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Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast… Stratford, Anna L; Fry, Christopher J; Desilets, Curtis; Davies, Alastair H; Cho, Yong Y; Li, Yvonne; Dong, Zigang; Berquin, Isabelle M; Roux, Philippe P; Dunn, Sandra E Nov 27, 2008

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Available online http://breast-cancer-research.com/content/10/6/R99Open AccessVol 10 No 6Research articleY-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cellsAnna L Stratford1, Christopher J Fry2, Curtis Desilets2, Alastair H Davies1, Yong Y Cho3, Yvonne Li1, Zigang Dong3, Isabelle M Berquin4, Philippe P Roux5 and Sandra E Dunn11Laboratory for Oncogenomic Research, Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada2Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, USA3Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA4Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA5Department of Pathology and Cell Biology, Faculty of Medicine, Institute for Research in Immunology and Cancer, P.O. Box 6128, Station Centre-Ville, Université de Montréal, Montreal, QC H3C 3J7, CanadaCorresponding author: Sandra E Dunn, sedunn@interchange.ubc.caReceived: 26 May 2008 Revisions requested: 24 Jun 2008 Revisions received: 25 Nov 2008 Accepted: 27 Nov 2008 Published: 27 Nov 2008Breast Cancer Research 2008, 10:R99 (doi:10.1186/bcr2202)This article is online at: http://breast-cancer-research.com/content/10/6/R99© 2008 Stratford et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractIntroduction Basal-like breast cancers (BLBC) frequentlyoverexpress the epidermal growth factor receptor (EGFR) andsubsequently have high levels of signaling through the MAPkinase pathway, which is thought to contribute to theiraggressive behavior. While we have previously reported theexpression of Y-box binding protein-1 (YB-1) in 73% of BLBC,it is unclear whether it can be regulated by a component of theMAP kinase signaling pathway. Phosphorylation of YB-1 at theserine 102 residue is required for transcriptional activation ofgrowth-enhancing genes, such as EGFR. Using Motifscan weidentified p90 ribosomal S6 kinase (RSK) as a potentialcandidate for activating YB-1.Methods Inhibition of RSK1 and RSK2 was achieved usingsiRNA and the small molecule SL0101. RSK1, RSK2, activatedRSK and kinase-dead RSK were expressed in HCC1937 cells.Kinase assays were performed to illustrate directphosphorylation of YB-1 by RSK. The impact of inhibiting RSKon YB-1 function was measured by luciferase assays andchromatin immunoprecipitation.Results Using an in vitro kinase assay, RSK1 and RSK2 wereshown to directly phosphorylate YB-1. Interestingly, they weremore effective activators of YB-1 than AKT or another novel YB-1 kinase, PKCα. Phosphorylation of YB-1 (serine 102 residue)is blocked by inhibition of the MAP kinase pathway or byperturbing RSK1/RSK2 with siRNA or SL0101. In immortalizedbreast epithelial cells where RSK is active yet AKT is not, YB-1is phosphorylated. Supporting this observation, RSK2-/- mouseembryo fibroblasts lose the ability to phosphorylate YB-1 inresponse to epidermal growth factor. This subsequentlyinterfered with the ability of YB-1 to regulate the expression ofEGFR. The RSK inhibitor SL0101 decreased the ability of YB-1to bind the promoter, transactivate and ultimately reduce EGFRexpression. In concordance with these results the expression ofconstitutively active RSK1 increased YB-1 phosphorylation, yetthe kinase-dead RSK did not.Conclusions We therefore conclude that RSK1/RSK2 arenovel activators of YB-1, able to phosphorylate the serine 102residue. This provides a newly described mechanism wherebyYB-1 is activated in breast cancer. This implicates the EGFR/RSK/YB-1 pathway as an important component of BLBC,providing an important opportunity for therapeutic intervention.IntroductionBasal-like breast cancers (BLBC) are clinically challengingcases that are not amenable to current targeted therapies dueto the absence of estrogen receptor or HER-2 expression.BLBC: basal-like breast cancers; DMEM: Dulbecco's modified eagle medium; EGF: epidermal growth factor; EGFR: epidermal growth factor recep-Page 1 of 12(page number not for citation purposes)tor; ELB: egg lysis buffer; ERK: extracellular signal-regulated kinases; FBS: fetal bovine serum; MAP: mitogen-activated protein; MEF: mouse embryo fibroblast; PKCα: protein kinase C alpha; PMA: phorbal 12-myristate 13-acetate; RIPA: radio immunoprecipitation assay; RSK: p90 ribosomal S6 kinase; S102: serine 102 residue; siRNA: small interfering RNA; YB-1: Y-box binding factor-1.Breast Cancer Research    Vol 10 No 6    Stratford et al.Treatment therefore depends on aggressive chemotherapy,yet relapse rates and overall survival are poor. Identification ofpotential therapeutic targets is an ongoing challenge.Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is overexpressed in a number of cancertypes, including breast cancer [1,2], prostate cancer [3], bonecancer [4], lung cancer [5,6], colon cancer [7], muscle cancer[8] and, most recently, pediatric brain tumours [9]. In particu-lar, we have shown YB-1 to be expressed in a high proportionof BLBC [1], where it is associated with high rates of relapse[10]. Overexpression of YB-1 in breast cancer cells results inan increase in monolayer and enhanced anchorage independ-ent growth [11]. Further, a study by Bergmann and colleaguesdemonstrated that targeted expression of YB-1 in the mam-mary gland of mice resulted in tumour formation with 100%penetrance [12]. Conversely, we find that suppressing YB-1using RNA interference inhibits tumour cell growth in vitro [1]and in vivo [13]. The role of YB-1 in promoting growth ofbreast cancer cells stems from its original identification as aDNA binding protein, interacting with the regulatory elementsof epidermal growth factor receptor (EGFR), HER-2 [14] andc-MYC [15].In the succeeding 20 years since these findings, many moregrowth-promoting genes have been identified as YB-1 targets,including topoisomerase II [7], DNA polymerase alpha andproliferating cell nuclear antigen (PCNA) [16] to name just afew examples. The question that arises is how YB-1 becomesactivated to induce the expression of these genes so centralto the development of cancer.We previously demonstrated the importance of phosphoryla-tion at the serine 102 residue (S102) to the functions of YB-1[1,2]. This site lies in the highly conserved cold-shock domainand is key for YB-1 nuclear localization and its ability to trans-form cells [11]. Recent studies have provided evidence for thevital role of phosphorylation this residue plays in the binding ofYB-1 to, and the regulation of, the EGFR promoter and subse-quent protein production [1,2]. In short, we have shown MCF-7 breast cancer cells overexpressing YB-1 have elevated lev-els of EGFR mRNA and protein [2]. Subsequently we reportedthat YB-1 bound the EGFR promoter in BLBC cells in a S102phosphorylation-dependent manner [1]. Several studies havealso implicated the importance of S102 phosphorylation inpromoting translation [17,18]. Phosphorylation of S102 istherefore important for activating the transcriptional and trans-lational control imparted by YB-1.We previously demonstrated that AKT binds directly to YB-1and phosphorylates the S102 site [11], an observation subse-quently confirmed in NIH3T3 cells [18]. A recent study byBasaki and colleagues showed that serum stimulated YB-1nuclear localization in ovarian cells and, further, this transloca-The Phosphatidylinositol-3 kinase (PI3K) pathway may not bethe major contributor to growth in BLBC. EGFR is expressedin at least 50% of BLBC [20] and was recently used as one offive markers to identify aggressive BLBC [21]. We previouslyfound that, by inhibiting EGFR with Iressa, we could slow thegrowth of BLBC cells [1]. Since this receptor signals throughthe MAP kinase pathway, we questioned whether otherkinases are able to phosphorylate this key residue. We there-fore took a bioinformatics approach to identify potential candi-dates, and determined that p90 ribosomal S6 kinase (RSK)may also phosphorylate YB-1 at S102 [22]. RSK1 to RSK4are members of the AGC serine/threonine superfamily ofkinases [23] that lie downstream of the MAP kinase pathway.RSKs are a direct substrate of ERK [24], but also requirephosphorylation by phosphoinositide-dependent proteinkinase-1 (PDK-1) [25] and subsequent autophosphorylationsteps [26].The importance of RSK family members in diseases such ascancer is just being appreciated. Of the four isoforms, RSK1and RSK2 are the most well characterized, and overexpres-sion has been associated with multiple cancer types such asprostate cancer [27] and those of hematologic malignancies[28]. Recent studies showed that RSK3 may actually be atumour suppressor in ovarian cancer [29], and RSK4 differedfrom the other isoforms in that it was expressed at low levelsand was constitutively active [30]. In breast cancer, a smallstudy carried out by Smith and colleagues found that bothRSK1 and RSK2 expression levels were elevated in ~50% oftumours compared with control cases (n = 12 controls, n = 48cancers) [31]. We questioned whether RSK1 or RSK2 mayplay a role in BLBC because they lie in the MAP kinase path-way, which is commonly activated in this type of breast cancerdue to overexpression of EGFR. In light of studies showingthat RSK phosphorylates other transcription factors such ascreb, c-fos [32] and the estrogen receptor [33], we contendedthat it may play an important role in regulating YB-1.Materials and methodsCell lines and reagentsThe SUM149, HCC1937, MDA-MB-231 and MDA-MB-468cells were used as models of BLBC; all are estrogen receptornegative, progesterone receptor negative and HER-2 negative[34]. SUM149 cells were purchased from Asterand (AnnArbor, MI, USA) and were cultured as previously described[1]. MDA-MB-231 and MDA-MB-468 (both American TypeCulture Collection, Manassas, VA, USA) cells were grown inDMEM (Gibco/Invitrogen, Burlington, ON, Canada) supple-mented with 10% FBS and 100 units/ml penicillin/streptomy-cin. HCC1937 cells (kind donation from WD Foulkes, McGillUniversity, QC, Canada) were cultured in RPMI-1640 mediasupplemented with 5% FBS, 10 mM HEPES, 4.5 g/l glucose(Sigma, Oakville, ON, Canada), 1 mM sodium pyruvate(Sigma) and 100 units/ml penicillin/streptomycin.Page 2 of 12(page number not for citation purposes)tion was prevented by inhibiting AKT [19].Available online http://breast-cancer-research.com/content/10/6/R99HTR-YB#5 (HTRY) are human mammary epithelial cellsimmortalized with HPV16, and express YB-1 if induced withtetracycline [35]. These were maintained in the same media asSUM149 cells supplemented with 10 ng/ml epidermal growthfactor (provided by author IMB). RSK1/RSK2 specific inhibitorSL0101 (Toronto Research Chemicals Inc., North York, ON,Canada) was dissolved in methanol [31,36,37], andPD098059 (Cell Signaling Technologies, Danvers, MA, USA),phorbal 12-myristate 13-acetate (PMA) (Sigma) and epider-mal growth factor (EGF) were dissolved in dimethylsulfoxide(DMSO).Growth factor stimulation and drug treatmentsSUM149 cells were seeded at a density of 4 × 105 cells in asix-well plate. Cells were subsequently serum-starved for 24hours prior to 6 hours treatment with vehicle, PD098059 (20μM) or SL0101 (50 μM). Treated cells were stimulated withthe following growth factors for 15 minutes before harvesting;5% FBS/Ham's/F12 (serum stimulation), EGF (25 ng/ml) andPMA (50 ng/ml), lysed in egg lysis buffer (ELB) and subjectedto western blot analysis [2]. In all other experiments,HCC1937, MDA-MB-231 and HTRY cells were treated with100 μM SL0101 and the SUM149 cells with 50 μM for 6hours. The experiment was performed three times.Protein extraction and western blot analysisProtein was extracted from log-growing cells in ELB [2], sup-plemented with protease and phosphatase inhibitors, andquantified using the Bradford assay (Biorad, Hercules, CA,USA). Immunoblotting was performed as previously described[2]. Specific proteins were detected using the following anti-bodies: EGFR, 1:1,000 (Stressgen, San Diego, CA, USA);ERK, 1:1,000 (p44/42 MAP kinase; Cell Signaling Technol-ogy, Danvers, MA, USA); RSK1, 1:1,000 (Santa Cruz Biotech-nology, Santa Cruz, CA, USA); RSK2, 1:500 (Santa CruzBiotechnology); YB-1, 1:2,000 (Abcam, Cambridge, MA,USA); P-ERK, 1:500 (Cell Signaling Technology); P-RSKS380,1:1,000 (Cell Signaling Technology); P-YB-1S102, 1:1,500(Cell Signaling Technology, Danvers, MA, USA); Vinculin,1:1,000 (Upstate, Temecula, CA, USA); and Pan-actin,1:1,000 (Cell Signaling Technology). Densitometry was per-formed where appropriate.RSK/AKT kinase assayA synthetic peptidomimetic of the YB-1 S102 region wasmanufactured by Sigma with the sequence PRKYLRSVG-COOH. Kinase assays for RSK1, RSK2, AKT1 and PKCαwere carried out on the peptide and activity was comparedwith an optimized control target (100% activity) (SignalChem,Richmond, BC, Canada). Control target sequences were asfollows: RSK, KRRRLASLR; AKT1, CKRPRAASFAE; PKC,KRREILSRRPSYR.The kinase assay reactions consisted of active protein kinasebuffer) (40 μM), radiolabeled 33P-ATP (50 μM in kinase assaybuffer; 25 mM MOPS, 12.5 mM β-glycerol phosphate, 25 mMMgCl2, 5 mM ethylene glycol tetraacetic acid (EGTA), 2 mMethylenediamine tetraacetic acid, 0.25 mM dithiothreitol) to afinal volume of 25 μl. Assays were performed at 30°C for 60minutes, and then the reaction mixture was dotted on phos-phocellulose P81 paper and the radioactivity measured. Activ-ity greater than 5% of the optimized positive control isconsidered highly significant.RSK1/YB-1 kinase assay from cell lysatesMCF-7 cells stably expressing Flag-YB-1 were serum starvedfor 16 hours prior to being lysed in radio immunoprecipitationassay (RIPA) buffer. As described above, 500 μg lysate wasprecleared with protein G agarose for 2 hours. YB-1 was thenimmunoprecipitated from the cells by overnight incubation at4°C with 5 μg anti-Flag M2 antibody (Sigma) followed by 2hours of incubation with protein G agarose. Complexes werethen collected by centrifugation and washed firstly in Tris-buff-ered saline/1% NP40 and then once in modified wash buffer(100 mM Tris, pH 7.4, 50 mM NaCl, 1.5 mM MgCl2, 1 mM eth-ylenediamine tetraacetic acid, 0.5% NP40). YB-1 was isolatedfrom protein G through incubation in 0.1 M glycine, pH 3.5, for5 min at room temperature. Kinase assays were performed forRSK1 as described above.Co-immunoprecipitationLog-growing SUM149 cells were lysed in RIPA buffer supple-mented with protease inhibitors. Cell lysates were subjectedto a Bradford assay for quantification and 500 μg protein wasused in subsequent immunoprecipitations. For YB-1 pull-down, lysates were precleared with 60 μl PrecipHen beads(previously described [2]) for 2 hours at 4°C with rotation, andthe supernatants then incubated with IgY or chicken anti-YB-1 antibodies (5 μg) overnight at 4°C with rotation. Immuno-complexes were collected on PrecipHen beads after incuba-tion at 4°C for 3 hours, by centrifugation. The beads werewashed once with PBS/1% NP40, twice with wash buffer(100 mM Tris, pH 7.4, 100 mM NaCl, 1.5 mM MgCl2, 1 mMethylenediamine tetraacetic acid, 0.5% NP40) and the pro-teins eluted by boiling in 5× loading dye for 5 minutes.Similarly, for total RSK1/RSK2 immunoprecipitations, lysates(500 μg) were precleared with 35 μl protein G agarose for 2hours prior to incubation with either control IgG or RSK1 orRSK2 antibodies (5 μg) (Santa Cruz Biotechnology) for 16hours at 4°C with rotation. Immunocomplexes were retrievedthrough the addition of protein G agarose for 2 hours. Immu-noprecipitated proteins were resolved on acrylamide gels andimmunoblotted as described above. Horseradish peroxidaseprotein A was used as the secondary antibody to avoid detec-tion of denatured immunoglobulins (1:2,000; Amersham Bio-sciences, Piscataway, NJ, USA).Page 3 of 12(page number not for citation purposes)(250 ng/assay), substrate (optimized, YB-1 peptide or assayBreast Cancer Research    Vol 10 No 6    Stratford et al.RSK1 and RSK2 siRNA transfectionSUM149 cells (4 × 105/well) were transfected with 20 nMsiRNA (Qiagen, Mississauga, ON, Canada) using Hiperfect(Qiagen). The fast-forward protocol was followed asdescribed by the manufacturer. RSK1 and RSK2 siRNAsequences were as previously described [38].Transient transfectionHCC1937 cells were seeded at a density of 4 × 105 in a six-well plate 24 hours prior to transfection. Cells were trans-fected with 2 μg plasmid DNA using 10 μl Lipofectamine2000/well as per instructions, and were lysed at 24 hours.Plasmid constructs for RSK overexpression studies wereempty vectors (pRK7 and pKH3), pKH3-avRSK1, pKH3-mRSK2, pRK7-Myr-avRSK1 and pKH3-avRSK1(K112/464R)(kinase-dead) as previously described [39]. The experimentwas carried out three times.RSK2-/- mouse embryo fibroblastsWild-type and RSK2-/- mouse embryo fibroblasts (MEFs) werecultured as described previously [40], and were stimulatedwith EGF (10 ng/ml) and cell lysates collected at 5, 15, 30, 60and 120 minutes (kind donation from Dr YY Cho, University ofMinnesota, Austin, MN, USA). Two sets of samples were ana-lyzed.Luciferase assaySUM149 cells were plated in six-well plates (4 × 105 cells/well) and transfected with a luciferase construct containingthe first 1 kb of the EGFR promoter (pER1) (kind gift fromAlfred C. Johnson US National Cancer Institute, Bethesda,MD, USA – previously described in [1,41]). Cells were trans-fected with a total of 1.5 μg DNA using Lipofectamine 2000(Invitrogen). To account for transfection efficiency, cells wereco-transfected with a renilla-expressing plasmid (pRL-TK,10:1 luciferase:renilla; Promega). After 18 hours, cells weretreated with vehicle or SL0101 (50 μM) for 6 hours prior toharvesting in 1 × passive lysis buffer (Promega). Luciferaseactivity was measured and normalized to the renilla readingfrom the same sample.Chromatin immunoprecipitationSUM149 cells (1 × 107 cells) were treated with vehicle,PD098059 (20 μM) or SL0101 (50 μM) for 6 hours.Crosslinks were established between protein and DNA follow-ing 15 minutes of incubation with 1% formaldehyde. Cellswere washed and collected by centrifugation. Chromatinimmunoprecipitation with anti-P-YB-1 antibody (gift from Dr PMertens, University Hospital RWTH – Aachen, Aachen, Ger-many) was carried out as described previously [1,2]. Theresulting DNA was amplified using the EGFR2a primers (pre-viously described [1,2]).ResultsYB-1 is phosphorylated by the MAP kinase pathwayWhile we have previously established that AKT can interactand phosphorylate YB-1S102 [11], it is unclear whether otherkinases are also able to perform this function. Serum-starvedSUM149 cells were stimulated with 5% FBS/growth media,EGF or the tumour promoter PMA. All of these stimuli acti-vated signaling through the MAP kinase/ERK pathway and ledto the induction of P-YB-1S102 (Figure 1a). The activation of theMAP kinase/RSK/YB-1 cascade was completely reversible bypretreating the cells with the MEK inhibitor PD098059 (Figure1b). SUM149 cells secrete amphiregulin, resulting in activa-tion of EGFR even in serum-free conditions [42]. We thereforealso treated the cells with the EGFR inhibitor Iressa. Asexpected, inhibiting EGFR signaling with Iressa decreased P-YB-1S102 (Figure 1c). By screening a panel of BLBC cell lines,we noted that YB-1 was activated at varying levels but, inter-estingly, the level of phosphorylation did not always correlatewith the expression of P-AKTS473. RSK was activated in all celllines including the MDA-MB-231 cells. These cells do notexpress P-AKTS473; however, the level of P-YB-1S102 is com-parable with that of the SUM149 cells, which express acti-vated AKT as well as P-RSKS380 (Figure 1d). Similarly, in theimmortalized normal breast cell line HTRY, P-RSKS380 is alsoelevated along with P-YB-1S102. These cells also do notexpress activated AKT (Figure 1e). It therefore appears thatactivation of the MAP kinase pathway can also lead to theinduction of P-YB-1S102. This is of particular importance inBLBC given the role of EGFR signaling in this particular typeof breast cancer.RSK phosphorylates YB-1 at serine residue 102To further explore the role of the MAP kinase pathway in thephosphorylation of YB-1S102 we next investigated the effect ofmodulating RSK, which lies downstream of ERK, either phar-macologically or genetically. Initially, using an in vitro kinaseassay, we show that RSK1 and RSK2 are able to directlyphosphorylate an YB-1 S102 peptide that mimics the regionsurrounding the S102 site (Table 1). The activity of RSK1 andRSK2 towards the YB-1 target peptide was 80% and 78%compared with the activity of these kinases towards the opti-mized positive control target, respectively (Table 1). Interest-ingly, this was greater than the activity of AKT1 towards theYB-1 target (7% of optimized control activity) (Table 1). Theactivity of AKT1, however, was still considered significant inthis assay. Of note, the YB-1 target peptide was also phos-phorylated by PKCα (Table 1). Weak RSK1 kinase activitywas also detected when using flag-tagged YB-1 immunopre-cipitated from stably expressing MCF-7 cells as a substrate(data not shown). In this case the salts required for the proteinisolation compromised the level of activity.We also found that, following immunoprecipitation of endog-enous YB-1 from log-growing cells, RSK1 is present in thePage 4 of 12(page number not for citation purposes)complex (Figure 2a, left). Similarly, by performing the reverseAvailable online http://breast-cancer-research.com/content/10/6/R99experiment, immunoprecipitation of RSK1, YB-1 was detected(Figure 2a, right). We were unable to determine an interactionof RSK2 with YB-1 due to a lack of suitable antibody for thisapplication. Since RSK2 could not be detected following YB-1 immunoprecipitation, we believe the interaction between thetwo proteins maybe weaker. This prompted us to investigatethe consequence of inhibiting RSK1 or RSK2 on YB-1 phos-phorylation. Following suppression of RSK1 expression withsiRNA for 72 hours, the level of P-YB-1S102 was greatlyreduced in SUM149 cells (Figure 2b). The loss of RSK2 alsoresulted in a decrease in YB-1 phosphorylation, although to alesser degree than that by RSK1. Simultaneous knockdown ofRSK1 and RSK2 produced an effect on the level of P-YB-1S102 greater than either gene knockdown alone (Figure 2b).The levels of total YB-1 and actin remained unchanged (FigureRSK2 or a constitutively active RSK1 (myr-RSK) for 24 hoursinduced P-YB-1S102 in HCC1937 cells (Figure 2c) comparedwith cells transfected with the empty vectors pKH3 and pRK7(myr-RSK empty vector). The kinase-dead RSK1 mutant, how-ever, was unable to phosphorylate YB-1 at S102 (Figure 2c).Taking an alternative genetic approach, we turned to usingMEFs that have a homozygous deletion for RSK2 [40]. Loss ofRSK2 prevented the induction of P-YB-1S102 following EGFstimulation in a time-dependent manner, as compared with thewild-type MEFs (Figure 2d). ERK and RSK were still phospho-rylated in response to EGF in the RSK2-/- MEFs (data notshown). Interestingly, the YB-1 downstream target geneEGFR could be induced in the wild-type cells after 120 min-utes; however, this was not the case in the RSK2-/- cells (rela-tive intensity of EGFR expression compared with wild-typeFigure 1Y-box binding factor-1 is phosphorylated by the MAP kinase pathway binding factor-1 is phosphorylated by the MAP kinase pathway. (a) Stimulation of SUM149 cells (SS) with serum, epidermal growth fac-tor (EGF) and phorbal 12-myristate 13-acetate (PMA) (15 min) results in the phosphorylation of Y-box binding factor-1 (YB-1) at the serine 102 res-idue (S102). There is no change in total YB-1 levels. Phosphorylation of ERK indicates activation of the MAP kinase pathway. Total ERK and vinculin indicate equal loading. (b) Inhibition of MAP kinase signaling with PD098059 results in the loss of growth-factor induced P-YB-1S102 (n = 3). (c) Treating SUM149 cells with Iressa (2 μM) results in a decrease in P-YB-1S102. (d) SUM149, MDA-MB-231, HCC1937 and MDA-MB-468 breast cancer cell lines were compared for expression level of P-RSKS380, P-AKTS473, P-ERKThr202/Tyr204 and P-YB-1S102. The MDA-MB-231 cells express high levels of P-YB-1 in the absence of P-AKTS473; however, they do express P-RSKS380. (e) Immortalized human mammary epithelial cells (HTRY) express P-RSKS380, P-ERKThr202/Tyr204 and P-YB-1S102, but not P-AKT. DMSO, dimethylsulfoxide.Page 5 of 12(page number not for citation purposes)2b). In a complementary study, introducing exogenous RSK1, cells at each time point given under blot) (Figure 2d).Breast Cancer Research    Vol 10 No 6    Stratford et al.We then used the RSK1/RSK2 specific inhibitor SL0101 [31]to confirm these findings. SL0101 was used at concentrationsin line with previous studies in MCF-7 cells [31]. Followingtreatment of SUM149 cells with SL0101 (50 μM) for between6 and 16 hours, we observed a reduction in P-YB-1S102 at alltime points whilst the YB-1 level remained constant (Figure3a). This finding was confirmed in the HCC1937, MDA-MB-231 and HTRY cells treated for 6 hours with SL0101 (100μM) (Figure 3b,c). Likewise, pretreating SUM149 cells withSL0101 prevented the stimulation of P-YB-1S102 by serum,EGF or PMA after 6 hours compared with cells treated withthe vehicle (methanol) control (Figure 3d). P-RSKS380 is phos-phorylated by the C-terminal kinase, and SL0101 inhibits theN-terminal kinase activity. One therefore cannot measure theeffect of SL0101 by studying P-RSKS380.Inhibition of RSK functionally inactivates YB-1We have previously established the importance of phosphor-ylation of YB-1S102 for its transcriptional activity in breast can-cer [2], and in particular the regulation of EGFR in BLBC.Firstly, we performed a reporter assay using a 1 kb EGFR–luci-ferase construct that contains an YB-1 binding site at -968base pairs [1]. Knocking down YB-1 with siRNA or inhibitingsignaling with PD098059 decreased the EGFR promoteractivity by ~80% (P < 0.001) (Figure 4a), while inhibition fur-ther downstream with the RSK inhibitor SL0101 decreasedEGFR reporter activity by 30% (P = 0.02) (Figure 4a). Con-sistent with this observation, PD098059 and SL0101 pre-vented P-YB-1(S102) from binding to the EGFR promoterbased on chromatin immunoprecipitation (Figure 4b).Inhibition of RSK2 by siRNA in SUM149 cells (Figure 4c) ledto a decrease in EGFR expression. This downregulation wasmirrored in HTRY and MDA-MB-231 cells following treatmentwith SL0101 (Figure 4d); densitometric analysis for MDA-MB-231 gave a 35% decrease. We thereby conclude that there isto the EGFR, which in turn leads to activation of the MAPkinase/RSK pathway resulting in phosphorylation of YB-1 atS102. Activated AKT and PKCα also have the ability to acti-vate YB-1. Following this, P-YB-1S102 binds to and transacti-vates the EGFR gene, further fueling the growth potential ofBLBC (Figure 4e).DiscussionWe reveal for the first time that phosphorylation of YB-1 at theS102 location is not only carried out by the PI3K cascade butthat signaling through the MAP kinase pathway can also acti-vate this transcription factor. This is particularly relevant inBLBC, where EGFR is overexpressed in over one-half of thecases. More specifically it is the serine/threonine kinasesRSK1 and RSK2 that are able to phosphorylate YB-1 at thekey S102 residue in BLBC cells. Not only do we identify RSK1and RSK2 as proteins that can directly interact and phospho-rylate YB-1, but they have a much greater efficiency towardsthe target than AKT1 does. In fact, we also identified PKCα ashaving greater kinase activity towards YB-1 than AKT1, a find-ing that warrants future investigation. Phosphorylated RSK isalso expressed in cell lines where we find abundant P-YB-1S102 and a lack of active AKT; in particular the MDA-MB-231cells and the immortalized human mammary epithelial cells,where we were unable to detect any P-AKTS473. The RSK1/RSK2-specific inhibitor SL0101 [31,43], as well as RSK1-tar-geted or RSK2-targeted siRNA, were able to reduce the phos-phorylation of YB-1 at S102 even following induction by theclassic tumour promoter PMA. Furthermore, we observed areduced level of P-YB-1S102 in RSK2-/- MEFs. Finally, inhibitionof RSK prevented P-YB-1S102 binding to the EGFR promoterand ultimately reduced the protein expression of this receptortyrosine kinase.Our data are consistent with a recent study by Hoadley andcolleagues reporting that EGFR and genes encoding compo-nents of the MAP kinase pathway were associated with thebasal-like subtype, while AKT1 was not [44]. Interestingly, wefound in our four BLBC cell lines that ERK2 expression waspredominantly expressed over ERK1. This is in concordancewith the analysis observed by Hoadley and colleagues, whichshows expression of ERK2 was increased in the BLBC clus-ter, but this was not the case for ERK1 [44]. It is thus conceiv-able that ERK2 may activate RSK and therefore YB-1 in basal-like tumours. In this context it is also of interest that we in factfind YB-1S102 to be a better substrate for RSK1 and RSK2than AKT1. ERK2 may also directly phosphorylate YB-1 andtherefore promotes its ability to transactivate target genes. Insupport of this idea, ERK2 promotes the transactivation of vas-cular endothelial growth factor by YB-1 [45]. This occurswhen ERK2 phosphorylates the N-terminal region of YB-1; theregion of the protein required for gene transactivation [16].More recently, we identified a putative ERK phosphorylationsite at serine 36 in this same region of the protein using MotifTable 1Activity of RSK1, AKT1 and PKCα against the Y-box binding factor-1(YB-1) serine 102 residue peptide compared with the optimized positive control substrateKinase Activity against YB-1 peptide compared with control (%)RSK1 80 ± 1.04RSK2 78 ± 0.78AKT1 7 ± 0.7PKCα 19 ± 1.14The p90 ribosomal S6 kinases RSK1 and RSK2 phosphorylated a peptide that mimics the serine 102 residue region of YB-1 with 80% and 78% efficiency compared with the positive control substrate, respectively. Both AKT1 and PKCα were also able to phosphorylate the YB-1 peptide – 7% and 19%, respectively, compared with the positive control. Activity for control substrates for each kinase is normalized to 100%. A change > 5% is considered highly significant in this assay.Page 6 of 12(page number not for citation purposes)a feed-forward signaling pathway in BLBC where EGF binds Scanner [22]; however, this has not been validated experimen-Available online http://breast-cancer-research.com/content/10/6/R99tally. While speculative at this point, if ERK does phosphor-ylate the transactivating domain of YB-1 this could explain whyinhibiting ERK activity with PD098059 was better thanSL0101 at suppressing EGFR reporter activity. In theory,inhibiting ERK2 would directly decrease phosphorylation ofYB-1 at S36 at the N-terminal and indirectly block RSK fromphosphorylating S102. These studies indicate that the MAPkinase pathway would have broad effects on YB-1.While the emphasis of this study has been on BLBC, EGFR isequally important in promoting growth signals in other types ofHer-2 to engage signaling through either the MAPK or AKTpathways, which perhaps also involves RSK as well as AKT.This obviously could be important in stimulating the growth ofbreast cancer cells harboring amplified Her-2. Beyond breastcancer, we suspect that the relationship between RSK andYB-1 could be important in other malignancies. A study byCho and colleagues demonstrated that RSK2 was a trans-forming gene, since stable expression in skin cells increasedthe colony number in anchorage-independent conditions [40].Conversely, knockdown of RSK2 reduced colony formationeven in the presence of constitutively active oncogenic RasFigure 2p90 ribosomal S6 kinase phosphorylates Y-box binding factor-1 at the serine 102 residueal S6 kinase phosphorylates Y-box binding factor-1 at the serine 102 r sidue. (a) p90 ribosomal S6 kinase RSK1 is detected by immunoblotting following immunoprecipitation (IP) with Y-box binding factor-1 (YB-1) in SUM149 cells. Immunoprecipitation with IgY antibody was used to account for nonspecific binding (left). YB-1 is detected by pulling down and immunoblotting for RSK1. Immunoprecipitations performed with IgG antibody were used to account for nonspecific binding. Secondary detection was performed with horseradish peroxidase protein A (right). WB, western blot. (b) Transfection of SUM149 cells with RSK1, with RSK2 or with RSK1 and RSK2 siRNA for 72 hours reduces P-YB-1S102 while total YB-1 remains unchanged. Actin acts as a loading control (n = 3). (c) HCC1937 cells transfected with RSK1 or activated RSK (Myr-RSK1) express elevated levels of P-YB-1S102 compared with the control vector pKH3 (pRK7 for myr-RSK). A kinase-dead form of RSK (RSK1 KD) failed to induce P-YB-1S102 and was comparable with the control (n = 3). (d) RSK2-/- mouse embryo fibroblasts (MEFs) stimulated with epidermal growth factor (EGF) for a designated amount of time contain less P-YB-1S102 than the wild-type mice. Epidermal growth factor receptor (EGFR) is also reduced, unlike RSK1 that was expressed at a comparable level in both sets of MEFs. The RSK2 immunoblot confirms the genotype of the mice, and actin was used a loading control. The relative expression levels of EGFR in the RSK2-/- MEFs compared with wild-type MEFs are shown under the EGFR blot (n = 2). Ctrl, control.Page 7 of 12(page number not for citation purposes)breast cancer. For example, EGFR forms heterodimers with [40]. Other studies implicate RSK2 in transmitting the prosur-Breast Cancer Research    Vol 10 No 6    Stratford et al.Page 8 of 12(page number not for citation purposes)Figure 3Pharmacological inhibition of p90 ribosomal S6 kinase decreases Y-box binding factor-1 phosphorylationacological inhibition of p90 rib somal S6 kinas  d cr ases Y- ox binding factor-1 phosph rylation. (a) Inhibition of p90 ribosomal S6 kinases RSK1/RSK2 with SL0101 (50 μM) in SUM149 cells resulted in decreased growth-factor induced P-YB-1S102 over a time course of 6 to 16 hours. Immunoblot with densitometric analysis below. All changes are statistically significant (P < 0.01). (b) After 6 hours of treatment with SL0101 (100 μM), P-YB-1S102 was decreased in HCC1937 and MDA-MB-231 cells while Y-box binding factor-1 (YB-1) remained constant. (c) Treatment of HTRY cells with SL0101 (100 μM) decreased P-YB-1S102 in a dose-dependent manner. (d) Treatment of SUM149 cells with SL0101 (50 μM) reverses the phosphorylation of YB-1 induced by stimulation with growth factors. SL0101 has no effect on total YB-1. Vinculin was used as a load-ing control (n = 3). SS, stimulation of SUM149 cells; EGF, epidermal growth factor; PMA, phorbal 12-myristate 13-acetate.Available online http://breast-cancer-research.com/content/10/6/R99Page 9 of 12(page number not for citation purposes)Figure 4Inhibiting p90 ribosomal S6 kinase modulates Y-box binding factor-1 transcription factor abilityibiting p90 ribosomal S6 kinase modulates Y-box binding factor-1 transcription f ctor ability. Inhibiting p90 ribosomal S6 kinase (RSK) modulates the ability of Y-box binding factor-1 (YB-1) to act as a transcription factor for epidermal growth factor receptor (EGFR). (a) EGFR pro-moter activity in SUM149 cells was reduced by 80% following knockdown of YB-1 or treatment with PD098059 (***P < 0.001) and by 30% (*P = 0.02) following treatment with SL0101 (50 μM). (b) Binding of P-YB-1S102 to the EGFR promoter is reduced in the SUM149 cells following treat-ment with PD098059 (lane 4 compared with lane 3 (vehicle)) or SL0101 (50 μM) (lane 11 compared with lane 10 (vehicle)). IgG immunoprecipita-tion acts as a negative control. Input samples show amplification of the region in the cross-linked cells prior to immunoprecipitation (n = 2). DMSO, dimethylsulfoxide. (c) Transfection with RSK2 siRNA for 72 hours led to a decrease in EGFR expression in SUM149 cells. (d) Treatment of immor-talized breast mammary epithelial cells (HTRY) (10 hours) or MDA-MB-231 cancer cells (12 hours) with SL0101 results in loss of P-YB-1S102 and a concomitant reduction in EGFR. (e) Model demonstrating the positive feedback loop generated on the activation of YB-1 by EGFR. Ligand binding to the receptor activates signaling pathways such as MAP kinase, resulting in the phosphorylation of RSK. Once the kinase is fully activated, it phos-phorylates YB-1 at S102 – subsequently allowing YB-1 to play a role in promoting translation and to enter the nucleus as a transcription factor. AKT and PKCα can also activate YB-1 following growth factor stimulation. On binding to inverse CAAT boxes, YB-1 promotes the transcription of genes such as EGFR – resulting in increased surface expression of the receptor. Ctrl, control.Breast Cancer Research    Vol 10 No 6    Stratford et al.vival and proliferative signals from oncogenic mutant receptortyrosine kinase FGFR3 in multiple myeloma, resulting in celltransformation [28,46]. Interestingly, YB-1 has been impli-cated in the survival and progression of multiple myeloma cells– the expression correlating with rapid proliferation and poordifferentiation [47]. We therefore postulate a model whereRSK is activated through aberrant tyrosine kinase signaling,resulting in the subsequent phosphorylation of YB-1. In thisway the cell will be influenced by any number of a diverse col-lection of genes that YB-1 has been shown to regulate, suchas EGFR [1,14], Her-2 [14], topoisomerase II [5,7] and themultidrug resistance gene [48,49]. This regulation in fact mayresult in a positive feedback loop in the case of genes such asEGFR.Beyond regulating transcription, YB-1 also promotes transla-tion, alternative splicing, RNA transport and DNA repair[17,18,50-52]. Whether phosphorylation of YB-1 at S102 isimportant for these events is not known. Interestingly, RSKitself promotes translation through several mechanisms[23,39,53,54]; therefore, the role of these two proteins actingtogether in this process needs to be further investigated.ConclusionWe conclude that RSK1 and RSK2 are able to phosphorylateYB-1S102, providing a newly described mechanism wherebythis transcription factor is activated in breast cancer. In fact,RSK activates YB-1 more effectively than AKT and may there-fore be the major facilitator of YB-1 function in BLBC. Interestin developing small molecules against RSK has increased overthe past 2 years, and we believe this could be an importantopportunity for therapeutic intervention. As RSK has neverbefore been associated with BLBC, we therefore introduce anew mechanistic understanding and potentially a therapeuticstrategy for treating this aggressive disease.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsALS drafted the manuscript and performed experimentsunless stated otherwise. CJF and CD made the phospho-YB-1S102 antibody. AHD performed Flag-YB-1 for the kinaseassay. YL carried out the Iressa treatment. YYC and ZD pro-vided samples from EGF-stimulated RSK2-/- MEFs. IMB pro-vided the HTRY cells. PPR provided the RSK constructs andconceptual suggestions. SED conceived the studies and wasinvolved in editing the manuscript.AcknowledgementsThe authors are very grateful to Dr J Sanghera and Mr R Li at Signal-Chem (Richmond, BC, Canada) for carrying out the RSK1, AKT1 and PKCα kinase assays on the YB-1 peptide. Research in the laboratory of SED is supported by National Cancer Institute of Canada (NCIC), the Family Research Institute. Research in the laboratory of PPR is sup-ported by a Terry Fox Foundation grant obtained through the NCIC and a Career Development Award from the Human Frontier Science Pro-gram Organization. PPR holds a Canada Research Chair in Signal Transduction and Proteomics. IMB is supported by an R01.References1. Stratford AL, Habibi G, Astanehe A, Jiang H, Hu K, Park E, ShadeoA, Buys TPH, Lam W, Pugh T, Marra MA, Nielsen TO, Klinge U,Mertens PR, Aparicio S, Dunn SE: Epidermal growth factorreceptor (EGFR) is transcriptionally induced by the Y-boxbinding protein-1 (YB-1) and can be inhibited with Iressa inbasal-like breast cancer providing a potential target for ther-apy.  Breast Cancer Res 2007, 9:R61.2. 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