cyclin-dependent kinase inhibitor 1A (P21) À1.325 Up AF091432 NM_009830 Ccne2 Cyclin E2 1.2 Down 7 X75483 NM_009828 Ccna2 Cyclin A2 1.2 Up 8, 9 L49507 NM_009831 Ccng1 Cyclin G1 À2.65 Up 10-12 U20735 NM_008416 Junb JunB oncogene À1.45 Up 13, 14 J04596 NM_008176 Cxcl1 Chemokine (C-X-C motif) ligand 1 À2.125 Up 15 J04596 NM_008176 Cxcl1 Chemokine (C-X-C motif) ligand 1 À2.275 Up X71922 NM_010514 Igf2 Insulin-like growth factor-II À1.025 Up 16-18 U88328 NM_007707 Socs3 Suppressor of cytokine signaling 3 À2.4 Up 19-25 D76440 NM_010882 Ndn Necdin 1.525 26 L22472 NM_007527 Bax Bcl2-associated X protein À0.95 Up M83649 NM_007987 Tnfrsf6 Tumor necrosis factor receptor superfamily, member 6 À2.425 Up M83649 NM_007987 Tnfrsf6 Tumor necrosis factor receptor superfamily, member 6 À1.475 Up X54542 NM_031168 IL6 Interleukin-6 À1.25 Up or down 27-31 X82786 XM_133912 Mki67 Antigen identified by monoclonal antibody Ki-67 1.5 Up Cell adhesion, signal transduction, and cell-cell signaling AF064749 NM_009935 Col6a3 Procollagen type VI, a3 1.45 Up 32, 33 X52046 NM_009930 Col3a1 Procollagen type III, a1 1.175 Up Z35168 XM_136081 Col4a5 Procollagen type IV, a5 1.175 Up 34 L19932 NM_009369 TGFb1I Transforming growth factor-h induced À2.625 Up L07803 NM_011581 Thbs2 Thrombospondin 2 À1.05 Up 35 X13986 NM_009263 Spp1 Secreted phosphoprotein 1 À1.475 Up 36, 37 L32838 NM_031167 Il1rn Interleukin-1 receptor antagonist À3.225 Up 38 X77952 NM_007932 Eng Endoglin 1.175 Up 39 AJ009862 NM_011577 Tgfb1 Transforming growth factor-h1 À2.85 Up 40-43 Z18280 NM_010934 Npy1r Neuropeptide Y receptor Y1 À2.6 Up 44, 45 X04480 NM_010512 Igf1 Insulin-like growth factor-I À2.575 Up 17, 46-48 M19681 NM_011333 Ccl2 Chemokine (C-C motif) ligand 2 À1.675 Up 49-52 Transcription factors X61800 NM_007679 Cebpd CCAAT/enhancer-binding protein, y À1.425 Down M88299 NM_008900 Pou3f3 POU domain, class 3, transcription factor 3 1.175 V00727 NM_010234 Fos FBJ osteosarcoma oncogene À3.175 Up 53, 54 L35949 NM_010426 Foxf1a Forkhead box F1a À3.75 55 M31885 NM_010495 Idb1 Inhibitor of DNA binding 1 À1.025 Down 56-60 AB021961 NM_011640 Trp53 Transformation-related protein 53 À3.3 Up 61 AB021961 NM_011640 Trp53 Transformation-related protein 53 À3.35 Up Transporter X81627 XM_130171 Lcn2 Lipocalin 2 À3.375 Up 62 M35523 ND Crabp2 Cellular retinoic acid–binding protein II À2.6 Up 40, 41 X15789 NM_013496 Crabp1 Cellular retinoic acid–binding protein I À3.075 Down 40, 41 X03505 NM_011315 Saa3 Serum amyloid A3 À3.525 Up 63-65 U75215 NM_018861 Slc1a4 Solute carrier family 1 (glutamate/neutral 1.3 Up 66, 67 amino acid transporter), member 4 U49430 NM_007752 Cp Ceruloplasmin À1.55 Up 68-70 X57349 NM_011638 Trfr Transferrin receptor 1.05 Up 71-74 Xenobiotic metabolism X99347 NM_008489 Lbp Lipopolysaccharide-binding protein À1.75 Up 75-78 Metabolism related L35528 NM_013671 Sod2 Superoxide dismutase 2, mitochondrial À1.55 Down 79-81 AB001607 NM_008968 Ptgis Prostaglandin I2 (prostacyclin) synthase À1.425 Up 82 M88242 NM_011198 Ptgs2 Prostaglandin endoperoxide synthase 2 À1.325 Up 83, 84 M34141 NM_008969 Ptgs1 Prostaglandin endoperoxide synthase 1 À1.3 Up 85

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Table 1. Sixty-six significantly differentially expressed genes between Egr1-null and WT MEFs and related by the Egr1- regulatory pathway (Cont’d)

Cluster RefSeq no. Symbol Gene name and type Affymetrix References expression

AJ006033 NM_007802 Ctsk Cathepsin K À2 Up 86, 87 M96827 NM_017370 Hp Haptoglobin À1.6 Up or down 88, 89 X72307 XM_131908 Hgf Hepatocyte growth factor À3.35 Up 90-94 X66402 NM_010809 Mmp3 Matrix metalloproteinase 3 À1.35 Up 95 M21285 NM_009127 Scd1 Stearoyl-CoA desaturase 1 À1.25 Up 96 M63335 NM_008509 Lpl Lipoprotein lipase À2Up42 Miscellaneous X58196 NM_023123 H19 H19 fetal liver mRNA 4.7 Down 97 AJ242778 NM_021327 Tnip1 Tumor necrosis factor AIP3-interacting protein 1 À1.1 X59520 NM_031161 Cck Cholecystokinin À5.6 Up 98-102 X70296 NM_009255 Serpine2 Serine (or cysteine) proteinase inhibitor, clade E, member 2 À4.275 Up 103, 104 U15012 NM_010284 Ghr Growth hormone receptor À1.425 105, 106 U20257 NM_009626 Adh7 Alcohol dehydrogenase 7 (class IV), A or j polypeptide 2.275 Up 107-109 AF038939 NM_008817 Peg3 Paternally expressed 3 À2.65 Up 110, 111 AF004833 XM_130305 Tfpi Tissue factor pathway inhibitor À2.525 Down 112, 113 M33960 NM_008871 Serpine1 Serine (or cysteine) proteinase inhibitor, clade E, member 1 1.125 Up 114-119 J03482 NM_015786 Hist1h1c Histone 1, H1c 1.125 Up 120, 121 X04653 NM_010738 Ly6a Lymphocyte antigen 6 complex, locus A À1.75 Up 122-128 Z12171 NM_010052 Dlk1 y-like 1 homologue (Drosophila) 1.45 Up 129, 130 X92411 NM_009011 Rad23b RAD23b homologue (Saccharomyces cerevisiae) 1.1 Up 131 U01915 NM_011623 Top2a Topoisomerase (DNA) IIa 1.125 Down 132-134 Unknown U90435 NM_008027 Flot1 Flotillin 1 À1.3 Up 135, 136

NOTE: The Egr1-regulatory pathway is summarized in Figs. 1 and 2. Listed here is a subset of 66 of 143 named significantly differentially expressed genes between Egr1-null and WT MEFs that are mapped to a single regulatory network (Fig. 2; the 143 genes are listed in Supplementary Table S1). For each gene, the direction of regulation is given as fold change (Affymetrix expression ratio of Egr1-null to Egr1 WT MEFs) that is negative for genes down-regulated in null cells and therefore negative for Egr1-up-regulated genes. A gene was included in the table when all Affymetrix GeneChip intensities satisfied the ‘‘present call’’ criterion and all four comparisons (duplicate WT versus duplicate Egr1-null MEFs) yielded z2-fold change in net fluorescence relative to WT MEFs. The Affymetrix ratio is the average of the ratio in all four comparisons. The genes Bax, GADD45, and Id1 are exceptions to the 2-fold rule. The Affymetrix ratios are 1.9, 1.5, and 1.9, respectively, but are differentially expressed in all four comparisons and were confirmed by Q-PCR. Further results are shown in Supplementary Table S1. The numbers in column 7 refer to citations given with Supplementary Table S1, which lists the complete 143 differentially expressed gene set.

Thus, p53 is predicted to be a fourth major node among the Egr1- each case, extracts of WT MEF but not Egr1-null MEF formed a regulatory relationships observed here (Fig. 3). This is consistent strong single band in EMSA assays. The band is similar to that with previous studies in which it was inferred that p53 was formed with authentic Egr1 and is not mimicked by Sp1, another required for the ability of Egr1 to regulate replicative senescence of transcription factor that binds to GC-rich sequences (data not MEFs in extended culture (18). shown). Moreover, in both cases, the band was disrupted by To test whether Egr1 is a direct regulator of p53, we examined inclusion of anti-Egr1 antisera, a known and specific property of the promoter of p53 for potential Egr1-binding sites. There are at anti-Egr1 on Egr1-DNA complexes (8, 9). The above result confirms least five potential Egr1-binding sites within 2 kb upstream of the Egr1 binding to p53 in vitro. ATG start site of mouse p53 gene that are consistent with the To test whether Egr1 and not other Egr1 family members are consensus sequence 3V-GCGGGGGCG-5V. To determine whether involved in binding to p53 and to test whether the interaction Egr1 bound to these sites, preliminary EMSAs were carried out occurs under the conditions that prevail in living cells, we examined using synthetic double-stranded deoxyoligonucleotides (dsDNA) whether it was possible to recover Egr1 bound to chromatin in WT and recombinant Egr1 for five potential Egr1-binding sites of the MEFs that had been cross-linked in the living state by addition of mouse p53 promoter (see Materials and Methods). Three sites were formaldehyde as described (refs. 17, 33, 34; Fig. 5). As a positive not able to bind Egr1 in parallel experiments, indicating that sites A control, human prostate DU145 cells (29) were analyzed in parallel. and B were specific. Sites A and B exhibited strong binding and The precipitated Egr1-DNA complexes were analyzed by PCR for the were examined in more detail. presence of p53 promoter sequences using primers specific for Because genetically defined MEFs were available, nuclear domains in the p53 promoter that had putative Egr1-binding sites extracts of both WT MEFs and Egr1-null MEFs as sources of (see Materials and Methods). Anti-Egr1-treated chromatin but not endogenous Egr1-containing extracts and control extracts and nonimmune control rabbit IgG precipitated DNA gave a readily their ability to bind dsDNA of each site were compared (Fig. 4). In detectable p53-specific band after PCR amplification (Fig. 5), thus www.aacrjournals.org 5137 Cancer Res 2005; 65: (12). June 15, 2005

skin tumors (Fig. 6A). The tumors of the WT group appeared with an average time of tumor onset of 12.5 weeks after the start of the protocol, which led to tumor development in >60% of the mice (Fig. 6C). In contrast, Egr1-null mice exhibited a uniform and much earlier time of onset of 8 weeks with a similar prevalence (Fig. 6C). Indeed, over half of the tumors appeared before any eruption of the skin or notable defect of the WT animals. The difference in the time of appearance is significant (P < 0.005). We examined the histologic appearance of the tumors (Fig. 6B), which confirmed the presence of papilloma similar to those described for p53-null and p21-null mice (35–37). No differences in the histologic appearance of any of the tumors were noted. Thus, these observations strongly indicate that the absence of Egr1 in genetically defined mice predisposes them to enhanced skin carcinogenesis.

Discussion Early growth response 1 is a growth suppressor in primary Figure 1. Quantitative-PCR confirms Affymetrix GeneChip results and the expression of other genes differentially expressed between WT and Egr1-null mouse embryo fibroblasts. In most human tumors, such as MEFs. RNA expression levels were quantified by real-time PCR using the breast cancer, fibrosarcoma, and glioblastoma, Egr1 is described to ABI7900 Sequence Detection System. Total RNA (0.5 Ag) from WT or Egr1-null be tumor suppressor gene (9–11) that is required for maximal MEFs was reverse transcribed into cDNA and amplified using the SYBR Green PCR Master Mix and specific primers for each cDNA as described (see Materials sensitivity to irradiation (17, 38). However, the tumor-suppressing and Methods). The relative amounts of each gene amplification products were role seems to be tissue specific, because recent studies implicated a calculated by reference to standard curves and then normalized to the relative tumor growth-promoting role of Egr1 in prostate cancer progres- amounts of GAPDH as detected in the same run. Fold change in RNA expression in Egr1-null MEFs compared with WT MEFs. Asterisks, genes sion (27, 34, 39). Higher levels of Egr1 were found in prostate cancer reported previously to be differentially expressed (18). (26, 40, 41) and Egr1 is growth promoting for vascular smooth muscle cells and for rat kidney tumor cells (42–44). In our previous study, we therefore investigated the role of Egr1 by use of a defined indicating the presence of Egr1-p53 promoter complex in vivo.No genetic difference of primary MEFs from WT and Egr1-null mice signal was obtained from Egr1 immunoprecipitates that were generated by Lee et al. (19) and showed that deletion of Egr1 leads prepared using Egr1-null cells, further indicating that specific Egr1- to a striking phenotype, including complete bypass of senescence p53 promoter complexes only occur in WT MEFs (Fig. 5). Moreover, and apparent immortal growth consistent with loss of a suppressor a PCR product similar to that from the WT MEF ChIP products was gene (18). To identify genes responsible for the growth-suppressing observed when human DU145 prostate carcinoma cells were used together with primers specific for the human p53 promoter. These results provide strong support for the conclusion that WT Egr1 indeed binds the p53 promoter in vivo. The sum of observations above suggests that Egr1 binds directly to p53 in vitro and in vivo and that this effect may be responsible for the increase in p53 and its target genes in WT MEFs compared with Egr1-null cells. Early onset of tumors in mice deficient in early growth response 1. The in vivo Egr1-binding properties provide a mechanistic basis for the strong up-regulation of p53 observed in WT MEFs and for the regulation of p53-dependent growth and DNA damage responses by Egr1 in WT MEFs in culture. It might be expected, therefore, that Egr1 plays a regulatory role over p53 in vivo and that Egr1-null mice would share phenotypic properties with p53-null mice, such as increased sensitivity to skin carcinogens (35–37). To test the role of Egr1 in vivo, we carried out a two-step carcinogenesis assay in WT, heterozygous, and Egr1- null mice (Fig. 6). In this assay, the shaved posterior skin of mice was treated once with the mutagen and tumor initiator DMBA (7.8 mmol/L) followed by treatment with the tumor promoter TPA (1.0 mmol/L) thrice weekly for 18 weeks for the three mouse genotypes (WT, heterozygous, and Egr1 null). Control groups treated by only one of the two agents or by the two vehicles of these agents were also included for each of the three genotypes. Tumor formation and growth was monitored in the 11 groups of Figure 2. Three genes that are directly regulated by Egr1 define branch points treated mice for 18 weeks following the treatment (144 mice in all). leading to downstream regulation of 40 of the 66 informative genes, whereas one gene, IL6, is a known direct regulator of Egr1 (for references, see Table 1 Only 3 of 11 groups of the treated mice, WT, heterozygous, and null and Supplementary Table S1) and is associated with regulation of 22 of treated with both DMBA and TPA, developed visible and palpable the 66 informative genes.

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Figure 3. Regulatory relationships of Egr1 in MEFs. Sixty-six of the 143 known significantly differentially expressed genes (Table 1) are either direct or indirect targets of Egr1 as reported. A network that accommodates all 66 direct and indirect regulatory associations was constructed. For any relationship, an arrowhead indicates up-regulation and a barred link indicated down-regulation based on the literature (for references, see Supplementary Table S1). The observed relationship is given in terms of the Affymetrix-based expression analysis in Table 1 and Supplementary Table S1. role of Egr1, we did gene expression analyses using mouse (58), cyclin E2, known to be aberrantly expressed in human cancer Affymetrix microarrays in WT and Egr1-null MEFs. Noteworthy, (59), or cyclin A2, a major regulator of the cell cycle progression many of the genes down-regulated in Egr1-null MEFs are involved required for progression to S phase (60), were up-regulated Egr1- in growth inhibition or apoptosis, whereas genes that were up- null cells. These changes correlate well with the observed regulated stimulate growth and play a role in matrix formation phenotype of Egr1-null MEFs of rapid growth on explant and a (Table 1; Supplementary Table S1). In this regard, p53 and p53 complete insensitivity to the growth arrest of ‘‘replicative marker genes, such as p21Cip1/Waf1 (45, 46), GADD45 (47, 48), Bax senescence.’’ Thus, unstimulated low-passage MEFs seem to (49) and Fas (50, 51) were down-regulated in Egr1-null MEFs. In express many of the factors that regulate growth. addition, TGF-h1, known as a potent growth inhibitor of epithelial The regulatory map derived here explains not only the presence proliferation (52), induced hig-h3, known as a growth inhibitor of genes as downstream events but also their existence in many (53), serum amyloid A3, known as an inhibitor of proliferation (54), reported interactions across nodes and as upstream regulators of and cyclin G and thrombospondin 2, known as inhibitors of cell Egr1. These relationships are shown in the extended map of Fig. 3. cycle progression (55, 56) were also down-regulated. On the other Early growth response 1 regulates growth through the p53- hand, genes, such as collagen type VI, known to mediate the three- MDM2-p19ARF pathway and transforming growth factor-B1. In dimensional organization of fibronectin in the extracellular matrix our previous study, we proposed that Egr1 represents a novel of cultured fibroblasts (57), FGF-inducible 15,knowntobe upstream gatekeeper of the p53 tumor suppressor pathway and expressed during fibroblast growth factor-4–induced proliferation thereby has an important effect on the regulation of cellular growth www.aacrjournals.org 5139 Cancer Res 2005; 65: (12). June 15, 2005

studies using antibodies to native Egr1. These results support the conclusion that in MEFs the nuclear regulation of the p53 promoter by Egr1 is an important mechanism regulating the level of p53 protein. This mechanism likely is the basis of the gatekeeper function (18) of Egr1 in cell cycle regulation in MEFs. The expression analysis studies carried out here also revealed additional potential growth control mechanisms. First, in addition to p53, the TGF-h1 pathway might be another important component involved in Egr1-dependent growth control. TGF-h1 RNA is down-regulated in Egr1-null MEFs (Table 1; Fig. 2) and up- regulated in Egr1-null MEFs infected with an Egr1 retrovirus (18). It has been shown that TGF-h1 is a direct target of Egr1 (3, 61). TGF- h1, in turn, may participate in the regulation of Egr1-dependent growth suppression through the induction of p21Cip1/Waf1 (ref. 62; Table 1) or by induction of p27Kip1 (63, 64), which represents an important mediator of the permanent cell cycle arrest (65). Second, we have observed recently that PTEN also is a direct target of Egr1 and is increased on reexpression of Egr1 (17). PTEN expression also results in an up-regulation of p27Kip1 (66). Intriguingly, p27Kip1 connects the TGF-h1- and p53-MDM2-p19ARF pathways by its ability to facilitate the assembly of the cyclin D-CDK4 complex (67), thereby inactivating pRb and releasing E2Fs, which in turn can induce p19ARF (68, 69). Of these possibilities, the steady-state RNA expression differences for the 266 significant changes provide some indication of the mechanism operating in MEFs (Fig. 3). Neither PTEN nor p27Kip1 is significantly changed, whereas the p53 and TGF-h1- regulated gene p21Cip1/Waf1 are significantly up-regulated. Moreover, the p21Cip1/Waf1-regulated gene, cyclin E, is significantly down- regulated. This configuration of results argues that growth

Figure 4. Egr1 binds to two p53 promoter sequences in vitro. Two synthetic regulation of MEFs is dominated by the p53-dependent control of dsDNA representing two different Egr1-binding sites (A and B) in the promoter of CDK2-cyclin E complex. p53 gene were examined by EMSA using nuclear extracts of WT or Egr1-null Additional p53-regulated genes observed here, such as top- MEFs following stimulation of cells by addition of serum. Bottom, forward strand sequence of each synthetic probe representing the consensus. Only oisomerase IIa, XPC/Rad53, and XPC stabilizer (Fig. 3), may play a extracts of WT cells form retarded migrating complexes, which occur in parallel role in the p53-dependent DNA damage response characterized by with the complex formed using a positive control Egr1-binding sequence cell cycle arrest and enhanced DNA repair (70–72). The absence of (3V-GCGGGGGCG-5V). The unbound excess radiolabeled probe migrated out of the gel and is not shown. A, EMSA result for Egr1-binding site A in the p53 the p53-dependent mechanisms provides an explanation for the promoter. B, EMSA result for Egr1-binding site in the p53 promoter. lack of growth arrest and the lack of a DNA damage response by Egr1-null MEFs (18). Early growth response 1–null mice exhibit enhanced (18). However, the exact mechanism of Egr1-dependent regulation tumorigenesis. We reasoned that, if Egr1 is a major regulator of p53 remained unknown. In this study, we show that Egr1 directly of the expression of p53, it might be expected that Egr1-null mice binds the p53 promoter in vitro and in vivo using EMSA and ChIP would share aspects of the phenotype of p53-null mice (i.e., a experiments, respectively. We have observed that Egr1 binds to the greatly increased rate of tumorigenesis for a variety of p53 promoter at two distinct sites (Fig. 4). This is in accordance malignancies on aging or on challenge by skin carcinogenesis; with studies that showed that Egr1 transactivates the human p53 refs. 35–37). However, accelerated or spontaneous tumorigenesis gene promoter of human melanoma cells (14, 15). In this case, is not a feature of Egr1-null mice (73, 19), which may be related however, the binding by Egr1 was associated with p53-mediated to the known compensatory properties of other Egr1 family apoptosis stimulated by thapsigargin. Thus, the regulation of p53 in members (19, 74). The two-stage carcinogenesis experiment in the melanoma cells does not clearly provide a general explanation of Egr1 WT and Egr1-null mice shows the early onset of tumors in the growth regulation and DNA damage response roles of Egr1 Egr1-null mice, consistent with the expected phenotype of p53- observed in MEFs. On the other hand, in WT MEFs, Egr1 is required null mice. The effect is striking in that the group of Egr1-null for the normal level of expression of p53 mRNA and protein and for mice uniformly developed one or more palpable and visible skin the expression of p53 target genes. Moreover, Egr1 is required for tumors in over half the group before any mice of the WT group the p53-dependent development of replicative senescence of exhibit lesions. Histologically, all tumors appeared similar and primary cultures (18), apoptosis in response to UV irradiation exhibited the typical features of hyperplastic and hypertrophic (17), and p53-dependent cell cycle arrest that follows DNA damage. epithelium with markedly increased epidermal thickness, prom- Here, we find that WT cells that express Egr1 exhibit increased p53 inent rete pegs, and florid hyperkeratosis. Infiltrative epithelial protein and numerous p53 target gene products. Moreover, the cells were not seen. The significantly increased time of onset of mouse p53 promoter contains at least two putative Egr1-binding tumor formation is the first observation of increased tumor sites. These sites were confirmed by EMSA studies and by ChIP susceptibility in Egr1-null mice.

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Figure 5. Egr1 binds to the p53 promoter in vivo. ChIP assay was carried out with WT, Egr1-null, and human DU145 cell lines treated with TPA as a positive control. After formaldehyde cross-linking, the Egr1-binding DNA fragments were recovered by immunoprecipitation using Egr1 antibodies. The cross-links were reversed and the recovered DNA population was analyzed by PCR with primers designed for detecting p53. Egr1(+/+), WT MEFs; Egr1(À/À), Egr1-null cells. An amplified PCR fragment is visible in the DNA immunoprecipitated with Egr1 antibody in both mouse and human p53 promoters. There is no visible p53 band in the control DNA without any antibody. The genomic input DNA shows that the primers work well for amplification of p53 promoter. This experiment has been replicated at least four times with independently prepared MEFs.

Interleukin-6 is a major node in mouse embryo fibroblasts indicating a role in progression. Moreover, a variety of studies and may explain the role of early growth response 1 in have linked Egr1 with an oncogenic role in prostate cancer (26, 27, prostate cancer. Egr1 expression is regulated in part by the IL-6 39–41; reviewed in ref. 29). Egr1 mRNA and protein are elevated in signal transduction pathway (32, 75). The role of IL-6 in prostate prostate cancer in proportion to grade and stage and tumor cancer has drawn considerable attention (76). Autocrine-derived progression in the TRAMP model of prostate cancer is slowed in IL-6 is a potent growth factor for androgen-independent cells, hybrid Egr1-null TRAMP mice. Moreover, systemic treatment of promotes colony formation, and confers resistance to apoptosis. TRAMP mice with antisense Egr1 reduces Egr1 expression and Circulating levels are elevated in metastatic prostate cancer, tumorigenicity (39). These mice lack functional p53 as do a

Figure 6. Egr1-null mice develop skin tumors earlier than WT and heterozygous mice. A two-stage carcinogenesis experiment was carried out using groups of WT, heterozygous, and Egr1-null mice treated with DMBA (200 AL of 2.0 mg/mL) and TPA (20 AL of 0.61 mg/mL) for 18 weeks (11 groups, 144 mice). A, three treatment groups of mice (WT, heterozygous, and Egr1 null) treated with DMBA and TPA developed tumors. A fully developed tumor in the Egr1-deficient mouse treated with DMBA and TPA is shown above before necropsy. B, tumors developed in the WT, heterozygous, and Egr1-null mice show the characteristics of papilloma tumors. After the completion of two-stage carcinogenesis experiment, necropsy was done and the tumors were paraffin embedded and sectioned for histologic examination. The sections reveal exuberant epithelial hyperproliferation and hyperkeratosis with the body tumor separated from the normal skin by a neck. P, papilloma; E, normal epidermis; ND, normal dermis; NT, neck of the tumor; K, keratin; HPE, hyperproliferative epithelium. C, Egr1-null mice develop tumors earlier than the WT or heterozygous mice. After the application of TPA, the onset of the tumors was monitored in the mice for the following 18 weeks. The Egr1-null mice developed tumors around week 8 compared with the WT or heterozygous mice, which developed tumors around 12.5 weeks (P < 0.005). HTZ, heterozygous; KO, Egr1 null. Number in parentheses, number of mice in the respective group.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Research significant fraction of human prostate cancers. Thus, one unifying Taken together, our results suggest that Egr1 exerts its growth although speculative explanation is that the role of IL-6 in human control function by directly interacting with p53 promoter in MEFs prostate cancer works in part by induction of Egr1, which functions leading to activation of the p53 pathway and furthermore as an oncogene in this setting. establishes the role of Egr1 as a tumor suppressor in whole Several additional reported Egr1-regulated pathways were not animals. observed as significantly altered in Egr1-null cells. Thus, there is no representation of the inflammatory roles of Egr1, such as by Acknowledgments differential regulation of thrombospondin, tissue factor, and the Received 10/27/2004; revised 2/25/2005; accepted 4/9/2005. members of the platelet-derived growth factor family as has Grant support: Deutscher Akademischer Austauschdienst and AACR 2002 been observed in inflammatory settings (77, 78). This may be Scholar-in-Training award (A. Krones-Herzig), USPHS/NIH grants CA76173 (D. Mercola) and CA67888 (E.D. Adamson), and Department of Defense BCRP grant because the basal tissue culture conditions examined exclude DAMD17-01-005 (E.D. Adamson). proinflammatory responses. Thus, considerable additional char- 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 acterization may be necessary to achieve complete understand- with 18 U.S.C. Section 1734 solely to indicate this fact. ing of Egr1. We thank P. Charnay, N. Mackman, and J. Milbrandt for Egr1-null mice.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Early Growth Response 1 Acts as a Tumor Suppressor In vivo and In vitro via Regulation of p53

Anja Krones-Herzig, Shalu Mittal, Kelly Yule, et al.

Cancer Res 2005;65:5133-5143.

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