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Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1)… Stratford, Anna L; Habibi, Golareh; Astanehe, Arezoo; Jiang, Helen; Hu, Kaiji; Park, Eugene; Shadeo, Ashleen; Buys, Timon P; Lam, Wan; Pugh, Trevor; Marra, Marco; Nielsen, Torsten O; Klinge, Uwe; Mertens, Peter R; Aparicio, Samuel; Dunn, Sandra E Sep 17, 2007

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Available online AccessVol 9 No 5Research articleEpidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a potential target for therapyAnna L Stratford1, Golareh Habibi1, Arezoo Astanehe1, Helen Jiang1, Kaiji Hu1, Eugene Park1, Ashleen Shadeo2, Timon PH Buys2, Wan Lam2, Trevor Pugh3, Marco Marra3, Torsten O Nielsen4, Uwe Klinge5, Peter R Mertens6, Samuel Aparicio7 and Sandra E Dunn11Laboratory for Oncogenomic Research, Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada2Department of Cancer Genetics and Developmental Biology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada3Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada4Genetic Pathology Evaluation Centre of the Prostate Research Centre, Vancouver General Hospital and British Columbia Cancer Agency, Vancouver, British Columbia, Canada5Department of Applied Medical Engineering, Helmholtz Institute, RWTH Aachen University, Aachen, Germany6Departments of Nephrology and Clinical Immunology, University Hospital Aachen, RWTH Aachen, Germany7Molecular Oncology and Breast Cancer Program, British Columbia Cancer Research Centre, Vancouver, British Columbia, CanadaCorresponding author: Sandra E Dunn, sedunn@interchange.ubc.caReceived: 16 May 2007 Revisions requested: 26 Jul 2007 Revisions received: 9 Aug 2007 Accepted: 17 Sep 2007 Published: 17 Sep 2007Breast Cancer Research 2007, 9:R61 (doi:10.1186/bcr1767)This article is online at:© 2007 Stratford et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractIntroduction Basal-like breast cancers (BLBCs) are veryaggressive, and present serious clinical challenges as there arecurrently no targeted therapies available. We determined theregulatory role of Y-box binding protein-1 (YB-1) on epidermalgrowth factor receptor (EGFR) overexpression in BLBC, and thetherapeutic potential of inhibiting EGFR. We pursued this inlight of our recent work showing that YB-1 induces theexpression of EGFR, a new BLBC marker.Methods Primary tumour tissues were evaluated for YB1protein expression by immunostaining tissue microarrays, whilecopy number changes were assessed by comparative genomichybridization (CGH). The ability of YB-1 to regulate EGFR wasevaluated using luciferase reporter, chromatinimmunoprecipitation (ChIP) and gel shift assays. The impact ofIressa on monolayer cell growth was measured using anArrayScan VTI high-throughput analyser to count cell number,and colony formation in soft agar was used to measureanchorage-independent growth.Results YB-1 (27/37 or 73% of cases, P = 3.899 × 10-4) andEGFR (20/37 or 57.1% of cases, P = 9.206 × 10-12) areexpressed in most cases of BLBC. However, they are nottypically amplified in primary BLBC, suggesting overexpressionowing to transcriptional activation. In support of this, wedemonstrate that YB-1 promotes EGFR reporter activity. YB-1specifically binds the EGFR promoter at two different YB-1-responsive elements (YREs) located at -940 and -968 usingChIP and gel shift assays in a manner that is dependent on thephosphorylation of S102 on YB-1. Inhibiting EGFR with Iressasuppressed the growth of SUM149 cells by ~40% inmonolayer, independent of mutations in the receptor. Moreimportantly anchorage-independent growth of BLBC cell lineswas inhibited with combinations of Iressa and YB-1suppression.Conclusion We have identified for the first time a causal link forthe expression of EGFR in BLBC through the induction by YB-1 where it binds specifically to two distinguished YREs. Finally,inhibition of EGFR in combination with suppression of YB-1presents a potential opportunity for therapy in BLBC.Page 1 of 14(page number not for citation purposes)BAC = bacterial artificial chromosome; BLBC = basal-like breast cancer; CGH = comparative genomic hybridisation; ChIP = chromatin immunopre-cipitation; ck5/6 = cytokeratin 5/6; DMSO = dimethyl sulphoxide; EGFR = epidermal growth factor receptor; EMSA = electrophoretic mobility shift assay; ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; IHC = immunohistochemistry; PR = progesterone receptor; SMRT = submegabase resolution tiling; TMA = tumour tissue microarray; YB-1 = Y-box binding factor-1; YRE = YB-1 responsive element.Breast Cancer Research    Vol 9 No 5    Stratford et al.IntroductionIdentifying molecular targets for aggressive types of breastcancer is a milestone in the pursuit of individualized therapies.Gene-expression profiling of primary tumours has led to thefollowing subcategories: luminal A, luminal B, the human epi-dermal growth factor receptor 2 (HER2) and the basal-likesubtypes [1]. Our attention was drawn to the basal-like sub-type, because these tumours do not respond to available tar-geted therapies and patients often die within two years ofdiagnosis [1,2]. Approximately 16% of all breast cancers arebasal-like [3]; this corresponds to 46,400 women among the~290,000 women in North America who will be diagnosedwith breast cancer each year. What sets these tumours apartis that unlike many breast cancers, basal-like tumours do notexpress the estrogen receptor (ER) or progesterone receptor(PR), nor do they have amplified HER2. In the clinic, thesetumours are often referred to as 'triple negative'. Women withtriple negative tumours are not eligible for treatments that tar-get ER (tamoxifen, aromatase inhibitors) or HER2 (trastuzu-mab). Instead they are treated with conventionalchemotherapies, which have limited efficacy and many sideeffects. Therefore, it is critically important to identify alternativetherapeutic strategies for basal-like breast cancer (BLBC).We recently found that the transcription factor, Y-box bindingprotein-1 (YB-1), protein is commonly expressed in ER-nega-tive breast cancers [4], and loss of this receptor is one of thehallmarks of BLBC [3,5]. More recently, YB-1 was pulled outof a screen from the BLBC cell line SUM149 in an attempt toidentify genes that promote malignant transformation andtumour cell growth [6]. It has also been shown recently thatepidermal growth factor receptor (EGFR) is highly expressedin approximately 50% of BLBCs [7]. Interestingly, YB-1 wasoriginally isolated as a transcription factor that bound toenhancer sites on the EGFR gene, a finding that could explain,at least in part, why it promotes the growth of breast tumourcells [8]. In keeping with this possibility, Berquin et al.expressed YB-1 in mammary epithelial cells and observed aconcomitant induction of EGFR [6]. We demonstrated inMCF-7 (ER-positive breast cancer cells) that overexpressionof YB-1 leads to an increase in the levels of EGFR mRNA andprotein [4]. This depends on the phosphorylation of YB-1 atS102 [4]. The YB-1 S102 site is located in the DNA-bindingdomain, suggesting that the effect on EGFR expression waslikely to be through transcriptional regulation. We demon-strated that Akt binds directly to YB-1 and phosphorylates theS102 site, an observation that was subsequently confirmed inNIH3T3 cells [9]. We now believe that Akt is one of severalkinases capable of phosphorylating the S102 site of YB-1. Insupport of this idea, inhibition of the kinase mTOR withrapamycin also inhibits YB-1 phosphorylation [9]. To under-stand this further, we demonstrated that YB-1 binds directly tothe EGFR promoter within the first 1 kb of the transcriptionstart site, and this occurs in a phosphorylation-dependentwe found that YB-1 is strongly correlated with EGFR in pri-mary breast tumours by screening a tissue microarray of ~490cases [4]. More recently, we have confirmed this observationin a cohort of ~2,222 primary breast tumours. In this study,YB-1 and EGFR are once again tightly correlated (P = 1.414× 10-24; data not shown).As both YB-1 and EGFR are expressed in BLBC, we ques-tioned whether there was a relationship between these twogenes in this particular subtype of breast cancer. First, wedetermined whether the overexpression was caused by geneamplification, and then further dissected the regulatory rela-tionship between the two. Finally, we addressed whetherinhibiting EGFR with Iressa (also referred to as ZD1839 orgefitinib) would slow the growth of BLBC.Materials and methodsTumour tissue microarrays and cluster analysisPatients in this cohort and their tumours have been previouslydescribed [10], as have the staining conditions for YB-1,HER2, ER and PR [10]. EGFR and CK5/6 staining was per-formed according to Nielsen et al. [7]. In total, we had inter-pretable data on these proteins from 285/438 total breastcancer cases. For our analysis, YB-1 scored as 0 or 1 wasconsidered negative, and 2 or 3 was considered positive. Datawas filtered to exclude patients who were missing diagnosticor survival information. Results were considered statisticallysignificant with P < 0.05. The data was analysed using SPSSsoftware (Chicago, Illinois, USA).Comparative genomic hybridizationTen formalin-fixed and paraffin-embedded archival BLBCcases from the Vancouver General Hospital archival TMA438series were identified based on a distinct immunohistochemi-cal (IHC) staining pattern (ER-, HER2-, PR-, CK5/6+). Tissuecores (1.5 mm) extracted from the source blocks were treatedwith xylene and ethanol, as described by Garnis et al. [11].Samples were placed into DNA lysis buffer comprised of 10mM Tris, 50 mM NaCl, 1 mM EDTA, 0.5% SDS placed at55°C, and digested with proteinase K (Invitrogen, Carlsbad,California, USA) over a period of 48 to 72 h. DNA wasextracted as previously described, RNase-treated, and quanti-fied by ND-1000 Full Spectrum UV/Vis Spectrophotometer(Nanodrop, Wilmington, Delaware, USA) [11]. The ten BLBCspecimens were assayed for genetic alterations using awhole-genome tiling path bacterial artificial chromosome(BAC) array in comparative genomic hybridization (CGH)experiments as previously described [12]. The submegabaseresolution tiling set (SMRT) array contained 32,433 overlap-ping BACs-derived DNA segments that provide tiling cover-age over the human physical genome map. All clones werespotted in triplicate, resulting in 97,299 elements over twosides. Hybridizations were scanned using a CCD-based imag-ing system (Arrayworx eAuto, Applied Precision; Issaquah,Page 2 of 14(page number not for citation purposes)manner [4]. Consistent with these preclinical developments, Washington, USA) and analyzed using SoftWoRx TrackerAvailable online Analysis software as previously described [13,14]. Datawas filtered and breakpoints were identified as previouslydescribed by Baldwin et al. [15]. Clones with standard devia-tions between replicate spots of >0.075 and with signal-to-noise ratios of <3 were filtered from raw data. Genomic break-point boundaries were defined by aCGH-Smooth softwareand visual inspection. Log 2 signal intensity ratio thresholdswere used to determine regions of gain and loss, with >0.5representing a gain and <-0.5 representing a loss.Characterization of YB-1 and EGFR in basal-like breast cancer cells in vitro184 htert cells were obtained from J. Carl Barrett at the USNational Institute of Health, and were cultured as previouslydescribed [16]. SUM149 cells, selected because theyexpress markers of BLBC [17,18], were purchased fromAstrand (Ann Arbor, Michigan, USA) and were grown accord-ing to the manufacturer's recommendation. The cells were cul-tured in F-12 (Ham's) media (Gibco/Invitrogen, Burlington,Ontario, USA) supplemented with 5 μg/ml insulin (Sigma,Oakville, Ontario, Canada) 1 μg/ml hydrocortisone (Sigma),10 mM HEPES (Sigma), 5% fetal bovine serum (Gibco/Invit-rogen), and 100 units/ml of penicillin/streptomycin (Gibco/Inv-itrogen). MDA-MB-468 cells were obtained from the ATCCand cultured in Dulbecco's modified Eagle's medium, 10%FBS and 100 units/ml penicillin/streptomycin. HCC1937breast cancer cells, also triple negative [19], were cultured inRPMI-1640 media supplemented with 5% FBS, 10 mMHEPES, 4.5 g/L glucose (Sigma), 1 mM sodium pyruvate(Sigma) and 100 units/ml penicillin/streptomycin. Cells weremaintained at 37°C in 5% CO2 and passaged every 2 days.Proteins were isolated from log growing 184 htert, SUM149and HCC1937 cells using an ELB buffer [4]. YB-1, EGFR andactin were detected by immunoblotting. The YB-1 polyclonalantibody (courtesy of Colleen Nelson, University of BritishColumbia, Vancouver, Canada) was used at a dilution of1:10,000. The EGFR monoclonal (clone 6F1, StressGen, SanDiego, California, USA) and actin (Sigma, St Louis Missouri,USA) antibodies were diluted 1:1000.Chromatin immunoprecipitationSUM149 cells were plated at a density of 1 × 107 in a 150 mmdish and YB-1-promoter complexes were isolated by chroma-tin immunoprecipitation (ChIP) as previously described [4].The primers to each of the potential YB-1 binding sites werepreviously described [4]. The EGFR promoter was amplified(40 cycles) using primers that span regions within the first 2kb upstream of the start site. The input DNA was diluted four-fold before amplification.Serial ChIP to determine YB-1 phosphorylation statusTo determine whether YB-1 is serine phosphorylated at theEGFR promoter, complexes were isolated as described abovein 10 mmol/L DTT at 37°C for 30 min with agitation. The eluatewas diluted 1:50 with buffer (20 mmol/L Tris (pH 8.1), 150mmol/L NaCl, 2 mmol/L EDTA, and 1% Triton X-100) and re-immunoprecipitated with 5 μg of anti-phosphoserine antibody(StressGen) overnight at 4°C. Secondary immunocomplexeswere incubated with salmon sperm DNA/protein A agarose for2 h at 4°C. Subsequent steps followed the ChIP protocoldescribed previously by [4] and PCR was performed withprimers to the EGFR 2a site as described above. To test fornon-specific binding species, matched IgY and IgG were incu-bated with an equal amount of SUM149 cross-linked DNA.The sample was then processed as described above andamplified with primers to EGFR 2a. The input DNA was alsointroduced as a positive control.ChIP was also performed using a phospho-YB-1 (S102) anti-body (in collaboration with Peter Mertens, Germany). The pep-tide sequence and supportive data demonstrating thespecificity of the antibody was recently described by us [20].The immunoprecipitation was carried out as described abovefor YB-1 with protein G-agarose used in place of PreciPhenbeads and rabbit IgG instead of IgY.Electrophoretic mobility shift assay (EMSA)Nuclear and cytoplasmic protein was extracted from log-grow-ing SUM149 cells, MDA-MB-468 or HCC1937 cells using theNE-PER nuclear and cytoplasmic extraction reagents (PierceBiotechnology, Rockford, Illinois, USA) following the manufac-turer's protocol. Briefly, cells were centrifuged to obtain apacked cell volume and lysed in ice cold CER I with proteaseinhibitors. Following 5 min on ice, ice-cold CER II was addedand samples centrifuged at 13,000 g for 10 min. Cytoplasmicprotein was retained and the pellet re-suspended in ice-coldNER with protease inhibitors. The sample was incubated onice for 40 min with frequent mixes and then centrifuged at13,000 g for 10 min. The supernatant containing nuclear pro-tein was stored. Proteins were quantified using the BradfordAssay. EMSAs were carried out using the Lightshift Chemilu-minescent EMSA kit (Pierce Biotechnology), following themanufacturer's protocol. 5' Biotin-labelled complementary oli-gonucleotides with the following sequences, wild-type (-979to -937) TTCACACATTGGCTTCAAAGTACCCATGGCT-GGTTGCAATAAACAT, -968 mutant 5'-TTCACACCCCCGCTTCAAAGTACCCATGGCTGGTT-GCAATAAACAT, -940 mutant 5'-TTCACACATTGGCTTCAAAGTACCCATGGCTGGTT-GCCCCAAACAT and double mutant 5' -TTCACACCCCCGCTTCAAAGTACCCATGGCTGGTT-GCCCCAAACAT were annealed to form double strandedDNA. Binding reactions consisted of 1 × binding buffer, 50ng/μl poly dIdC, 20 fmol Biotin-labeled DNA and 5 μg nuclearprotein in a 20 μl reaction. Competition reactions included 16pmol unlabelled oligonucleotide (800-fold excess), and 1 μgchicken anti-YB-1 antibody was included to determine YB-1Page 3 of 14(page number not for citation purposes)with the chicken YB-1 antibody and then eluted by incubation involvement. An antibody to CREB (1 μg) was introduced as aBreast Cancer Research    Vol 9 No 5    Stratford et al.negative control. The protein was incubated with theunlabelled oligonucleotide or the antibody for 20 min beforethe addition of the biotin-labelled oligonucleotide. The sam-ples were incubated for 20 min at room temperature. The reac-tion mixture was run on a 6% non-denaturing polyacrylamidegel and transferred to a positively charged nylon membrane(Amersham Biosciences, Little Chalfont, UK). DNA wascrosslinked to the membrane at 120 mJ/cm2 using a UV-lightcrosslinker (Stratalinker, Stratagene, La Jolla, California, USA)and detected using chemiluminescence (PierceBiotechnology).Nuclear extraction of primary BLBC tumoursTissue slices from six BLBC tumour specimens were obtainedfrom the British Columbia Cancer Agency, Canada. Nuclearfractions were extracted using the NE-PER nuclear and cyto-plasmic extraction reagents as described above. Since tissuewas limited the samples were pooled before the nuclearextraction step. Electrophoretic mobility shift assays were car-ried out as described above with 10 μg protein.EGFR luciferase assayTo determine whether YB-1 has a direct effect on EGFR pro-moter activity the normal breast cell line, 184 htert, was trans-fected with a 1 kb EGFR promoter construct [21] (courtesy ofAlfred C Johnson US National Cancer Institute, Massachu-setts, USA), a renilla expression vector, pRL-TK (Promega,Madison, Wisconsin, USA), and a YB-1 expression plasmid, aYB-1 S102 mutant (A102) or empty vector. The cells wereplated in 6-well plates (4 × 105 cells/well) and transfected witha total of 1.5 μg DNA using lipofectamine 2000 (Invitrogen).Cells were harvested 24 h post-transfection in 1 × PLB buffer(Promega), and luciferase activity measured. All luciferasemeasurements were normalized to the renilla reading from thesame sample. To carry out the inverse experiment the Fast-For-ward Protocol provided with the HiPerFect Transfection Rea-gent (Qiagen, Mississauga, Ontario, USA) was used toachieve knockdown of YB-1 in SUM149 and HCC1937 cellsusing small interfering RNA (siRNA) (for control and YB-1siRNA sequences see [4]). Briefly, cells were seeded at 4 ×105 cells/well of a 6-well plate in 2 ml media shortly beforetransfection. siRNA was diluted to 100 μl in serum-free mediato achieve a final concentration of 5 nM (SUM149) or 20 nM(HCC1937), and 3 μl HiPerFect was added. Samples werevortexed, incubated at room temperature for 10 min, and thenadded drop-wise to the cells. At 48 h the cells were re-platedin 6-well plates (4 × 105 cells/well and transfected with thepER1, pRL-TK and empty vector and harvested at 24 h post-transfection as described above. Cell lysates were also col-lected at the time of re-plating to ensure successful knock-down of YB-1. The experiments were performed in triplicate ontwo separate occasions. The results are reported as the aver-age of two experiments.Cell viability following treatment with IressaSUM149 breast cancer cells were plated in 96-well plates (5× 103 cells/well) and incubated for 24 h at 37°C in the growthmedia described above. Cells were treated with Iressa (iso-lated from tablets purchased from Astra Zeneca and kindlyprovided by Ching-Shih Chen, Ohio State University, USA) atthe following concentrations; 0, 0.25, 0.5, 1 and 2 μM withdimethyl sulphoxide (DMSO) as vehicle control. Cell numberwas ascertained after 72 h treatment. Cells were washed inPBS and then incubated with Hoechst dye (1 μg/ml) for 15mins. Nuclei counts/well were determined using the Array-Scan VTI high throughput analyser. Statistical analyses werecarried out using the Student t test with significance acceptedwhen P < 0.05.Growth in soft agarSUM149 cells were plated at a density of 2.5 × 104 in a 24-well plate in 0.6% agar, as previously described [10] and sup-plemented with Iressa in the cell layer (concentrations asabove). HCC1937 cells were treated with CTRL and YB-1siRNA for 48 hours and then plated at a density of 10 × 103 in0.6% agar. At the time of seeding the agar was supplementedwith Iressa (0.25 to 2 μM) as described earlier. Coloniesdeveloped over 30 days and were then counted. Each experi-ment was performed in replicates of four and repeated twice.EGFR sequencing from SUM149 cellsGenomic DNA was isolated from 2 × 107 SUM149 cells usingphenol chloroform extraction followed by alcohol precipitation(modified from [22]). DNA was quantified in a UV spectropho-tometer. EGFR exons 1 to 28 were amplified by PCR andsequenced using standard techniques used by the BritishColumbia Cancer Agency Michael Smith Genome SciencesCentre. PCR primers were designed using human genome ref-erence sequence acquired from the UCSC Genome Browser[23] ([24]) and the Primer3 program [25]. The PCR primersequences are listed in Additional file 1. Each PCR reactionwas performed on 10 ng of SUM149 DNA and the productswere visualized on a 2% agarose gel. PCR products werecleaned up using Ampure magnetic beads (Agencourt, Bev-erly, Massachusetts, USA) and sequenced using a standardBigDye Terminator v3.1 cycle sequencing chemistry andApplied Biosystems (Foster City, California, USA) 3730 × lDNA Analyzer.ResultsYB-1 and EGFR amplification is not common in BLBC, indicating changes in transcriptional controlBreast tumour tissue microarrays were profiled to evaluate thefrequency to which EGFR and YB-1 are expressed in triplenegative breast cancers. Such tumours express YB-1 andEGFR in 73% and 57.1% of the BLBC cases, respectively(Table 1). Representative immunohistochemical images forboth EGFR and YB-1 are shown in Figure 1. As indicated byPage 4 of 14(page number not for citation purposes)the arrowheads, YB-1 was expressed in the cytoplasm as wellAvailable online the nucleus. Although we have established that YB-1 andEGFR are frequently expressed in triple-negative breast can-cers, it is not clear why this occurs. One possibility is thatthese genes are both amplified during the development ofBLBC. To study this, we isolated DNA from 10 primary BLBCsand evaluated them for copy number changes by array CGHusing a genome-spanning tiling path array (SMRT) [26]. Copynumber changes were not observed at the YB-1 locus(1p34.2) or the EGFR locus (7p13-11.2) in 10/10 and 9/10cases, respectively (Figure 2). A borderline 10 Mb segmentalgain was present in one (referred to as BLC9) of the 10 casesat 7p13-11.2 encompassing many gene loci including EGFR(Figure 2). The lung cancer adenocarcinoma cell line(HCC827), known to have amplified EGFR, was evaluated asa positive control (Figure 2). Overall neither YB-1 nor EGFRwere commonly amplified, suggesting expression is increasedowing to promoter activation.YB-1 regulates the expression of EGFR in BLBCTo perform functional investigations into the role of YB-1 andEGFR in BLBC, we tested the SUM149 and HCC1937 celllines, which have a basal phenotype [17-19,27]. Initially thelevels of YB-1 and EGFR were compared between 184 htert(immortalized breast epithelial cells) and the cancer cells.SUM149 and HCC1937 cells had high levels of YB-1 andEGFR compared with the 184 htert cells (Figure 3a). Buildingon the observation that YB-1 binds to the EGFR promoterwithin the first 1 kb of the start site [4], we then investigatedTable 1YB-1 is highly expressed in triple negative breast cancerMarker Correlation Likelihood ratio valueYB-1 P = 3.899 × 10-4 12.58N = 27/37 (73%)EGFR P = 9.206 × 10-12 46.491N = 20/37 (57.1%)Y-box binding protein 1 (YB-1) is expressed in 73% of triple negative breast cancers in the TMA438. Epidermal growth factor receptor (EGFR) is expressed in 57.1% of these cases.Figure 1Epidermal growth factor receptor (EGFR) and Y-box binding protein 1 (YB-1) are detected in basal-like breast cancer specimens on a tumour tissue microarr ymicroarray. (a) EGFR-negative staining (40×). (b) Brown cells indicate EGFR positivity (40×), a segment of the core is magnified at 200×. (c) YB-1-negative staining (40×). (d) Brown staining indicates YB-1 positivity (40×), which is detected in both the nucleus and cytoplasm (arrowheads 200×).Page 5 of 14(page number not for citation purposes)Breast Cancer Research    Vol 9 No 5    Stratford et al.whether there was a causal link between YB-1 and the expres-sion of EGFR in the SUM149 and HCC1937 cells. First, wehave determined that YB-1 was able to stimulate EGFR pro-moter activity using a luciferase reporter construct containingthe first 1 kb of the EGFR promoter. Immortalized breast cells(184 hterts) confirmed not to express YB-1 (Figure 3a) trans-fected with a hYB-1 plasmid increased EGFR luciferase activ-ity 1.5-fold compared with the control cells (P = 0.04, N = 6)(Figure 3b). Interestingly, when cells were transfected with theYB-1 mutant (A102) that could no longer be phosphorylatedat S102, there was a significant attenuation in reporter activitycompared with control cells (P = 0.013, N = 6) (Figure 3b).We then addressed whether silencing the high levels of YB-1in the SUM149 and HCC1937 cells would attenuate EGFRreporter activity. YB-1 was knocked down with siRNA for 48 hand then transfected with the EGFR reporter. Under theseconditions, we observed a 78% and 77% loss in EGFRreporter activity in SUM149 and HCC1937 cells, respectively(P = 4.53 × 10-5 and P = 5.98 × 10-7, N = 6) (Figure 3c,d).Therefore, through gain-of-function and loss-of-function stud-ies we showed that YB-1 transactivates the EGFR promoter,and that this occurs in a manner that is dependent on theS102 DNA binding site.Figure 2Basal-like breast tumours do not exhibit amplifications for epidermal growth factor receptor (EGFR) or Y-box binding protein 1 (YB-1) t exhi it a plifications for epidermal growth factor receptor (EGFR) or Y-box binding protein 1 (YB-1). Primary breast tumours were evaluated for genetic amplifications using SMRT array CGH. DNA was isolated from ten primary basal-like breast tumours and genomic profiles were generated by submegabase resolution tiling array comparative genomic hybridisation. There was no obvious gain of copy number on chromosomes 1 or 7, representing the loci for YB-1 and EGFR, respectively. The exception to this trend was BLC9, where there was a large amplicon on chromosome 7. The lung adenocarcinoma cell line HCC827 was included as a positive control of EGFR amplification.Page 6 of 14(page number not for citation purposes)Available online demonstrated that YB-1 can transactivate EGFR wenext determined whether YB-1 interacted with the EGFR pro-moter in the basal-like breast cancer cells to further confirmbinding observed in breast cancer cell lines that were notbasal-like [4], and to address whether this occurs in a mannerthat is dependent on S102 phoshorylation using a newlydeveloped antibody directed at YB-1(S102) [20]. Using theprimer sets previously described [4] we show that, in SUM149and most strongly at the 2a site (Figure 4a, lane 2). This inter-action is also observed in the basal-like MDA-MB-468 cellsthat we have previously reported [20]. Binding did not occurin the SUM149 cells in the regions designated 2b and 3 (Fig-ure 4a, lanes 3 and 4). We confirmed that binding was specificand did not bind to the IgY alone (Figure 4a, lanes 5 to 8), andthat the primers could amplify genomic input DNA (Figure 4a,lanes 9 to 13) compared with the negative controls in whichFigure 3Y-box binding protein 1 (YB-1) regulates the expression of epidermal growth factor receptor (EGFR) in basal-like breast cancer cells. (a) The levels of YB-1 and EGFR proteins were compared between immortalized breast epithelial cells, 184 htert, SUM149 and HCC1937 basal-like breast can-cer cells. Actin was evaluated as a control for equal protein input. (b) 184 htert cells were transfected with an EGFR promoter (1 kb) luciferase con-struct (pER1), a control renilla plasmid (pRL-TK) and either flag-EV or flag-YB-1 or flag-YB-1(A102). Luciferase and renilla activity were measured after 24 hours. YB-1 induced EGFR promoter activity by 1.5-fold (P = 0.04, N = 6), whereas the A102 mutant did not. (c) SUM149 cells were treated with YB-1 small interfering RNA (siRNA) (5 nM) for 48 h. The cells were then transfected with the EGFR reporter for 24 h and compared with the empty vector. Loss of YB-1 expression resulted in a 78% decrease in EGFR reporter activity (P = 4.53 × 10-5, N = 6). Inset: evidence that siRNA targeting YB-1 causes a decrease in expression of the protein. Actin was used as a loading control. (d) The same experiment was repeated using HCC1937 cells treated with 20 nM YB-1 siRNA for 48 h. Loss of YB-1 expression resulted in a 77% reduction in EGFR promoter activity (P = 5.98 × 10-7, N = 6).(d)Page 7 of 14(page number not for citation purposes)cells, YB-1 binds to the EGFR promoter within the first 1 kb, no DNA was added to the amplification reaction (Figure 4a,Breast Cancer Research    Vol 9 No 5    Stratford et al.lanes 13 to 16). This binding pattern is in keeping with our pre-vious work showing that YB-1 binds to the EGFR promoterwithin the first 1 kb in a manner that was dependent on phos-phorylation at S102 [4]. As the phosphorylation status of YB-1affected its ability to transactivate EGFR, we assessedwhether this was also the case in the interaction between theYB-1 and 2a site of the promoter. We therefore questionedwhether YB-1 is serine phosphorylated when it binds to the 2asite. To address this, we initially developed serial ChIP proto-col, whereby YB-1 was initially used to pulldown protein–DNAcomplexes, and the resulting samples were then immunopre-cipitated with an antibody to phospho-serine. Using thismethod we were able to show that YB-1 is serine phosphor-ylated when it binds to the 2a site (Figure 4b). More recently,we have had the opportunity to test a new polyclonal antibodyraised against YB-1(S102) specifically [20]. In this case, bind-ing to the 2a site is also observed (Figure 4c) further support-ing the idea that YB-1 is serine phosphorylated at S102 whenit binds to the EGFR promoter.The ability of YB-1 to bind to the EGFR promoter specificallyat the 2a region was further confirmed using gel shift assays.Nuclear extracts from SUM149, MDA-MB-468 andHCC1937 cells were incubated with a biotin-labelled oligonu-cleotide probe spanning -979 to -934 of the EGFR promoter(Figure 5a). MDA-MB-468 and HCC1937 cells were used asan additional basal-like cancer cell lines as they are triple neg-ative and they overexpress EGFR. Compared with theunbound probe (Figure 5b, lanes 1, 5 and 10), the introductionof the nuclear extract from all cell lines produced intense bind-ing to the EGFR promoter (Figure 5b, lanes 2, 6 and 11) thatcould be competitively inhibited with unlabelled probe (Figure5b, lanes 3, 7 and 12). Co-incubation of the nuclear extractwith a YB-1 antibody caused a supershift (Figure 5b, lanes 4,8 and 13), an effect not observed when an unrelated CREBantibody was used in the same reaction (Figure 5b, lanes 9and 14); therefore, we validated our ChIP results bydemonstrating that YB-1 binds directly to the EGFR promoter.We have also been able to show that YB-1 binds to the 2aregion of the EGFR promoter in primary BLBC cancer sam-ples (Figure 5c, lane 2). This interaction could be competed offwith unlabelled oligo (Figure 5c, lane 3) and supershiftedusing the YB-1 antibody (Figure 5c lane 4). To further dissectYB-1 binding within the 2a region we designed biotin-labelledoligonucleotides in which the YB-1-responsive elements(YREs) were mutated at -968, -940 or both sites (Figure 5a).Losing either of the YREs resulted in less YB-1 binding com-pared with the wild-type EGFR promoter sequence (Figure5d). These data verify that the -968 and -940 binding sites arebona fide YREs. Together these data show that YB-1 is ableto bind to the first 1 kb of the EGFR promoter, and this leadsto transactivation in a phosphorylation dependent manner.Figure 4Y-box binding protein 1 (YB-1) binds to the epidermal growth factor receptor (EGFR) promoterreceptor (EGFR) promoter. (a) Chromatin immunoprecipitation was performed on SUM149 cells. YB-1 binds to the EGFR promoter in the basal-like cells where the 2a loci is the preferred binding site (lane 2). Weak binding was also detected with the 1b primers (lane 1). No bind-ing was observed in the 2b or 3 sites (lanes 3 to 4), nor was there any non-specific binding detected in the IgY negative controls (lanes 5 to 8). Input DNA was diluted fourfold and amplified to demonstrate that the primer produced an expected product (lanes 9 to 12). The no input controls (lanes 13 to 16) are presented to show a lack of non-specific amplifications. (b) Serial ChIP was performed by sequentially pulling down YB-1 and then immunoprecipitating with a phospho-serine anti-body. This demonstrated that at least some of the YB-1 is serine phos-phorylated when bound to the EGFR 2a site. YB-1 binds to the 2a site (lane 1) as expected. Similarly, the phospho-serine antibody pulls down a complex that can be amplified with the 2a primers (lane 2). Re-ChIP with the YB-1 antibody and subsequently with the phospho-serine anti-body also bound to EGFR at the 2a site (lane 3). A phospho-serine YB-1 complex bound to the 2a site on EGFR (lane 3). Species-matched IgG and IgY controls were included to show that the binding was spe-cific (lane 4). The input DNA and no DNA controls were also included (lanes 5 and 6). (c) ChIP was carried out using a phospho-YB-1 anti-body (S102), and binding was detected for the EGFR 2a region (lane 4). There was no binding observed when immunoprecipitation was per-formed using IgG as a control (lane 3). Input DNA was diluted fourfold Page 8 of 14(page number not for citation purposes)and amplified to demonstrate that the primer produced an expected product (lanes 5 and 6). Lane 1 is the DNA ladder.Available online 5Y-box binding protein 1 (YB-1) binds to specific sites within the epidermal growth factor receptor (EGFR) promoter. (a) Sequence of the EGFR2a oligonucleotide used in the gel shift assays (-979 to -934). Highlighted sequences are the potential YB-1 binding sites. The substitutions made in the two mutants are given under the wild-type sequence. (b) Direct evidence for YB-1 binding to the EGFR promoter using gel shift assays. Nuclear extract from SUM149, MDA-MB-468 or HCC1937 cells were incubated in the presence of the EGFR oligonucleotide spanning -979 to -934. There was no binding in the absence of protein (lanes 1, 5 and 10), whereas the addition of the nuclear extract (lanes 2, 6 and 11) resulted in strong bind-ing that could be inhibited with the unlabelled oligonucleotide (lanes 3, 7 and 12). The addition of a YB-1 antibody caused a supershift (lane 4, 8 and 13) that did not occur when the non-related CREB antibody was used (lanes 9 and 14). (c) Nuclear extracts from 6 primary BLBC samples were pooled and used in a gel shift assay for the EGFR 2a site. Lane 1 contains EGFR2a biotin-labelled oligo only. Binding to the probe is evident in lane 2, which was competed off in lane 3 and supershifted with a YB-1 antibody in lane 4. A CREB antibody was used to demonstrate specificity of the supershift (lane 5). (d) Validation of putative YB-1-responsive elements on the EGFR promoter. SUM149 nuclear extracts were incubated with either Page 9 of 14(page number not for citation purposes)wild-type (lane 1) or mutant biotin oligo nucleotides (lanes 3, 4, and 5). A competition reaction was carried out against the wild-type (lane 2). nuclear extract bound to the wild-type sequence (lane 1), but was unable to bind the mutants (lanes 3, 4 and 5).Breast Cancer Research    Vol 9 No 5    Stratford et al.Inhibiting EGFR suppresses the growth of BLBC cellsAs there are several commercially available EGFR inhibitorsavailable (such as Iressa and erlotinib), we questionedwhether targeting this receptor tyrosine kinase would be effec-tive in cells in which it is highly expressed. Monolayer cellgrowth could be inhibited by up to 40% when SUM149 cellswere treated with Iressa (0 to 2 μM) for 72 h (Figure 6a); how-ever, more interestingly, if we grew SUM149 cells in anchor-age-independent conditions then formation of colonies, andtherefore the ability of the cells to transform, was completelyabolished in the presence of as little as 0.25 μM Iressa com-pared with vehicle-treated cells (control 1,867 ± 363, 0.25 to2 μM Iressa 0 ± 0) (Figure 6b). These concentrations areachievable in patients [28] and have previously been shown toinhibit MAP kinase signalling [29]. To confirm this observation,we also found that low doses of Iressa inhibited signallingthrough the MAP kinase pathway (data not shown). To ascer-tain whether this sensitivity was inherent to other BLBC celllines we repeated the same experiment in HCC1937 cells,and somewhat surprisingly these cells were still able to formcolonies in anchorage-independent conditions in the pres-ence of up to 2 μM Iressa. Similarly, the MDA-MB-468 basal-like breast cancer cells are insensitive to Iressa initially but canbe sensitized by targeting PI3 kinase with LY294002 [30]; anobservation that we independently confirmed (data notshown). In a separate study, LY294002 has been shown toinhibit phosphorylation of YB-1 [9]. This is in keeping with ourprevious studies demonstrating that YB-1 is phosphorylatedby Akt in response to PI3 kinase activation [10]. We thereforequestioned whether knocking down YB-1 in HCC1937 cellsbefore treating with Iressa would be effective at reducing theability of these cells to grow in soft agar. The suppression ofYB-1 alone caused a 42% reduction in the number of coloniescompared with control (P = 0.0008), but there was furthersignificant decreases in colony number with the addition of aslittle as 0.25 μM Iressa (P < 0.001 for all concentratons)(Fig-ure 6c). Thus, our studies indicate that although some BLBCcells may be sensitive to Iressa, for others the inhibition of YB-1 may be necessary to sensitize the cells to drug.We were rather surprised that the SUM149 cells were so sen-sitive to the drug. An obvious explanation would be that thesecells express activating mutations in EGFR that would makethem sensitive to Iressa, as has been described for lung can-cer [31]. We therefore sequenced EGFR but unexpectedly didnot find such mutations. All 28 exons coding for this gene wereamplified by PCR and sequenced. Activating mutations suchas L858R or delL747-P753insS that have previously beenreported to be associated with Iressa sensitivity [31] were notfound. However, we did identify five single-nucleotide poly-morphisms (SNPs) in exons 12, 13, 15 and 20 (Additional file2). There was one homozygous non-translated SNP(rs712830), three heterozygous synonymous SNPs(rs17290005, rs17290162 and rs17337198), and one heter-dbSNPs have been previously identified for EGFR ([32]),although their functional significance is not yet known. TheSNP of most interest is R521K, located on exon 13, becauseit results in an amino acid change located in the extracellulardomain of the receptor [33].We concluded that irrespective of 'activating mutations' inEGFR, Iressa inhibits the growth of basal-like breast cancercells. In some cases, co-targeting EGFR and YB-1 may benecessary to optimally inhibit the growth of these aggressivebreast cancer cells. Given these data, we concluded thatinhibiting EGFR and YB-1 significantly slows the growth ofBLBC cells.DiscussionIt has previously been reported that both YB-1 and EGFR arehighly expressed in aggressive forms of breast cancer [4,7]. Inthis study we show that although these proteins are a featureof BLBC, neither gene is overexpressed owing to amplifica-tion. In further studying YB-1 as a transcription factor, weshow that it transcriptionally induces EGFR in basal-like celllines, which could lead to the increased expression observed.Importantly, we have been able to pinpoint that YB-1 bindsspecifically to YREs located at -968 and -940. On preciselyidentifying the bona fide YREs on the EGFR promoter, wedemonstrate for the first time that binding to this region occurswhen YB-1 is phosphorylated at S102. The high levels of bothEGFR and YB-1 in BLBC begs the question of whether eitherof them are potential therapeutic targets. Based on the poorsurvival rates previously reported [1,2] it is clear that the BLBCsubtype represents a very aggressive form of the disease, andEGFR is a rational target for the treatment of BLBC. In fact,since it was reportedly associated with this subtype of breastcancer in 2004 [7], the use of EGFR in classifying basal-liketumours by immunohistochemistry has become widelyaccepted [34,35].We show for the first time that the EGFR inhibitor Iressa sup-presses the growth of SUM149 cells, a model for BLBC, invitro at concentrations achievable in patients [28]. This is notthe case for other BLBC models, as no inhibition of anchor-age-independent growth was evident in the HCC1937 cellswhen they were treated with Iressa alone. This insensitivity isalso reported in MDA-MB-468s [30] and MDA-MB-231 cells,another triple negative cell line with high levels of EGFRexpression [36,37]. Why the SUM149 cells alone are sensi-tive to the drug is not clear. Several studies suggest that acti-vating mutations in EGFR are predictive of whether inhibitors,such as Iressa, would be effective in patients with lung cancer[31,38]. The same could be true for breast cancer, but it is notknown whether BLBCs harbour such mutations. However, wedid sequence the entire EGFR gene from SUM149 cells anddid not find activating mutations previously described for lungcancer. Whether the SNP at R521K influences sensitivity toPage 10 of 14(page number not for citation purposes)ozygous non-synonymous SNP (rs11543848). These Iressa is not known, and warrants further investigation.Available online factor that may influence the sensitivity to EGFR inhib-itors is the level of expression of the target itself, and also thepresence of alterations in downstream signalling independentof receptor activation. For example, both the HCC1937 [19]and MDA-MB-468 cells [39] are PTEN null, resulting inincreased propagation of the PI3-kinase pathway. She et al.have previously shown that by inhibiting the PI3-kinase path-way with LY294002 they can sensitize cells to Iressa [30], andwe also found that by suppressing the expression of YB-1,HCC1937 cells we were able to increase the effect of Iressa.Why YB-1 sensitizes BLBC cells to Iressa is an interestingquestion. YB-1 has been shown to regulate the MDR1 gene[40,41], and thus the P-glycoprotein pump, a member of theABC family of transporters. This pump is involved in the effluxof many drugs, and has been associated with resistance tomany chemotherapeutic agents [42]. We recently performeda ChIP on chip analysis of YB-1 target genes in SUM149 cells,and identified ~15 ABC transporter family members that wereFigure 6Inhibiting epidermal growth factor receptor (EGFR) suppresses the growth of basal-like breast cancer cells r  t  r t  f basal-like breast cancer cel s. (a) Inhibition of EGFR with Iressa (0.25, 0.5, 1 and 2 μM) blocks the growth of basal-like breast cancer cells by up to 40% when the cells were treated for 72 h (0.5 μM P = 0.02, 1 μM P = 0.02, 2 μM P = 0.07). Each experiment was performed in replicates of six on two separate occasions. (b) Anchorage-independent growth was measured by counting colonies formed after 4 weeks exposure to Iressa or vehicle control. Representative images of colonies following each treat-ment are shown, with average colony number/well shown underneath. The ability to form colonies was completely lost in the presence of concentra-tions of Iressa as low as 0.25 μM in SUM149 cells. (c) The ability of HCC1937 cells to form colonies was not effected by Iressa alone; however, knockdown of YB-1 significantly reduced the number of colonies (P < 0.001). The addition of Iressa further reduced the number of colonies. This was highly significant at all concentrations (P < 0.001). Statistical analysis carried out using students t-test; *P < 0.05, **P < 0.01.Page 11 of 14(page number not for citation purposes)which is downstream of phospho-Akt [10], using siRNA in the putatively bound by YB-1, including ABCG2, ABCA5 andBreast Cancer Research    Vol 9 No 5    Stratford et al.ABCC3. Studies carried out by Özvegy-Laczka et al. showedthat multidrug transporters such as ABCG2 may be involvedin the resistance to tyrosine kinase inhibitors such as Iressa bymodulating the uptake and extrusion of these drugs to andfrom cells [43]. In fact, they specifically show that ABCG2, butnot mutant ABCG2, protects the lung cancer cell line A431from Iressa-induced growth inhibition [44]. A more recentstudy [45] also confirms these findings with the demonstrationof decreased intracellular accumulation of low concentrationsof Iressa (0.1 μM to 1 μM) and higher efflux with 1 μM Iressa.Although further work is required to ascertain the mechanisminvolved, the suppression of YB-1 expression could indirectlyincrease the levels of these inhibitors in the cells, allowingthem to bind to their target and reduce cell growth.Not withstanding that SUM149 cells are sensitive to Iressa,suggesting that some BLBCs may be also, we recognize thatacquired resistance to inhibitors such as Iressa is a commonproblem. There are many studies that implicate the overactiva-tion of alternative signalling pathways, such as the insulin-likegrowth factor 1 pathway [46] and MET receptor amplification,leading to the activation of ERBB3–Akt pathway [46]. Alterna-tively, downstream pathways can become constitutively acti-vated, an example being KRAS, which has been reported inlung and colon cancers [47-50]. Given this problem ofacquired resistance, and the fact that many BLBC cases willnot be sensitive, using Iressa in combination with an inhibitorfor a downstream component may provide more long-termbenefits.Although we have established an association between YB-1and EGFR in BLBC, it is likely that this transcription factor reg-ulates the expression of other proteins linked to BLBC. Forexample, YB-1 regulates proliferating cell nuclear antigen(PCNA) and topoisomerase IIα [51], both of which areexpressed in BLBC [52]. In colorectal carcinomas, YB-1 andtopoisomerase IIα are co-ordinately expressed [53]. Likewise,similar expression patterns are reported in lung cancer [54]and synovial sarcomas [55]. More direct evidence for thisassociation is supported by Shibao et al. who reported thatknocking down YB-1 with antisense attenuates topoisomer-ase IIα reporter activity [53]. These and other YB-1 targetgenes are yet to be confirmed in BLBC. If PCNA and topoi-somerase IIα are YB-1-responsive genes in BLBC, it wouldexplain why the expression of this transcription factor is clearlyassociated with poor survival, based on work previously doneby us [4] and others [56]. There are currently no commerciallyavailable inhibitors to YB-1. However, as YB-1 transactivatesmany growth-promoting genes, and we have shown that it canincrease sensitivity to approved agents in BLBC, the questionof whether it would also be a potent therapeutic target for thisaggressive type of breast cancer is being actively pursued inour laboratory.ConclusionWe conclude from our data that YB-1 has a role in EGFR geneexpression in BLBC. Furthermore, we demonstrate thattumour cell growth can be attenuated by blocking EGFR,alone or in combination with YB-1 inhibition, providing newpossibilities for the treatment of this highly aggressive disease.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsALS carried out the luciferase assays, EMSA, phospho-YB-1ChIP, growth assays and soft agar and was involved in draftingthe manuscript. GH carried out the TMA, AA carried out thewestern blots on HCC1937 cells, HJ carried out the ChIP, KHwas involved in acquisition of data for the growth assays, EPcarried out the western blot characterising the SUM149 andHCC1937 cells, AS, TPHB and WL performed the arrayCGH, TON was involved in the TMA, UK and PRM made thephospho-YB-1 antibody, SA provided the primary BLBC tis-sue and SED conceived the studies and was involved in draft-ing the manuscript.Additional filesAcknowledgementsWe would like to thank Steven Yip for his assistance in obtaining the tis-sues for the array CGH analyses. In addition, we would like to acknowl-edge that the Genetic Pathology Evaluation Center is supported, in part, by an unrestricted educational grant from Sanofi-Aventis. This research The following Additional files are available online:Additional file 1A table showing PCR primers for 28 exons of EGFR. Forward primer sequences were prefixed with a 21M13 sequencing tag, TGTAAAACGACGGCCAGT and reverse primer sequences were prefixed with an M13R sequencing tag, CAGGAAACAGCTATGAC. The primers (21M13 and M13R) were then used in the corresponding sequencing reaction.See file 2A table showing sequence analysis of EGFR from the SUM149 cells. Variants were identified in exons 1, 12, 13, 15, and 20. The variants in exons 12, 13, 15 and 20 relate to SNPs that have been previously reported for EGFR.See 12 of 14(page number not for citation purposes)was supported by grants through the Canadian Breast Cancer Available online Alliance: Translational Acceleration Grant II and a National Cancer Institute of Canada.References1. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, DengS, Johnsen H, Pesich R, Geisler S, et al.: Repeated observationof breast tumor subtypes in independent gene expressiondata sets.  Proc Natl Acad Sci USA 2003, 100:8418-8423.2. 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