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Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic… Hu, Kaiji; Law, Jennifer H; Fotovati, Abbas; Dunn, Sandra E Feb 6, 2012

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RESEARCH ARTICLE Open AccessSmall interfering RNA library screen identifiedpolo-like kinase-1 (PLK1) as a potentialtherapeutic target for breast cancer that uniquelyeliminates tumor-initiating cellsKaiji Hu, Jennifer H Law, Abbas Fotovati and Sandra E Dunn*AbstractIntroduction: Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence oftumor-initiating cells (TICs), molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapyand radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore,we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.Methods: A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened againstthe TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancercell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including itsexpression in breast cancer cell lines, effect on the CD44high/CD24-/low TIC subpopulation, growth inhibition,mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.Results: Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231(TNBC), BT474-M1 (ER+/HER2+, a metastatic variant), and HR5 (ER+/HER2+, a trastuzumab-resistant model) cells andshowed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44highsubpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growthinhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed inbreast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. ThePLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44high/CD24-/low populationcompared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggestingthat it could potentially be used to prevent relapse.Conclusion: Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TICsubpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatmentof TNBC as well as other subtypes of breast cancer.* Correspondence: sedunn@interchange.ubc.caLaboratory for Oncogenomic Research, Departments of Pediatrics,Experimental Medicine, and Medical Genetics, Child and Family ResearchInstitute, University of British Columbia, 950 W. 28th Ave, Vancouver, BritishColumbia, V5Z 4H4, CanadaHu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22© 2012 Dunn et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.IntroductionTriple-negative breast cancer (TNBC) is considered themost aggressive breast cancer subtype because it is asso-ciated with the greatest probability of early relapse anddeath [1-3]. It is estimated that more than 1 millionwomen are diagnosed with breast cancer annually, andTNBC accounts for about 15% of those cases [4]. Theyare challenging clinically for a number of reasons. Theydo not express the estrogen receptor (ER), progesteronereceptor, and human epidermal growth factor 2 (HER2).Therefore, patients are not candidates for targeted agents,such as antiestrogens and trastuzumab, that afford thegreatest survival benefit for eligible patients. The prog-nosis for patients with this type of tumor is very poor,not only because hormonal therapy and treatment withtrastuzumab are inapplicable, but also because thesetumors seem to be more aggressive than other breastcancer subtypes [5].Although it is highly sensitive to chemotherapy, theprogression-free time of TNBC, however, is generallyshort, and has greater recurrence rates than those ofnon-TNBC tumors during the first and third years aftertheir initial diagnosis, as well as a higher 5-year mortalityrate [3,4]. The high rates of early relapse indicate that thetumor cells rapidly adapt to the insult of chemotherapyby inducing resistance mechanisms. In addition, theadverse side effects of traditional chemotherapy are inevi-table for patients with TNBC, which leads to the notablemorbidity associated with treating this particular breastcancer subtype. Thus, identifying specific molecular tar-gets against TNBC is timely and essential.No currently accepted therapeutic target is known forTNBC, unlike some other subtypes of breast cancer [4].ER-expressing breast tumors, for instance, can be treatedwith tamoxifen and aromatase inhibitors, and HER2-expressing ones can be treated with trastuzumab. Ongoingstudies are searching for new drug targets against TNBC.One such development is the inhibition of poly (ADP-ribose)-polymerase 1 (PARP1) [4,6]. PARP1 plays a vitalrole in repairing DNA damage together with othermechanisms that involve BRCA1 and BRCA2. The combi-nation of the mutation of BRCA and PARP inhibitionattributed to so-called synthetic lethality [6,7]. Theimpressive clinical phase II results involving these criteriahave led to a definitive phase III study [4]. Although this ispromising, BRCA1 and BRCA2 mutations account forslightly more than 10% of breast cancers that are triple-negative [8]. Other therapeutic targets under developmentfor TNBC include epidermal growth factor receptor(EGFR), mammalian target of rapamycin (mTOR), theRAS-mitogen-activated protein kinase signaling pathway(Raf/Mek/MAP), and Src tyrosine kinase [4,9]. However,some of these proposed targets are applicable only inmore-specific subgroups of TNBC, and the ways to tacklethe tumor-initiating subpopulation, which is believed to bethe root cause of the relapse of cancer, have not been fullystudied. For breast cancer, it has been proposed that thesubpopulation cells of CD44high/CD24-/low have cancerstem cell properties [10,11]. Such cancer stem cells ortumor-initiating cells (TICs) are resistant to traditionalchemotherapies and are considered to be responsible forcancer relapse [10-13]. It has been reported that treatmentwith traditional chemotherapies leads to enriched TICsboth in vitro and in vivo [14-17]. Thus targeting the bulkcancer cell population, as well as TICs, should be consid-ered at the early stage of the search for therapeutic targets.Kinases play an essential role in the processes of pro-tein phosphorylation and are deregulated in many dis-eases, such as cancer. Numerous studies have provedthat many kinases are critical in cancer cell survivalunder both in vitro and in vivo conditions [18-20].Kinases are eminently the most treatable with drugs.Some new drugs of kinase inhibitor, such as imatinib(Gleevec), fasudil, and rapamycin, have been successfullydeveloped and applied clinically for treatment of a vari-ety of cancers [21,22]. For TNBC, it has been shownthat several kinases could be targeted for growth inhibi-tion, including MAP kinase, Src tyrosine kinase(PDGFR, EGFR, IGF-1R, and HGFR), RSK kinases[4,9,23,24]. More important, targeting kinases resultingin growth inhibition of TICs of different cancers hasbeen reported [25,26]. Prochownik et al. [13,27] foundthat CGP74514A and rottlerin, which are kinase inhibi-tors of CDK1/cyclin B and PKC, respectively, can selec-tively inhibit cancer stem cells isolated from the breastcancer cell line MCF7. The availability of a large kinasesmall interfering RNA (siRNA) library provides an excel-lent tool for an unbiased genome-wide screen for activekinases as potential therapeutic targets against not onlythe bulk cancer cells but also TICs.In this study, we first performed a genome-wide humankinase siRNA library screen against a TNBC cell lineSUM149 for growth inhibition. A panel of selected activekinases was then further tested on four different breastcancer cell lines to confirm the spectrum of growth inhi-bition. Several kinases that also inhibited the tumor-initi-ating CD44high population in SUM149 after siRNAtreatments were identified and tested directly againstsorted CD44high/CD24-/low cells of SUM149. The mostimpressive kinase lead was polo-like kinase 1 (PLK1).Therefore, we focused on PLK1 inhibition as the bestpotential therapeutic lead for TNBC by showing that it ishighly expressed in breast cancer cell lines, and its inhibi-tion by PLK1 siRNA as well as BI 2536, an ATP-competi-tive inhibitor designed to inhibit PLK1 [28], killed theCD44high/CD24-/low population and induced apoptosis.Hu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 2 of 15Combined treatment with drugs and BI 2536 greatlyinhibited the growth of TNBC. Therefore, it offers poten-tial as a better therapeutic target for TNBC.Materials and methodsCell cultureSUM149 cells were purchased from Astrand (Ann Arbor,MI, USA) and cultured in F-12 (Ham) media (Gibco/Invitrogen, Burlington, ON, Canada) supplemented with5 μg/ml insulin (Sigma-Aldrich, Oakville, ON, Canada),1 μg/ml hydrocortisone (Sigma-Aldrich), 10 mM HEPES(Sigma-Aldrich), and 5% fetal bovine serum (FBS; Gibco/Invitrogen). MDA-MB-231 and MCF7 were purchasedfrom ATCC and cultured in Dulbecco Modified Eaglemedium (DMEM, Gibco/Invitrogen) with 10% FBS.BT474-M1, a metastatic variant of BT474, was a gift ofDr. Mien-Chie Hung (MD Anderson Cancer Center,Houston, TX, USA). HR5, which is derived from BT474and is resistant to trastuzumab, was from Dr. CarlosArteaga (Vanderbilt-Ingram Cancer Center, Nashville,TN, USA). They were both cultured in DMEM-F12 (1:1)with 10% FBS. AU565, HCC1937, and T47D (ATCC)were cultured in RPMI-1640 media supplemented with5% FBS (Gibco/Invitrogen), 10 mM HEPES, 4.5 g/L glu-cose, 1 mM sodium pyruvate, and 100 units/ml penicil-lin/streptomycin (Sigma-Aldrich). All the cells wereincubated at 37°C with 5% CO2, and subcultured twiceweekly during the experimental period.Kinase siRNA libraryThe siRNA library (Vision 2) of 691 human kinases waspurchased from Qiagen (Toronto, ON, Canada). Two dif-ferent sequences of siRNA target each of the genes in thelibrary. The siRNA stock samples were diluted to workingstocks at 2 μM on arrival by following the manufacturer’sinstructions and stored at -20°C before use.Kinase siRNA library screenThe screening methods were previously described [19]. Inbrief, SUM149 cells were seeded (5,000 cells/well) into 96-well plates (BD; Becton Dickinson, Franklin Lakes, NJ,USA) overnight. The cells were transfected with siRNA inLipofectamine RNAiMAX (Invitrogen) at 5 nM for72 hours. Cells were then fixed in 2% paraformaldehyde(Sigma-Aldrich) with nuclear dye, Hoechst 33342 (1 μg/ml) (Sigma-Aldrich). After a gentle wash with phosphate-buffered solution (PBS), the cells were kept in fresh PBS,and the plates were kept at 4°C in the dark before analysison the ArrayScan high-content screening system (HCS;Thermo Fisher Scientific, Pittsburgh, PA, USA). Twentyview fields per well were scanned and analyzed. Thescreen was repeated once to confirm the activity of siR-NAs. Cells treated with Lipofectamine RNAiMAX alonewithout siRNA served as controls. Additionally, scrambledsiRNAs and green fluorescent protein siRNAs, which wereincluded in the library, served as internal references ineach assay plate. Apoptosis was identified by nuclear mor-phology and Hoechst dye intensity by the HCS system[19], which allows simultaneously acquiring quantitativecellular data and images of each individual cell sample.Growth inhibition was calculated as a percentage of thecontrol. To focus on the most important kinases, onlythose siRNAs that were active for both sequences andshowed a minimum of 30% inhibition compared with con-trol were considered to be active in the screen.Effect of the active kinases on the growth of differentbreast cancer cell linesA panel of 28 active kinases was selected from the hit list,based on their activity and classes, and silenced by theircorresponding siRNAs in four breast cancer cell lines,MDA-MB-231, SUM149, BT474-M1, and HR5. Cell linesMDA-MB-231 and SUM149 are TNBC, whereas the lattertwo are HER2 positive. Unless otherwise stated, all growthassays in the study were done in replicates of three or fiveand repeated at least once to confirm the activity.Effect of the selected kinases on CD44 high subpopulationof SUM149SUM149 cells were treated with the selected siRNAs at5 nM, as described in a previous section. After 72 hours oftreatment, the cells were fixed in 2% paraformaldehydewith nuclear dye, Hoechst 33342, at room temperature for30 minutes. The cells were then washed gently 3 timeswith PBS and stained with 40 μl/well of mouse anti-human CD44-PE conjugated antibody (BD Biosciences,Mississauga, ON, Canada; 1:100 dilution) at room tem-perature for 1 hour in the dark. The samples were thenwashed with PBS and kept at 4°C in the dark before analy-sis with the HCS system for the CD44high cells survivingthe siRNA treatments.Effect of the selected kinases on sorted CD44high/CD24-/low TIC subpopulation of SUM149SUM149 cells were cultured and sorted for theCD44high/CD24/-low subpopulation as described [14] totest directly the effect of the active kinases on TICs.Sorted cells were seeded at 5,000 cells/well into 96-wellculture plates (BD) and cultured overnight. The siRNAsof the 12 selected kinases were then added as describedearlier. Cells treated with Lipofectamine RNAiMAXalone without siRNA served as controls. Additionally,scrambled siRNAs were included in the experiments,and served as internal reference in each assay plate. Thetreatment lasted for 72 hours. The treated cells werethen fixed and stained with Hoechst dye, and thegrowth inhibition was analyzed with the HCS system, asdescribed in previous sections.Hu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 3 of 15PLK1 expression in different breast cancer cell lines andits correlation to CD44PLK1 protein expression in eight breast cancer cell lines,SUM149, MDA-MB-231, BT474, HR5, HR6, MCF7,HCC1937, and AU565, was investigated with Westernblot, as previously described [29]. In brief, proteins wereisolated from log-phase growing cells of these six cell linesby using an ELB buffer [24]. PLK1 (Abcam, Cambridge,MA, USA; 1:2,000 dilution) and actin (Cell Signaling, Pick-ering, ON, Canada; 1:5,000 dilution) were detected withimmunoblotting.To confirm the silencing efficacy of PLK1 siRNA onPLK1 expression, SUM149 and MBA-MB-231 wereseeded into six-well culture plates (BD) at 350,000 cells/well in 2 ml corresponding media. PLK1 and control siR-NAs were added to achieve 5 nM final concentration, andLipofectamine RNAiMAX alone without siRNA served asthe control. The sample plate was then incubated for72 hours. After harvesting the cells and extracting the pro-teins, PLK1 expression was detected with immunoblotting(1:2,000 for PLK1 and 1:5,000 for actin), as describedearlier.To explore the possible connection between PLK1 andCD44, SUM149 cells were seeded onto eight-chamberslides (BD), washed with PBS, fixed with 2% formaldehydefor 20 minutes, rinsed twice with PBS, and then incubatedwith PBS containing 0.1% Triton X-100 (Sigma-Aldrich)for 30 minutes. Next, the slides were washed with PBS andincubated with mouse anti-CD44 (BD Biosciences; 1:200dilution) and rabbit anti-PLK1 (LifeSpan Bioscience Inc.,Seattle, WA, USA; 1:400 dilution) antibodies diluted inbuffer containing 10% bovine serum albumin and 2% goatserum overnight at 4°C in a humidified container. Afterwashing 3 times with PBS, glass slides were incubatedwith Alexa Fluor 546 anti-mouse and Alexa Fluor 488anti-rabbit antibodies (Invitrogen; 1:1,000 dilution) for1 hour, washed 3 times, and then mounted by using Pro-long Gold (Invitrogen) with 4ʹ,6-diamidino-2-phenylin-dole (DAPI; Invitrogen). Cells were observed with a ZeissAX10 microscope and photographed by using an OlympusDP72 digital camera. All cells in three randomly selectedview fields (×10 magnification) were surveyed for CD44and PLK1 expression, and the percentage of CD44high cellsthat were also PLK1high was calculated.PLK1 activity after inhibition by BI 2536 (a known PLK1small-molecular inhibitor)The effect of PLK1 inhibitor on PLK1 activity was studiedwith an immunofluorescence method. SUM149 cells wereseeded on glass coverslips in six-well dishes and treatedwith dimethyl sulfoxide (DMSO) or BI 2536 at 25 nM or100 nM for 72 hours. Fixed cells were then stained withrabbit anti-phospho-cyclin B1 (S133) (Cell Signaling; 1:200dilution), which is a known downstream substrate of PLK1[26]. This was followed by secondary antibody and imageacquisition, as described earlier.For quantitative analysis of PLK1 activity, SUM149 cellswere seeded at 3,000 cells/well overnight and treated withDMSO or BI 2536 at 10 to 100 nM in 96-well plates for72 hours. Fixed cells were then stained with the cyclin B1antibody, as described earlier, except that Hoechst wasused, and the cells were kept in PBS before analyzing withthe HCS system.Growth inhibition of BI 2536 on different breast cancercells and TICsPrior studies reported that BI 2536 is highly selective forPLK1 when tested against 1,000 related kinases [28]. BI2536 (Sigma-Aldrich) was prepared in DMSO and testedagainst seven cell lines, SUM149, MDA-MB-231, BT474-M1, HR5, MCF7, AU565, and T47D. Each cell line wasseeded at 3,000 cells/well and incubated overnight. Cellswere then treated with BI 2536 at concentrations of 1 to100 nM in the medium for 72 hours. Propidium iodide(PI, Sigma-Aldrich) and Hoechst dye solution were added40 minutes before the end of treatments to each well at afinal concentration of 1 μg/ml for each dye. The sampleplates were then scanned live with the HCS system.Growth inhibition was calculated as a percentage of thecontrol without the DMSO and the drug, and the samplestreated with DMSO alone served as a reference. Toaddress whether a longer period of treatment wouldincrease the efficacy of the drug compound, SUM149 cellswere treated with BI 2536 for 10 days. The methods werethe same as stated earlier, except that the seeding densitywas only 1,000 cells/well, and the media with BI 2536were later replaced with fresh media containing BI 2536 atdays 4 and 7 of the treatments.To determine whether BI 2536 has a similar inhibitoryeffect on TICs as do the PLK1 siRNAs, sorted CD44high/CD24-/low cells of SUM149 were seeded at a density of3,000 cells/well in 96-well plates. They were then treatedwith BI 2536 at concentrations ranging from 1 to 100 nMfor 72 hours.Mammosphere assays were performed with SUM149, aswell as with MDA-MB-231 cells, which highly expressesCD44 in about 90% of its population, in ultra-low attach-ment six-well culture plates (Corning, Lowell, MA, USA)in complete Mammocult media (StemCell Technologies,Vancouver, BC, Canada), as previously described [30].DMSO control or BI 2536 (10 nM or 25 nM) was addedat time of seeding (5,000 cells/well). Serial passaging wasperformed as per Subculture of Mammospheres protocol(StemCell Technologies). In brief, after 7 days in culture,mammospheres were counted, collected in a conical tube,and centrifuged at 350 g for 5 minutes. Pellets were tritu-rated with trypsin-EDTA (Invitrogen) to break up mam-mospheres to single cells. Cold PBS with 2% FBS wasHu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 4 of 15added, and cells were centrifuged at 350 g for 5 minutes.Pellets were resuspended in Mammocult media, and cellcounts were performed. The mammosphere assay wasreseeded by using the same cell densities and treatmentsas described earlier.Chemotherapeutic drugs like paclitaxel (Taxol), doxoru-bicin (Dox), and 5-fluorouracil (5FU) had been reported toinduce resistance of cancer cells, and to this is probablyattributed their induction of TICs in the surviving popula-tion [14,15,31,32]. To determine whether drug treatmentfollowed by BI 2536 could overcome the TICs, character-ized as CD44high/CD24-/low, SUM149 cells were seeded at1,000 cells/well in 96-well plates overnight. Taxol, Dox, or5FU (Sigma-Aldrich) at different concentrations were thenadded the following day, and the plates were incubated for72 hours. One of the plates was then fixed and stained forHoechst, CD44 APC (BD Biosciences; 1:50 dilution) andCD24 FITC (BD Biosciences; 1:10 dilution) antibodies, asdescribed earlier, and analyzed with an HCS system forgrowth and CD44high/CD24-/low cells. The medium in thesecond plate was removed and washed once with freshmedium. Then the medium with BI 2536 at different con-centrations was added to the plate and incubated foranother 4 days. The plate was fixed and analyzed withHCS, as described.Detection of apoptosis caused by BI 2536 on differentbreast cancer cell linesTo investigate apoptosis caused by BI 2536 on breast can-cer cells of SUM149, MDA-MB-231, BT474-M1, and HR5,the cells after drug treatment were stained with PI or phos-pho-H2AX for quantification of apoptosis [14,19]. In brief,PI and Hoechst were added to cell wells at a final concen-tration of 1 μg/ml each, 40 minutes before the end of the72-hour treatments. The sample plates were then scannedlive with the HCS system. For phospho-H2AX, which is anearly indicator of apoptosis [14,19], treated cells were fixedwith 2% paraformaldehyde and Hoechst dye for 30 minutesfollowed by permeabilization with Triton X-100 (FisherScientific, Nepean, ON, Canada) and blocking with bovineserum albumin (Sigma-Aldrich) [19]. They were then incu-bated with mouse anti-human phospho-H2AX (Abcom;1:100 dilution) for 1 hour at room temperature. This wasfollowed by rabbit anti-mouse Alexa Fluor 488 antibody(Invitrogen; 1:100 dilution). The cells were gently washedwith PBS after each procedure. The sample plates werefinally analyzed, and images were taken by the HCS system.ResultsThe siRNA library screen identified active kinases thatsignificantly inhibited the growth of TNBC cell lineSUM149In the initial screen, 85 of the 691 kinases in total wereidentified to be significantly growth inhibitory (> 30%growth inhibition) on SUM149 cells once they weresilenced by 5 nM siRNAs for 72 hours under the experi-mental conditions (Table 1; Table 1 of Additional file 1).These active kinases (about 12.3% of the kinome librarytested) comprised a wide range of classes and functionalgroups, indicating that the cancer cell growth could beregulated through multiple genes and pathways. Of sig-nificant importance are the cell cycle-related kinases,MAP kinases, and protein kinases, as many identifiedactive kinases belong to these groups. The critical rolesthey played in SUM149 cell growth and the strong sen-sitivity to siRNA silencing indicate their potential astherapeutic targets for TNBC. PLK1, in particular, is oneof the most active kinases identified in the screen. Thegrowth inhibition on SUM149 is more than 80%, withsignificant apoptosis of the cells under the experimentalconditions.The active kinases showed a broad spectrum of growthinhibition on different breast cancer cell linesAlthough the initial kinase siRNA library screen wasdone on SUM149 cells, most of the 28 selected activekinases, once silenced by their corresponding siRNAs,showed a strong and broad spectrum of inhibitory effecton the growth of all four cell lines tested, SUM149,MDA-MB-231, BT474-M1, and HR5 (Figure 1). A fewexamples of such kinases are PLK1, GCK, SKP2, PLAU,RPS6KA2, PI4K2B, and LOC392265. In particular, thesekinases are significantly active on HR5, a trastuzumab-resistant model. The results indicated that these kinasesoffer potential applications not only in TNBC but alsoin other subtypes of breast cancer.The active kinases reduced the CD44high subpopulationand inhibited the growth of sorted CD44high/CD24-/lowcells of SUM149 after siRNA knockdownSUM149 cells consist of about 5% CD44high cells undernormal culture conditions. Of the 28 kinases tested, abouthalf of them significantly reduced the number of CD44highin the surviving population of SUM149 after siRNA treat-ments compared with the control (Figure 2A). In particu-lar, 12 kinases, CSNK2A2, GCK, MAP3K4, PDGFRA,PIK3C2G, PLAU, PLK1, SKP2, RPS6KA2, IHPK1, MAP-K8IP3, and UCK1, are the most active ones. It is notedalso that deoxyguanosine kinase (DGUOK), conversely,significantly induced CD44high cells after siRNA silencing.When these 12 kinases were tested directly on TICs ofsorted CD44high/CD24-/low cells of SUM149 by silencingthem with corresponding siRNAs at 5 nM for 72 hours,all of them, as expected, significantly inhibited the growthof the TICs compared with control (Figure 2B). Theresults confirmed our earlier observation of the reducednumber of CD44high cell in SUM149 after siRNAHu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 5 of 15treatments of these 12 kinases (Figure 2A). PLK1, onceagain, had the most significant inhibitory effect on TICs.PLK1 is commonly expressed in breast cancer cells, andits expression is correlated positively to CD44Analysis with Western blot confirmed that PLK1 is com-monly expressed in all eight breast cancer cell lines tested(Figure 3A). In particular, SUM149, MDA-MB-231, andHCC1937 are TNBC. Also, a siRNA silencing experimentconfirmed the specific knockdown of PLK1 in bothSUM149 and MDB-MB-231 cell lines (Figure 3B).PLK1 is known to be highly associated with cell prolif-eration [28,31]. We therefore addressed whether itresides within the CD44high subpopulation. By immuno-fluorescence, PLK1 was positively correlated to theexpression of CD44, in that most (89% ± 14%) ofCD44high cells were also PLK1high, whereas the CD44lowcells failed to express high levels of PLK1 (Figure 3C).The high PLK1 in CD44high cells may help maintain TICsand the ongoing proliferation of the tumor-initiatingpopulation. The results could partially explain our obser-vation that the CD44high subpopulation of SUM149 grewfaster than did CD44-/low cells (unpublished data).BI 2536 inhibited PLK1 activity, which led to theaccumulation of phospho-cyclin B1 in SUM149 cellsBoth qualitative and quantitative studies showed that PLK1inhibition by BI 2536 at 25 nM or higher concentrationsled to aberrant accumulation of phospho-cyclin B1 in thenuclear area of SUM149 cells (Figure 3D and 3E). The sig-nificant accumulation started 24 hours after treatment with100 nM but not 10 nM BI 2536 (data not shown).Table 1 Partial list of the active kinases identified in the siRNA library screenAccessionnumberSymbol Brief description Growthinhibition(%)NM-001786 CDC2 Cell-division cycle 2, G1 to S and G2 to M 68aNM-033487 CDC2L1 Cell-division cycle 2-like 1 (PITSLRE proteins) 71NM-001274 CHEK1 CHK1 checkpoint homolog (S. pombe) 61NM-001896 CSNK2A2 Casein kinase 2, alpha prime polypeptide 62aNM-001929 DGUOK Deoxyguanosine kinase 61NM-000162 GCK Glucokinase (hexokinase 4, maturity-onset diabetes of the young 2) 62NM-153273 IHPK1 Inositol hexaphosphate kinase 1 55NM-001569 IRAK1 Interleukin-1 receptor-associated kinase 1 59XM-498294 LOC392265 Similar to cell-division protein kinase 5 (tau protein kinase II catalytic subunit) (TPKII catalytic subunit)(serine/threonine-protein kinase PSSALRE)67NM-005922 MAP3K4 Mitogen-activated protein kinase kinase kinase 4 57NM-015133 MAPK8IP3 Mitogen-activated protein kinase 8 interacting protein 3 56NM-006206 PDGFRA Platelet-derived growth factor receptor, alpha polypeptide 63NM-006212 PFKFB2 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 60NM-012395 PFTK1 PFTAIRE protein kinase 1 53NM-000294 PHKG2 Phosphorylase kinase, gamma 2 (testis) 65NM-018323 PI4K2B Phosphatidylinositol 4-kinase type-II beta 51aNM-004570 PIK3C2G Phosphoinositide-3-kinase, class 2, gamma polypeptide 69aNM-002651 PIK4CB Phosphatidylinositol 4-kinase, catalytic, beta polypeptide 56NM-181805 PKIG Protein kinase (cAMP-dependent, catalytic) inhibitor gamma 65NM-002658 PLAU Plasminogen activator, urokinase 57NM-005030 PLK1 Polo-like kinase 1 (Drosophila) 86aNM-006254 PRKCD Protein kinase C, delta 60NM-016457 PRKD2 Protein kinase D2 60NM-021135 RPS6KA2 Ribosomal protein S6 kinase, 90 kDa, polypeptide 2 50NM-005983 SKP2 S-phase kinase-associated protein 2 (p45) 61NM-003318 TTK TTK protein kinase 64NM-031432 UCK1 Uridine-cytidine kinase 1 63NM-006296 VRK2 Vaccinia-related kinase 2 55aApoptosis is at least 5% more than the control for both siRNA sequences of the kinase, as measured by HCS system, based on nuclear properties (morphologyand Hoechst dye intensity) of the cells. Note: Growth inhibition resulted from siRNA silencing of SUM149 cells at 5 nM for 72 hours. Data shown here are theaverage results of both siRNA sequences of the kinase from two independent screens. For a complete list of active kinases identified in the siRNA library screen,refer to Additional file 1.Hu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 6 of 15PLK1 small-molecule inhibitor BI 2536 is as active as PLK1siRNA against different breast cancer cell lines and TICsand induces apoptosisLike its siRNA counterpart, PLK1 small-molecule inhibitorBI 2536 showed a significant growth-inhibitory effect onthe cells of the seven different breast cancer cell linesunder experimental conditions (Figure 4A). The activeconcentrations are as low as 1 to 5 nM with 80% to 90%growth reduction at 10 to 25 nM for most cancer cell linesafter a 72-hour treatment. In particular, HR5, a trastuzu-mab-resistant cell line, is similarly sensitive to BI 2536 asis BT474-M1. A longer period of treatment of SUM149cells with BI 2536 killed almost all cells at concentrationsof 25 nM or higher (Figure 4B). More important, treat-ment with BI 2536 significantly inhibited the growth ofsorted TICs of SUM149 compared with control (Figure4C), further supporting its potential application in breastcancer.In mammosphere assays on both SUM149 and MDA-MB-231 cells, BI 2536 treatment led to significant reduc-tion of the sphere formation (Figure 4D). The resultsfurther confirmed our earlier observation of the inhibitoryeffect of BI 2536 on TICs on monolayer models.Similar to PLK1 siRNA, BI 2536 also caused significantapoptosis at 10 to 100 nM in all four cell lines tested, acharacteristic for PLK1 inhibition (Figure 4E through G).The loss of PLK1 activity triggered apoptosis in up to70% of BT474-M1 cells that remained at the end of the72-hour treatment. SUM149 had relatively fewer cells leftat the end point and also fewer apoptotic cells compared020406080100CCDC2L1CHEK1CDC2CSNK2A2DGUOKGCKIRAK1MAP3K4PDGFRAPFKFB2PIK3C2GPIK4CBPHKG2PFTK1PLAUPLK1PRKCDSKP2RPS6KA2 TTKVRK2IHPK1PKIGMAPK8IP3PRKD2PI4K2BUCK1LOC392265Growth inhibition (%)MDA-MB-231 020406080100CCDC2L1CHEK1CDC2CSNK2A2DGUOKGCKIRAK1MAP3K4PDGFRAPFKFB2PIK3C2GPIK4CBPHKG2PFTK1PLAUPLK1PRKCDSKP2RPS6KA2 TTKVRK2IHPK1PKIGMAPK8IP3PRKD2PI4K2BUCK1LOC392265Growth inhibition (%)020406080100CCDC2L1CHEK1CDC2CSNK2A2DGUOKGCKIRAK1MAP3K4PDGFRAPFKFB2PIK3C2GPIK4CBPHKG2PFTK1PLAUPLK1PRKCDSKP2RPS6KA2 TTKVRK2IHPK1PKIGMAPK8IP3PRKD2PI4K2BUCK1LOC392265Growth inhibition (%)BT474-M1 HR5 A. D. B. C. 020406080100CCDC2L1CHEK1CDC2CSNK2A2DGUOKGCKIRAK1MAP3K4PDGFRAPFKFB2PIK3C2GPIK4CBPHKG2PFTK1PLAUPLK1PRKCDSKP2RPS6KA2 TTKVRK2IHPK1PKIGMAPK8IP3PRKD2PI4K2BUCK1LOC392265Growth inhibition (%)SUM149 * * * * Figure 1 Growth inhibition of the 28 active kinases on different breast cancer cells after siRNA silencing. (A) Percentage growthinhibition of SUM149 by the kinases after siRNA silencing at 5 nM for 72 hours. C, control without siRNA. *PLK1. (B) Percentage growthinhibition of BT474-M1 by the kinases. (C) Percentage growth inhibition of MDA-MB-231 by the kinases. (D) Percentage growth inhibition of HR5by the kinases. Data are presented as mean ± SD of two independent tests. Kinases in the figure: CDC2L1, cell-division cycle 2-like 1 (PITSLREproteins); CHEK1, CHK1 checkpoint homologue (S. pombe); CDC2, cell-division cycle 2, G1 to S and G2 to M; CSNK2A2, casein kinase 2, alpha primepolypeptide; DGUOK, deoxyguanosine kinase; GCK, glucokinase (hexokinase 4, maturity-onset diabetes of the young 2); IRAK1, interleukin-1receptor-associated kinase 1; MAP3K4, mitogen-activated protein kinase kinase kinase 4; PDGFRA, platelet-derived growth factor receptor, apolypeptide; PFKFB2, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2; PIK3C2G, phosphoinositide-3-kinase, class 2, g polypeptide; PIK4CB,phosphatidylinositol 4-kinase, catalytic, b polypeptide; PHKG2, phosphorylase kinase, g 2 (testis); PFTK1, PFTAIRE protein kinase 1; PLAU,plasminogen activator, urokinase; PLK1, polo-like kinase 1 (Drosophila); PRKCD, protein kinase C, delta; SKP2, S-phase kinase-associated protein 2(p45); RPS6KA2, ribosomal protein S6 kinase, 90 kDa, polypeptide 2; TTK, TTK protein kinase; VRK2, vaccinia-related kinase 2; IHPK1, inositolhexaphosphate kinase 1; PKIG, protein kinase (cAMP-dependent, catalytic) inhibitor g; MAPK8IP3, mitogen-activated protein kinase 8 interactingprotein 3; PRKD2, protein kinase D2; PI4K2B, phosphatidylinositol 4-kinase type-II b; UCK1, uridine-cytidine kinase 1; LOC392265, similar to cell-division protein kinase 5 (Tau protein kinase II catalytic subunit) (TPKII catalytic subunit) (serine/threonine-protein kinase PSSALRE).Hu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 7 of 15A. B. 01020304050607080C ScCDC2L1CHEK1CDC2CSNK2A2DGUOKGCKIRAK1MAP3K4PDGFRAPFKFB2PIK3C2GPIK4CBPHKG2PFTK1PLAUPLK1PRKCDSKP2RPS6KA2TTKVRK2IHPK1PKIGMAPK8IP3PRKD2PI4K2BUCK1LOC392265No. of CD44high cells per view field (10x)020406080100C ScCSNK2A2GCKMAP3K4PDGFRAPIK3C2GPLAUSKP2RPS6KA2IHPK1MAPK8IP3UCK1PLK1Growth inhibition (%)(CD44high/CD24-/low)* * Figure 2 Effect of the active kinases on CD44high and the sorted CD44high/CD24-/low populations of SUM149. (A) Number of CD44highcells (per view field, ×10 magnification) of SUM149 after kinase silencing by siRNA at 5 nM for 72 hours. C, control without siRNA; Sc, scramblesiRNA. *PLK1. (B) Growth inhibition of the sorted CD44high/CD24-/low population of SUM149 by siRNA silencing of different kinases at 5 nM for 72hours. Data are presented as mean ± SD of two independent tests. Kinases in the figure: CDC2L1, cell-division cycle 2-like 1 (PITSLRE proteins);CHEK1, CHK1 checkpoint homologue (S. pombe); CDC2, cell-division cycle 2, G1 to S and G2 to M; CSNK2A2, casein kinase 2, alpha primepolypeptide; DGUOK, deoxyguanosine kinase; GCK, glucokinase (hexokinase 4, maturity-onset diabetes of the young 2); IRAK1, interleukin-1receptor-associated kinase 1; MAP3K4, alpha mitogen-activated protein kinase kinase kinase 4; PDGFRA, platelet-derived growth factor receptor,alpha polypeptide; PFKFB2, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2; PIK3C2G, phosphoinositide-3-kinase, class 2, gammapolypeptide; PIK4CB, phosphatidylinositol 4-kinase, catalytic, beta polypeptide; PHKG2, phosphorylase kinase, gamma 2 (testis); PFTK1, PFTAIREprotein kinase 1; PLAU, plasminogen activator, urokinase; PLK1, polo-like kinase 1 (Drosophila); PRKCD, protein kinase C, delta; SKP2: S-phase kinase-associated protein 2 (p45); RPS6KA2, ribosomal protein S6 kinase, 90 kDa, polypeptide 2; TTK, TTK protein kinase; VRK2, vaccinia-related kinase 2;IHPK1, inositol hexaphosphate kinase 1; PKIG, protein kinase (cAMP-dependent, catalytic) inhibitor gamma; MAPK8IP3, mitogen-activated proteinkinase 8-interacting protein 3; PRKD2, protein kinase D2; PI4K2B, phosphatidylinositol 4-kinase type-II beta; UCK1, uridine-cytidine kinase 1;LOC392265, similar to cell-division protein kinase 5 (tau protein kinase II catalytic subunit) (TPKII catalytic subunit) (serine/threonine-protein kinasePSSALRE).Hu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 8 of 15with the other three cell lines, probably because the massapoptosis occurred earlier. This was confirmed by time-course experiments at earlier times (Figure 4F), in whichapoptosis peaked at about 48 hours after BI 2536 treat-ments. Together, the results from nuclear morphology(nuclear fragmentation and/or condensation), phospho-H2AX detection (an earlier indicator of apoptosis), andPI uptake (a late apoptosis indicator) clearly demon-strated the apoptosis in breast cancer cells caused by BI2536.An unfortunate consequence of chemotherapies usedto treat breast cancer is that they induce TICs [15,17].Here we show that Taxol, Dox, and 5FU inhibited can-cer cell growth, while at the same time, they induced ahigher proportion of CD44high/CD24-/low cells fromabout 2% in the controls to about 6% to 20% in the sur-viving populations after a 72-hour exposure (Figure 5Aand 5B). After the induction of CD44high/CD24-/low cellsby these drugs, we subsequently exposed the cells to BI2536 for an additional 4 days. The sequential treatmentled to almost complete cell death (Figure 5C). Thisdemonstrates that although resistant cells exist after thedrug treatments, they remain sensitive to BI 2536 at lowconcentration. Most important, BI 2536 can be used toovercome chemotherapy-induced TICs and suggested apotential to prevent relapse.DiscussionThe key functions of kinases in signal transduction forall organisms make them very attractive targets for ther-apeutic interventions in many diseases, including can-cers [18,21,23]. Several kinase inhibitors have been usedfor the treatment of cancer, such as imatinib, gefitinib,erlotinib, fasudil, and rapamycin [21,22]. Genome-widegene-library screens have proved an excellent tool inidentifying such biologic targets [18-20,33]. In thisSUM149 PLK1 HR5 MDA-MB-231BT474 HR6 AU565 MCF7 HCC1937 Actin A. B. Control Scramble  siRNA PLK1 siRNA Control Scramble  siRNA PLK1 siRNA MDA-MB-231 SUM149 PLK1 Actin C. DMSO 25 nM 100 nM BI 2536 p-Cyclin B1 Merge (DAPI) D. E. CD44 CD44 PLK1 PLK1 CD44 PLK1 DAPI 0100200300400500600700800DMSO 10 25 50 100BI 2536 (nM)p-cyclin B1 intensity (72h)Figure 3 PLK1 expression, its association with CD44, and its activity after BI 2536 inhibition. (A) PLK1 expression in different breastcancer cells by immunoblotting. (B) PLK1 expression after PLK1 siRNA silencing in two TNBC (triple-negative breast cancer) cell lines, SUM149and MDA-MB-231. (C) PLK1 is positively associated with CD44 expression with immunofluorescent assay. (D) Immunofluorescent images showingthe accumulation of phospho-cyclin B1 in SUM149 cells after BI 2536 treatment (72 hours). DMSO, dimethyl sulfoxide; DAPI, 4ʹ,6-diamidino-2-phenylindole. (E) Quantitative analysis of phospho-cyclin B1 in SUM149 cells after BI 2536 treatment (72 hours).Hu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 9 of 15Control BT474-M1 Hoechst PI merge BI-2536 Control MDA-MB-231 BI-2536 Hoechst p-H2AX merge G. p-H2AX PI 020406080100C D 1 5 10 25 50 100BI 2536 (nM)Apoptosis (%, SUM149)6h24h48hA. B. C. F. 020406080100C D 1 5 10 25 50 100BI 2536 (nM)Growth inhibition (%, 72h)(CD44high/CD24-/low)020406080100C D 1 5 10 25 50 100BI 2536 (nM)Apoptosis (%, 72h)SUM 149M DA-M B-231HR5BT474-M 1E. D. -20020406080100C D 0.1 1 5 10 25 50 100BI 2536 (nM)Growth inhibition (%, 10d)(SUM149)-20020406080100C D 1 5 10 25 50 100BI 2536 (nM)Growth inhibition (%, 72h)SUM 149M DA-M B-231HR5BT474-M 1AU565T47DM CF70204060801001201st 2nd 1st 2ndNumber of passagingMammosphere (%)DM SO10 nM25 nM* * * SUM149 MDA-MB-231 Figure 4 Effect of PLK1 inhibition on breast cancer cell growth, apoptosis, and TICs. (A) Percentage of growth inhibition of differentbreast cancer cells by a PLK1 small-molecule inhibitor BI 2536 at different concentrations for 72 hours. C, control (medium only). D, dimethylsulfoxide only. (B) Percentage of growth inhibition of SUM149 cells by BI 2536 at different concentrations for 10 days. (C) Percentage of growthinhibition of sorted CD44high/CD24-/low of SUM149 by BI 2536 for 72 hours. (D) Percentage of mammosphere reduction in SUM149 and MDA-MB-231 by BI 2536. *Significant difference from the control (Student t test, P < 0.05). (E) Percentage of apoptosis of different breast cancer cellscaused by BI 2536 after 72 hours. (F) Time-course apoptosis of SUM149 treated with BI 2536 for 6, 24, and 48 hours. (G) Apoptotic images of thecells after BI 2536 treatment, as revealed by HCS (high-content screening) instrument (×10) with phospho-H2AX antibody or PI (propidiumiodide) intake. Data are presented as mean ± SD of two independent tests. TICs, tumor-initiating cells.Hu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 10 of 15A. B. C. BI 2536 25 nM     Hoechst                   CD24                     CD44                      Merge 020406080100120C DMSO Taxol5nMTaxol2nMDox100nMDox50nM5FU1mM5FU0.1mMGrowth (%)010203040C DMSO Taxol5nMTaxol2nMDox100nMDox50nM5FU1mM5FU0.1mMCD44high/CD24-/low(%)020406080100120C DMSO Taxol5nMTaxol2nMDox100nMDox50nM5FU1mM5FU0.1mMGrowth (%)Figure 5 Effect of combined treatment of Taxol, Dox, or 5FU followed by BI 2536 on SUM149 cells. (A) Taxol, Dox, and 5FU inhibited thegrowth of SUM149 after 72 hours. C, control (medium only). (B) Drug treatment led to a higher percentage of CD44high/CD24-/low TIC (tumor-initiating cell) subpopulation in surviving SUM149 cells after 72 hours. Top image panel: CD44high/CD24-/low cells (arrows) after 5FU treatment (0.1mM, 72 hours) as viewed by HCS (high-content screening) instrument (×10). (C) Combined treatment with the drugs (Taxol, Dox, or 5FU) for 72hours, followed by BI 2536 for another 96 hours, significantly reduced cell growth, even though the drugs induced a higher percentage of theCD44high/CD24-/low subpopulation. All data are presented as mean ± SD of two independent tests. DMSO, dimethyl sulfoxide; Taxol, paclitaxel;Dox, doxorubicin; 5FU, 5-fluorouracil.Hu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 11 of 15study, we screened a human kinase siRNA libraryagainst a TNBC cell line, SUM149, for in vitro growthinhibition. As a result, 85 kinases, including PLK1, wereidentified to be strongly inhibitory against the cellgrowth once they were silenced by corresponding siR-NAs. The diverse functional groups of the kinases iden-tified in this study demonstrate their important roles inregulating the growth of breast cancer cells. In particu-lar, about one fourth of the identified kinases were pre-viously proposed to be the targets or already are inclinical trials for breast cancers (Additional file 1).AURKB, BUB1B, CHEK1, EPHB6, GSK3, MAPKs,MYLK, NEKs, PDGFRA, PLAU, PLK1, PKC, RSK, SKP2,and TTK are just a few of them [21,22,34-38]. KinasesBUB1, CHEK1, IRAK1, TTK, RYK, and VRK2, identifiedin this study, for example, have been reported to behighly overexpressed in ER-negative breast tumors andwere critical for the growth of either ER-negative onlyor both ER-positive and -negative breast cancer cells[9,23]. These studies validate our approach of a gen-ome-wide gene library screen in target discovery forTNBC. In addition, most of the 28 active kinases thatwere selected for further study showed a broad spec-trum of activity, not only on TNBC, but also on otherER/HER2-positive breast cancer groups. Thus our studyprovides a broad basis of potential therapeutic targets,not only to TNBC, but also to other subtypes of breastcancers.Cancer relapse has long been a clinical problem in breastcancer treatment. Recent theories and evidence havepointed to cancer stem cells or TICs for the root cause.The cancer stem cell hypothesis proposed that tumors aredriven by a cellular component that retains stem cell prop-erties, including self-renewal, tumorigenicity, and multili-neage differentiation capacity [11,12]. In breast cancer,several subpopulations, such as CD44high/CD24-/low,CD133/PROM/prominin, and ALDEFLUOR+, have beenshown to contain highly enriched cancer stem cells[10,15,39]. Targeting such a subpopulation, as well as thebulk cancer population, could lead to complete cure of thecancer diseases. In this study, after identifying the activekinases, we questioned whether any of these kinases hadalso played a role in TICs. When we analyzed theCD44high population in the surviving cells after siRNAtreatment, 12 of these 28 selected kinases significantlyreduced the population of CD44high cells. This led to thetest of these 12 kinases directly against a sorted CD44high/CD24-/low subpopulation of SUM149. As expected, theyinhibited the growth of the sorted TICs. The confirmationof the anti-TIC subpopulation is particularly significant,given the accepted role of TICs in drug resistance andcancer relapse. The involvement of kinases in TICs of dif-ferent cancers has been reported [16,25-27], and our studyprovides new evidence for further exploration on thesekinases and TICs, in particular, for better breast cancertherapy.PLK1 is one of the four mammalian PLK family mem-bers. Its prime role in mammalian cells is the control ofmitotic progression, particularly the regulation of proteinsthat are involved in metaphase-anaphase transition andmitotic exit. The activity and concentration of this kinaseare crucial for the precise regulation of cell division [40].PLK1 was reported to be overexpressed in a broad spec-trum of cancer types, and its expression often correlateswith poor patient prognosis [19,40,41]. PLK1 has longbeen established as a marker for cellular proliferation [42].Its levels in non-small-cell lung cancer tumors correlateinversely with survival, indicating that PLK1 may haveprognostic value [43]. This was later confirmed in multiplecancer types [40]. PLK1 expression has also been shownto be a reliable marker for identifying a high risk of metas-tasis in malignant melanomas [44]. In a cluster analysis of82 normal and malignant breast specimens with cDNAarray, PLK1 was found overexpressed to various extents ina subgroup of patient tumors, designated class A, whichcontains a higher proportion of patients with metastasesand a greater risk of recurrence [45,46]. Given this, itwould be important to evaluate the potential for PLK1inhibitors in patients with metastatic disease as a futuredirection. Numerous studies have now established thatPLK1 is a prime target for drug development in prolifera-tive diseases such as breast cancer [37,40]. Inhibition ofPLK1 leads to mitotic arrest, interruption of cytokinesis,and apoptosis in susceptible tumor cell populations.In this study, the expression of PLK1 in different breastcancer subtypes was confirmed, and its inhibition led togrowth inhibition and apoptosis on all breast cancer celllines tested, indicating a broad application in breast can-cer treatment. The sensitivity to PLK1 depletion has beenlinked to p53 status in cancer cells, although conflictingreports exist [19,40]. In this study, AU565 (ER- andHER2+), which has a wild-type p53, is equally sensitive toPLK1 inhibition as MDA-MB-231 (TNBC), which is p53mutant. Similarly, of the three slightly less-sensitive celllines, SUM149 (TNBC) is p53 mutant, whereas MCF7and T47D (ER+ and HER2-) are both p53 wt. The resultsindicate that sensitivity to PLK1 inhibition may not belinked directly to p53 status. Although a normal cell linewas not included in the study for comparison, numerousstudies, both in vivo and even clinical trials, have estab-lished that PLK1 inhibition by siRNA or BI 2536 is welltolerated, with neutropenia being the main side effect[26,28,39,47-49]. PLK1 inhibitors seem also to have anadvantage over mitotic inhibitors such as the taxanes orvinca alkaloids, because they do not induce the neuro-toxicity, as do these earlier inhibitors [50,51]. Combina-tion of PLK1 siRNA with chemotherapeutic drugs alsoenhanced the sensitivity toward Taxol and trastuzumabHu et al. Breast Cancer Research 2012, 14:R22http://breast-cancer-research.com/content/14/1/R22Page 12 of 15(Herceptin) in a synergistic manner [32]. Most important,our study represents the first attempt to associate PLK1with TICs in breast cancer. Of the 28 selected kinases inour focused studies, PLK1 is the leading candidate, basedon its activity in inhibiting cancer cell growth, and in par-ticular, its activity against the TICs once silenced bysiRNA or by the small-molecule inhibitor, BI 2536. Fill-more and Kuperwasser [15] reported that current che-motherapeutic agents for breast cancer, such as Taxoland 5FU, actually induced TICs. This is indeed the casefor Taxol, Dox, and 5FU, under our test conditions. Inaddition, when these drug treatments were followed withBI 2536, few cells survived, even though they inducedCD44high/CD24-/low cells under the experimental condi-tions. Interestingly, Gleixner et al. [52] recently reportedthat inhibiting PLK1 with BI 2536 could override imati-nib resistance in chronic myeloid leukemia. Whether thisis related to the activity of PLK1 on TICs of the diseaseremains to be explored.Although PLK1 is the focus of our study for its signifi-cant growth inhibition on breast cancer, availability ofsmall-molecular inhibitors, and the safety data in clinicaltrials of different cancer treatment [28,40,49], severalother active kinases identified in this study deservefurther study for their roles in TICs in breast cancer,such as SKP2 and PLAU (uPA), which inhibited thegrowth of sorted CD44high/CD24-/low cells of SUM149.Indeed, uPA/PAI-1 is the only biomarker to have beenconferred with LOE-1 as a definitive prognostic markerof poor disease outcome in early breast cancer [53].Furthermore, the guidelines of the American Society ofClinical Oncology also consider the components of theuPAS to be promising targets for future therapeutic stu-dies [53]. The first inhibitors of uPA have now beentested in oncology trials worldwide, and one of the com-pounds, WX-671, has received US FDA approval for aphase II trial in metastatic breast cancer in combinationwith chemotherapy [53,54]. Evidence exists that uPA ishighly expressed in CD44+ cells [55]. Conceptually, thisfits with the idea that TICs are invasive [12], and assuch, they are found in circulating tumor cells frompatients [56]. High levels of uPA are also associatedwith breast cancer relapse, which again could underpinthe idea that its expression in TICs is associated withdrug resistance. SKP2 is overexpressed in a subset ofbreast carcinomas (ER- and HER2-) and might play arole in the development of resistance to anti-estrogens[34]. Overexpression of SKP2 is associated with resis-tance to preoperative doxorubicin-based chemotherapyin primary breast cancer [36]. Further confirmation ofthis effect on TICs could help define better therapeuticstrategies. It should be noted also that our primaryscreen targets the overall growth inhibition of SUM149rather than the TICs; it is possible that some kinasescould be missed from the hit list if they are active onlyon the TICs, but not or weakly active on the bulk of thecancer cell population.ConclusionsThe inhibition of PLK1 led to significant growth inhibi-tion, either alone or in combination with other drugs,on different breast cancer cells and TICs, making thempromising therapeutic targets in the treatment of TNBCand other breast cancers.Additional materialAdditional file 1: Complete list of active kinases identified in siRNAlibrary screen. Table 1. Active kinases identified in the siRNA libraryscreen. The table lists the accession numbers, symbols, and briefdescription of the kinases identified in the library screen as well as thegrowth inhibition (percentage) of the kinases after siRNA silencing at 5nM for 72 hours under the test conditions.Abbreviations5FU: 5-fluorouracil; DAPI: 4ʹ,6-diamidino-2-phenylindole; DMEM: DulbeccoModified Eagle medium; DMSO: dimethyl sulfoxide; Dox: doxorubicin; ER:estrogen receptor; FBS: fetal bovine serum; HCS: high content screening;HER2: human epidermal growth factor 2; PBS: phosphate-buffered solution;PI: propidium iodide; siRNA: small interfering RNA; Taxol: paclitaxel; TICs:tumor-initiating cells; TNBC: triple-negative breast cancer.AcknowledgementsWe thank Ms. Sukhi Sandhu for her technical assistance. The research projectwas supported by funding from CIHR (SED).Authors’ contributionsKH performed siRNA screens, growth assays, cyclin B1 assays, apoptosismeasurements, and prepared the manuscript. JL performed mammosphereformation and immunoblotting assays. AF performed immunofluorescenceof PLK1 and cyclin B1. SED conceived the study, arranged research funding,and prepared the manuscript. All authors read and approved the finalmanuscript.Competing interestsThe authors declare that they have no competing interests.Received: 26 August 2011 Revised: 12 January 2012Accepted: 6 February 2012 Published: 6 February 2012References1. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR,Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenchikov A, Williams C,Zhu SX, Lønning PE, Børreson-Dale AL, Brown PO, Botstein D: Molecularportraits of human breast tumours. Nature 2000, 406:747-752.2. 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