deregulation of the cell cycle by breast tumor kinase (brk)

Deregulation of the cell cycle by breast tumor kinase (Brk)

Edward Chan1 and Anjaruwee S. Nimnual2

1 Department of Pediatric Hematology/Oncology, State University of New York at Stony Brook, Stony Brook, New York 117942 Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794

Brk is a cytoplasmic nonreceptor tyrosine kinase that is overexpressed in breast tumors but undetectable in normal or benign

mammary tissues. Brk promotes proliferation of human mammary epithelial cells and tumor growth in a mouse model, but the

role of Brk in cell cycle regulation is not known. In this study, we describe the mechanism of Brk-induced deregulation of the

cell cycle. We provide evidence that Brk antagonizes the transcriptional activity of the transcription factor FoxO family of proteins

by inhibiting its nuclear localization. As a result, the cell cycle inhibitor p27, a FoxO target gene, is down-regulated. This event is

accompanied by G1/S cell cycle progression of quiescent cells. As p27 is a key regulator of the G1/S cell cycle checkpoint,

these data suggest that perturbation of p27 expression induced by Brk causes S phase entrance. Deregulation of the cell cycle is

a key event in neoplasia, and thus, the mechanism presented here likely contributes to breast cancer development.

Breast tumor kinase (Brk, PTK6) is a cytoplasmic nonrecep-tor tyrosine kinase associated with breast tumors. Whereasnormal mammary tissues or benign lesions express low orundetectable levels, �65% of breast tumors express Brk, with27% of the tumors overexpressing Brk by at least 5-fold.1 Brkis also expressed in other cancers including metastatic mela-nomas, and colon and prostate tumors.2 Considered to bedistantly related to the Src-family protein tyrosine kinases,Brk contains SH2, SH3, and tyrosine kinase domains, and iscapable of autophosphorylation.3 Brk promotes proliferationof human mammary epithelial cells and tumor growth in amouse model.4,5 Studies show that Brk is co-overexpressedwith and promotes the mitogenic signaling of epidermalgrowth factor receptors (EGFR).5,6 Brk interacts with EGFRand ErbB3 and, as a consequence, sensitizes the mitogeniceffect of EGF and activates the ErbB3/PI3-kinase/Akt path-way, respectively.6–8 A recent study shows that Brk and ErbB2genes coamplify in human breast cancers and the interactionbetween Brk and ErbB2 enhances the intrinsic kinase activityof Brk and mitogenic signaling of ErbB2.5 Furthermore, Brk

activates the Ras and Rac signaling pathways and insulin re-ceptor substrate-4 (IRS-4), phosphorylates and activates signaltransducer and activator of transcription (STAT) 3 andSTAT5b, key mediators of cytokine and growth factor signal-ing, and activates the MAPK pathway.8–13 Together, thesedata suggest that complex mitogenic signaling pathways areinvolved in the regulation of cell proliferation by Brk.

Cell cycle deregulation plays an important role in neopla-sia. Progression of the cell cycle from quiescence to mitosis istightly regulated at multiple steps by cyclins and cyclin-de-pendent kinases (CDKs). CyclinD/CDK4/6 and CyclinE/CDK2 complexes activate the transcription factor E2F result-ing in the expression of genes necessary for G1 to S phaseprogression. The kinase activity of CDKs is inhibited byCDK inhibitors (CDKIs), which bind to the cyclin/CDKcomplexes and block cell cycle progression.14 The CDKIp27(Kip1) (hereafter called p27), whose expression and activ-ity are modulated by mitogenic signaling, connects the extrac-ellular environment to cell cycle regulation. In the absence ofmitogens, the cellular level and activity of p27 are upregulatedleading to cell cycle arrest.15 In many types of cancer, p27 isderegulated through mechanisms, such as gene expression,proteolysis and functional inactivation.15 In this study, weinvestigated the role of Brk in the regulation of the cell cycle.We found that Brk downregulates the expression of p27through a mechanism that involves the modulation of the sub-cellular localization of the transcription factor FoxO3a, a p27transcription regulator. As a consequence, the cell cycle isderegulated and quiescent cells enter S-phase in a growth fac-tor-independent manner. These data provide insight into themechanism of cell cycle deregulation induced by Brk, whichmay contribute to the development of breast cancer.

Material and MethodsCell culture, constructs and reagents

The breast cancer cell line MDA-MB-231 was purchased fromAmerican Type Culture Collection. The cells were maintained

Key words: breast tumor kinase, Brk, p27, Cell cycle, forkhead

Additional Supporting Information may be found in the online

version of this article.

Grant sponsor: Department of Defense Congressionally Directed

Medical Research Programs; Grant number: W81XWH-05-1-0448

(A.S.N.); Grant sponsor: American Cancer Society, Mentored

Research Scholar Grant; Grant number: 117718-MRSG-09-172-01-

CCE (E.C.)

DOI: 10.1002/ijc.25263

History: Received 16 Sep 2009; Accepted 28 Jan 2010; Online

16 Feb 2010

Correspondence to: Anjaruwee S. Nimnual, Department of

Molecular Genetics and Microbiology, State University of New York

at Stony Brook, Stony Brook, NY 11794, USA, Tel: 631-632-8802,

Fax: 631-632-8891, E-mail: [email protected]

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Int. J. Cancer: 127, 2723–2731 (2010) VC 2010 UICC

International Journal of Cancer

IJC

as described by the manufacturer. cDNAs encoding humanBrk (generous gift from Dr. Todd Miller) were subclonedinto pCGN vector containing HA-tag epitope. The plasmidwas transfected into the cells utilizing lipofectamine-2000(Invitrogen). siRNAs targeting Brk (ON-TARGET plus smartpool predesigned PTK6, # L003166-00), FoxO3a (ON-TAR-GET plus smart pool #L003007-00-0005) and control (ON-TARGET plus siControl nontarget pool, #D001810-10) werepurchased from Dharmacon. The siRNAs were transfectedinto cells utilizing Dharmafect (Dharmacon). siRNA targetingp27 (Stealth Select RNAiTM siRNA, # 1299003) and control(Stealth Select RNAiTM siRNA negative control, # 12935-400)were purchased from Invitrogen and transfected into cellsutilizing Lipofectamine 2000.

MTT assay

Cell viability was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetricassay according to the manufacturer’s protocol (Roche Diag-nostics). The cells were seeded in 96-well tissue culture dishesat a density of 1 � 104 cells per well. Eighteen hours afterseeding, cells were transfected with plasmids or siRNAs, asindicated. Forty-eight hours after transfection, the cells weretreated with MTT reagent I followed by 4 hr incubation at37�C, subsequently, MTT reagent II was added and the incu-bation continued overnight. Cell number was determined byspectrophotometry utilizing an ELISA plate reader at 590 nm.

Flow cytometry and cell cycle analysis

The cells were labeled with 10 lM BrdU 45 min before tryp-sinization. The cell pellets were washed with PBS and fixedin 70% ethanol and stored at 4�C. Cells were collected bycentrifugation. The cells were denatured with 2M HCl, neu-tralized with 0.1M sodium borate and incubated with anti-BrdU antibody (BD biosciences), followed by fluorescence-conjugated goat-anti mouse (Invitrogen). The cell pelletswere resuspended in 10 lg/ml of propidium iodide contain-ing RNase and the suspension was incubated in the dark atroom temperature for 30 min. The cell suspension was ana-lyzed for DNA content on a FACScan flow cytometer (Bec-ton Dickinson). The percent of cells in different phases of thecell cycle was analyzed with CELLQUest software (BectonDickinson)

DNA synthesis (BrdU incorporation)

Cells were serum-starved for 30 hr before incubation withBrdU (10 lM) for 10 hr. Subsequently, the cells were fixedwith ethanol/acetic acid (95:5) and immunostained withBrdU antibody (Sigma), followed by Alexa-conjugated goat-anti mouse (Invitrogen) and DAPI (Sigma). In brief, after fix-ation, cells were rehydrated in PBS, washed with TBST(20 mM Tris, 150 mM NaCl and 0.1% Tween20) and dena-tured in 2N HCl. Subsequently, cells were neutralized in0.1M sodium borate (pH 8.5) and rinsed with TBST beforeincubation with BrdU antibody for 1 hr, and secondary anti-

body for 1 hr. DAPI was added at the last 15 min. The imagewas captured by a Zeiss Axiovert 200M microscope (Zeiss).

Cell extraction

Cells were lysed in lysis buffer containing 150 mM NaCl,10 mM Tris-HCl (pH 7.4), 1% Triton X-100, 10% glycerol,1 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadateand protease inhibitors. The cell lysates were centrifuged at14,000 rpm for 10 min and the supernatant was treated with5� Laemlli buffer. The proteins were resolved by SDS-PAGE.

Quantitative RT-PCR

Total RNA from cells was isolated by TRIzol (Invitrogen)and purified by RNeasy Mini Kit and RNase-free DNase Set(Qiagen) according to the manufacturer’s protocols. Reversetranscription was carried out using a SuperScript Preamplifi-cation Kit (Life Technologies) on 1 lg of total RNA aliquots.PCR was performed using capillary LightCycler (Roche, Indi-anapolis, IN) with the following p27 primers: 50-TGG CCAGGA TTG CTA CAG TTG-30 and 50-CGC CGC GGA CATCAT CTT-30

Immunofluorescence staining

Cells grown on coverslips were fixed for 1h with 3.7% form-aldehyde. After fixation, the cells were washed with PBS, per-meabilized with 0.1% Triton X-100, rinsed with PBS andblocked with 2% BSA before incubation with FoxO3a anti-body for 1 hr, and Rhodamine-conjugated goat anti-rabbit(Invitrogen) for 1 hr. The images were captured using a ZeissAxiovert 200M digital deconvolution microscope (Zeiss).

Subcellular fractionation

Cells were lysed in hypotonic buffer (10 mM HEPES pH7.4,10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM DTT, 10mM NaF, 1 mM sodium orthovanadate and protease inhibi-tors). Nuclei were sedimented by centrifugation (2,000 rpm,10 min) and the cytoplasmic fraction was retained. Thenuclei were extracted in the hypotonic buffer containing 1%NP40 and 400 mM NaCl. The nuclear and cytoplasm frac-tions were resolved by SDS-PAGE.

ResultsBrk promotes cell proliferation

We first assessed the role of Brk in the proliferation ofMDA-MB-231, an estrogen receptor- and ErbB2-negativebreast carcinoma cell line expressing detectable levels of en-dogenous Brk. The cells were transfected with siRNA target-ing Brk (siBrk) to suppress the expression of Brk or scramblesiRNA (siSc) as a control. Forty-eight hours after transfec-tion, cell proliferation was analyzed by MTT assay. As shownin Figure 1a, suppression of Brk caused a significant decreasein cell proliferation. We did not observe cell death in the cul-ture suggesting that this decrease was not due to loss of cellviability. Similarly, knockdown of endogenous Brk in thebreast carcinoma cell lines T47D and SKBr3 resulted in a

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2724 Deregulation of the cell cycle by BrK


decrease in cell number of 60 and 50%, respectively, (datanot shown) indicating that breast cancer cells require Brk forcell proliferation. We next examined whether elevated levelsof Brk would enhance the cell proliferation rate. MDA-MB-231 cells were transfected with pCGN HA-tagged Brk(pCGNBrk), BrkK219M, a kinase defective mutant of Brk(pCGNBrk-KD), or empty vector. Forty-eight hours aftertransfection, the MTT assay was performed. Overexpressionof Brk enhanced cell proliferation, whereas BrkK219M hadno effect on cell proliferation (Fig. 1b). Together, these resultsindicate that Brk plays an important role in the proliferationof these cells and the mechanism of the regulation is depend-ent on the kinase activity of Brk.

Brk induces G1 to S phase progression

To maintain normal cell proliferation, progression throughthe cell cycle is tightly regulated at multiple steps. Growthfactor stimulation transiently downregulates the activity ofcell cycle inhibitors allowing cells to exit quiescence and enterthe cell cycle. Several oncogenes induce cell cycle progressionin the absence of mitogens by deregulating the cell cyclecheckpoint machinery. Brk has previously been implicated inG1/S phase transition4 and has been implicated as an onco-gene. Therefore, we investigated whether Brk was able toinduce S-phase entry in a growth factor-independent manner.Bromodeoxyuridine (BrdU) incorporation assay was per-formed to monitor DNA synthesis. MDA-MB-231 cells weretransfected with pCGNBrk or vector, and the cells were se-rum starved before BrdU addition. Subsequently, cells werefixed, immunostained and scored for cells that enteredS phase. As demonstrated in Figures. 2a and 2b, 70% of cells

transfected with Brk had incorporated BrdU, compared with18% of control cells. This data suggests that Brk induces G1/Sphase progression in a growth factor-independent manner. Toconfirm that Brk induces G1/S phase progression, FACS analy-sis was performed. Cells were transfected with pCGNBrk orcontrol vector and serum starved for the indicated times.Subsequently, the cells were double labeled with BrdU andpropidium iodide before flow cytometry. As shown in Figures2c and 2d, the S-phase population of Brk-transfected cells is�2-fold higher than that of control cells, with Brk promotingG1 to S phase progression while control cells entered quies-cence. These results support the immunostaining data thatBrk induces cell cycle progression in the absence of serum.The population of Brk-transfected cells entering S-phasedoes, however, progressively decline over time (Figures 2cand 2d). By 48 h, less than 20% of Brk-expressing cells are inS-phase, while less than 5% of control cells were detected inS-phase (data not shown). Together, these data suggest thatBrk plays a key role in S phase progression by promoting Sphase entry and delaying S phase exit in a growth-factor in-dependent manner.

Brk inhibits p27 expression

We next investigated the pathway that connects Brk to cellcycle regulation. The CDKI p27 plays a key role in the regu-lation of the G1/S cell cycle checkpoint and it is controlledby growth factor levels. Therefore, we investigated whetherBrk regulates p27. MDA-MB-231 cells were transfected withpCGNBrk or vector, followed by serum-starvation to inducequiescence. Subsequently, p27 expression levels were observedby immunoblot analysis. Overexpression of Brk induced a

Figure 1. Brk promotes cell proliferation. MDA-MB-231 cells were transfected with siBrk or siSc (Dharmacon) (a) or pCGNBrk, pCGNBrk-KD

or vector (b). Forty-eight hours after transfection, the MTT colorimetric assay was performed according to the manufacturer’s protocol

(Roche Diagnostics). Graph represents the number of viable cells expressed relative to control. Data are the mean of three independent

experiments, þ/� SD. (c) Cells were transfected with siBrk, siSc, pCGNBrk, pCGNBrk-KD or vector, as indicated, and were harvested in

Laemmli buffer 48 hours after transfection. Whole cell lysates were subjected to SDS-PAGE followed by immunoblot analysis with Brk and

actin antibodies (Santa Cruz biotechnology). Actin was used to indicate equal protein loading. Transfection efficiency, as verified by GFP

expression, was 80–90% (data not shown).

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Chan and Nimnual 2725


significant decrease in p27 levels indicating that Brk down-regulated p27 (Figs. 3a and 3b). To further examine the effectof Brk on p27 expression in a physiological setting, p27 levelswere examined in cells in which Brk was knocked down.siBrk was transfected into MDA-MB-231 cells followed by se-rum-starvation. Suppression of endogenous Brk led to anincrease in the levels of p27 (Figs. 3c and 3d) demonstratingthat Brk mediated p27 downregulation in a physiological set-ting. We next examined the effect of Brk on the mRNA levelsof p27 by performing quantitative PCR. Cells were trans-fected with Brk or control vector, followed by serum starva-tion before RNA extraction and quantitative PCR analysis.As shown in Figure 3e, p27 mRNA levels were significantlylower in cells expressing Brk compared with control cellsindicating that Brk downregulated p27 at the mRNA level. Inaddition to being regulated at the transcriptional level, p27 is

also regulated by ubiquitin-mediated degradation. Previouslyit has been shown that Src induces phosphorylation of tyro-sine 88 of p27 which promotes the SCF-Skp2-dependent deg-radation of p27.16 Therefore, we examined whether Brk hadan effect on p27 degradation and phosphorylation. MDA-MB-231 cells were transfected with Brk or vector, followed byserum-starvation. The cells were treated with the proteasomeinhibitor MG-132 for 6 hr before analyzing the levels of p27.Treatment with MG-132 did not alter the effect of Brk onp27 suggesting that Brk does not downregulate p27 by proteindegradation (Figs. 3f and 3g). Furthermore, analysis of tyro-sine phosphorylation in cells transfected with Brk revealedthat Brk did not affect the tyrosine phosphorylation of p27(Supporting Information Fig. S1). As tyrosine-88 is the princi-pal phosphoacceptor site of p27,16 this result suggests that,unlike Src, Brk does not play a role in p27 phosphorylation.

Figure 2. Brk induces DNA synthesis under serum-starved conditions. MDA-MB-231 cells were transfected with pCGNBrk or vector. Eighteen

hours after transfection, cells were serum-starved for 30 hr before incubation with BrdU (10lM) for 10 hr. Cells were fixed and

immunostained with BrdU antibody (BD Biosciences), followed by Alexa-conjugated goat-anti mouse (Invitrogen) and DAPI (Sigma). The

image was captured by a Zeiss Axiovert 200 M microscope (Zeiss). Cells that had incorporated BrdU were scored and graphed. (a), BrdU-

positive (green) and DAPI-stained (blue). (b) The percentage of BrdU-positive cells relative to the number of total cells. At least 200 cells

per transfection were counted. Data are the mean of three independent experiments, þ/� SD. (c and d) MDA-MB-231 cells were

transfected with pCGNBrk or vector. Eighteen hours after transfection, cells were serum-starved for 12, 24, or 36 hr, as indicated and BrdU

(10 lM) was added to the cell culture 45 min before fixation. The fixed cells were stained with anti-BrdU antibody followed by

fluorescence-conjugated goat-anti mouse (Invitrogen) and propidium iodide (PI). The cell cycle was analyzed by FACS. (c) Representative

flow cytometric histograms of the cells labeled with PI and fluorescence. G1- and S-phase cells are located in lower left and top right þ top

left panels, respectively. (d) Graph represents the percentage of S-phase cells determined from the dual parameter histograms. Data are

the mean of three independent experiments, 6 SD. Gray and black columns represent vector and Brk-transfected cells, respectively.

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Brk inhibits nuclear localization of FoxO3a

Expression of p27 is activated by the transcription factorforkhead boxO (FoxO) family, which is comprised of 4 mem-bers, FoxO1, FoxO3a, FoxO4 and FoxO6. The nuclear local-ization of FoxO proteins is pivotal to their transcriptionalactivities, and is negatively regulated by Akt-mediated proteinphosphorylation.17 The phosphorylated FoxO proteins bindto the chaperone protein 14-3-3, which disrupts the associa-tion of FoxOs with DNA. Subsequently, 14-3-3-bound FoxOproteins are exported into the cytoplasm and, as a conse-quence, the proteins fail to activate gene expression. As Brkhas been shown to activate the PI3-kinase/Akt pathway,6 wehypothesized that Brk downregulates p27 by inducing the nu-clear exclusion of FoxO proteins. To test this, we examinedthe effect of Brk on FoxO subcellular localization. We usedFoxO3a (FKHRL1) as it is a major FoxO family proteinexpressed in MDA-MB-231. pCGNBrk or vector was trans-fected into MDA-MB-231 cells and subsequently, the cellswere serum starved. Following serum starvation, the cells

were harvested and subjected to subcellular fractionation fol-lowed by SDS-PAGE and immunoblot analysis. Expression ofBrk induced a marked reduction in the nuclear fraction ofFoxO3a (Figs. 4a and 4b). However, we did not detect a sig-nificant increase of the cytoplasmic fraction of FoxO3a in thepresence of Brk. As FoxO proteins are degraded in the cyto-plasm, we predicted that this may be the reason that therewas no significant change detected in levels of the cytoplas-mic FoxO3a. To test this, we treated cells with the protea-some inhibitor MG-132. MDA-MB-231 cells were transfectedwith pCGNBrk or vector, followed by serum-starvation. Thecells were treated with MG-132 for 3 hr before analyzing thelevels of FoxO3a. Treatment with MG-132 led to elevatedlevels of cytoplasmic FoxO3a in both control and Brk trans-fected cells, indicating that FoxO3a is degraded in the cytosoland that Brk induces the sequestration of FoxO3a in thecytoplasm (Supporting Information Fig. S2). Brk had noeffect on FoxO3a gene expression, as the mRNA levels ofFoxO3a were similar in control and Brk-transfected cells

Figure 3. Brk inhibits p27 expression. MDA-MB-231 cells were transfected with pCGNBrk or vector (a, b), or siSc or siBrk (c,d). Eighteen

hours after transfection, cells were serum-starved for 30 hr before being harvested in lysis buffer. The cell lysates were subjected to SDS-

PAGE followed by immunoblot analysis with antibodies against p27 (Santa Cruz biotechnology), Brk and actin. Graphs represent p27

expression relative to control. (e) Analysis of mRNA levels. Cells were transfected as in (a, b) and RNA was extracted. The amount of p27

mRNA was analyzed by quantitative RT-PCR and normalized to the housekeeping gene S26 ribosomal subunit. Graph represents p27 mRNA

levels relative to control. (f,g) cells were transfected with pCGNBrk or vector, serum-starved for 30 hr and treated with MG-132 (10 lM) for

the last 6 hr. Cells were harvested in lysis buffer and subjected to immunoblot analysis as described in (a, b). Graph represents p27

expression relative to control. Data are the mean of three independent experiments, 6 SD.

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(data not shown). To determine if Brk altered the subcellularlocalization of FoxO3a, MDA-MB-231 cells were cultured onglass-coverslips and cotransfected with CMV-GFP andpCGNBrk. Subsequently, cells were serum starved, fixed andstained with FoxO3a antibody. The localization of FoxO3awas observed by fluorescence microscopy. As demonstratedin Figure 4c, the majority of FoxO3a localized in the nucleiof control cells, however, in Brk-transfected cells, indicatedby GFP expression, FoxO3a was excluded from the nucleusand present in the cytoplasm. This result supports the frac-tionation data that Brk induces nuclear exclusion of FoxO3a.

We next investigated whether the PI3-kinase/Akt pathwaymediated the Brk-induced nuclear exclusion of FoxO3a.MDA-MB-231 cells were transfected with pCGNBrk or vec-

tor and serum starved. The cells were incubated with thePI3-kinase inhibitor LY294002 for 14 hr before subcellularfractionation. Treatment with LY294002 inhibited the antago-nistic effect of Brk on FoxO3a localization in the nucleussuggesting that the regulation of FoxO3a localization by Brkis mediated by the PI3-kinase pathway (Fig. 4d and 4e).

p27 and FoxO3a play an important role in the

regulation of cell proliferation by Brk

We next asked if Brk promotes cell proliferation throughdownregulation of the FoxO3a/p27 pathway. MDA-MB-231cells were transfected with siSc, sip27, and/or siRNA targetingFoxO3a (siFoxO3a) together with pCGNBrk or vector, asindicated. Forty-eight hours after transfection, cells were

Figure 4. Brk inhibits nuclear localization of FoxO3a. (a) MDA-MB-231 cells were transfected with pCGNBrk or vector. Eighteen hours after

transfection, cells were serum-starved for 30 hr before subcellular fractionation. The nuclear and cytoplasm fractions were subjected to SDS-

PAGE, followed by immunoblot analysis with antibodies against FoxO3a (Santa Cruz). A fraction of the samples was analyzed by

immunoblotting with antibodies against the nuclear marker c-Jun (Santa Cruz), and the cytosol marker MEK1/2 (Santa Cruz). (b) Graph

represents the nuclear and cytoplasm fractions of FoxO3a expressed as fold over control. Data are the mean of 3 independent experiments;

þ/� SD. (c) Cells were cotransfected with CMV-GFP and pCGNBrk. Eighteen hours after transfection, cells were serum starved for 30 hr and

fixed with 3.7% formaldehyde. Cells were permeabilized and incubated with FoxO3a antibody followed by Rhodamine-conjugated goat anti-

rabbit (Invitrogen). The image was captured by a Zeiss Axiovert 200M digital deconvolution microscope (Zeiss). Arrows indicate GFP expressing

cells (top panels). Higher magnification of representative control and Brk expressing cells are shown. DAPI staining reveals cell nuclei. (bottom

panels). (d,e) Cells were transfected with the indicated plasmids, and serum starved for 30 hr. During the final 14 hours, cells were treated

with 10lM LY294002 (Sigma). Subsequently, subcellular fractionation was performed, and the nuclear fraction was subjected to SDS-PAGE

and immunoblot analysis. Graph represents the nuclear fraction of FoxO3a. Data are the mean of 3 independent experiments, 6 SD.

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subjected to the MTT assay. As shown in Figure 5a, expres-sion of Brk led to a significant increase in cell proliferation,compared to control (siSc þ vector). Knockdown of eitherp27 or FoxO3a enhanced cell proliferation but to a lesserextent than that of Brk expression, suggesting that there is anadditional pathway(s) mediating Brk induction of cell prolif-eration. Codepletion of both p27 and FoxO3a produced aneffect similar to depletion of either one alone suggesting alinear pathway of FoxO3a to p27. Furthermore, expression ofBrk in cells depleted of either p27 or FoxO3a or codepletedof p27 and FoxO3a produced an effect on cell proliferationsimilar to Brk expression alone, indicating a linear signalingpathway from Brk to FoxO3a and subsequently to p27.

As our data indicates that the FoxO3a/p27 pathway is notthe sole pathway responsible for Brk inducing cell prolifera-tion, we assessed the contribution of FoxO3a and p27 inBrk-induced cell proliferation. MDA-MB-231 cells werecotransfected with siSc, sip27 or siFoxO3a together withpCGNBrk or vector, as indicated. Forty-eight hours aftertransfection, cells were subjected to the MTT assay. Expres-sion of Brk led to a 2.3-fold increase in cell number comparedwith vector control (Fig. 5b). However, in a p27-knockdownbackground, the effect of Brk was reduced to 1.5-fold. Sincethe proliferative effect of Brk is markedly reduced in cells inwhich p27 was depleted, it indicates that p27 plays a signifi-cant role in the regulation of cell cycle by Brk. Correspondingto the p27-knockdown result, the proliferative effect of Brkwas also decreased by 1.5-fold in cells in which FoxO3a wasknocked down (Fig. 5b). These results indicate a significantrole of both FoxO3a and p27 in the mitogenic signaling path-way of Brk. Taken together these data suggest that downregu-lation of the FoxO3a/p27 pathway plays an important role inthe mechanism of Brk-induced cell proliferation.

DiscussionBrk promotes cell proliferation and has been associated withmultiple mitogenic pathways. However, the role of Brk in thecell cycle is still elusive. Here, we present a mechanism ofBrk-induced cell cycle progression. We demonstrate that Brkdownregulates the cell cycle inhibitor p27 by altering FoxO3asubcellular localization. p27 is a key component of the G1/Scell cycle checkpoint and downregulation of p27 promotes Sphase entry. The progression of the cell from G1 to S phase,induced by Brk, is independent of growth factors suggestinga role for Brk in malignant transformation. Downregulationof p27 occurs through multiple mechanisms. One majormechanism is the activation of Skp2-mediated ubiquitina-tion-dependent degradation.18 Surprisingly, our experimentsindicated that Brk does not down-regulate p27 via the ubiqui-tin-proteasome pathway (Figs. 3f and 3g). Further study isneeded to understand how Brk fails to induce p27 degrada-tion. Our data indicates that FoxO3a appears to be a majorcomponent in p27 down-regulation by Brk, as the effect ofBrk on p27 expression is significantly abrogated in cells trans-fected with siRNA targeting FoxO3a (Supporting Information

Figure 5. p27 and FoxO3a play an important role in the regulation

of cell proliferation by Brk. MDA-MB-231 cells were transfected

with siSc, sip27, and/or siFoxO3a together with pCGNBrk or vector,

as indicated, for 48 hr before analysis by the MTT assay. (a) Graph

represents the cell number expressed relative to control. Data are

the mean of three independent experiments, þ/� SD. (b and c)

MDA-MB-231 cells were transfected with sip27, siFoxO3a, or siSc

together with pCGNBrk or vector for 48 hr before analysis by the

MTT assay (b) or extraction of protein followed by SDS-PAGE and

immunoblotting (c). (b) Graph represents the number of cells

expressed relative to control. Data are the mean of three

independent experiments, þ/� SD. (c) Whole cell lysates were

subjected to SDS-PAGE followed by immunoblot analysis with p27,

FoxO3a (Santa Cruz) and actin antibodies.

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Fig. S3). Furthermore, our results indicate that both FoxO3aand p27 are essential targets of Brk in promoting cell prolif-eration since the ability of Brk to affect cell proliferation issignificantly reduced in FoxO3a or p27 knockdown cells.However, the effect of Brk on cell proliferation is not abol-ished by p27 suppression which indicates additional key fac-tors in Brk signaling. Such factors may include the Ras/MAPK pathway and cyclinE/cdk2 activity, which have beenshown to mediate the effect of Brk in enhancing ErbB2-induced cell proliferation and tumorigenesis in mice.5

The regulation of FoxO3a subcellular localization by Brkis inhibited by LY294002 indicating that the mechanism ismediated by the PI3-kinase/Akt pathway. This data is sup-ported by previous reports demonstrating that Akt phospho-rylates FoxO proteins, which promotes their binding to theadaptor protein 14-3-3.17 As a result, FoxOs are exportedfrom the nucleus. The relationship between Brk and Aktneeds further elucidation since, depending on the cellularcontext, Brk can activate or inhibit Akt activity.6,19,20 Addi-tionally, the mechanism of Brk-induced activation of the PI3-kinase pathway in a growth factor-independent manner iscomplex and needs further investigation. A previous studyshowed that Brk elevated the levels of tyrosine phosphorylationof ErbB3 and induced association of ErbB3 with the p85 subu-nit of PI3-kinase in quiescent HB4a cells.6 This data suggests

that the physical interaction of Brk with ErbB3 may induce aconformational change that is sufficient for ErbB3 to recruitPI3-kinase. However, ErbB3 may not be the sole mediatorlinking Brk to Akt activation since Brk also influences otherPI3-kinase activators including Ras and EGFR pathways.5,6,9

Brk has multiple substrates which may impact the role ofBrk in cancer. For example, Brk promotes cell proliferationupon interacting with EGF receptors and STAT3, but inducescell cycle arrest when bound to the polypyrimidine tract-binding (PTB) protein-associated splicing factor PSF.5,12,21

Therefore, it is essential to define the signaling cascade ofBrk and understand the regulation of its substrate selectionto control cancer development in cells overexpressing Brk.

Our findings indicate that Brk modulates the subcellularlocalization of FoxO proteins and down-regulates the expres-sion of p27. FoxO proteins regulate the expression of genesinvolved in all phases of the cell cycle as well as in apoptosis,and their role is crucial in preventing neoplasia. Therefore,the mechanism presented here is likely to play an importantrole in cancer development.

AcknowledgementsThe authors thank Dr. Laura Taylor for invaluable comments and insightfuldiscussions; Ms. Mei-ling Chin and Mr. James Keller for technical expertise;and Dr. ToddMiller for the Brk genes.

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4. Harvey AJ, Crompton MR. Use of RNAinterference to validate Brk as a noveltherapeutic target in breast cancer: Brkpromotes breast carcinoma cellproliferation. Oncogene 2003;22:5006–10.

5. Xiang B, Chatti K, Qiu H, Lakshmi B,Krasnitz A, Hicks J, Yu M, Miller WT,Muthuswamy SK. Brk is coamplified withErbB2 to promote proliferation in breastcancer. Proc Natl Acad Sci USA 2008;105:12463–8.

6. Kamalati T, Jolin HE, Fry MJ, CromptonMR. Expression of the BRK tyrosinekinase in mammary epithelial cellsenhances the coupling of EGF signalling toPI 3-kinase and Akt, via erbB3phosphorylation. Oncogene 2000;19:5471–6.

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specific substrate of breast tumor kinase.Oncogene 2006;25:4904–12.

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breast tumor kinase. J Biol Chem 2005;280:1982–91.

20. Haegebarth A, Bie W, Yang R, CrawfordSE, Vasioukhin V, Fuchs E, Tyner AL.

Protein tyrosine kinase 6 negativelyregulates growth and promotes enterocytedifferentiation in the small intestine. MolCell Biol 2006;26:4949–57.

21. Lukong KE, Huot ME, Richard S. BRKphosphorylates PSF promoting itscytoplasmic localization and cell cyclearrest. Cell Signal 2009;9:1415–22.

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deregulation of the cell cycle by breast tumor kinase (brk)

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