Proc. Natl. Acad. Sci. USAVol. 93, pp. 11831-11836, October 1996Medical Sciences

Transcription factor EGR-1 suppresses the growth andtransformation of human HT-1080 fibrosarcoma cellsby induction of transforming growth factor (81

(cancer/tumor suppression)

CHAOTING LIu*, EILEEN ADAMSONt, AND DAN MERCOLA*t§*Sidney Kimmel Cancer Center, 3099 Science Park Road, San Diego, CA 92121; tBurnham Institute, La Jolla, CA 92037; and tThe Center for MolecularGenetics, University of Califomia at San Diego, La Jolla, CA 92093

Communicated by Pedro Cuatrecasas, Warner-Lambert Co., Ann Arbor, MI, August 5, 1996 (received for review April 22, 1996)

ABSTRACT The early growth response 1 (EGR-1) geneproduct is a transcription factor with roles in differentiationand growth. We have previously shown that expression ofexogenous EGR-1 in various human tumor cells unexpectedlyand markedly reduces growth and tumorigenicity and, con-versely, that suppression of endogenous Egr-1 expression byantisense RNA eliminates protein expression, enhancesgrowth, and promotes phenotypic transformation. However,the mechanism of these effects remained unknown. The pro-moter of human transforming growth factor .31 (TGF-f81)contains two GC-rich EGR-1 binding sites. We show thatexpression of EGR-1 in human HT-1080 fibrosarcoma cellscauses increased secretion of biologically active TGF-p1 indirect proportion (rpe8a0n = 0.96) to the amount of EGR-1expressed and addition of recombinant human TGF-p1 isstrongly growth-suppressive for these cells. Addition of mono-clonal anti-TGF-j81 antibodies to EGR-1-expressing HT-1080cells completely reverses the growth inhibitory effects ofEGR-1. Reporter constructs bearing the EGR-1 binding seg-ment of the TGF-181 promoter was activated 4- to 6-foldrelative to a control reporter in either HT-1080 cells thatstably expressed or parental cells cotransfected with anEGR-1 expression vector. Expression of AEGR-1, a mutantthat cannot interact with the corepressors, nerve growthfactor-activated factor binding proteins NAB1 and NAB2, dueto deletion of the repressor domain, exhibited enhancedtransactivation of 2- to 3.5-fold over that of wild-type EGR-1showing that the reporter construct reflected the appropriatein vivo regulatory context. The EGR-1-stimulated transacti-vation was inhibited by expression of the Wilms tumor sup-pressor, a known specific DNA-binding competitor. Theseresults indicate that EGR-1 suppresses growth of humanHT-1080 fibrosarcoma cells by induction of TGF-f31.

The mitogenic stimulation of many cell types is accompaniedby rapid induction of immediate-early genes or primary re-sponse genes that occurs without prior protein synthesis (1, 2).Many of these genes encode transcription factors such as Fosand Jun gene family members, c-myc, NF-KB, and others thatare known to be important in regulating subsequent steps incell growth and differentiation. Searches for additional growthregulatory genes have led to the recognition of the earlygrowth response 1 gene product (EGR-1) as a rapidly appear-ing gene product in response to treatment of sensitive cells withserum [Zif/268 (3) and krox-24 (4)], nerve growth factorinducible-A [NGFI-A (5)], tetraphorbol ester induced se-quence-8 [TIS8 (6)], radiation (7, 8), and other stimuli (8).EGR-1 is a member of a zinc-finger gene family of at least four(Egr-1, -2, -3, and -4) transcription factors characterized by the

presence of three Cys2-His2 type zinc fingers in the C-terminalportion of the molecules that confer DNA binding properties(9, 10). EGR-1 preferentially binds GC-rich regulatory ele-ments (GCEs) with the sequence 5'-GCGT/GGGGCG-3'(10) or 5'-TCCT/ACCTCCTCC-3' (11, 12).GCEs occur in the promoter regions of numerous genes

including a provocative array of growth regulatory genes suchas platelet-derived growth factor A (PDGF-A) (12-14),PDGF-B (11), c-myc (12), c-Ki-ras (12), transforming growthfactor (31 (TGF-f31) (15), insulin-like growth factor 1 receptor(16), epidermal growth factor receptor (17), and other geneswith roles in proliferation and/or transformation. Moreover,the GCE sequence and extended forms (18-20) are knownbinding sites of the Wilms tumor suppressor gene product,WT1, by virtue of four adjacent zinc-finger motifs, three ofwhich are highly homologous to those of EGR-1 (18, 19). Lossof function ofWT1 is believed to play an important role in thedevelopment of a significant proportion of Wilms tumors (1, 2,18, 19). WT1 binds to GCE sequences of a number ofpromoters including its own promoter (20) and usually acts asa strong repressor (10, 13, 16-24) although in some circum-stances activation has been observed (25, 26). In contrast,EGR-1 is usually activating (4, 18, 25, 27). However, for avariety of promoters, this simple pattern is complicated by thepresence of adjacent or overlapping GC-rich Sp-1 sites (11, 21,28). Increased expression of EGR-1 is associated with activa-tion of the PDGF-B promoter by binding to overlapping Sp-1sites (11) but is associated with antagonism of the Sp-1-stimulated transcriptional activity of adenosine deaminase(28). Similarly, WT1 competes with Sp-1 for binding to thepromoter of the colony-stimulating factor gene leading torepression of transcription (21). These and other observationsillustrate cell-type-specific responses to EGR-1 (11, 14, 28).An important example that may be relevant to growth

control in many cell types is the regulation of the TGF-f31 gene(15, 29-32). TGF-(31 commonly has growth inhibitory effectson cells of epithelial origin but is stimulatory or promotesbiphasic effects depending on the concentration for some cellsof mesenchymal origin (30-35). The expression of TGF-,31, inturn, is regulated in part by the presence of several GC-richelements (15). WT-1 is a strong repressor of the transcriptionactivity of the human TGF-f31 promoter and inhibits secretionof latent TGF-,B1 by monkey kidney CV-1 cells (15). This effectis specifically counteracted in a dose-dependent manner bycoexpression of EGR-1. This observation suggests, as onepossibility, that EGR-1 may have growth-suppressive proper-

Abbreviations: GCE, GC-element, DNA binding site of EGR-1 andWT1; CM, conditioned medium; TGF-,B1, transforming growth factor(31; rhTGF-p1, recombinant human TGF-,B1; PDGF, platelet-derivedgrowth factor; CAT, chloramphenicol acetyltransferase.9To whom reprint requests should be addressed. e-mail: 74361.2163@compuserve.com.

11831

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

11832 Medical Sciences: Liu et al.

ties by stimulating secretion of TGF-31, which in turn acts asan autocrine or paracrine agent to inhibit proliferation.We have previously shown that expression of exogenous

EGR-1 in various human tumor cells markedly reduces growthand tumorigenicity of human fibrosarcoma HT1080 cells (36,37). Here we show that stable expression of EGR-1 byHT-1080 cells leads to both increased secretion of TGF-131 andto decreased growth in proportion to the amount of EGR-1expressed. Conversely, the growth inhibitory effects of stablyexpressed EGR-1 on HT-1080 cells is completely reversed byaddition of anti-TGF-f31 antibodies. Moreover, reporter con-structs bearing the GCE-containing segment of the TGF-p1promoter confirm that EGR-1 activates and WT-1 inhibits thetransactivation potential of the TGF-,B1 promoter in HT-1080cells. These results provide evidence that one potential regu-latory mechanism of EGR-1 is control of the expression ofgrowth regulatory molecule TGF-f31. This effect entirely ac-counts for the growth suppressive effect of EGR-1 on HT-1080cells.

MATERIALS AND METHODSCells and Cell Culture. HT-1080 cells and subclones (H4,

H4N, H4E2, H4E3, H4E6, H4E9, H4LHCX, H4LHC-C, andH4LHC-AC) were prepared and maintained as described (36,37). NIH 3T3 cells and A549 cells were maintained in DMEMsupplemented with 5% fetal calf serum and grown a humid-ified environment of 10% CO2. Cell numbers were determinedby Coulter counting similar to previous studies (36, 37).ELISA Assay. Conditioned medium (CM) was prepared

from HT-1080 cells and derivative lines by replacing thenormal medium of confluent cultures withDMEM followed bytwo additional changes over a 6- to 24-h period followed by afinal change at 2 ml per 35-mm culture dish and incubation fora further 24 h. The resulting CM was processed fresh or frozenat -70°C, and the number of conditioning cells was deter-mined by harvesting and counting cells (Coulter counter). CMwas clarified by centrifugation, "activated" by acidificationwith 1 M HCl to pH <3.0 for 15 min, neutralized with an equalvolume of 1 M NaOH, assayed by ELISA (Promega) in parallelwith multiple serial dilutions of a known amount of recombi-nant human TGF-f31 (rhTGF-131), and processed according tothe manufacturer's specifications.

Chloramphenicol Acetyltransferase (CAT) Assay. Test cellswere seeded into 60- or 100-mm tissue culture plates such thatthey were 80% confluent at the time of transfection in 18-24h. Transfections of EGR-1-expressing clones were carried outby addition of calcium phosphate-precipitated DNA mixturesconsisting of 1 ,ug of pRSV-,BGal (C. Van Beveren, SidneyKimmel Cancer Center) and 1 ,tg of reporter [phTG7-1-3 (15)]or control reporter [phTG7-4 (15)] devoid of TGF-l31 regu-latory sequences. For parental or control clones, cotransfec-tions using the same constructs and EGR-1 expression vectors[pCMVEgr-1 or AEgr-1 (pA284-330EGR-1) (45)] or a WT1expression vector (pCB6-WT1) or empty vector. pBluescript(Stratagene) was added in an amount sufficient to provide aconstant total DNA of 10 jig (60-mm dish) or 20 ,ug (100-mmdish). After an overnight exposure of the cells to the DNAprecipitates, the cells were washed and incubated an additional48 h prior to lysis in "reporter lysis buffer" (Promega). Thesamples were divided and used for determination of totalprotein (Bio-Rad), B-galactosidase synthesis by determinationof enzyme activity, and CAT synthesis as determined byenzyme activity by addition of [3H]acetyl-CoA for 2 h andextraction of the resulting [3H]acetylchloramphenicol productin a toluene/7 M urea mixture followed by scintillation count-ing as described (39). All results were normalized to theamount of f3-galactosidase synthesis as a correction for anyvariability of cell numbers and transfection efficiency amongthe wells.

RESULTSEGR-1 Stimulates Expression of TGF-f31 by Human HT-

1080 Fibrosarcoma Cells. To determine whether EGR-1 in-creases the secretion of TGF-31, we carried out ELISA assayson CM from a series of characterized clonal lines of HT-1080cells that exhibit graded amounts of stable expression ofEGR-1 (37) (Fig. 1A). Parental HT-1080, H4 (the subclone ofHT1080 used to prepare the EGR-1-expressing cells), andcontrol cells (H4N) that express the neomycin phosphotrans-ferase gene alone do not express detectable levels of EGR-1whereas the clones H4E9, H4E4, H4E2, and H4E3 stablyexpress various amounts of wild-type EGR-1 under the controlof the human cytomegalovirus promoter-enhancer with rela-tive steady-state levels of 100, 91, 32, and 26%, respectively(Fig. 1A). An additional clone, H4E6, transfected with theEGR-1 expression vector exhibits neomycin resistance but failsto express of EGR-1 (Fig. 1A) and serves as a control. Asadditional controls, we prepared clonal lines that express adifferent selection principle, hygromycin-B phosphotransfer-ase, either alone (H4pLHCX) or as part of a colinear constructwith the DNA-binding domain of EGR-1, termed C domain,in sense (H4pLHC-C) and antisense (H4pLHC-AC) orienta-tion (37). All these lines were used to examine the level of basaland EGR-1-induced expression of TGF-f1.

Quantitative ELISA analysis of CM revealed that moderatelevels of TGF-13l are expressed by all of the HT-1080 controlderivatives with an average of 481 ± 240 pg per ml per 24 h per106 cells similar to previous studies of HT-1080 cells (40). Bycomparison, the average for four primary cultures of humandermal fibroblasts determined in parallel is 119 ± 44 pg per mlper day per 106 cells (0. Dorigo, personal communication). Incontrast to the control cells, the clonal lines with gradedexpression of EGR-1 secrete increased amounts of TGF-/31 inproportion to the expression of EGR-1 with a maximal level of1150 pg per ml per 24 h per 106 cells (Fig. 1B). The results forthree replicate experiments are summarized in Fig. 1B, whichsuggests a linear relationship between TGF-,31 secretion abovecontrol levels and EGR-1 expression. Indeed, none of theaveraged observations of the three determinations deviatesfrom linearity by more than the 0.90 confidence interval. Thus,the TGF-j31 secretion is highly correlated (rpreason = 0.956)with steady-state EGR-1 expression.

Since the proliferation of these clones decreases continu-ously with increasing expression of EGR-1 (37), it may beexpected that proliferation would also decrease linearly withincreasing TGF-,B1 secretion. The results (Fig. 1C) show thatproliferation of HT-1080 cells is strongly negatively correlated(rpearson = -0.92) with the amount of secreted TGF-f31.Moreover, since this relationship extends to the clonal lines ofminimum proliferation potential (H4E9, Fig. 1C), EGR-1-stimulated TGF-,B1 secretion appears to be sufficient to ex-plain all of the growth-inhibitory effects of EGR-1.We confirmed that the CM of EGR-1-expressing cells

contains increased amounts of functional TGF-,1 by assayingCM from EGR-1-expressing cells and control cells on humanTF-1 erythroleukemia cells, which are highly sensitive toTGF-13 (41). As for the ELISA, these results showed that H4E9secreted about 234% more TGF-,1 than the average of thecontrol clones. CM of H4E9 or H4E4 cells significantlyinhibited the proliferation of HT-1080 or H4 cells by up to 70%compared with control CM (H4 CM) (P < 0.01, all compar-isons). The growth inhibition produced by these CM weredirectly proportion to the measured TGF-31 content by eitherELISA (rpearOn = 0.96) (Fig. 1) or bioassay (rpearson = 0.94)(unpublished results). Since HT1080 fibrosarcoma cells are ofpresumed mesenchymal origin, a cell type commonly stimu-lated by TGF-131 (30-35), we directly compared the responseof HT1080 and H4 cells after addition of authentic rhTGF-,31to the response of NIH 3T3 cells, which are known to be

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 11833

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1100

1000

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5000 20 40 60 80 100 1500 2000 2500 3000

RELATIVE AMOUNT EGR-I (%) PROLIFERATION (cells/cm*'2/5d x 10's-3)

FIG. 1. The secretion of TGF-j31 is directly proportional to the amount of stably expressed EGR-1 and inversely related to proliferation. (A)Western blot analysis of steady-state expression of EGR-1 by lines of H4 cells transfected with an expression vector for wild-type EGR-1 (fromref. 37). The relative expression of EGR-1 was determined by digitalization of the band intensities at the characteristic molecular mass of EGR-1,84 kDa, yielding 100, 91, 32, 26, 0, 0, and 0% for H4E9, H4E4, H4E2, H4E3, H4E6, H4N (neomycin-resistant empty-vector control), and H4,respectively. (B) The TGF-,31 concentration of CM from the same clonal lines (A) was determined by ELISA. (C) The proliferation of the sameclones as examined inA and B was determined by integration of growth curves (41) expressed as cells per cm2 of culture plate x 10-3 previouslydetermined by direct cell counting in triplicate daily for 5 days (37) and plotted as a function of the observed TGF-,B1 secretion. The curves in Adefine ±0.95 confidence intervals about the straight line. r = Pearson correlation coeficient. A is reproduced by permission of the AmericanAssociation of Cancer Research.

stimulated by TGF-,B1 (42). These observations showed thatthe addition of rhTGF-,B1, indeed suppressed the growth ofHT1080 cells by >50% in the same experiments where NIH3T3 exhibited 200-300% increased growth (unpublished re-sults). The sum of observations support the conclusion thatEGR-1 suppresses HT1080 cells by induction of TGF-131. Thisconclusion leads to the prediction that addition of anti-TGF-A31 antibody to EGR-1-expressing cells should reversethe growth suppressive effects of EGR-1.

Anti-TGF-611 Antibodies Increased the Proliferation ofEGR-l-Expressing HT-1080 Cells. To test whether secretedTGF-,B1 inhibits the growth of EGR-1-expressing cells, pro-liferation rates were examined in the presence and absence ofa well-characterized antibody known to specifically neutralizeTGF-,B1 (43). Addition of this antibody to the EGR-1-expressing HT-1080 clones lead to increased growth and theeffect was most marked for the clone with the greatestexpression of EGR-1, H4E9 (Fig. 2). The average of fourexperiments shows a 84 ± 6.2% increase in proliferation overuntreated or rhTGF-,B1-treated H4E9 cells. The size of theincrease is significant (P < 0.001) and is in close agreementwith the expected increase if the entire growth suppressiveeffect of EGR-1, 55% (Fig. 3 and ref. 37) were accounted forby the TGF-j31 [i.e., 100 x (1.00 - 0.55)Go/0.55G. = 82%,where Go is the uninhibited growth of HT-1080 cells]. Thisresult has been observed in two other replicate experiments. Incontrast, the antibody has only a very small effect on parentalHT1080 or H4N cells, which express little or no EGR-1 (Fig.1A) and secrete little TGF-A31 (Fig. 1B). Similarly, the antibodyalso had only a small effect on A549 human lung cells (Fig. 2).These cells are known to be strongly inhibited by TGF-A31 (28)as also observed here (Fig. 2), showing that the antibody doesnot confer nonspecific growth promoting properties to controlcells and that the culture conditions do not lead to significantTGF-13l-like modulation of growth. These results support theconclusion that the EGR-1-stimulated secretion of TGF-l31completely accounts for the growth suppression observed inEGR-1-expressing cells.

EGR-1 Activates the TGF-j81 Promoter in HT-1080 Cells.To determine whether EGR-1 can enhance TGF-13l expres-sion by activation of the TGF-,B1 gene, a series of transienttransfection studies were carried out using a 119-bp segmentthat contains the two GCEs of the human TGF-31 promoter

._

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a

E0z

Anti-TGBp1

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HT1 080 H4E9 A549FIG. 2. Specific anti-TGF-,B1 antisera enhances the growth of

EGR-1-expressing HT-1080 cells. The proliferation of the indicatedcells was determined by direct cell counting (Coulter counter) inquadriplicate 4 days after seeding 24-well tissue culture plates with theindicated cells at 25 x 103 cells per cm2. Antisera to 0.2% (solid bars)or rhTGF-,31 to 1 ng/ml (hatched bars) or vehicle alone (control, openbars) on were added on days 1-3 of culture. For each condition, theaverage cell density ± o was normalized by dividing by the control(vehicle alone) density of the respective cell type. H4E9 cells +anti-TGF-,31 antiserum (43) lead to an increased in proliferationation(Coulter counter) of 84 ± 6.2% (P < 0.001, t test).

Medical Sciences: Liu et aL

Proc. Natl. Acad. Sci. USA 93 (1996)

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+WTr vector

j El Control-Reporter+wr-1 vector

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H4E4 H4 E9 H4Neo

HT1080 Clones

FIG. 3. HT-1080 cells that stably express EGR-1 show enhancedactivation of the TGF-,B1 promoter. All cells were cotransfected withpj3Gal and a CAT report construct bearing 119 bp of the humanTGF-f31 promoter including the two GCEs or a control construct withthe TGF-,13 promoter sequences deleted. Where an expression vectorfor WT1 was used, total DNA was maintained constant by reductionof carrier DNA (hatched bars). f3-Galactosidase and CAT was subse-quently determined. In this example, all assays were carried out in100-mm dishes with a total of 20 ,ug ofDNA and all CAT activities arenormalized to the ,3-galactosidase activities. Relative EGR-1 expres-sion is 100, 91, and 0% for H4E9, H4E4, and H4N (H4Neo) clones,respectively.

as the regulatory elements of a CAT gene reporter construct(15). All values are compared with a control reporter constructwith the GCE-bearing segment deleted. First, the EGR-1expressing clones were examined. All EGR-1 expressing clonesactivated the TGF-,31 reporter relative to various null controlclones. In the example shown in Fig. 3, both EGR-1 expressingclones, H4E4 and H4E9, substantially activated the TGF-f31reporter by 4.4 (n = 7)- and 3.5 (n = 6)-fold, respectively, overthe H4N control clone albeit in the opposite order than thatexpected on the basis of relative EGR-1 expression of 91 and100%. The results are the average of six experiments and thedifference is significant (P = 0.016). This effect may be relatedto the fact that clone H4E9 expresses several aberrant lowmolecular weight forms of EGR-1 (Fig. 1A) that may interferewith the transcriptional activity of full-length EGR-1. In contrast,no activation was observed in the H4N control cells (Fig. 3).Moreover, cotransfection of the reporters with an expressionvector for WT1, a specific suppressor of the TGF-,31 promoter(15), abolished activation by the EGR-1 expressing cells (Fig. 3),showing that the activation seen in these cells specifically re-

flected expression of exogenous EGR-1.Second, unaltered parental HT-1080 cells or subclone H4

were used for cotransfection studies with reporter and expres-sion vectors for EGR-1 (Fig. 4). Again substantial activationwas only seen in the presence of the EGR-1 expression vector.The results are the average of four replicate experiments andeven the lowest average activation, 2.3-fold observed usingHT-1080 cells is significantly greater than any of the controlvalues (P < 0.027). In contrast, cotransfection of an emptyvector plasmid with the TGF-,31 reporter or with the controlreporter construct did not lead to activation in either cell line.The results, therefore, support the conclusion that the sub-stantial activation observed in clones of stably transfectedHT1080 cells (Fig. 3) was not related to clonal selection butdue to the expression of EGR-1.

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ReporterControl-ReporterEmpty VectorEgr-1 vector

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FIG. 4. Transient expression ofEGR-1 in HT-1080 cells specificallyactivates the TGF-,B1 promoter. The indicated cells were cotransfectedwith p,3Gal, an EGR-1 expression vector, the TGF-l1-CAT reporterconstruct, or a control construct without Egr-1 sequences as indicatedby the combination matrix. f-Galactosidase and CAT was subse-quently determined. In this example, all assays were carried out in100-mm dishes with a total of 20 ,ug ofDNA and all CAT activities arenormalized to the f3-galactosidase activities.

Finally, it has been reported recently that transactivationpotential of EGR-1 may be limited by interaction with specificcorepressors, termed nerve growth factor-activated factorbinding proteins NAB1 (38) and NAB2 (44), which interactwith a repressor domain of EGR-1 located in the region ofresidues 284-330 (44, 45). We tested whether the transactiva-tion activities observed here using wild-type EGR-1 werelimited by the presence of any repressor activity in HT-1080cells. Cotransfection assays were carried out with altered formsof EGR-1 that either lacked the repressor domain of EGR-1,AEGR-1 (45), or contained a point mutation, 1293F, thatinterferes with the interaction of EGR-1 with the corepressors(44). For example, in a series of CAT assays (n = 7) in whichthe transactivation potential of wild-type EGR-1 was com-pared with the AEGR-1, which is unable to interact withrepressor factors (45), revealed a modest but consistent (P <0.005) 2- to 3.5-fold increase in transactivation potential (Fig.5), very similar to previous studies with AEGR-1 (45). More-over, we observed very similar results when either the TGF-,B1reporter or a minimum reporter CAT construct containing aconsensus EGR-1 binding sequence was coexpressed withAEGR-1 in NIH 3T3 cells (data not shown). These resultssuggest that, as for a variety of other cells types previouslytested (38), a low but readily detectable repressor activity ispresent in HT1080 and the H4 subclone (Fig. 5). Relief of thisactivity by the use of altered forms of EGR-1 that are unableto interact with corepressors leads to increased transactivationof the TGF-f31 reporter construct. We conclude that the lowertransaction activities observed when wild-type EGR-1 is ex-pressed with the reporter system used here (Figs. 3 and 4) are

11834 Medical Sciences: Liu et al.

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Proc. Natl. Acad. Sci. USA 93 (1996) 11835

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FIG. 5. Transient expression of a mutant 8EGR-1 that does notinteract with corepressors exhibits increased transactivation activitycompared with wild-type EGR-1. The indicated cells were cotrans-fected with either the wild-type EGR-1 expression vector or 8EGR-1expression vector (p6284-330EGR-1) (45) and either the TGF-,B1-CAT reporter construct or a control construct without the EGR-1binding sequences. As a further control, the empty expression vectorwas cotransfected with the TGF-,B1 reporter and control reporter andall results have been normalized to the background activation observedwith the TGF-f31 control reporter. In this example, all assays werecarried out in six-well tissue culture plates using 9 ,ug of total DNA perwell. CAT activity was subsequently determined. The experiment wasreplicated seven times.

due to the sensitivity of the system to the complete regulatorycontext governing expression of TGF-,B1 by EGR-1.

DISCUSSIONStable expression of EGR-1 in HT-1080 cells reduces theproliferation rate and maximum cell density by more than60%, suggesting that the expression of EGR-1 leads to restoreddensity-dependent growth arrest (37). Expression of wild-typeEGR-1 also reduced tumorigenicity and tumor growth by 40%.The effects on tumor growth are likely to be underestimatessince we have observed that expression of EGR-1 in xenograftsis unstable and is often absent in large tumors (37). Similaralbeit less dramatic effects have been observed in PDGF-B/v-sis-transformed NIH 3T3 cells (36), human breast carcinomacells ZR-75-1, human glioblastoma cells U251, and humanosteosarcoma cells Saos-2 (37). These observations suggestthat, although derived as a primary response gene or earlygrowth response gene, EGR-1 has growth suppressive prop-erties under certain circumstances.

In the case of HT-1080 cells, the growth suppressive prop-erties may be related to a direct activation of the TGF-f31promoter. HT-1080 cells have been shown to express TGF-,Breceptors (30) and we have observed that the proliferation ofHT-1080 cells is negatively regulated by rhTGF-,B1. Moreover,expression of EGR-1 in HT-1080 cells leads to increasedsecretion of TGF-31 in amounts that are highly correlated(rpearson = -0.92) with the decreased proliferation. Conversely,specific anti-TGF-,B1 antibody reverses the effect. Expressionof a reporter that is regulated by a 119-bp sequence of theTGF-f31 promoter in cells that stably express EGR-1 but not

in control cells leads to significant activation of the reporter.The EGR-1-dependent activation is blocked by coexpressionof WT1, a known suppressor of the endogenous TGF-131promoter. Similarly, the cotransfection studies employing tran-siently expressed EGR-1 further support this conclusion.Further, the nuclear factors NAB1 and NAB2 are believed tomodulate the transactivation potential of Egr-1 by, in part,interacting with the EGR-1 at the repressor domain (38, 44).Inhibition of this mechanism by deletion, AEGR-1 (45), oralteration of the NAB1 binding domain of EGR-1 (38) leadsto enhanced transactivation activity of up to 17-fold greaterthan that of wild-type EGR-1 (38). For AEGR-1, we observeda modest but highly reproducible 2- to 3.5-fold increase intransactivation potential over wild-type EGR-1, suggestingthat a small but readily detectable amount of repressor activitymay be present in HT-1080 cells. Low levels of repressoractivity combined with the increased expression of wild-typeEGR-1 may lead to increased expression of TGF-,31 thataccounts for the suppression of growth and transformation ofstably transfected clonal lines of HT1080 cells. The enhancedactivation observed with AEGR-1 also suggest that the re-porter construct used here accurately reflects the normal invivo regulatory context. Nevertheless, it is emphasized that itremains a hypothesis that the mechanism of induction ofTGF-,31 secretion and growth suppression by EGR-1 involvesdirect binding of EGR-1 to the TGF-,31 gene promoter.

Lesions in the TGF-f31 growth regulatory system have beenincreasingly recognized as important in the development ofseveral human tumor types (46-49). For example, in heredi-tary nonpolyposis colon carcinoma, defects in the DNA repairsenzymes lead to an accumulation of errors of microsatilliteDNA (mutator phenotype) (46). This results in loss of expres-sion of TGF-,B1 receptor subunit RII, which is essential forTGF-,B1-dependent growth regulation. Moreover, the TGF-,3receptor RI subunit is also absent in certain sporadic coloncarcinomas (48) and prostate carcinoma cell lines (47). Also,about 30% of pancreatic cancers show homozygous loss of acandidate suppressor gene at chromosome 18q21.1 that issimilar in sequence to a Drosophila melanogaster gene (Mad)implicated in a TGF-,B1-like signaling pathway (49). Thesignificance is emphasized by the recent observations thatexpression of wild-type TGF-131 receptor subunit in eitherbreast carcinoma (50) or colon carcinoma cells (46) that aredevoid of functional wild-type TGF-,B1 receptors suppressestumorigenicity. Thus, lesions that inactivate any step of theTGF-,31 growth regulatory pathway might be expected tooccur in transformed cells.

Since EGR-1 is a positive transactivator of TGF-,B1, it maybe important that EGR-1 is low or undetectable in most humantumors that have been investigated (51-53). For example, in aseries of 101 nonsmall cell lung carcinomas, Slamon andcoworkers (51) observed that the level of expressed EGR-1 isless than in adjacent normal tissue in more than 72% of cases.In the case of acute myeloid leukemia (AML), a small regionof 2.8 megabases within chromosome band 5q31 has beenresolved as the minimally deleted region in a series of 120 cases(52). This region was not deleted in nine other patients withAMLwho did not have abnormalities of chromosome 5 and is believedto be the site of a suppressive principle (52). This region containsthe Egr-1 gene, suggesting that it is a major candidate for the lossof a tumor-suppressive principle (52).Although the expression ofEGR-1 has been most commonly

observed after treatment of cells with mitogenic stimuli (1-6),a consistent requirement for cell cycle progression has not beobserved, leaving the role of EGR-1 in growth control unclear.Our results provide an example of one mechanism of growthcontrol by EGR-1.

We thank Seong-Jin Kim for providing TGF-,B1 reporter constructs,Jeffrey Milbrandt and Vikas Sukhatme for providing expression

Medical Sciences: Liu et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

vectors of mutant Egr-1 constructs, Katherine Flanders for a gift ofanti-TGF-,1 antibody, and A. Mire-Sluis for providing TF-1 cells. Wethank Vikas Sukhatme and Ruo-Pan Huang for advice, help, andproviding an expression vector for WT1. N. Dean kindly provided theA549 cells. This work was supported in part by Grants CA 63783(D.M.) and CA 67888 (E.A.) from the National Institutes of Health,by the Sidney Kimmel Fellowship Program, and by J. Morris.

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