Tumors caused by overexpression and forced activation of Stat5 in mammary

epithelial cells of transgenic mice are parity-dependent and developed in aged,

postestropausal females

Tali Eilon1, Bernd Groner2 and Itamar Barash1*

1Institute of Animal Science, ARO, The Volcani Center, Bet-Dagan, Israel2Georg Speyer Haus, Institute for Biomedical Research, Frankfurt/M, Germany

In transgenic mice overexpressing Stat5 or a constitutively acti-vated Stat5 variant (STAT5ca), we show for the first time thatparity is required for the development of tumors in postestro-pausal females. Tumors were detected in glands of multiparoustransgenic female mice after latency period of 14 months, butrarely in their age-matched virgin (AMV) counterparts. This pe-riod was not affected by distinguishable tumor pathologies andwas not dependent upon transgenic Stat5 variant. To associateStat5 deregulation, parity and the postestropausal tumor occur-rence with mammary cancer formation, the activities of endoge-nous and transgenic Stat5 were measured in the glands of agedmultiparous and AMV females. No differences in phosphorylatedStat5 (pStat5) levels were found between the 2 cohorts. However,promoter sequences comprising the Stat5 binding sites from thecyclin D1 or the bcl-x genes associate differentially with acetylatedhistone H4 in aged multiparous and AMV STAT5ca transgenicfemales. Individual epithelial cells varied greatly with respect tothe presence of nuclear pStat5. A small subset of epithelial cells, inwhich pStat5 and cyclin D1 were co-expressed, was exclusivelypresent in the multiparous glands. Changes in chromatin struc-ture might persist past the reproductive life time of the multipa-rous mice and contribute to the transcription of the cyclin D1 geneby activated Stat5. This may cause the detectable expression ofcyclin D1 and add to the process of tumorigenesis.' 2007 Wiley-Liss, Inc.

Key words: transgenic mice; mammary gland; breast; Stat5; cancer;parity

Epidemiological studies have established a correlation betweenparity and the incidence of breast cancer in women.1–3 Parity has aprotective effect, which depends on an early age of full-term preg-nancy and is augmented by the total number of pregnancies.4,5 Incontrast, first pregnancy in women at their 30s is considered a lifetime risk factor.2,6 A transient increase in risk for breast cancer af-ter delivery has also been found and augmented in women at their30s.7,8 The diversity between the protective and inductive effectsof parity on breast cancer was mainly associated with the be-direc-tional double edged action of sex steroid hormones. Estrogen andprogesterone could induce breast cancer by growth promotingeffect on premalignant or preexisting malignant cells and protectfrom the disease by exerting persistent changes during a criticalperiod of adolescence with long terms effects on cell proliferationand stem cells population.9–14 The detailed mechanisms regulatingthe action of sex steroid hormones remain elusive, but most likelyinvolve signaling pathways that govern the balance between apo-ptosis, proliferation and differentiation. Recently, the mammarymicroenvironment was also associated with the transient increasein breast cancer risk. It was proposed that remodeling of the mam-mary gland during involution induces proinflammatory andwound-healing signals that are pro-oncogenic and contribute tocancer promotion and progression.15

Mammary development during puberty results in a network ofducts covering the entire fat pad. This ductal tree is preserved dur-ing the life span of the nulliparous female.16 When the animalsbecome pregnant mammary development proceeds. Epithelialcells proliferate rapidly and form secondary and tertiary brancheswithin the ductal network. Cells differentiate into lobulo-alveolarstructures, acquiring different characteristics from the ductal cells.These cells produce large quantities of milk during the suckling

period. Upon weaning the epithelial cells undergo apoptosis andthe gland involutes. The remaining ducts resemble the morphol-ogy found in the virgin animals. These processes are synchronouswith the reproductive cycle. They are precisely repeated duringsubsequent pregnancies and are controlled by ovarian and pituitaryhormones. These hormones trigger well known intracellular signaltransduction pathways. Their de-regulation leads to uncontrolledproliferation and cancer. c-myc and cyclin D1, both mediators ofthe mitogenic effect of estrogen,17–22 can act as oncogenes in theirown right when expressed as transgenes in mice.

Expression of individual contributors is usually not sufficientfor cellular transformation and tumor formation. Cooperatingevents are required as reflected by the lag in tumor cell appear-ance. Tumors develop frequently past the end of the fertile period.Eighty percent of breast cancers were diagnosed in women above50 years of age.23 At this time, which is long after the peak of ste-roid hormone action, breast cancer and breast cancer related mor-tality significantly increases.5,24

The signal transducer and activator of transcription (Stat5) is acytoplasmic signaling molecule and a transcription factor whichconfers the effects of cytokines, polypeptide hormones and growthfactors into transcriptional activity. In the mammary gland, itmediates the action of prolactin. The mechanism has been studiedin detail. It is initiated by the recruitment of Stat5 to the activatedreceptor and is followed by phosphorylation and dimerization ofStat5, translocation into the nucleolus and binding to specificDNA sequence elements.25–27 Stat5 fulfills several additionalfunctions in mammary epithelial cells. It controls epithelial cellproliferation, cell survival during pregnancy and involution, dif-ferentiation and specific gene expression during the lactation pe-riod.28–31 Stat5 is also involved in mammary tumorigenesis. Over-expression and forced activation of Stat5 in the mammary glandsof multiparous transgenic female mice resulted in tumors: highlydifferentiated papillary and micropapillary adenocarcinomas wereobserved. The average tumor incidence was 8–9% of the popula-tion of the transgenic strains. In one particular Stat5 transgenicstrain, 22% of the animals developed tumors.32 As pointed outabove, Stat5 is a major contributor to mammary development andfunction,28,33–35 but very much like estrogen and progesterone,can also assume a role in tumorigenesis.

Abbreviations: AMV, age-matched virgin; BLG, b-lactoglobulin; cdk,cyclin-dependent kinase; ER, estrogen receptor; H&E, hematoxylin & eo-sin; Jak, Janus kinase; PR, progesterone receptor; RB, retinoblastoma pro-tein; RT-PCR, reverse-transcription PCR; Stat, signal transducer and acti-vator of transcription; STAT5, transgenic Stat5; STAT5ca, constitutivelyactive Stat5 transgene; TAD, transactivation domain.Grant sponsor: Israel Science Foundation, Israel Academy of Science;

Grant number: 706/04; Grant sponsor: Deutsche Forschungsgemeinschaft(DFG); Grant number: GR536/11-1.*Correspondence to: Institute of Animal Science, ARO, The Volcani

Center, P.O. Box 6, Bet-Dagan 50250, Israel. Fax:1972-8-9475075.E-mail: barashi@agri.huji.ac.ilReceived 1 February 2007; Accepted after revision 30 May 2007DOI 10.1002/ijc.22954Published online 19 July 2007 in Wiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 121, 1892–1902 (2007)' 2007 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

In this study, we demonstrate that overexpression and forcedactivation of Stat5 in the mammary glands of transgenic micecauses mammary tumors after a rather long latency period.Tumors appeared on the average in animals of 14 months of age.The latency period was similar for the different pathological phe-notypes and independent of the transgenic Stat5 variant. Mam-mary tumorigenesis was strictly dependent on parity. It seems thatthe effects of estrogen and progesterone exposure have long-termconsequences for tumor formation, which are linked to gene acti-vation by Stat5. Such effects might be reflected by histone modifi-cations at the Stat5 binding sites in the cyclin D1 and bcl-x genepromoters. Activated Stat5 and the expression of cyclin D1 coin-cided in a small number of luminal epithelial cells in postestro-pausal multiparous glands, but not in their age-matched virgin(AMV) counterparts. We propose that these cells might preservethe effects of parity on gene expression. They could form a reser-voir of cells prone to tumor development.

Material and methods

Plasmids and constructs

Two Stat5-based constructs were employed (Fig. 1): (i) the wild-type version of ovine Stat5a, termed STAT5, linked to an influenzavirus epitope in order to distinguish it from the endogenous Stat538;(ii) constitutively activated STAT5, termed STAT5ca, comprisingsequences from 3 genes: amino acids 1–750 from ovine Stat5, 677–847 from human Stat6, and 757–1,129 from mouse Janus kinase(Jak) 2.39 These constructs were inserted into the b-lactoglobulin(BLG) gene multiple-cloning site as previously described.30 Toavoid biphasic expression, the natural BLG ATG translation-initia-tion codon, as well as a second potential initiation codon in BLGexon 1, were converted into noninitiating ATT and ATC sequences.BLG sequences between the first part of BLG exon 2 and the middleof BLG exon 6 (including the TAG termination codon) weredeleted and replaced with a multiple-cloning site.

Generation and identification of transgenic mice

Mice used in this study were of the FVB/N strain. Plasmids con-taining sequences of BLG/STAT5 and BLG/STAT5ca weredigested by SalI and the appropriate fragments were prepared andmicroinjected into fertilized mouse oocytes. Transgenic animalswere identified by Southern-blot analysis of genomic DNA pre-pared from tail biopsies using a probe encompassing 3 kb of theBLG gene promoter. The mice were housed for up to 18 monthsunder a 12-hr light/dark cycle and were kept under the rules estab-lished by the Israeli National Committee for Animal Care. Micewere permitted ad libitum access to food and water and visuallyinspected for tumors twice a week.

Chromatin immunoprecipitation

The inguinal and thoracic mammary glands from 18-month-oldmice were excised, washed in PBS and cut into �1-mm3 pieceswith fine scissors. The tissue pieces were fixed with 1% (v/v)formaldehyde for 30 min at 37�C and chromatin immunoprecipita-tion (ChIP) was performed with a ChIP Assay Kit (Upstate, LakePlacid, NY) according to the manufacturer’s protocol. Briefly, tis-sue pieces were washed, collected and sonicated on ice (SONICSVibra cell, Newtown, CT) in 500 ll SDS lysis buffer. DNA wassheared to lengths of between 200 and 1,000 bp and ChIP was per-formed with anti-acetyl histone H4 polyclonal antibodies(Upstate) diluted 1:200 as previously described.40 DNA-proteincrosslinking was reversed at 65�C for 4 hr in the presence of 5 MNaCl and DNA was recovered by phenol/chloroform extractionand ethanol precipitation.

RNA extraction and reverse transcription

Total RNA was isolated from the inguinal and thoracic mam-mary glands with TRIZOL (Gibco/BRL, Gaithersburg, MD) aspreviously described.40 DNase-I-treated aliquots (0.2 lg) were

reverse-transcribed using a superscript II reverse transcriptase(RT) kit and oligo(dT) primers (Invitrogen, Carlsbad, CA) accord-ing to the manufacturer’s protocol.

Real-time PCR

Real-time PCR analyses of DNA recovered from the ChIP, orsynthesized by RNA RT, were performed in an ABI Prism 7700(Foster city, CA) in a 20-ll reaction volume containing 1 ll ofcDNA (10 ng), 10 ll of SYBR Green PCR Master Mix (AppliedBiosystems, Foster City, CA) and 10 lM primers. The thermal-cycle conditions consisted of 2 min at 50�C, 10 min at 95�C, and 40cycles of 15 sec at 95�C and 1 min at 60�C. The primers weredesigned so that the PCR would yield a single product without anyprimer dimerization, the product being verified using a dissociationprotocol. The amplification curves for the regions (70–92 bp) fromthe promoters of bcl-x, cyclin D1, c-myc, cyclin D2 and 16S, aswell as for the coding regions from cyclin D1 and bcl-xL, gene wereparallel. The results were calculated as DDCT, as described in UserBulletin no. 2 for the ABI prism 7700 sequence detection system.

The following primers were used to amplify: the Stat5-bindingsite in the bcl-x gene promoter; 50-TCTGCTCGTACACTGA-

FIGURE 1 – Parity-dependent tumorigenesis was detected in BLG/STAT5- and BLG/STAT5ca-transgenic mice. (a) Tumor incidence inmultiparous and virgin transgenic mice. (b) Individual latency periodsfor hyperplasia and tumor development. Averages (mean 6 SEM)exclude hyperplasia. Note: no tumors were developed in comparablysized multiparous transgenic cohorts (at least 66 mice) of transgenicstrains BLG/STAT5-10 and BLG/STAT5ca-11, which carry the trans-gene but do not express the protein,32 in over 60 multiparous femalescarrying the BLG/luciferase gene, which were maintained at our dis-posal,36,37 or in at least 40 wild-type FVB/N transgenic mice (virginand multiparous) of the same age.

1893PARITY-DEPENDENT STAT5-INDUCED TUMORIGENESIS

TAATTGG-30 and 50-GTGTTCTGCATATGTTGGATT-30; region1 in the bcl-x gene promoter; 50-CTCTCCAGCACACACCAAAT-30 and 50-CTGTTGGCCACAGAGTTCAT-30; region 2 in the bcl-xgene promoter; 50-ACAACTAGCGGTGTTGTGG-30 and 50-GATTCGGAAAGACCACTCC-30; the Stat5-binding site in thecyclin D1 gene promoter; 50-CTCAACGAAGCCAATCAAGA-30and 50-AATCGCTGCAAAGTTATTAGTCG-30; region 1 in thecyclin D1 gene promoter; 50-TGGCATCTTCGGGTGTTAC-30and 50-AGCGTCCCTGTCTTCTTTCA-30 region 2 in the cyclinD1 gene promoter; 50-TGTTGAAGTTGCAAAGTCCTG-30 and50-CGCGGAGTCTGTAGCTCTCT-30 the Stat5-binding site in thec-myc gene promoter 50-ATCTGGGTTGGCAATTCACT-30 and50-CAAGGCTAGACGCGAGAATA-30 region 1 in the c-mycgene promoter; 50-CAGGGTACATGGCGTATTGT-30 and 50-TTGCCCTGCGTATATCAGTC-30; the Stat5-binding site in thecyclin D2 gene promoter; 50-CGAGCCATTTCCTAGAAAGC-30and 50-CCCTGCATCTGCTGACAA-30; the coding region fromthe bcl-XL gene; 50-GGATGGCCACCTATCTGAAT-30 and 50-TGTTCCCGTAGAGATCCACA-50; the coding region from thecyclin D1 gene; 50-GGTGAACAAGCTCAAGTGGA-30 and 50-TGGAGAGGAAGTGTTCGATG-30; the coding region from theSTAT5ca gene; 50-AGAGACCAAGCAGACTTCCAG-30 and 50-CAAGCTTATCGATACCGTAGACA-30; the coding region fromthe endogenous Stat5 gene; 50-TGGACTCCATGCTTCTCTTG-30and 50-GCAAAGCCATTGTTGGTATAG-30; the mouse S16rRNA; 50-GCTAAACGAGGGTCCAACTGTCT-30 and 50-GCTCCATAGGGTCTTCTCGTCTT-30.

Immunoblot analysis

Proteins from intact mammary glands or mammary tumorswere extracted with extraction buffer composed of 10 mM Tris-HCl pH 7.4, 50 mM NaCl, 5 mM EDTA, 0.1% (w/v) BSA, 1%Triton, 50 mM NaF, 1 mM Na3VO4,1 mM EGTA, 1 mM PMSF, 1mM aprotinin, and subjected to SDS-PAGE. Fractionated proteinswere transferred to nitrocellulose filters and equal amounts of pro-tein/sample were confirmed by staining the blot with Ponceau S(Sigma, St. Louis, MO). The expression levels of selected proteinswere determined according the signals generated with their re-spective antibodies: anti-HA (Abcam, Cambridge, UK, diluted1:500), anti-Stat5 (Cell Signaling Technology, Beverly, MA,diluted 1:750) and anti-pStat5 (Abcam, diluted 1:500). Rabbitanti-rat IgG complexed with horseradish peroxidase (HRP), orgoat anti-mouse IgG complexed with HRP served as secondaryantibodies (Zymed, San Francisco, CA, diluted 1:5,000) and sig-nals were generated with Super Signal, West Pico Chemilumines-cent Substrate (Pierce, Rockford, IL) according to the manufac-turer’s protocol. Signals in the linear range of development werequantitated using Gel-Pro Analyser software ver.3.0 (MediaCyber-netics, Silver Spring, MD).

Histopathology and immunohistochemistry

Mammary or tumor biopsies were fixed with Bouin’s solution,dehydrated in a graded series (50–100%) of ethanol, cleared in xy-lene and embedded in paraffin. Sections of 4 lm were processedfor hematoxylin & eosin (H&E) staining. For pStat5 analysis,Bouin-fixed paraffin-embedded sections were dewaxed, dehy-drated through graded ethanols, treated with 0.3% hydrogen per-oxide for 30 min, boiled in 0.1 M sodium citrate buffer for 20 min,blocked and reacted for 1 hr with anti-pStat5 antibodies diluted1:100. Sections were then washed and reacted with EnVision rea-gent (Dako, Glostrup, Denmark) containing the HRP-labeled sec-ondary antibody. Signals were generated with diaminobenzidine(DAB) substrate (Vector Laboratories, Burlingame, CA).

Immunofluorescence staining

For immunofluorescence double-staining, Bouin-fixed paraffin-embedded sections were dewaxed, dehydrated through gradedethanols and boiled in 0.1 M sodium citrate buffer for 20 min. Thesections were then washed again with TBST, treated with 0.3 M

glycine for 10 min, washed, blocked, and reacted overnight at 4�Cwith a mixture of mouse anti-pStat5 monoclonal antibody and rab-bit anti-cyclin D1 polyclonal antibody (Cell Signaling, Danvers,MA), both diluted 1:100. Signals were generated after incubationfor 1 hr with a mixture of the appropriate secondary antibodies, ei-ther goat anti-rabbit IgG conjugated to FITC (Sigma) or goat anti-mouse IgG labeled with Alexa Fluor 555 (Molecular Probes,Eugene, OR), both diluted 1:100. Stained sections were mountedin Fluoromount (DBS, Pleasanton, CA) and visualized using aLeica fluorescent microscope (Vertrieb, Bensheim, Germany).

Results

Stat5-induced mammary tumorigenesis is parity dependent

The effect of parity on Stat5 induced mammary tumorigenesiswas studied in transgenic mice carrying 1 of 2 Stat5 variants: awild type, ovine Stat5, very similar to mouse Stat5a, and a consti-tutively activated Stat5 variant. The expression of both constructsin transgenic mice was directed to the mammary glands by regula-tory sequences of the BLG milk-protein gene.30 Introduction ofthe wild-type BLG/STAT5 and its activated variant BLG/STAT5ca into the mouse genome results in pregnancy-dependentexpression in the mouse mammary glands. The expression of theStat5 transgenes mimics the pattern of the BLG gene expressionand occurs mainly during pregnancy and lactation. We demon-strated earlier that the transgene-encoded Stat5 is expressed at asimilar level as the endogenous Stat5 during lactation,30 anddescribed the effects of Stat5 transgene expression on mammarygland development, function and cell survival.30,31 Mammarytumors developed in multiparous BLG/STAT5- and BLG/STAT5ca-transgenic females from all expressing strains. Notumors were observed in control mice or in mice in which the trans-gene was present, but silent.32 In the current study we examined theinteractions between deregulated Stat5 expression and parity onmammary tumorigenesis. Nulliparous virgin females from thetransgenic strains BLG/STAT5-6 and BLG/STAT5ca-8 wereobserved for 18 months and compared to transgenic females of thesame strains which were allowed to mate and nurse their pups.

Figure 1 shows that there is a significant difference between themultiparous transgenic females and their AMV counterparts withrespect to their mammary tumor incidences. Fifteen percent of themultiparous females carrying the wild-type BLG/STAT5 and 20%of the animals expressing the constitutively activated BLG/STAT5ca variant developed tumors. These mice were much moresusceptible to tumorigenesis than their AMV counterparts (Fig.1a). In fact, only 1 mammary epithelial tumor developed in 44 or42 virgin transgenic mice of lines BLG/STAT5-6 or BLG/STAT5ca-8, respectively. This is only about 10% of the tumors thatdeveloped in the respective multiparous colonies. In addition tomammary epithelial cell tumors, a limited number of lymphomaswere also detected in the transgenic line BLG/STAT5-6, thus con-firming earlier results.32 In contrast to the parity-dependent, Stat5-induced epithelial cell tumors, equal numbers of lymphomas weredetected in multiparous and AMV transgenic females of lineSTAT5-6 (2 in each cohort). This suggests that the combined effectof parity and Stat5 deregulation is limited to the epithelial cells.

The periods required for tumor development in the individualtransgenic females were also monitored (Fig. 1b). The average la-tency period for tumor development in line BLG/STAT5-6 (13months) and in line BLG/STAT5ca-8 (15 months) did not differsignificantly (p < 0.05). This analysis excludes 2 hyperplasias,detected in the BLG/STAT5ca multiparous cohort at 3 and 6months of age. The latency periods for the rare tumors whichdeveloped in virgin females were not different from those in theirmultiparous counterparts. Except for the hyperplasias, whichdeveloped during the fertile period and were characterized by pap-illary projections into the alveolar lumen, no significant differen-ces were observed in tumor latencies among the 4 main pheno-types: carcinoma, adenocarcinoma, papillary adenocarcinoma andlymphoma, though the number of tumors of each phenotype was

1894 EILON ET AL.

limited (Fig. 2). Histological characterization of these phenotypeshas been previously demonstrated.32 In this previous study wereported tumor incidence rates of 9.2 and 18.6% for the multipa-rous transgenic mice of lines BLG/STAT5-6 and BLG/STAT5ca-8, respectively, when compared to 15 and 20%, in the currentstudy. The differences may result from the limited size of the pop-ulations analyzed, but more probably from the limitation of the ex-perimental period in the current study to 18 months. This age limi-tation was not exerted earlier.

Endogenous and transgenic Stat5 expression inpostestropausal mice

The levels of Stat5 expression and activity, represented by thephosphorylation status of tyrosine 694, are elevated during latepregnancy and lactation and drastically reduced during involution.To gain insights into the role of parity in Stat5 dependent transfor-mation, we studied the expression and activation levels of Stat5 inthe mammary glands of the aged BLG/STAT5 and BLG/STAT5camultiparous mice and their AMV transgenic counterparts (Fig. 3).The Stat5 transgene was tagged with an epitope to allow its immu-nological detection and the distinction from the endogenous Stat5.Immunoblot analysis confirmed Stat5 expression in the mammaryglands of lactating females from line BLG/STAT5-6 (Fig. 3a).Extracts from BLG/STAT5ca-8 in which the STAT5ca was nottagged served as a negative control. About 3.5 6 0.4- and 4.1 60.7-fold lower levels of Stat5 expression were detected in multipa-rous post-menopause transgenic mice and their AMV counterparts,respectively. No significant differences (p < 0.05, n 5 5) in signalintensities of the HA epitope were detected when individual micefrom the 2 cohorts were compared (not shown). Similarly, no sig-nificant differences between the multiparous and AMV mice werenoted when the combined expressions of the native and transgenicStat5s were measured with antibodies recognizing both forms.

Activated pStat5 was detected with antibodies specific for thephosphotyrosine residue at position 694 (Fig. 3a). This residue ispresent in both transgenic Stat5 variants. Phosphorylation of Tyr694 is essential for Stat5 dimerization and translocation into thenucleus. pStat5 levels were lower in aged mice when compared tothose of Stat5. Indeed, a more pronounced decline in the levels ofactivated Stat5 when compared to the levels of total Stat5 hasbeen observed in studies following Stat5 levels and activity duringmammary development and involution.35 No significant differen-

ces (p < 0.05, n 5 5) were observed in pStat5 levels of individualmultiparous mice when compared to AMV mice (not shown).

The expression of the constitutively activated STAT5ca wasdetermined in the mammary glands of the transgenic mice by real-time PCR. The primers used distinguish it from the endogenousStat5. Signals of the amplified STAT5ca cDNAs were also visual-ized by hybridization to 32P-labeled cDNA probes encompassingthe sequences distinguishing it from wild type Stat5. Figure 3bpresents the intensity of STAT5ca signals from pooled samples ofthe 2 cohorts. As noted for the native STAT5 transgene, no signifi-cant differences (p < 0.05, n 5 5) were observed for STAT5caexpression in individual multiparous transgenic mice and theirAMV counterparts. Putative differences in cell composition mayexist in the mammary glands of the 2 cohorts. Our data suggestthat this difference do not significantly affect the levels of Stat5expression in the glands of the multiparous and AMV females.

We confirmed the results by an in situ analysis of Stat5 expressionand activity in mammary tissues of 18-month-old multiparous andAMV STAT5ca transgenic mice (Fig. 4). H&E stained alveolarstructures from mammary gland of lactating mice are shown in Fig-ure 4a. The multiparous involuted glands (Fig. 4b) resemble struc-tures of terminal ductal lobular units found in the human breast.41

FIGURE 2 – Comparable latency periods were measured for the de-velopment of tumors with distinct phenotypes in transgenic mice car-rying the BLG/STAT5 and BLG/STATca transgenes. Mean 6 SEMare presented. Hyperplasia was detected after a significantly (p <0.05) shorter period. The number of tumors with distinct phenotypesin relation to the overall number of tumors in the multiparous or theirAMV transgenic females is indicated below the respective bars. Mul,multiparous; Vir, virgin.

FIGURE 3 – Endogenous and transgenic Stat5 variants are expressedin the mammary glands of menopausal multiparous and AMV trans-genic mice with no differences between the 2 cohorts. (a) Representa-tive immunoblot analysis. Equal amounts of extracted protein werepooled from 5 individual mice, separated by SDS-PAGE, blotted ontofilters and reacted with antibodies to the HA epitope, Stat5, or pStat5.Equal amounts of protein/lane were confirmed by staining the blotwith Ponceau S. (b) Real-time PCR quantitation of STAT5ca and en-dogenous Stat5 expression was visualized by analysis of equal levelsof cDNA, combined from 5 individual mice, on agarose gels andhybridization with respective 32P-labeled probes.

1895PARITY-DEPENDENT STAT5-INDUCED TUMORIGENESIS

These structures are surrounded by a sheet of myoepithelial cellsresting on the basement membrane. The morphology of the meno-pausal virgin gland appeared somewhat different and was comprisedmainly of clustered ducts (Fig. 4c). In both the virgin and multipa-rous menopausal mice the glands contained unilocular adipocytes.

Immunohistochemical analyses of activated Stat5 expressionwere performed with specific antibodies recognizing the tyrosinephosphorylated Stat5, pStat5 (Figs. 4d–4f). Sections from a glandof lactating wild type mice served as a positive control andrevealed the nuclear localization of pStat5 in virtually all of the

epithelial cells lining the alveoli (Fig. 4d). This complements anearlier study which reports that 80% of the normal luminal breastepithelial cells contain Stat5a in the cytoplasm and nucleus.42 Nu-clear pStat5 was also detected in the luminal epithelial cells, butnot in the myoepithelial cells, of the multiparous and the AMVmice (Figs. 4f and 4g). However, in the mammary sections ofthese mice, pSTAT5 expression varied drastically between neigh-boring cells. Variegated expression was demonstrated also for theepitope-tagged transgenic STAT5 (Fig. 5), indicating that thetransgene encoded STAT5 is expressed in the aged mice. Taken

FIGURE 4 – Variable expression of pStat5 in the mammary glands of estroapausal multiparous and AMV transgenic mice. Paraffin sections frommammary glands of 6-day-lactating wild-type (a) or menopausal multiparous (b) and virgin (c) transgenic mice of line BLG/STAT5ca-8 werestained with H&E to demonstrate the morphology of the gland. Alveolar structures, some of which are filled with engorged secreted material, aredemonstrated in a. Structures reminiscent of the lobuloalveolar structures are demonstrated in b and clustered epithelial ducts are shown in (c). (d–g)Immunohistochemical analysis of pStat5. (d) pStat5 is detected in most epithelial cells composing the alveoli of the normal lactating gland. (e) Sec-tion processed as in (a), but not reacted with specific antibody. (f) Irregular brown staining for pStat5 (endogenous and transgenic) in mammary sec-tion of a multiparous gland. (g) Irregular pStat5 staining in mammary section of a virgin gland. Scale bar5 50 lm. Inset magnification:33. Resultsrepresent more than 3 independent analyses. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

1896 EILON ET AL.

together, the above studies confirmed the residual expression andactivity of Stat5 (endogenous and transgenic) in the mammaryglands of aged, female mice.

Histones associated with the Stat5 binding sites of selected genepromoters differ in their modifications in multiparous females andtheir AMV counterparts

During the periods of pregnancy and lactation, elevated levels oftransgenic and endogenous Stat5 expression were detected in themultiparous females, a feature not found in the virgin animals.30

These differences may persist in other forms and affect tumor devel-opment in the multiparous females at later stages of life, in which nosignificant deviation between the multiparous and virgin mice interms of transgenic and endogenous Stat5 expression or activitieswere detected. To gain insights into the mechanisms which mightcontribute to the persistence of the parity effects, we analyzed dif-ferences in histone modifications between the 2 cohorts.

In addition to packaging DNA, chromatin is known to play aregulatory role: it can restrict or enhance DNA interactions withthe protein factors and promote or inhibit transcription. Openchromatin is accessible to the transcription machinery, while aclosed conformation prevents the access of regulatory pro-teins.43,44 Acetylation of lysine residues in the N-terminal tail ofhistone H3 and H4 reduces their positive charge and weakens theiraffinity for DNA. This, in turn, facilitates DNA accessibility andtranscription-factor binding. It has been suggested that chromatinpatterns predestine future cell differentiation and deter cell lineageinfidelity. These mechanisms might also be involved in neoplastictransformation.45

The interactions of particular DNA sequences with acetylatedhistone H4 were compared between multiparous and AMV mice.We chose the cis-regulatory Stat5 binding sites in the promotersof genes involved in the regulation of cell fate and transformation.Stat5 binding sites have been located in the promoters of the bcl-x,46 cyclin D1,47 cyclin D248 and c-myc49,50 genes. These siteswere identified in the mouse gene promoters (Fig. 6) and ChIPanalysis was carried out to measure the interactions with acety-lated histone H4 in the multiparous and in their AMV counter-parts. Adjacent promoter regions in the respective genes were ana-lyzed as controls.

Three regions, each covering 70–90 bp of the bcl-x and cyclinD1 gene promoters, were analyzed. Two regions of c-myc werealso analyzed, as well as the region containing the Stat5-bindingsite in the cyclin D2 gene promoter. In the latter, repetitive DNAsequences limited the choice of sequences available for analyses.Chromatin was sheared and the fragments were immunoprecipi-tated with an antibody specific for the acetylated histone H4. Thecoprecipitated DNA was recovered and amplified with gene pro-moter specific primers.

Significantly (p < 0.05) higher levels of DNA comprising theStat5-binding sites from the promoters of the bcl-x and cyclin D1genes of the virgin BLG/STAT5ca transgenic mice were detectedwhen compared with the same regions from multiparous mice.This indicates that histone H4 bound to the promoter regions ana-lyzed is less acetylated in the multiparous mice than in the virginmice. In contrast, no significant differences in promoter acetylatedhistone interactions were observed for other selected regions inthe promoters of these genes or for the regions encompassing theStat5 binding sites and other sites in the c-myc and cyclin D2genes.

Does the decreased binding of acetylated histone H4 to theStat5 binding sites in the cyclin D1 and bcl-x promoters in themammary glands of multiparous STAT5ca transgenic femalesaffect the expression of these genes and how do the expressionlevels compare to the AMV counterparts? To answer these ques-tions, real-time PCR analysis was carried out and the cyclin D1and bcl-xL expression levels were compared in the 18-month-oldtransgenic mice. The apoptosis inhibitor bcl-xL is known as aStat5 target gene.49 Primers specific for the intragenic sequencesof the junction between exon 2 and 3 of the bcl-x gene wereselected to amplify bcl-xL and distinguish the transcript from bcl-xs or bcl-xb.51 No significant differences (p < 0.05, n 5 5) incyclin D1 or bcl-xL expression were measured by real-time PCRwhen individual samples from mammary glands of multiparousand AMV mice were analyzed (not shown). This may suggest thatonly a subset of the cells is affected by the parity effect.

Immunohistochemical localization of activated Stat5 andcyclin D1

Immunofluorescene experiments were carried out to support thenotion that only a subset of cells is involved in mediating the par-ity effect and to establish a link between Stat5 deregulation andgene expression at the individual cell level. Antibody staining wasused to localize the expression of pStat5 (green) and cyclin D1(red) in the same sections and mammary glands from menopausalSTAT5ca multiparous and AMV transgenic females were com-

FIGURE 5 – Variegated expression of transgenic STAT5 in mam-mary gland epithelial cells of postestropausal multiparous and AMVBLG/STAT5 transgenic mice. Paraffin sections from estropausal mul-tiparous (a) and virgin (b) glands were reacted with antibodies to theHA epitope, followed by FITC-labeled anti-rabbit IgG. Nuclei werestained with DAPI (c, d). Some nonspecific staining was detected inthe lumen of the ducts in the multiparous sections (a, e). The morphol-ogy of the multiparous (g) and virgin (h) glands is also demonstrated.Scale bar 5 10 lm. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

1897PARITY-DEPENDENT STAT5-INDUCED TUMORIGENESIS

pared (Fig. 7). Localization of pStat5 in the individual epithelialcells lining the lobuloalveolar or ductal structures was observed,thus confirming the data presented in Figure 4, which showspSTAT5 nuclear translocation. The immunofluorescence imaging

also indicates a high variability pStat5 expression among individ-ual epithelial cells. Only a few cells express high levels of thephosphorylated protein while most of the epithelial cells compos-ing the ductal or alveolar structures do not (Figs. 7a and 7e andinsets to Figs. 7d and 7h).

Cyclin D1 expression was probed in the same sections. In mostcells, its expression was below the detection limit. However, onlyin the sections from multiparous females alveolar epithelial cellswith high cyclin D1 expression were detected (Figs. 7b and 7f andinsets to Figs. 7d and 7h). Merging the images showed that thecells strongly expressing pStat5 coincide with cyclin D1 express-ing cells (Figs. 7c and 7g and insets to Figs. 7d and 7h). By screen-ing 5 microscopic fields (403) from slides prepared from 3 mice,we found that the number of cells co-expressing high levels ofpStat5 and cyclin D1 was 0.5 6 0.3/microscopic field. Sectionsfrom the AMV BLG/STAT5ca transgenic females also containedpStat5 expressing cells (Figs. 7i and 7m and insets to Figs. 7l and7p), but cyclin D1 expression was not found (Figs. 7j and 7n).Only nonspecific red staining of intraluminal secreted materialwas noted.

No epithelial cells overexpressing cyclin D1 could be observedalso in sections from aged, wild type female multiparous gland(Fig. 8). The lack of cyclin D1 overexpression in these mice char-acterizes both ducts and lobuloalveolar structures. These resultslink the presence of pStat5 and the expression of cyclin D1 in thetransgenic multiparous glands. They indicate that a small subset ofcells persists in the postestropausal female gland, in which theeffect of Stat5 on cyclin D1 induction is being maintained. Thissubset of cells could represent a reservoir of cells, which couldprogress to tumors.

Discussion

Stat5 is usually ubiquitously expressed. Its activation is depend-ent on extracellular signals, cytokine or growth factor receptor rec-ognition and the activation of receptor associated tyrosine kinases.The persistent activation of Stat5 has been shown to be associatedwith human cancer and its overexpression and forced activation inmammary epithelial cells of transgenic mice causes mammarytumors.32 These results are now being complemented by our ob-servation that tumor development in transgenic mice occurs late,during the postestropausal period, and does not coincide with thepeak of Stat5 expression or activation. In addition, we found thatparity is a prerequisite for Stat5-induced tumorigenesis. ActivatedStat5 in the mammary gland of transgenic mice caused tumorsalmost exclusively in multiparous females, but rarely in theirAMV counterparts. Carcinomas, adenocarcinomas, papillaryadenocarcinomas developed in the mammary glands of BLG/STAT5 and BLG/STAT5ca transgenic mice with an average la-tency period of 14 months. The similar time periods needed for tu-mor development indicate that the phenotypic tumor manifestationis not a direct consequence of Stat5 activation. Transcription regu-lated by Stat5 activation has to be supplemented by additional cel-lular alterations, possibly by genetic mutations or epigeneticevents.32 The data also indicate that parity-dependant tumor for-mation is not a consequence of forced activation of Stat5 or the ki-nase domain which accompanies the STAT5ca variant, but is dueto the Stat5 overexpression. Previously, we have shown that trans-genic Stat5 doubled overall Stat5 expression during lactation, buthas lower contribution during other stages of the reproductivecycle.30 This variation may be sufficient to induce long-termtumorigenic effect, especially due to the lack of direct correlationbetween BLG/STAT5ca expression level in lactating females andits tumorigenic effect.32 In tissue culture experiments, the effectsof STAT5ca are mainly restricted to the regulation of genes withStat5 binding elements.39 In transgenic mice, overexpression andforced activation of Stat5 in the mammary gland have effects oncell proliferation and apoptosis, with the effects of STAT5ca beingslightly more pronounced.29 However, we cannot formally rule

FIGURE 6 – Distinct differences between BLG/STAT5ca multipa-rous mice and their AMV counterparts in promoter-acetylated histoneinteractions at the Stat5 binding site of the cyclin D1 and bcl-x genepromoters. Binding of different regions of gene promoters containinga Stat5-binding site to acetylated histone H4 was examined by ChIPanalysis as described in Materials and Methods. The amount of DNAreleased from the complexes was determined by real-time PCR analy-sis and compared between multiparous and AMV mice of line BLG/STAT5ca-8. Five animals from each cohort were analyzed. Student’st-test was performed to compare DDCT values between the 2 groupswhich are presented as bar graphs. Bars with significant differences ofp < 0.05 between DDCT values are marked by asterisks.

1898 EILON ET AL.

out that the kinase domain in Stat5ca might have additionaleffects.

The observation that Stat5 caused tumors almost exclusively inmultiparous females and only rarely in their AMV counterpartsimplies a number of interesting questions: Which mechanismsconfer the parity-dependent tumorigenesis in Stat5 transgenicmice? Why do the beneficial effects of parity on protection fromtumor formation change to a tumor promoting effect? Why do thetumors arise at a time when Stat5 signaling and estrogen signalingare way beyond their peak?

Basal Stat5 expression and activity have been detected in virginmice52 and last beyond the termination of the fertile period. How-ever, Stat5 activity is highest during pregnancy and lactation, atwhich time it exerts its main effects on epithelial cell proliferation,differentiation and survival. Our results indicate that while Stat5effects and parity are linked, both are not sufficient to acutelycause the transformation of mammary epithelial cells, but mediatesusceptibility to carcinogenesis to a later time point. Developmen-tal biology has been confronted in other contexts with such phe-nomena. It has been found that early developmental cues caninfluence cell fates not only acutely, but also at much later stages.

These cues are thought to leave marks at distinct gene loci that aremaintained throughout successive rounds of mitosis by establish-ing a cell type specific chromatin pattern.44 We took up this con-cept and compared the association of the acetylated histone H4with particular promoter sequences in multiparous mice and theirAMV counterparts. We chose specific cis-regulatory Stat5-bindingsites in the promoter regions of genes involved in cell proliferationsurvival, and possibly tumorigenesis. Cyclin D1, cyclin D2, c-mycand bcl-x are candidate genes able to mediate the effects of Stat5and parity on mammary tumorigenesis.

Our results show that there is a differential association of acety-lated histone H4 with the Stat5 binding sites of the bcl-x andcyclin D1 gene promoters when chromatin samples from multipa-rous glands of BLG/STAT5ca mice were compared to their AMVcounterparts. Stat5 is able to recruit histone acetyltransferases byinteracting with factors such as CBP/p300 via its transactivationdomain.53 However, transcriptional induction by Stat5 has notonly been associated with histone acetyltransferase activity, butalso with deacetylase activity.54,55 In addition, histone acetylationhas been linked to a number of additional regulatory processes56

so that the simple correlation of transcriptional induction and

FIGURE 7 – Co-localization of highly expressed pStat5 and cyclin D1 in a small subset of epithelial cells stained in the multiparous BLG/STAT5ca-transgenic glands, but not in their AMV counterparts. Paraffin sections from multiparous (a–c, e–g) and virgin (i–k, m–o) glands ofpostestropausal females were reacted with antibodies to pStat5 and Cyclin D1, followed by FITC-labeled anti-rabbit IgG (green) and Alexa 555-labeled goat anti-mouse IgG (red) to discriminate between the proteins. The green channel shows only those cells that express pStat5 while thered channel shows anticyclin D1 staining. The anti-pStat5a detects both endogenous and transgenic pStat5. The merged images show nearly per-fect overlap, indicating that pStat5 and cyclin D1 expression are co-localized only in the multiparous gland. Some nonspecific staining wasdetected in the lumen of the ducts in the virgin sections (j, k). The morphology of the multiparous (d, h) and virgin (l, p) glands is also demon-strated and the black arrows indicate that staining is restricted to the lobuloalveolar and ductal epithelial cells. Scale bar 5 25 lm. Insets (33)magnify the indicated structures in the ‘‘Merge’’ column. White arrows point towards the magnified structures.

1899PARITY-DEPENDENT STAT5-INDUCED TUMORIGENESIS

acetylated histone H4 association is probably not justified. Theaccessibility of transcription factor binding sites in particular genepromoters has been shown to be distinguishable among differentcell types. Chromatin immunoprecipitation has been used to iden-tify different binding sites for serum response factor in embryoniccells, muscle cells and neurons and correlation with differentialgene expression has been shown.57 Thus, cyclin D1 and bcl-xLmight mediate the role of Stat5 in cell-fate regulation. The ChIPexperiments were performed with intact tissues rather than withdispersed cells to resemble the in vivo conditions and avoid altera-tion in gene expression profile due to cell dissociation.58 Differ-ence in cell composition between the multiparous and AMV mam-mary gland could affect this quantitative analysis. By monitoringStat5 interactions with adjacent sites to Stat5 binding domains anddifferent gene promoters, the specific role of parity on chromatinstructure at the Stat5 binding domains in the cyclin D1 and bcl-xcould be validated.

The link between the differential association of acetylated his-tone H4 with particular promoter regions and the effects of paritywas established with the cyclin D1 gene. In its gene promoter, theStat5 binding site is located relatively close to the transcriptionstart site. Progression of eukaryotic cells through the cell cycle iscontrolled by 2 families of G1 cyclins: the D-type (D1, D2 and

D3) cyclins and E-type (E1 and E2) cyclins and their associatedcyclin dependent kinases (cdks).59,60 Cyclin D1 involvement inmammary development and tumorigenesis has been demonstrated.Cyclin D1 null mice are unable to undergo normal mammary de-velopment,21,61 while cyclin D1 overexpression in the mammarygland results in excessive cell proliferation and mammary adeno-carcinomas.62

In our experiments, a small subset of cells was detected onlyin the multiparous transgenic mice in which high levels of acti-vated Stat5 in the nucleus and elevated transcription of cyclin D1were detectable. This suggests that parity leaves a mark on theepithelial cells and exposes or sensitizes them to Stat5 effects.The parity effect could be possibly mediated through persistentchanges in chromatin structure, reminiscent of a mammary epi-thelial cell population, which has been identified in the parousmammary gland. This population does not undergo apoptosis andgives rise to alveolar cells during subsequent pregnancies.39,63

The partially committed cells are absent from the nulliparous vir-gin gland and share properties of multipotent mammary stemcells. The high levels of cyclin D1 expression coincide and possi-bly depend also on high Stat5 expression. Our results suggestthat in a small number of epithelial cells, the expression of theconstitutively active Stat5 escaped the developmental-dependent

FIGURE 8 – Cyclin D1 is overexpressed in a subset of mammary epithelial cells of postestropausal multiparous BLG/STAT5ca transgenicmice, but not in their wild type counterparts. Paraffin sections from aged wild type multiparous mammary gland (a) or from BLG/STAT5catransgenic counterpart (b) were reacted with antibodies to cyclin D1, followed by Alexa 555-labeled goat anti-mouse IgG. Nuclei were stainedwith DAPI (c, d). The morphology of the multiparous gland is also presented (e, f). Scale bar 5 15 lm. Insets 33. Arrows point towards themagnified structures. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

1900 EILON ET AL.

repression of the BLG regulatory sequences. In combinationwith the parity effect on chromatin, this could favor cyclin D1expression. It has already been shown that during cell trans-formation, regulatory sequences of the whey acidic proteinmilk protein gene become independent of lactogenic hormonecontrol.64

Stat5 binding sites in additional promoter regions were foundindistinguishable in their ability to associate with acetylated his-tone H4. No difference in association were found between multip-arous and AMV mice when the Stat5 binding site in the cyclin D2promoter was probed, suggesting that cyclin D2 is not subjected tothe chromatin-mediated tumorigenic effect of Stat5 deregulation.Indeed, the cyclin D2 promoter is methylated in mammary epithe-lial and breast cancer cells65 and the repressive effect of cyclin D2overexpression on mammary alveolargenesis and lactation hasbeen attributed to altered p27Kip1 protein level and inhibition ofcyclin D1 phosphorylation.66 In addition, breast carcinoma usuallyoriginates from luminal epithelial cells. These cells express cyclinD1 and cyclin D3, but not cyclin D2, which is exclusivelyexpressed in the basal epithelial cells.67–69 We have not detectedchanges in bcl-xL expression in the multiparous compared to thevirgin mice. Although we have no direct evidence, we do notexclude the possibility that an increased epithelial cell populationwith a prolonged half life could be also involved in tumor devel-opment and that involution-induced persistent changes in the epi-thelial cell microenvironment may participate in Stat5 tumorigeniceffect.

Our study complements previous reports that have demon-strated biological and genetic differences between mammaryepithelial cells of the multiparous mice and their AMV counter-parts.3,41 In transgenic mice overexpressing Stat5 or a consti-tutively activated Stat5 variant, parity is required for the develop-ment of tumors in menopausal females. We associated this obser-vation with altered histone acetylation and possible changes in thechromatin structure encompassing the Stat5-binding site in thepromoters of the cyclin D1 and bcl-x genes. Only epithelial cellsin multiparous glands co-express high levels of pStat5 and cyclinD1. It is possible that the alterations in chromatin structure encom-passing the Stat5 binding site in the cyclin D1 promoter of themenopause parous gland have been initiated by parity and exposeor sensitize the cells to the deregulated Stat5 effect. The fractionof cells in which activated Stat5 induces the expression of cyclinD1 may serve as progenitors for the development of mammarycancer in the multiparous gland.

Acknowledgements

We thank Dr. Robert Cardiff (University of California, Davis)for pathological analyses of the tumors and Dr. M. Reichensteinfor helpful suggestions and technical support. This study was sup-ported by a grant from the Israel Science Foundation. Israel Acad-emy of Science; contract number: 706/04 to Dr. Itamar Barash anda grant number GR536/11-1 from the Deutsche Forschungsge-meinschaft (DFG) to Dr. Bernd Groner.

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