src3 phosphorylation at serine 543 is a positive independent prognostic factor in … · biology of...

Biology of Human Tumors

SRC3 Phosphorylation at Serine 543 Is a PositiveIndependent Prognostic Factor in ER-PositiveBreast CancerWilbertZwart1, KoenD. Flach1, BharathRudraraju2,TarekM.A.Abdel-Fatah3,OndrejGojis4,Sander Canisius1, David Moore5, Ekaterina Nevedomskaya1, Mark Opdam1,Marjolein Droog1, Ingrid Hofland1, Steve Chan4, Jacqui Shaw5, Ian O. Ellis6,R. Charles Coombes3, Jason S. Carroll7, Simak Ali3, and Carlo Palmieri2,8,9

Abstract

Purpose: The steroid receptor coactivator SRC3 is essentialfor the transcriptional activity of estrogen receptor a (ERa).SRC3 is sufficient to cause mammary tumorigenesis, and hasalso been implicated in endocrine resistance. SRC3 is post-translationally modified by phosphorylation, but these eventshave not been investigated with regard to functionality ordisease association. Here, we investigate the spatial selectivityof SRC3-pS543/DNA binding over the human genome and itsexpression in primary human breast cancer in relation withoutcome.

Experimental Design: Chromatin immunoprecipitation,coupled with sequencing, was used to determine the chromatinbinding patterns of SRC3-pS543 in the breast cancer cell lineMCF7 and two untreated primary breast cancers. IHC was usedto assess the expression of SRC3 and SRC3-pS543 in 1,650primary breast cancers. The relationship between the expression

of SRC3 and SRC3-pS543, disease-free survival (DFS), andbreast cancer specific survival (BCSS) was assessed.

Results: Although total SRC3 is selectively found at enhancerregions, SRC3-pS543 is recruited to promoters of ERa responsivegenes, both in the MCF7 cell line and primary breast tumorspecimens. SRC3-pS543 was associated with both improved DFS(P ¼ 0.003) and BCSS (P ¼ 0.001) in tamoxifen untreated high-risk patients, such a correlation was not seen in tamoxifen-treatedcases, the interaction was statistically significant (P ¼ 0.001).Multivariate analysis showed SRC3-pS543 to be an independentprognostic factor.

Conclusions: Phosphorylation of SRC3 at S543 affects its geno-mic interactions on a genome-wide level, where SRC3-pS543 isselectively recruited to promoters of ERa-responsive genes. SRC3-pS543 is a prognostic marker, and a predictive marker of responseto endocrine therapy. Clin Cancer Res; 22(2); 479–91. �2015 AACR.

IntroductionEstrogen receptor a (ERa) is a key transcription factor in

normal breast development and plays a central role in the path-ogenesis of approximately 70%of all breast cancers. Consequent-ly, ERa is recognized as the key therapeutic target in this group ofpatients (1). The p160 steroid receptor coactivator (SRC) familymember, SRC3 (also known as amplified in breast cancer 1; AIB1)is an essential coactivator for the transcriptional activity of ERa,which recruits SRC3 to chromatin (2). SRC3 is required forestrogen-driven proliferation of MCF7 breast cancer cells (3) andis critical for normalmammary development (3). Overexpressionof SRC3 can induce mammary tumorigenesis in mice (4), whileits absence protects against breast cancer development (5, 6).SRC3 was originally identified as a gene mapping to a region ofchromosome 20 (20q12–13), which is frequently amplified inbreast cancer and was shown to be amplified in 10% of breastcancers (7). SRC3 mRNA levels are significantly higher in breastcancer as comparedwith normalmammary tissue (8, 9) and SRC3protein levels are elevated in 16% to 83% (depending on thestudy) of human breast tumors (10).

SRC3 expression has been associated with decreased risk ofrelapse in patientswith breast cancerwhodid not receive adjuvantendocrine therapy (11). Conversely, in tamoxifen-treated patientsSRC3 expressionwas associatedwith poor outcome and increased

1Department of Molecular Pathology, The Netherlands Cancer Insti-tute, Amsterdam, the Netherlands. 2Department of Molecular andClinical Cancer Medicine, Institute of Translational Medicine, The Uni-versity of Liverpool, Liverpool, United Kingdom. 3Clinical OncologyDepartment, NottinghamUniversity City Hospital NHS Trust, Notting-ham, United Kingdom. 4Cancer Research UK Laboratories, ImperialCentre for Translational and Experimental Medicine, Division of Can-cer, Imperial College London, London, United Kingdom. 5Departmentof Cancer Studies and Molecular Medicine, University of Leicester,Leicester, United Kingdom. 6Division of Pathology, School of Molec-ular Medical Sciences, University of Nottingham, Nottingham, UnitedKingdom. 7Cancer Research UK Cambridge Institute, Cambridge,United Kingdom. 8Liverpool and Merseyside Academic Breast Unit,The Linda McCartney Centre, Royal Liverpool University Hospital,Liverpool, United Kingdom. 9Academic Department of MedicalOncology, Clatterbridge Cancer Centre NHS Foundation Trust,Wirral,United Kingdom.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

W. Zwart, K.D. Flach, and B. Rudraraju contributed equally to this article.

Corresponding Authors: Carlo Palmieri, University of Liverpool, The DuncanBuilding, Daulby Street, Liverpool L69 3GA, UK. Phone: 44-151-706-3616; Fax:44-151-706-5826; E-mail: [email protected]; andWilbert Zwart, E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-3277

�2015 American Association for Cancer Research.

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risk of relapse (11). Others have found no such association withtamoxifen treatment, although an increased risk was seen withhigh SRC3when expressed alongsideHER1,HER2, orHER3 (12).In a group of women of whom 82%were postmenopausal, SRC3expressionhas been associatedwith early recurrence on tamoxifentreatment (13), although high SRC3 in premenopausal womenwas an independent predictive factor of improved response totamoxifen (14). ERa-associated SRC3 is typically found atenhancer regions, where expression of genes proximal to sharedERa/SRC3 chromatin binding events correlates with poor out-come of ERa-positive patients with breast cancer treated withtamoxifen (15). This featurewas only found for geneswhere SRC3was selectively enriched as compared with the other two p160family members, SRC1 and SRC2.

Phosphorylation of SRC3 is required for it to function as apotent transcriptional activator, and SRC3 possesses multiplephosphorylation sites that may be functionally involved in thisprocess (16, 17). Estradiol treatment increases SRC3 phosphor-ylation, but the kinase responsible for this remains elusive.However, a number of kinases are known that induce phosphor-ylation at these residues of SRC3 (16, 17). In addition, dephos-phorylation of serine-101 and -102 regulates SRC3 protein sta-bility by preventing proteasome-dependent turnover (18). Ofnote, the C-terminal domain of SRC3 has weak histone acetyl-transferase activity raising the possibility that it may have a role inchromatin remodeling (10).

Previous studies have shown a requirement of SRC3 phosphor-ylation for SRC3 activity, including phosphorylation of SRC3 atserine residue 543 (SRC3-pS543; ref. 16). Furthermore, the regionaround S543 is evolutionarily conserved in mammals, birds, andreptiles, although no sequence homology around S543 is foundin SRC1or SRC2 (19). In addition, thus far no studies explored thegenomic consequences and clinical significance of SRC3 phos-phorylation. Given all the above, we decided to investigate thechromatin associations of SRC3 in vitro and in vivo when phos-phorylated at serine 543 (SRC3-pS543) together with its clinical

significance in ERa-positive breast cancer. To separate the prog-nostic from the predictive potential of SRC3 phosphorylation,tamoxifen-treated patients were compared with patients who didnot receive any adjuvant endocrine therapy.

Material and MethodsCell lines, plasmids, and antibodies

MCF7 and CHO cells were from the ATCC, which utilizes shorttandem repeat profiling with no authentication being performedby the authors. All experiments were performed within 10 pas-sages from the original stock. Cell lines were grown in DMEMwith 10% FCS and standard antibiotics. Wild-type SRC3 andmutant S543A plasmids were kindly provided by B.W. O'Malley(Baylor College of Medicine, Houston, Texas). The antibodiesused were SRC3 (IHC, BD Transduction Laboratories; 611105),SRC3 (ChIP-seq experiments, SC-9119; Santa Cruz), and ERa(SC-543; Santa Cruz). A rabbit polyclonal antiserum for SRC3-pS543 was generated by immunizing animals with the peptide537-LLSTLSSPGPKLDN-550, where the residue in bold is phos-pho-serine (Moravian Biotechnologies).

Chromatin immunoprecipitations (ChIP)ChIPs were performed according to the standard protocols

(20). Primary breast cancer specimens and proliferating MCF7breast cancer cells were used. Tissue was cryosectioned in 30 slicesof 30 mm, and subsequently defrosted in solution A (50 mmol/LHepes, 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5 mmol/L EGTA)supplemented with 1% formaldehyde. For MCF7 cells, formal-dehyde as added to the culturemedium at a final concentration of1%. After 10 minutes of fixation, 100 mmol/L Glycine as added,after which cells were washed in PBS in presence of proteaseinhibitor (Roche Diagnostics 11836145001) and phosphataseinhibitors (Roche Diagnostics: 04906837001) at concentrationsrecommended by the supplier. Tissue was homogenized, andnuclei were isolated, washed, and sonicated using a DiagenodeBioruptor as described before (21, 22). After sonication, the lysatewas cleared by centrifugation. For eachChIP 100 mL ofDynabeads(Invitrogen) was used, precoupled overnight with 10 mg of SRC3or SRC-pS543 antibody. Immunoprecipitation was performedovernight while rotating at 4 degrees. Subsequently, beads werewashed 10 times using RIPA buffer and reverse cross-linked at 65degrees for 6 hours. Supernatant was removed, cleaned using aphenol/chloroform column, and precipitated as described before(20). For standard immunoprecipitations, beads were resus-pended in sample buffer, boiled at 95�C for 5 minutes, and thesamples run onto 8% polyacrylamide gel for protein analysis forS543P, input SRC3, and immunoprecipitated SRC3. Immuno-precipitations were performed in the presence of protease inhib-itor (Roche Diagnostics 11836145001) and phosphatase inhibi-tors (Roche Diagnostics: 04906837001) using standard concen-trations recommended by the supplier. When indicated, sampleswere incubated with Lambda Protein Phosphatase (Millipore; cat# 14–405) for 30 minutes prior to ChIP (2,000 units). Primersequences for qPCR analysis are in Supplementary Table S1.

For MCF7 experiments, approximately 6 � 107 asynchronousproliferating cells were used, and two independent replicates wereperformed for each ChIP-seq, where only consensus peaks thatwere found in both the experiments were considered. For tumorspecimen ChIP-seq, fresh snap-frozen primary ERa-positive,HER2-negative untreated breast cancers were obtained from the

Translational Relevance

SRC3 is an essential coactivator for the transcriptionalactivity of ERa and is required for estrogen-dependent cellproliferation. Analogous to ERa, SRC3 is typically found atenhancer regions involved in long-range regulation of hor-monal-responsive genes. In this study, we establish thegenome-wide chromatin binding preferences for activatedSRC3 (SRC3-pS543) in ERa-positive breast cancer, as well asthe association between SRC3-pS543 expression and clinico-pathologic features and clinical outcomes in 1,650 primarybreast cancers. Our findings reveal that while ERa and totalSRC3 are predominately found in distal enhancers andintrons, SRC3-pS543 is predominately located at promotersof genes. SRC3-pS543 was associated with favorable clinico-pathologic features and was found to be an independentprognostic factor as well as a predictive marker with regardto tamoxifen treatment. These observations illustrate that highexpression of SRC3-pS543 can potentially identify a popula-tion of early ER-positive breast cancer with a good clinicaloutcome without receiving adjuvant therapy.

Zwart et al.

Clin Cancer Res; 22(2) January 15, 2016 Clinical Cancer Research480




Imperial CollegeHealthcareNHSTrust TissueBank,whichhas therequisite ethical approval.

Solexa sequencing and enrichment analysisChIP DNA was amplified as described (20). Sequences were

generated by the Illumina Hiseq 2000 genome analyzer (using50 bp reads), and aligned to the Human Reference Genome(assembly hg19, February 2009). Enriched regions of the genomewere identified by comparing the ChIP samples to mixed inputusing the MACS peak caller (23) version 1.3.7.1. Total read count,percentageof aligned reads, andnumber ofpeaks for eachChIP-seqis shown inSupplementaryTableS2. Forhistonemodificationdata,publicly available MCF7 ChIP-seq data for H3K4me1 (GEO:GSM588568), H3K4me2 (GEO: GSM822392), H3K4me3 (GEO:GSM945269), and H3K27Ac (GEO: GSM945854) were used. ForERaChIP-seq data, peaks from ref. 24were used. ChIP-seq data forSRC1, SRC2, and total SRC3 were from ref. 15. For heatmapvisualizations, Seqminer software was used.

Motif analysis, heatmaps, and genomicdistributions of bindingevents

ChIP-seq data snapshots were generated using the IntegrativeGenome Viewer IGV 2.1 (www.broadinstitute.org/igv/). Motifanalyses were performed through the Cistrome (cistrome.org),applying the SeqPos motif tool (25). For visualization purposes,data were represented in Motif Radar Plots (MRP). Motifs datafiles were first generated using Galaxy Cistrome with equal num-bers of peaks for each subset, randomly downsamples toward thelowest number. All motifs with a P value of 3 kb awayfrom the RefSeq transcription start site, it is considered distalintergenic.

Nottingham Tenovus Primary Breast Cancer SeriesPrimary operable breast cancer cases (n ¼ 1,650) from the

Nottingham Tenovus Primary Breast Carcinoma Series was uti-lized as described before (refs. 27, 28; Supplementary Table S3).Patients were under the age of 71 years (median, 55 years),diagnosed between 1986 and 1999, and treated uniformly in asingle institution. Patients within the good prognosis group[Nottingham Prognostic Index (NPI) � 3.4] did not receiveadjuvant therapy. Premenopausal patients within the moderate(NPI > 3.4 to� 5.4) and poor (NPI > 5.4) prognosis groups werecandidates for chemotherapy. Conversely, postmenopausalpatients with ERa-positive breast cancer with moderate or poorNPI were offered hormonal therapy, whereas ERa-negativepatients received chemotherapy. Clinical data were maintainedon a prospective basis with a median follow-up of 130 months(range, 2–311 months).

IHCBreast cancer tissue microarrays from 1,650 cases were immu-

nostained with SRC3 (dilution 1:20) and SRC3-pS543 (dilution:

1:2,000) antibodies overnight at room temperature, after antigenretrieval [citrate buffer (pH 6.0) and microwave (20 minutesat 750 W)]. Negative controls were performed by omission ofthe primary antibody. Nuclear staining was scored based on theH-score. Determination of the optimal cutoffs was performedusing X-tile bioinformatics software (Yale University, USA) andaccording to the histogram distribution of the H-score. For IHCblocking experiments, peptides were used, which contained thephospho-moiety (CLLSTLS-phosphoS-PGPKLDN) or the non-phospho-peptide (CLLSTLSSPGPKLDN).

The peptides were preincubated overnight at 100-fold excessover antibody prior to staining as described previously.

Statistical analysisData analysis was performed using SPSS (SPSS, version 17).

Where appropriate, the Pearson x2, Fisher exact, Student t, andANOVA one-way tests were used. Cumulative survival probabil-ities were estimated using the Kaplan–Meier method and differ-ences between survival rates were tested using the log-rank test.Multivariate analysis for survival was performed using the Coxhazard model. The proportional hazards assumption was testedusing standard log–log plots. HR and 95% confidence intervals(CI) were estimated for each variable, with P < 0.05 as statisticallysignificant. For multiple comparisons, a stringent P value(

and SRC3-pS543, 2,365 peaks were shared (for raw data visua-lizations, see Fig. 2C). Not all SRC3 binding events were phos-phorylated and 2,795 peaks for SRC3-pS543 were apparently notshared with total SRC3. However, there was weak total SRC3signal observed at these positions, which did not reach thedetection threshold for peak calling (Supplementary Fig. S3A andS3B). Based on these results, we conclude that differential bindingpatterns between SRC3-pS543 and total SRC3 should be consid-ered as "enriched" rather than "unique." P160 family membersshare substantial sequence homology. Even though the sequencesurrounding S543 is not conserved in SRC1 and SRC2, the SRC3-pS543 antibody might still detect the other two p160 members.Therefore, we determined potential overlap of SRC3-pS543enriched sites with SRC1 and SRC2 in MCF7 cells (15). Theoverlap of SRC3-pS543 enriched sites with SRC1 and SRC2 waslimited, rendering it unlikely that the SRC3-pS543 enriched sitescan be explained merely by cross-reactivity with one of the other

p160s (Supplementary Fig. S4). Differential chromatin bindingbetween SRC3-pS543 and total SRC3 could be validated by qPCRanalyses on asynchronous MCF7 cells (Supplementary Fig. S5A),using four primer sets that were designed for each subset ofbinding sites, that is, SRC3-pS543-enriched, total SRC3-enriched,and shared sites (Supplementary Table S1 for primer sequencesand Supplementary Fig. S5B for genome browser snapshots ofSRC3 and SRC3-pS543 ChIP-seq signal at these sites). To assesshormone dependency of SRC3-pS543 chromatin interactions,SRC3-pS543 ChIP-qPCR was performed on cells that were hor-mone deprived for 3 days, and subsequently treated for 3 hourswith vehicle, E2, or tamoxifen (Supplementary Fig. S6). SRC3-pS543/chromatin interactions at SRC3-pS543-enriched sites (thatwere not bound by ERa) were hormone independent and notaffected by ligand. For SRC3-pS543 sites shared with total SRC3,no chromatin binding was observed in the absence of hormoneand in tamoxifen-treated cells, but this interaction was actively

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Figure 1.Validation of phosphostate-specific antibody. A, CHO cells were transfected with pCDNA3, Flag-tagged wild-type (WT) SRC3 or a Flag-tagged SRC3 mutant inwhich serine at residue 543 has been substituted by alanine. MCF7 cells were used as control. The lysates were immunoprecipitated with SRC-3 antibody.Immunoblotting was performed for SRC-3 and phospho-S543. Note that the Flag-tagged SRC3 migrates slower on Western blot as compared with endogenousSRC3. B, MCF7 cell lysate was immunoprecipated with SRC-3 antibody. The beads were then divided into two halves, one untreated and the other treatedwith l-phosphatase. Immunoblottingwas performed for SRC-3 and phosphorylation state-specific antibody. C, CHO cells were grown in DMEMwith dextran-coatedcharcoal stripped FCS for 72 hours and then transfected with vector (pCDNA3) or SRC-3 for 24 hours. Cells were then treated with vehicle or increasingconcentrations of 17ß-estradiol (E2) for 1 hour before making lysates for immunoprecipitation. Western blotting was carried out with the antibodies indicated.MCF7 cells were used as control. D, representative tumor sections following preincubation with phospho-peptide and nonphospho-peptide and phosphatasetreatment prior to staining with SRC3-pS543 in breast cancers with negative, low/medium, and high expression of SRC3-pS543.

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induced by E2 treatment. Phosphatase treatment abrogated SRC3-pS543 enrichment at these sites (Supplementary Fig. S6).Genomicdistributions of ERa and SRC3 were comparable as reported byothers (ref. 29; Supplementary Fig. S7). To further illustrate func-tional interplay between ERa and SRC3-pS543, ERa was depletedfromMCF7 cells using Fulvestrant (Supplementary Fig. S6). SRC3-pS543/SRC3 shared, but not SRC3-pS543-enriched chromatininteractions were dependent on ERa levels, as shown by Fulves-trant-mediated ERa degradation (Supplementary Fig. S8).

In order to identify potential selectivity of transcription factorusage, motif analyses were performed on the sites that were eitheror not shared between SRC3-pS543 and ERa, and visualized in aMRP (Fig. 2D). For the shared events,motif enrichmentwas foundfor classical luminal breast cancer–selective transcription factors,including ESR1 and forkhead motifs. For SRC3-pS543-enriched

binding events, nomotifs for ESR1 or FOXA1were enriched, but aclear enrichment was observed for ELK, ELF, and ETV motifs (seealso Supplementary Table S4 for the total list).

Differential motif enrichment could form the basis of differ-ential gene expression programs. Therefore, GO pathway enrich-ment was assessed for genes with an SRC3/chromatin bindingevent proximal (

Because the SRC3-pS543 chromatin binding events only reca-pitulated a proportion of the total SRC3 binding events, genomicdistributions could deviate as well. As described before (15, 30),the majority of ERa and SRC3 binding events are found withinenhancers and introns (Fig. 2E). In contrast, SRC3-pS543 bindingwas clearly enriched at promoter regions and 50UTR, with pro-moter-bound SRC3-pS543 colocalizing with SRC3, ERa, andhistone modifications indicative for active gene transcription(H3K4me2, H3K4me3, and H3K27Ac; Supplementary Fig. S10).As expected, SRC3andERa signalwas foundcenteredon theSRC3-pS543 signal. Because transcription factors bind accessible chro-matin, histone marks indicative for active promoters (H3K4me3and H3K27Ac) are found around the SRC3-pS543 sites instead ofdirectly centered on them. Interestingly, this was not the case for

H3K4me2, which was clearly enriched on the SRC3-pS543 peak.The enhancer-selective histone mark H3K4me1 was not enrichedat SRC3-pS543 promoters. Even though less pronounced thanthe total of SRC3-pS543 peaks, the sites shared between ERaand SRC3-pS543 were also more promoter enriched (13.9%) ascompared with the ERa/SRC3 shared regions (4.9%). Further-more, SRC3-pS543 signal was highly enriched at promotersof E2-regulated genes (31) as compared with all Refseq genes(P < 0.0001; Fig. 2F).

SRC3-pS543 and ERa chromatin binding events in primarytumor tissue

Next, we determined phosphorylated SRC3/chromatin inter-actions in primary tumors (Fig. 3). ChIP-seq was performed on

Figure 3.SRC3-pS543 binding events inMCF7 cells and breast tumors. A, genomebrowser snapshot, ChIP-seq for SRC3-pS543 onMCF7 cells (green), tumor sample #1 (blue),and tumor sample #2 (purple). Y bar shows tag count. Genomic coordinates are indicated. B, Venn diagram of SRC3-S543P ChIp-seq for MCF7 cells (green),tumor sample #1 (blue), and tumor sample #2 (purple). Number of shared and unique sites is illustrated. C, heatmap visualization of SRC3-S543P ChIp-seq for MCF7cells (green), tumor sample #1 (blue), and tumor sample #2 (purple). Number of shared and enriched sites is illustrated. D, genomic distributions of sharedand unique SRC3-pS543 binding events for MCF7 cells, tumor sample #1, and tumor sample #2. E, genome browser snapshot, ChIP-seq on a primary breast tumor forERa (red) and SRC3-pS543 (blue). F, Venn diagram of ERa (red) and SRC3-pS543 (blue) binding events. G, heatmap analysis, depicting all shared andenriched binding events for ERa (red) and SRC3-pS543 (blue). Shown are all binding eventswith awindowof 5 kb around the binding site. H, genomic distributions ofthe shared and enriched binding events of ERa and SRC3-pS543. I, motif analyses of the shared and enriched binding events of ERa and SRC3-pS543. Z-scorefor each motif is shown in a MRP. Motif enrichment is shown for chromatin binding sites enriched for ERa (red), SRC3-pS543 (green), or shared betweenERa and SRC3-pS543 (blue). The radial data points represent the absolute value of Z-score.

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two independent ERþ/PRþ/HER2� breast tumor specimens asdescribed (21, 24), and directly compared with theMCF7 cell linedata. Peak intensities between the two tumors and the MCF7sample were comparable, and peaks are well conserved (Fig. 3A).Between the two tumors andMCF7 cells, 2,670 binding events ofSRC3-pS543 were shared (Fig. 3B, visualized in a raw heatmapin Fig. 3C). Furthermore, peaks were also found selectivelyenriched for only MCF7 (2,523 sites), tumor #1 (1,816 sites), ortumor #2 (4,631 sites). The SRC3-pS543 binding sites from allsamples were annotated for their genomic distributions, andagain enrichment for promoters and 50UTR regions was found(Fig. 3D). This was even more strikingly observed for bindingevents shared by all three samples, with 52% of all sites beinglocalized to promoters and 20% at 50UTR.

For tumor #1, the lysate was divided in two prior to immu-noprecipitation, and the secondhalf of the lysatewas used for ERaChIP-seq, enabling a direct comparison of SRC3-pS543 and ERabinding patterns within the same tumor (Fig. 3E). Between ERaand SRC3-pS543, 1,727 binding events were shared, although15,050 binding events were only found for ERa and 5,537binding sites were only found for SRC3-pS543 (Fig. 3F). Theshared and enriched binding patterns were not dictated by dif-ferent thresholds in peak calling algorithms, as shownbyheatmapanalyses (Fig. 3G). The genomic distributions of ERa in the tumorsample closely resemble what was found in the cell line (Fig. 2E)and previously reported in breast tumor tissue (21, 24), withapproximately 5% of ERa binding events being found at promo-ters, with the vast majority of sites located at distal enhancers andintrons (Fig. 3H). The SRC3-pS543 sites (either or not sharedwithERa), were highly enriched at promoters and 50UTR regions.

Motif analyses for the ERa binding events in the tumor spec-imen revealed enrichment in ERa, FOXA1, and AP-2 motifs, asexpected (refs. 30, 32; Fig. 3I). Enrichment for these specificmotifswas found both for ERa-enriched sites as well as for sites sharedbetweenERa and SRC3-pS543. Furthermore, practically allmotifsfound at SRC3-pS543–enriched sites were also found at theregions shared by ERa and SRC3-pS543 bounds, includingmotifsfor ELK, ETV, and ETS factors as were observed in the MCF7 cells(see Supplementary Table S6 for the total list).

Association of total SRC3 and SRC3-pS543 withclinicopathologic features

Total SRC3 and SRC3-pS543 expressionwas evaluated in 1,650breast carcinomas, with protein expression predominately nucl-ear (Supplementary Fig. S11). The total SRC3 mean histoscorewas 157 (interquartile range, 0–300) and for SRC3-pS543 meanhistoscore was 31 (interquartile range, 0–300). For subsequentanalysis, total SRC3 expression was categorized as high (abovemean,H-score >157) or low (belowmean,H-score 31) or low (belowmean, H-score � 30; Supplementary Table S3). A scatterplotanalysis of total SRC3 versus SRC3-pS543 in these tumors showsthat the relative levels between total SRC3 versus SRC3-pS543were strongly variable between tumors. Most importantly, a largeproportion of tumors were identified that were positive for totalSRC3, but negative for SRC3-pS543. Furthermore, no tumorscould be identified that were positive for the phospho-SRC3 butnegative for total SRC3, again highlighting specificity of theantibody (Supplementary Fig. S12).

Although no association on IHC between total SRC3 and ERawas found, there was a strong association for SRC3-pS543 with

ERa (P ¼ < 0.0001) and high expression of ERa-associatedproteins (progesterone receptor; P ¼ < 0.0001 and Bcl2; P ¼

Table 1. Patient characteristics and tumor biomarkers and correlation SRC3 and SRC3-pS543

VariablesSRC3

P valueSRC3-pS543

P valueLow (n ¼ 689) High (n ¼ 699) Low (n ¼ 680) High (n ¼ 836)Pathological parameters:Tumor size 0.096 0.50T1 a þ b (�1.0) 75 (9.7) 55 (8.8) 95 (9.0) 24 (10.5)T1 c (>1.0–2.0) 373 (48.3) 341 (54.6) 517 (49.2) 130 (56.8)T2 (>2.0–5.0) 304 (39.3) 271 (34.8) 415 (39.5) 68 (29.7)T3 (>5) 21 (2.7) 11 (1.8) 24 (2.3) 7 (3.1)

Lymph node stage 0.924 0.31Negative 475 (61.4) 382 (61.1) 627 (59.5) 152 (66.4)Positive 299 (38.6) 243 (38.9) 427 (40.5) 77 (33.6)

Tumor grade 0.173

and other validated prognostic factors; and absence of SRC3-pS543 signal correlated with recurrence (HR ¼ 1.4, CI 95%,1.1–2.5) anddeath frombreast cancer (HR¼ 1.6, CI 95%, 1.1–2.5;Table 2).

DiscussionCoactivators for ERa have been the focus of many clinical

and cell biologic studies. SRC3 is of particular interest,because the SRC3 gene is frequently amplified in breast cancer(7), and its expression was found to correlate with HER2expression levels and poor outcome (11). SRC3 is a memberof the p160 coregulator protein family (along with SRC1 andSRC2), which all have shared and preferred genome-wide

chromatin binding patterns, which are strongly induced byERa activation (15).

Here, we focus on one distinct phosphorylation event on SRC3,Serine 543 phosphorylation. This phosphorylation has spatialselectivity on a genomic scale, where chromatin binding wasselectively enriched at active promoter regions. This genomicpreference at promoters is on contrast to total SRC3, which ismainly found at enhancers. It is likely that SRC3 and SRC3-pS543antibodies have different sensitivities, and the level of overlapdetectedbetween these two signals is possibly anunderestimate ofthe actual proportion of SRC3 that is phosphorylated. Relativeexpression levels and antibody sensitivity are likely to alter thethreshold of detection in ChIP-seq experiments, forming a majorsource for false-negative signal. Because the strongest total SRC3

Table 1. Patient characteristics and tumor biomarkers and correlation SRC3 and SRC3-pS543 (Cont'd )

VariablesSRC3

P valueSRC3-pS543

P valueLow (n ¼ 689) High (n ¼ 699) Low (n ¼ 680) High (n ¼ 836)HER2 familyEGFR 0.82 0.604Negative 512 (82.0) 378 (77.6) 670 (80.5) 150 (84.4)Overexpression 113 (18.0) 109 (22.4) 162 (19.5) 32 (17.6)

HER2

sites are enhancer-selective, whereas the strongest SRC-pS543were mostly enriched at promoters, this genomic selectivity ofphosphorylated SRC3 to promoter regions cannot be merelyexplained by relative expression levels or antibody sensitivity.

Because ERa typically binds enhancers (30), the determinationof which ERa binding events are functionally orchestrating whatgenes has remained a challenge in the field. Because most ERabinding events are observed within a 20-kb window from the

A

SRC3-pS543 high expression (n = 227)

SRC3-pS543 low expression (n = 1,055)

Log rank = 11.0, P < 0.001

Nottingham whole series

(n = 1,282)ER+ (n = 924)


SRC3-pS543 low expression (n = 729)

Log rank = 8.7, P = 0.003

ER–high-risk (NPI>3.4) subgroup (n = 294)



Log rank = 0.006, P = 0.937

B C


SRC3-pS543 low expression (n = 1,055)

Log rank = 14.7, P < 0.0001



Log rank = 1.4, P = 0.001



Log rank = 0.14, P = 0.707

ER+ high-risk (NPI>3.4) subgroup who did not receive

Tamoxifen (n = 263)



Log rank = 4.4, P = 0.035



Log rank = 0.3 , P = 0.57

ER+ high-risk (NPI>3.4) subgroup who receive

tamoxifen (n=263)

ER+ low-risk (NPI 3.4. D, ERa-positive patients NPI < 3.4 who did not receive tamoxifen. E, ERa-positive patients NPI� 3.4 who did not receive tamoxifen. F, ERa-positivepatients NPI� 3.4 who received tamoxifen. G, BCSS based on SRC3-pS543 protein expression and tamoxifen treatment in the ERa-positive high-risk group with lowpSRC3-pS543. H, BCSS based on SRC3-pS543 protein expression and tamoxifen treatment in the ERa-positive high-risk group with high pSRC3-pS543.

Table 2. Multivariate analysis using Cox regression analysis confirms that SRC3-pS543 protein expression is an independent prognostic factor for both DFS andBCSS

VariableBCSS at 10 years DFS at 10 years

HR (CI 95%) P HR (CI 95%) P

S543 (low expression) 1.6 (1.1–2.5) 0.024a 1.4 (1.0–1.9) 0.038a

Tumor size 1.1 (1.0–1.3) 0.056 1.1 (1.0–1.2) 0.017�7a

Grade 0.018a 0.933G1 1.0 1.0G2 1.4 (1.3–3.1) 0.98 (0.7–1.4)G3 2.1 (2.6–5.7) 1.03 (1.2–2.0)

Lymph node stage 9.6 � 10�11a 1.6 � 10�10aNegative 1 1Positive (1–3 nodes) 1.7 (1.3–2.3) 1.4 (1.1–1.8)Positive (>3 nodes) 3.6 (2.5–5.3) 3.2 (2.3–4.4)

Endocrine therapy (no) 1.1 (0.8–1.4) 0.661 0.9 (0.7–1.2) 0.565Chemotherapy (no) 1.2 (0.9–1.7) 0.259 1.3 (1.0–1.7) 0.03Bcl2 expression (positive) 0.4 (0.3–0.6) 6.8 � 10�8a 0.5 (0.4–0.7) 9.2 � 10�7aKi67 expression (high) 1.5 (1.1–2.2) 0.022a 1.3 (1.0–1.7) 0.093a

ER expression (negative) 1.5 (1.1–2.1) 0.016a 1.4 (1.0–1.8) 0.036a

HER2 (overexpression) 1.7 (1.2–2.4) 0.002a 0.7 (0.5–1.0) 0.03a

aStatistically significant.

Zwart et al.





transcription start sites of responsive genes (33), this spacingaround the promoter is typically applied to provide an indicationof functionality. All p160 coactivators, including SRC3, showcomparable genomic features as compared with ERa and aretypically found at enhancers as well (15). Because the SRC3-pS543 phosphorylation is promoter-enriched, commonly sharedwith ERa and induced by estradiol, this phosphorylation eventcould be considered as a novel ERa functionality probe, highlight-ing ERa complexes that are actively involved in transcriptionalevents at gene promoters.

Smaller previous studies on SRC3 expression and its correla-tions with ERa and members of the EGFR/HER family havereported varying results (11–13). We find no correlation of SRC3with ERa but do find a correlation with HER2 and HER3.However, SRC3-pS543 does significantly correlate with ERa,potentially reflecting the role of E2/ERa in phosphorylation ofSRC3. Consistent with these results was the observed correlationof SRC3-pS543 with known estrogen-regulated genes, includingPR. Although previous studies have explored the influence ofSRC3 expression on patient outcome as well how itmay influenceendocrine therapy (11–13, 34–39), the current study is the largestand most well-characterized series to date, with a long medianfollow-up.We find no statistically significant association betweenSRC3 levels and DFS and BCSS in patients with ERa-positivebreast cancer treatedwith tamoxifen, consistentwith other reports(12, 13). Of note, in those women with ERa-positive tumors nottreated with tamoxifen, there is a trend to an improved outcomeand survival for SRC3-positive tumors, analogous to the firstreport relating SRC3 and response to endocrine therapy (11).The poor outcome seen in HER2-positive/SRC3-high tumors forpatients treated with tamoxifen is consistent with previouslypublished data (11, 12). One previous study on ERa-negativetumors, which utilized automated quantitative analysis in asubgroup of 133 patients, found SRC3 associated with a pooreroverall survival (38). However, in the current study of 377 ERa-negative tumors no correlations with recurrence or outcome werefound for SRC3. Of note, we show a significant correlation ofp-JNKandp-p38with SRC3-pS543. These kinases have previouslybeen described to phosphorylate SRC3-S543 (16), and activated(phosphorylated) JNK has previously be linked to endocrinetherapy resistance (37) (40), as well as poor outcome in breastcancer (41). The current study linking pJNK with high SRC3-pS543 and a good prognosis group is not comparable withthese previous studies for technical and methodological rea-sons. In the previous study, which investigated the expressionof pJNK in human breast cancer (39), differing reagents, scoringsystem, as well as a smaller number of cases (n ¼ 68) was used.Furthermore, 40% of the cases were ERa negative, and in thosethat were ERa positive no clinicopathologic correlation waspresented (39). Although the other studies either investigatedthe activity of JNK and not its expression in the context of breastcancers exposed to tamoxifen (37) or where levels of pJNK wereassessed via immunoblotting in the context of a MCF7 breastcancer xenograft model (38).

Our study is the first to explore the clinical significance of aphosphorylated form of SRC3. SRC3-pS543 correlates with animprovedDFS and BCSS for thewhole cohort and is a predictor ofoutcome in ERa-positive breast cancer. Furthermore, in high-riskER-positive disease it is demonstrated that SRC3-pS543 is pre-dictive of benefit with adjuvant tamoxifen. With no benefit beingseen with adjuvant tamoxifen in the presence of high SRC3-

pS543, although those tumors with low SRC3-pS543 hadimproved outcomeswith tamoxifen treatment. The lack of benefitobserved in high SRC3-pS543 tumors treated with tamoxifenindicates that the transcriptional events driven by SRC3-pS543at gene promoters of ERa responsive result in a phenotypewith anexcellent clinical outcome, this would be consistent with theclinicopathologic features associatedwith SRC3-pS543. Althoughinsufficiency or inability to enrich at these promoters as a result oflowor absent SRC3-pS543 results in amore aggressive phenotype,which can be abrogated by tamoxifen.

The patient group studied here is a well-characterized historicalcohort treated with or without tamoxifen. However, estrogenwithdrawal therapy using aromatase inhibitors (AI) is now astandard clinical practice in the adjuvant setting for postmeno-pausal breast cancer, and correlations of response to AIs with(phosphorylated) SRC3 levels need to be assessed within thissetting, particularly because the oncogenicity of SRC3 appearsmodulated by the hormonal milieu in mouse models (42).Currently, the translational arm of the Intergroup ExemestaneStudy (so-called Path-IES) is exploring the potential role of SRC3and pS543 in this context (43).

In summary, wehave found anovel biomarker for ERa-positivebreast cancer and provide mechanistic insights in its functionalinvolvement in the ERa transcription complex. SRC3-pS543phospho-specific antibodies could aid in identifying patientswitha functional ERa signaling pathway,whichpredicts for a favorableoutcome in the absence of adjuvant therapy andwhere tamoxifenmight better be withheld.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: W. Zwart, S. Chan, R.C. Coombes, J.S. Carroll, S. Ali,C. PalmieriDevelopment of methodology: W. Zwart, B. Rudraraju, T.M.A. Abdel-Fatah,O. Gojis, S. Chan, J. Shaw, R.C. Coombes, J.S. Carroll, S. Ali, C. PalmieriAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Zwart, K.D. Flach, B. Rudraraju, T.M.A. Abdel-Fatah, D. Moore, M. Opdam, I. Hofland, S. Chan, I.O. Ellis, R.C. Coombes,C. PalmieriAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Zwart, K.D. Flach, B. Rudraraju, T.M.A. Abdel-Fatah, S. Canisius, E. Nevedomskaya, M. Opdam, M. Droog, S. Chan, J. Shaw,I.O. Ellis, R.C. Coombes, J.S. CarrollWriting, review, and/or revision of the manuscript: W. Zwart, K.D. Flach,T.M.A. Abdel-Fatah, S. Chan, I.O. Ellis, R.C. Coombes, S. Ali, C. PalmieriAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Rudraraju, T.M.A. Abdel-Fatah, M. Opdam,S. Chan, C. PalmieriStudy supervision: W. Zwart, S. Chan, R.C. Coombes

AcknowledgmentsThe authors thank Gordon Brown (CRI) and Arno Velds (NKI) for

bioinformatics support as well as James Hadfield (CRI), Ron Kerkhoven(NKI) for Solexa sequencing, Professor Gerry Thomas, Sarah Chilcott-Burns,and Imperial College Healthcare NHS Trust, Human Biomaterials ResourceCentre (Tissue Bank). In addition, the authors thank Angie Gillies (Univer-sity of Leceister) and Core Facility of the Molecular Pathology Biobank of theNetherlands Cancer Institute for technical help with IHC and IHC validationexperiments.

Grant SupportW. Zwart is supported by a KWF Dutch Cancer Society/Alpe d'HuZes Bas

Mulder Award and a Veni grant from The Netherlands Organisation for

SRC3-pS543 in ER-Positive Breast Cancer

www.aacrjournals.org Clin Cancer Res; 22(2) January 15, 2016 489




Scientific Research (NWO). C. Palmieri was a recipient of a Clinician Scientistfellowship from Cancer Research UK, and is supported by Clatterbridge CancerCharity. O. Gojis is supported by a Translational Research Fellowship from theEuropean Society of Medical Oncology and J. Carroll was supported by an ERCstarting grant and an EMBOYoung investigator award. Thisworkwas supportedby Cancer Research UK, Cancer Research UK Cambridge Research Institute, TheNetherlandsCancer Institute, andA Sister's Hope. TheDepartment ofMolecularand Clinical Cancer Medicine, forms part of the North West Cancer Centre-

University of Liverpool, which is funded by North West Cancer Research.Funding is also received from the Clatterbridge Cancer Charity.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 21, 2014; revised July 11, 2015; accepted August 18,2015; published OnlineFirst September 14, 2015.

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SRC3-pS543 in ER-Positive Breast Cancer




2016;22:479-491. Published OnlineFirst September 14, 2015.Clin Cancer Res Wilbert Zwart, Koen D. Flach, Bharath Rudraraju, et al. Prognostic Factor in ER-Positive Breast CancerSRC3 Phosphorylation at Serine 543 Is a Positive Independent

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