Cancer cells acquire a drug resistant, highly tumorigenic, cancer stem-like phenotype through...

Cancer cells acquire a drug resistant, highly tumorigenic,cancer stem-like phenotype through modulation of thePI3K=Akt=b-catenin=CBP pathway

Kaijie He1, Tong Xu1, Yucheng Xu1, Alexander Ring2,3, Michael Kahn2,3 and Amir Goldkorn1,2,3

1 Division of Medical Oncology, Department of Internal Medicine, University of Southern California Keck School of Medicine, Los Angeles, California2 Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California3 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California Keck School of Medicine,

Los Angeles, California

Cancer initiation and progression have been attributed to newly discovered subpopulations of self-renewing, highly

tumorigenic, drug-resistant tumor cells termed cancer stem cells. Recently, we and others reported a new phenotypic plasticity

wherein highly tumorigenic, drug-resistant cell populations could arise not only from pre-existing cancer stem-like populations

but also from cancer cells lacking these properties. In the current study, we hypothesized that this newfound phenotypic

plasticity may be mediated by PI3K=Akt and Wnt=b-catenin signaling, pathways previously implicated in carcinogenesis,

pluripotency and drug resistance. Using GFP expression, Hoechst dye exclusion and fluorescence activated cell sorting (FACS)

of cancer cell lines, we identified and tracked cancer stem-like side populations (SP) of cancer cells characterized by high

tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly

reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem-like SP cells, whereas PI3K=Akt

activation conversely enhanced NSP to SP conversion. PI3K=AKT signaling was mediated through downstream phosphorylation

of GSK3b, which led to activation and accumulation of b-catenin. Accordingly, pharmacological or genetic perturbation of

GSK3b or b-catenin dramatically impacted conversion of NSP to SP. Further downstream, b-catenin’s effects on NSP-SP

equilibrium were dependent upon its interaction with CBP, a KAT3 family coactivator. These studies provide a mechanistic

model wherein PI3K=Akt=b-catenin=CBP signaling mediates phenotypic plasticity in and out of a drug-resistant, highly

tumorigenic state. Therefore, targeting this pathway has unique potential for overcoming the therapy resistance and disease

progression attributed to the cancer stem-like phenotype.

Drug resistance and cancer progression constitute the centralchallenge in the management of advanced malignancy.1

While the underlying biology of these phenomena has yet tobe fully elucidated, a growing volume of recent work hasestablished that cancer cell populations are quite heterogene-ous, and that therapy resistance and brisk tumorigenicityreside disproportionately in a relatively small subpopulationof cancer cells sometimes termed “tumor initiating cells” or“cancer stem cells.”2,3 As a result, numerous ongoing preclin-

ical and clinical studies aim to neutralize, differentiate oreradicate these subpopulations of cells to avert drug resist-ance and halt disease progression.4–8

While a general consensus has emerged regarding thetherapeutic desirability of eliminating highly tumorigenic,drug-resistant cancer stem-like cells, the precise biologicalorigin and function of these cells has generated significantuncertainty and controversy, reflecting the great variability ofmodels, markers and assays devised to isolate and studythem.2,3,9,10 Most early studies of the cancer stem-like pheno-type were conducted in models of hematopoietic malignancyand gave rise to the presiding theory that these cells—liketheir benign counterparts—were a well-circumscribed, self-renewing seed population that arose hierarchically from somecommon progenitor and in turn gave rise to cancer cell prog-eny.11,12 In contrast to this model, several recent studies dem-onstrated that cancer cells lacking putative cancer stem-likemarkers could nonetheless exhibit brisk tumorigenicity whenxenografted into mice, a finding attributed to variability ofmarkers and mouse models or to imperfect sorting.13–15 Stillother studies showed that under certain selective pressures(e.g., ectopic gene expression), cells lacking cancer stem-likeproperties could acquire this phenotype,16,17 much as had

Key words: side population, cancer stem-like cell, plasticity,

PI3K=Akt, b-catenin=CBP

Additional Supporting Information may be found in the online

version of this article.

Conflicts of interest: Dr. Kahn is a consultant and owns equity in

Prism Pharmaceuticals, a company that is developing PRI-724, a

CBP=catenin antagonist for oncology.

Grant sponsor: NIH=NCI; Grant number: K08 CA126983-05

DOI: 10.1002/ijc.28341

History: Received 12 Dec 2012; Accepted 31 May 2013; Online 20

Jun 2013

Correspondence to: Amir Goldkorn, M.D., 1441 Eastlake Avenue,

Suite 3440, Los Angeles, CA 90033, USA, E-mail: [email protected]

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Int. J. Cancer: 00, 00–00 (2013) VC 2013 UICC

International Journal of Cancer

IJC

been previously demonstrated with induced pluripotent stemcells (iPS).18 Collectively, these reports suggested that a can-cer stem-like phenotype might arise by more than one route,perhaps even emerging de novo in an existing cancer cellpopulation.

Recently, we reported that highly tumorigenic, drug-resistant cancer stem-like cells could indeed arise by directconversion from cancer cells lacking these properties.19 Spe-cifically, we used Hoechst staining and fluorescence-labelingof cancer cell lines to isolate and track “side populations”(SP) characterized by drug-resistance and high tumorigenicityin mouse transplantation experiments. Using this model, weobserved significant plasticity whereby SP cells differentiatedinto “non-SP” (NSP) cells at the start of each passage in cul-ture, followed by coordinated conversion of NSP cells backinto the SP phenotype towards late passage. This spontane-ous, cyclical interconversion suggested a high level of plastic-ity occurring without selection pressures such as ectopic geneexpression or drug treatment. Moreover, the rapid and coor-dinated nature of conversion was not consistent with selec-tion and expansion of rare stochastic events, but rather wasmore consistent with a population-wide adaptive behavior,perhaps in response to environmental signals. Indeed, a con-temporaneous study described a very similar observation ofspontaneous plasticity.20 These discoveries have cast the can-cer stem-like phenotype in a new light: From a biologicalperspective, a high degree of plasticity—if borne out in addi-tional models—would imply that a cancer stem-like cellsneed not necessarily arise hierarchically from founder cells;rather, any population of cancer cells—given the right cues—could give rise to a subpopulation of cells characterized byhigh tumorigenicity and drug resistance. From a clinical per-spective, this more functional and fluid definition of the can-cer stem-like state—one that accommodates bidirectionalplasticity—has important disease implications, because it sug-gests that novel therapeutic strategies must contemplate andaddress the potential replenishment of a highly tumorigenic,drug-resistant population. These biological and therapeuticconsiderations have motivated our efforts to identify signal-ing mechanisms and pathways which mediate this newfoundphenotypic plasticity.

The phosphoinositide-3-kinase (PI3K)=Akt pathway iscommonly activated and controls the survival and prolifera-tion of cancer cells; hence, it has been the target of intensedevelopmental therapeutic efforts.21 Several recent studieshave shown that pharmacological inhibition of this pathway

could effectively decrease the SP in cancer cell populations22,23

and sensitize cancer cells to chemotherapeutic drugs in com-bination treatment, thus effectively attenuating the drug-resistant, tumorigenic cancer stem-like phenotype.24,25 Thesepreliminary reports have underscored the therapeutic poten-tial of PI3K=Akt targeting and highlighted the underlyingmechanistic questions which remain unanswered: DidPI3K=Akt inhibition exert a direct inhibitory effect specificallyon cancer stem-like cells? Or, alternatively, did PI3K=Akt andtheir downstream mediators function as switches that regulatetransition in and out of a cancer stem-like phenotype, asobserved in our previous plasticity experiments?

To investigate these questions, we returned to the originalcancer cell line models in which we had made our plasticityobservations.19 We tracked drug-resistant, highly tumorigenicside population (SP) cells using fluorescence activated cellsorting (FACS) and GFP labeling, and we examined the roleof PI3K=Akt pathway and its downstream signaling media-tors in regulating plasticity between the NSP and SP (cancerstem-like) phenotypes. PI3K=Akt signaling was transducedvia inhibition of GSK3b, which induced accumulation of b-catenin, which in turn interacted with CBP, a transcriptionalcoactivator, to potentiate NSP to SP conversion. Accordingly,pharmacologic or genetic (siRNA) blockade of any of thesesignals abrogated NSP to SP conversion. These findingsdefine a novel role for PI3K=Akt=b-catenin-CBP pathway inthe regulation of a highly tumorigenic, drug resistant pheno-type. As such, members of this pathway play a direct mecha-nistic role governing plasticity in and out of a cancer stem-like state, and thus warrant intense study and therapeutictargeting.

Material and MethodsCell culture

MCF7 breast cancer and J82 bladder cancer cell lines weregenerously provided by collaborators at the University ofSouthern California (Michael Press and Peter Jones) andwere maintained at 37�C, 5% CO2 in DMEM (Mediatech)supplemented with 10% heat-inactivated fetal bovine serum(Omega), penicillin (100 U ml21, Invitrogen), and streptomy-cin (100 lg ml21, Invitrogen).

Treatment with pharmacological agents

Cells were treated with vehicle control or with pharmacologicalagents or siRNAs at various doses at 37�C and analyzed atmultiple time points (see Results for experimental details).

What’s new?

The ability of cancer cells to spontaneously convert to and from a cancer stem-like phenotype may be a major factor in drug

resistance and cancer progression. While a mechanistic explanation has been lacking, new findings reported here suggest

that this remarkable process is governed by the PI3K=Akt and b-catenin=CBP signaling pathways. Pharmacological inhibition

or genetic disruption of molecules in these pathways led to dramatic reductions in the phenotypic plasticity of breast and

bladder cancer cells in vitro. The results indicate that the pathways’ mitigation could be key to overcoming therapy resistance

and disease progression.

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2 PI3K=Akt=b-catenin=CBP Pathway Regulates Plasticity


LY294002, Akt inhibitor IV, Hoechst33342, verapamil and pro-pidium iodide were purchased from Sigma; BIO was purchasedfrom Millipore; ICG-001 and IQ-1 were available throughDr. Michael Kahn’s laboratory at the University of SouthernCalifornia. Antibodies to pan-Akt, phosphor-Akt (Ser473), andactive-b-catenin were purchased from Cell Signaling Technol-ogy; GAPDH was purchased from Millipore cooperation;b-catenin antibody was purchased from BD Bioscience; CBPand P300 antibodies were purchased from Santa Cruz Biotech-nology. siRNA to Akt and b-catenin were purchased from CellSignaling Technology; siRNA to CBP and P300 were purchasedfrom Dharmacon (On-target Plus Smart Pools). NegativesiRNA controls were from QIAGEN and Dharmacon, andhiPerfect transfection reagent was purchased from QIAGEN.

Lentivirus infection

Lentivirus was generated as previously described to delivereither GFP under a CMV promoter or control empty vec-tor.26 Briefly, 1 day prior to infection 2 3 105 J82 cells wereseeded in 10-cm plates, and on the next morning media wasreplaced by 3 ml virus supernatant plus 7 ml media supple-mented with 8 lg ml21 polybrene. After 8-hr incubation at37�C, the virus-containing media was replaced with freshmedia. Cells were observed for 48 hr to ensure >90% GFPexpression prior to fluorescence activated cell sorting (FACS)and side population studies.

Flow cytometry

Hoechst staining and FACS were conducted as described pre-viously.19 Briefly, adherent cancer cells (1 3 106 mL) weretrypsinized, counted, and resuspended in prewarmed 10%FBS DMEM media. Hoechst 33342 (Sigma–Aldrich) wasadded at a concentration of 5 lg mL21, incubated for 2 hr in37�C water bath and gently inverted several times during thecourse of incubation. Parallel sample aliquots were preparedin the presence of 50 lM verapamil (Sigma–Aldrich), anATP-binding cassette transporter family inhibitor, at roomtemperature for 10min before adding the Hoechst 33342 dye.Cells were centrifuged at 1,000 rpm for 5 min after incuba-tion and resuspended in ice-cold DMEM media. Propidiumiodide (Sigma–Aldrich) was added to the cells at a final con-centration of 2 lg mL21. Samples were incubated for at least5 min on ice before FACS sorting (FACSAria, BD Bioscien-ces) or analysis (FACSLSR-II, BD Biosciences).

Immunofluorescence microscopy

J82 and MCF7 (2 3 104) were plated on coverslips in 12-well plates, the next day, media were changed and drugs orDMSO control were added. After 72-hr treatment, cells werefixed in 10% formalin and stained as previously described26

with primary antibody against b-catenin, secondary antibodyconjugated with Alexor 647 (Invitrogen) and DAPI fornuclear visualization. Coverslips were mounted on slides, andb-catenin localization was analyzed using Zeiss Imager.Z1microscope with Axiovision software at 633 magnification.

siRNA transfection

siRNAs were transfected using QIAGEN HiPerFect transfec-tion reagent following manufacture’s fast-forward transfectionprotocol. Briefly, 2–3 3 105 cells were seeded in six-wellplates shortly before transfection. siRNA or control siRNAwere diluted in serum free medium, and incubated with 10lL HiPerFect reagent for 10 min at room temperature. Com-plexes were added drop-wise onto cells. Media were changedthe next morning, and cells were harvested 72 hr (for siAktand sib-catenin), or 5–6 days (for siCBP and siP300) post-transfection. Knockdown of endogenous protein levels weredetermined by western blot.

Western blot

Nearly 4–12% Bis=Tris precast gels (Invitrogen) were used tovisualize and quantify pan-Akt, pAkt, GSK3b, pGSK3b andb-catenin and active-b-catenin proteins. About 3–8%Tris=acetate gels (Invitrogen) were used for CBP and P300.Proteins were transferred using Invitrogen iBlot Dry Blottingsystem (8-min transfer). The membranes were blocked with5% milk or Odyssey blocking buffer (Li-COR), and incubatedwith primary antibodies, secondary antibodies labeled withIRDye infrared dyes and then detected with Infrared Imagingsystem (Li-COR).

Sphere formation assay

Sphere culture was done as previously described.27 About 104

cells were plated in a six-well low attachment plate (withultra-low attachment surface, Corning Incorporated). Cellswere grown in MEBM media (Lonza) supplemented withB27 (Stem Cell Technologies), 20 ng mL21 EGF, and 20ng mL21 basic FGF (Invitrogen), 4 lg mL21 insulin, and 1%penicillin=streptomycin. After 6 days, spheres were countedunder microscope visually.

Protein coimmunoprecipitation (Co-IP)

MCF7 and J82 cells were treated with 10 lM ICG001 inDMSO or vehicle control for 24 hr. Cell pellets were collectedand processed using the Pierce NE-PER Nuclear and Cytoplas-mic Extraction Kit (Thermo Scientific) according to the manu-facturer’s instructions. A total of 250 lg of nuclear lysate wasused per antibody and treatment condition for IP. The antibod-ies used were: CBP (A22, Santa Cruz Biotechnology, sc-369)and normal rabbit IgG (sc-2027). The next day, a 50% slurryof Immobilized Protein A Resin (GBioscience) was added toeach sample and incubated for 1 hr at 4�C under constantnutation. Resin beads were pelleted for 60 sec and boiled in 30lL Laemmle buffer for 5 min. The resin beads were removedfrom the denatured protein solution by spinning in filter col-umns (GE Healthcare). Western blot was done as described:primary anti-human b-catenin antibody (BD Bioscience) andgoat anti-mouse secondary antibody conjugated with HRP (sc-2005) were used. Chemiluminescent bands were visualizedusing GeneMate Blue lite films (Bioexpress).

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Quantitative reverse transcription-PCR assays

J82 and MCF7 cells were treated with ICG001 for 24 hr.Total RNA was isolated and reverse transcribed to cDNAusing RETROscript kit (Ambion). Real time PCRs were per-formed on MyIQ (BioRad) using Quanta B-R Syber GreenQPCR supermix (BioScience). Primers used for Survivin: FW:50-CTGTGGGCCCCTTAGCAAT-30; RW: 50-TAAGCCCGGGAATCAAAACA-30. Beta actin was used as the house keep-ing gene control. Primers for beta actin: FW: 50-AGAGCTACGAGCTGCCTGAC-30; RW: 50-AGCACTGTGTTGGCGTACAG-30.

Statistical analysis

All experiments were conducted in triplicate with error barsrepresenting standard deviation around the mean. Student’st test was used to determine statistical significance whencomparing mean values at one point in time.

ResultsConversion of NSP cells to SP cells is blocked by PI3K

pathway inhibition

We recently reported that drug-resistant highly-tumorigenicSP cells can arise by direct conversion of NSP cells,19 andPI3K=Akt signaling previously has been reported to play arole in maintaining side populations (SP) in cancer celllines.22,23 Therefore, we hypothesized that PI3K=Akt activa-tion may potentiate the direct NSP to SP conversion that wehad observed.19 To test this hypothesis, we applied a GFPmodel19 to track SP and NSP subpopulations over time.Briefly, J82 bladder cancer cells were infected with lentivirusexpressing either empty vector (EV) or GFP. J82EV andJ82GFP were FACS sorted into their respective SP and NSPsubpopulations as previously described and characterized (seeMethods and Ref. 19). These were recombined to create a“hybrid” J82 cell line (hJ82) consisting of �20% SPEV and80% NSPGFP (proportions that reflect the native state of J82).The cells were grown and tracked in cell culture in the pres-ence of either LY294002 (PI3K inhibitor) or vehicle (DMSO)control, then analyzed by FACS at 2-day intervals forHoechst dye efflux and GFP status.

hJ82 cells treated with DMSO control exhibited theexpected SP trend previously described by our group19: Inthe first 2 days after seeding, SP percentage in the wholepopulation dropped from 13.2 to 0.1% as SPEV cells (red)moved from the SP gate into the NSP gate (Figs. 1a and 1b).In subsequent days, SP% began to rise again gradually, andon day 6 SP% underwent rapid expansion from 1.5 to 25.2%(17 fold increase) as large numbers of NSPGFP cells (green)moved into the SP gate (Figs.1a and1b). In contrast, inhibi-tion of PI3K signaling with LY294002 resulted in a very dif-ferent pattern: The initial day 1–2 movement of SPEV cells(red) into the NSP gate still occurred as before, but the sub-sequent day 6–10 movement of NSPGFP cells into the SP gatewas abrogated (Figs.1a and 1b). Moreover, we found thatLY294002 treatment did not significantly alter the GFP% in

the whole population and therefore did not have a selectiveantiproliferative effect on SP or NSP cells (Fig.1c), furthersupporting a model wherein PI3K inhibition blocks conver-sion from NSP to SP.

Akt signaling regulates conversion to the SP phenotype

Downstream of PI3K, we characterized the impact of Aktupon the side population using the pharmacological agentAkt inhibitor IV. In both J82 and MCF7 cells, treatment withthis agent caused a reduction in Akt phosphorylation in adose dependent manner (minimal effect on total Akt) as wellas a dose dependent reduction in phosphorylation of GSK3b,a downstream mediator of PI3K=Akt signaling (Fig. 2a).These effects were associated with a highly significant near-total elimination of SP in both cell types (Fig.2a). To deter-mine whether Akt inhibition eliminated the SP by blockingconversion of NSP to SP, we used FACS to isolate NSP cellsand cultured only these cells with Akt inhibitor IV or DMSOcontrol. By day 3, regeneration of SP from NSP cells wasreduced by >90% and by 80% in J82 and MCF7 cells, respec-tively (p 5 0.009 and 0.01, respectively, Fig.2b). In addition,we conducted MTS proliferation assays on NSP and SP cellstreated separately with Akt inhibitor IV or DMSO control,and we confirmed that Akt inhibitor IV treatment did notdifferentially impact the proliferation rate of NSP vs. SP cells(Fig.2b). These two experiments (SP regeneration and MTSassays) using FACS-separated subpopulations support themodel wherein PI3K=Akt inhibition depletes SP via directinhibition of NSP to SP conversion rather than by anothermechanism such as differential effects on proliferation.

Similar results were observed with siRNA-mediated knock-down of Akt: Three days after treatment with Akt siRNA (50nM), Western blot demonstrated knockdown of total Akt pro-tein and of phosphor-Akt levels with a concomitant reductionin SP: �40% decrease in J82 and �60% decrease in MCF7compared to treatment with scrambled sequence siRNA con-trol (p 5 0.02 and p 5 0.03, respectively, Fig.2c).

To determine whether activation of PI3K=Akt pathway hasan opposite effect on SP, we used the pharmacological PTENinhibitor bpV(pic). PTEN normally inhibits PI3K=Akt signal-ing, and PTEN inhibition by bpV(pic) results in activation ofAkt signaling.22 As expected, bpV treatment for 3 days signifi-cantly increased SP by �40% (p 5 0.01) in MCF7 cells whichexpress PTEN, whereas bpV treatment had no effect on J82cells (p 5 0.3), which do not express PTEN28 (Fig.2d).

GSK3b and b-catenin signaling function downstream of

PI3K=Akt pathway in the regulation of NSP to SP

conversion

PI3K=Akt signaling is mediated downstream by GSK3b andb-catenin, which have been shown to impact mammary stemcell properties in mouse models and in human cancercells.25,29 GSK3b normally phosphorylates b-catenin at Ser33,Ser37 and Thr41, thereby promoting its degradation anddiminishing its nuclear transcriptional activity.30 To

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determine whether GSK3b=b-catenin signaling also regulatesthe conversion from NSP to SP, we treated J82 and MCF7cancer cells with BIO (6-bromoindirubin-30-oxime), a phar-macological inhibitor of GSK3b. As expected, BIO-treatedcells had markedly higher cytosolic and nuclear accumulationof b-catenin compared to DMSO-treated control cells (Fig.3a). These results are supported by Western blots using anantibody specific for unphosphorylated (Ser33=37=Thr41)active b-catenin, which show a dose-dependent accumulationof active b-catenin with BIO treatment (Fig.3b). BIO-mediated GSK3b inhibition and consequent accumulation ofb-catenin resulted in a significant increase of SP% after 3days (2.4-fold increase in J82, p 5 0.02 and 2.9-fold increasein MCF7, p 5 0.001, Fig.3c).

To further confirm that this BIO-induced SP expansion ismediated through direct conversion of NSP cells to SP cells,we isolated NSP cells by FACS, cultured them with or with-out BIO (or DMSO control), and monitored the regenerationof SP on subsequent days. BIO induced significant SP regen-eration within 7 days (p < 0.01) compared to DMSO control(Supporting Information Fig.1a). In another control experi-ment, BIO did not exert any differential impact on the prolif-eration rates of NSP vs. SP cells when the two populationswere isolated by FACS and treated separately in culture (Sup-porting Information Fig.1b). Taken together, these control

experiments confirm that GSK3b=b-catenin signaling inducesSP expansion by regulating direct conversion of NSP to SPcells rather than exerting differential proliferative effects onthe original SP and NSP populations.

To exclude nonspecific “off-target” effects of pharmacolog-ical agents and to further study the role of b-catenin in NSPto SP conversion, we used siRNAs to specifically knock downb-catenin (Fig.3d). Western blots confirmed effective knock-down of b-catenin protein (�70% in J82 and �80% inMCF7), and 72 hr after transfection with siRNA cells wereFACS-analyzed for SP and NSP phenotypes. As expected, SPwas significantly diminished after b-catenin knockdown inboth cell lines (�5-fold reduction in J82 and �10 fold reduc-tion in MCF7), whereas cells treated with control scrambledsiRNA were not affected (p > 0.1).

b-catenin-CBP interaction is required for

NSP to SP conversion

The b-catenin transcription co-activator CBP (CREB-bindingprotein) was recently shown to be required for b-catenin-mediated transcriptional activation of Survivin, a key anti-apoptotic step in cancer cell survival.31 Therefore, we rea-soned that conversion to SP, a phenotype marked by drugresistance and tumorigenicity, might be regulated throughb-catenin’s interaction with CBP. To test this hypothesis, we

Figure 1. Inhibition of PI3K signaling blocks NSP to SP conversion. (a) Treatment with LY294002 abrogates the increase in SP%. (b) FACS

plots: treatment with LY294002 abrogates transition of NSPGFP cells into the SP gate. (c) LY294002 does not alter the GFP% in whole popu-

lation and thus has no preferential affect on the proliferation rate of NSP cells (GFP1) vs. SP cells (GFP2). [Color figure can be viewed in

the online issue, which is available at wileyonlinelibrary.com.]

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Figure 2. Akt signaling regulates conversion to the SP phenotype. (a) Akt inhibitor IV dose response in J82 and MCF7: Upper panel: Akt

inhibitor IV prevents Akt phosphrylation and downstream GSK3b phosphorylation, whereas total Akt and GSK3b protein levels are unaf-

fected; Lower panel: Akt inhibitor IV treatment significantly reduces SP in J82 and MCF7 cells. (b) Left panel: FACS-isolated NSP cells con-

vert to SP, an effect that is abrogated by Akt inhibitor IV; Right panel: Proliferation rates of FACS-isolated NSP and SP are not differentially

affected by Akt inhibitor IV (after 3 days of treatment). (c) siRNA knockdown of total Akt and phosphor-Akt protein reduces SP in J82 and

MCF7 cells (Western bands are below their corresponding histogram bars). (D) PTEN inhibition with bpV(pic) increases SP in MCF7 cells

(PTEN wt), but does not affect SP in J82 (PTEN null).

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Figure 3. b-catenin signaling regulates conversion to side population. (a) GSK3b inhibition with BIO induces b-catenin accumulation.

(b) Western blots with dose-dependent increase in active-b-catenin in response to BIO treatment. (c) Induction of b-catenin accumulation

results in SP expansion. (d) siRNA knockdown of b-catenin protein decreases side population (Western bands are below their correspond-

ing histogram bars). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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used ICG-001, a pharmacological agent which specificallyinterferes with the b-catenin-CBP interaction.32 ICG-001 treat-ment decreased SP by �80% in J82 cells (p 5 0.02) and by�67% in MCF7 cells (p 5 0.01), respectively (Fig. 4a). More-over, ICG-001 treatment significantly inhibited sphere forma-tion in low-attachment plates, a characteristic cancer stem-likeproperty, in both J82 and MCF7 cancer cell lines (p 5 0.03and p 5 0.003, respectively, Fig.4b). As done with Akt andGSK3b inhibition, we conducted control experiments whereinFACS-isolated NSP cells were treated in culture with ICG-001vs. DMSO control, and we found that conversion of pure NSPto SP was inhibited by ICG-001 (Supporting InformationFig.2a). Furthermore, the proliferation rates of isolated SP andNSP subpopulations was not differentially affected by ICG-001treatment, further substantiating a conversion mechanismsrather than differential impact upon proliferation (SupportingInformation Fig.2b).

To further confirm the importance of b-catenin-CBPinteraction in regulating SP, we used siRNA knockdown totarget the CBP protein in J82 and MCF7 cells. Five days afterCBP depletion, SP was significantly reduced by 22% in J82cells (p < 0.05), and by 75% in MCF7 cells (p < 0.01)(Fig.4c), thus further implicating b-catenin-CBP interactionin regulation of the SP phenotype.

As pharmacological interference or knockdown of CBPinhibited conversion to SP, we tested whether activation of

CBP-b-catenin interaction would have the opposite effect,i.e., potentiation of the SP phenotype. P300 is a b-catenincofactor that is highly homologous to CBP, which has previ-ously been shown to initiate differentiation in stem progeni-tor cells in part by inhibiting expression of b-catenin targetgenes associated with pluripotency and proliferation (e.g.,Survivin, Oct4, and Sox2).8,31,33 It has been proposed thatb-catenin function is governed by an equilibrium between itsinteraction with CBP and p300, which potentiate a pluripo-tent versus a differentiated state, respectively.34 Accordingly,treatment with IQ-1, a small molecule pharmacological inhib-itor of the P300-b-catenin interaction, was shown to “tilt”the equilibrium towards a b-catenin-CBP pluripotent statemarked by heightened expression of b-catenin-CBP tran-scriptional target genes.33 Here, we used IQ-1 to inhibitb-catenin-p300 interaction and to potentiate a b-catenin-CBP interaction, and we found that treatment with IQ-1(1 lM) for 3 days significantly increased SP in both J82 andMCF7 cells relative to treatment with DMSO vehicle control(Fig.4d).

To further characterize the mechanism through whichICG-001 disrupted NSP to SP conversion, we performedimmunocytochemical stains of ICG-001-treated cells andfound no effect on overall b-catenin levels compared toDMSO control; moreover, ICG-001 did not inhibit BIO-induced accumulation of b-catenin (Fig. 5a). Though it did

Figure 4. b-catenin-CBP interaction regulates NSP to SP conversion. (a) Inhibition of b-catenin-CBP interaction with ICG-001 significantly

decreases SP. (b) ICG-001 reduces sphere formation in low-attachment plates. (c) siRNA knockdown of CBP reduces SP (Western bands are

below their corresponding histogram bars). (d) Potentiation of b-catenin-CBP interaction by IQ-1 increases SP.Can

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Figure 5. b-catenin-CBP signaling occurs downstream of b-catenin activation and trafficking. (a) Disruption of b-catenin-CBP interactionusing ICG-001 does not affect the accumulation of b-catenin induced by BIO. (b) Disruption of b–catenin-CBP interaction using ICG-001abrogates BIO-induced SP expansion. (c) Co-IP: b-catenin interacts directly with CBP, and their biochemical interaction is diminished byICG-001. (d) qRT-PCR demonstrates reduction in Survivin mRNA after 24-hr treatment with 10 lM ICG-001. [Color figure can be viewed inthe online issue, which is available at wileyonlinelibrary.com.]

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not alter these upstream events, ICG-001 co-treatment (for 3days) nonetheless significantly reduced BIO-mediated SPexpansion by �80 and 60% in J82 and MCF7, respectively,underscoring the important role of downstream b-catenin-CBP interaction in conversion to the SP phenotype (Fig.5b).Coimmunoprecipitation (co-IP) demonstrated a direct bio-chemical interaction between CBP and b-catenin which wasdiminished by �50% in J82 and MCF7 cells treated for 24 hrwith ICG-001 (Fig.5c), and qRT-PCR showed a significantreduction in transcript levels of Survivin—a transcriptional tar-get of b-catenin-CBP—with 24 hr ICG-001 treatment (Fig.5d).Collectively, these data further implicate the b-catenin-CBPtranscriptional complex in NSP to SP conversion.

DiscussionRecently we reported that the drug resistant highly tumori-genic side population (SP) is not a static discrete populationbut rather can spontaneously arise from the differentiatednon-side population (NSP) of cancer cells in a highly-orchestrated cyclical manner.19 Replenishment of SP fromthe bulk NSP suggests a phenotypic plasticity between thedrug resistant, highly tumorigenic state and the drug sensi-tive, low tumorigenic state. Moreover, it implies that hetero-geneity in cancer cell populations can arise not only throughDarwinian selection from a pre-existing palette of pheno-types, but also de novo from a phenotypically negative popu-lation that undergoes phenotypic conversion. In this model,differentiated cancer cells may revert to a “hardier” highlytumorigenic drug-resistant state under certain environmentalconditions. Such an ability carries strong implications forongoing therapeutic efforts to eradicate cancer stem-like cellsper se,35 because phenotypically negative cancer cells may becapable of functionally replenishing these hardy subpopula-tions by switching to a drug resistant tumorigenic state.17,36

In this study, we sought to elucidate the mechanisms whichgovern this phenotypic plasticity in our model, and thus toidentify potential candidates for novel therapeutic strategiesaimed at blocking regeneration of drug resistant, tumorigeniccancer stem-like cells.

PI3K=Akt signaling is one of the most commonly acti-vated pathways in cancer21 and potentiates tumorigenicityand drug resistance by regulating proliferative and anti-apoptotic mechanisms.7,27 Moreover, PI3K=Akt activationcan induce expansion of cancer stem-like populations(CD1331, CD441, or SP), whereas PI3K=Akt inhibition caninduce differentiation and depletion of CSC populations.27,37

These PI3K=Akt pathway effects have been attributed toinduction of self-renewal and proliferation directly in cancerstem-like cells. However, recent reports by our group andothers17,19,36 cast the role of PI3K=Akt in a new light: Per-haps beyond its intrinsic function in maintenance and prolif-eration of cancer stem-like cells, activation of PI3K=Akt indifferentiated, phenotypically negative cells can induce thesecells to convert to a highly tumorigenic, drug-resistant, can-cer stem-like phenotype, thus contributing to the plasticity

recently observed in our studies. To test this hypothesis, weused pharmacological agents and siRNA targeting to modu-late PI3K=Akt pathway and its downstream partners, and wemeasured the effects of these perturbations upon phenotypicplasticity in the side population (SP) model that we describedpreviously.19 Upstream, we found that PI3K=Akt inhibition(knockdown or pharmacological) dramatically reduced SP,whereas PI3K=Akt potentiation (via PTEN inhibition) led toSP expansion. By GFP-labeling the subpopulations, we deter-mined that PI3K=Akt perturbation was impacting SP byblocking the conversion of NSP cells to SP cells, an observa-tion that was further substantiated through experiments thatcontrolled for potential confounders like sorting contamina-tion or differential proliferation rates.

PI3K=Akt signaling recently has been shown to convergewith Wnt signaling at the common downstream partnerGSK3b to regulate b-catenin, itself a well-recognized media-tor of drug resistance and tumorigenicity.25,29,38–40 Therefore,we investigated whether these signals converge to controlplasticity in the NSP-SP model. We found that Akt depletionvia pharmacological inhibition or siRNA knockdown resultedin reduced Akt-mediated phosphorylation and thus reducedactivation of GSK3b. As GSK3b normally downregulatesb-catenin activity via phosphorylation and targeting for deg-radation, reduction of GSK3b activity via Akt-mediatedphosphorylation (or with the direct GSK3b inhibitor BIO)led to higher levels of dephosphorylated b-catenin, accumula-tion of cytosolic and nuclear active b-catenin, and a concom-itant expansion of SP. This effect is consistent with previousfindings that activation of b-catenin by BIO can help tomaintain pluripotency in embryonic stem cells as well as incancer cells.25,41 Conversely, b-catenin knockdown led to asignificant SP reduction in our model.

b-catenin transcriptional activity in cancer and embryonicstem cells can be regulated by CBP and p300, highly homolo-gous co-factors with differential functions: Interaction withCBP favors a more pluripotent, tumorigenic phenotype,whereas interaction with p300 favors initiation of a more dif-ferentiated state.32,33 Therefore, we investigated whether CBPand p300 played a role in b-catenin-mediated NSP to SPconversion. We found that attenuation of b-catenin-CBPfunction (via specific pharmacological inhibition or siRNAknockdown) significantly reduced NSP to SP conversion andsphere formation, whereas potentiation of b-catenin-CBP sig-naling had an opposite effect and expanded SP. Moreover,disruption of b-catenin-CBP interaction and transcriptionalactivity (by ICG-001) occurred downstream of Akt-GSK3b

signaling and abrogated the SP expansion that results fromBIO-induced b-catenin accumulation.

These studies illuminate a novel PI3K=Akt=GSK3b=b-catenin=CBP signal transduction pathway that is responsi-ble, at least in part, for coordinated transformation from a dif-ferentiated cancer cell state to a more drug resistant, highlytumorigenic cancer stem-like phenotype (Fig. 6). The modelintegrates into a single conceptual framework the seemingly

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disparate functions historically attributed to these proteins: Forexample, PI3K=Akt signaling has a well-documented role inmaintaining pluripotency and governing cell fate in variousstem cell models,42,43; at the same time, PI3K=Akt activation—often through PTEN silencing—is a recognized driver of can-cer proliferation and disease progression in multiple malignan-cies.23,29,44 Similarly, Wnt=b-catenin signaling is a key“stemness” factor in stem cell niches and embryonic develop-ment,33,41,45 and at the same time b-catenin signaling contrib-utes to aggressive progression of cancers such as melanomaand colorectal tumors.40,46 Moreover, both PI3K=Akt and

b-catenin signaling have been shown to activate antiapoptoticsurvival machinery and to promote drug resistance.47,48 Thus,both the PI3K=Akt pathway and the Wnt=b-catenin pathwayhave been implicated in (i) maintenance of “stemness” or plu-ripotency, (ii) activation of proliferation and cancer progres-sion, and (iii) potentiation of survival mechanisms and drugresistance—the three defining properties of the cancer stem-like phenotype.

Not surprisingly given their parallel effects, the two path-ways have recently been observed to converge directly in cer-tain noncancer models; for example, PI3K=Akt signaling wasshown to regulate the differentiation of human embryonicstem cells (hESC) and intestinal stem cells by activating ofGSK3b-mediated Wnt=b-catenin signaling.49,50 Our currentstudies demonstrate similar crosstalk in cancer cells, whereupstream PI3K=Akt activation is transduced via GSK3b toinduce active b-catenin accumulation, interaction with theCBP and p300 transcriptional cofactors, and regulation of acancer stem-like side population (SP) marked by high tumor-igenicity and drug-resistance. Moreover, we show that SPexpansion is not intrinsic; rather, PI3K=Akt=b-catenin=CBPsignaling predominantly acts within the non-SP cells andinduces them to convert synchronously in large numbers tothe SP phenotype.

Our findings thus integrate and contextualize the roles ofPI3K=Akt and b-catenin=CBP as signaling pathways whichdirectly cooperate to mediate phenotypic plasticity in cancercells. Specifically, expansion of a stem-like, tumorigenic,drug-resistant population may occur not only through Dar-winian selection of pre-existing cancer stem-like cells, butalso through activation of PI3K=Akt=b-catenin=CBP signal-ing in phenotypically negative cells that replenish a cancerstem-like population. Identifying these integrated roles ofPI3K=Akt and b-catenin=CBP in phenotypic plasticity thuscontributes to our understanding of how tumorigenic, drug-resistant cells arise. Continued investigation and validation ofthese novel mechanisms in additional models may ultimatelylead to more effective therapies aimed at blocking these path-ways and eliminating the “escape hatches” that replenish can-cer stem-like subpopulations.

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