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Tumor and Stem Cell Biology

Targeting Akt3 Signaling in Triple-Negative Breast Cancer

Y. Rebecca Chin1, Taku Yoshida2,3, Andriy Marusyk2,3, AndrewH. Beck1, Kornelia Polyak2,3,4, and Alex Toker1

AbstractTriple-negative breast cancer (TNBC) is currently the only major breast tumor subtype without effective

targeted therapy and, as a consequence, in general has a poor outcome. To identify new therapeutic targets inTNBC, we performed a short hairpin RNA (shRNA) screen for protein kinases commonly amplified andoverexpressed in breast cancer. Using this approach, we identified AKT3 as a gene preferentially required forthe growth of TNBCs. Downregulation of Akt3 significantly inhibits the growth of TNBC lines in three-dimensional (3D) spheroid cultures and in mouse xenograft models, whereas loss of Akt1 or Akt2 have moremodest effects. Akt3 silencingmarkedly upregulates the p27 cell-cycle inhibitor and this is critical for the ability ofAkt3 to inhibit spheroid growth. In contrast with Akt1, Akt3 silencing results in only a minor enhancement ofmigration and does not promote invasion. Depletion of Akt3 in TNBC sensitizes cells to the pan-Akt inhibitorGSK690693. These results imply that Akt3 has a specific function in TNBCs; thus, its therapeutic targeting mayprovide a new treatment option for this tumor subtype. Cancer Res; 74(3); 964–73. �2013 AACR.

IntroductionBreast cancer is the most common cancer among women

worldwide. On the basis of the gene expression profiling, thisdisease is categorized into three major subtypes: luminal,HER2þ/estrogen receptor–negative (ER�), and basal-like (1).Recent developments in endocrine therapy for the treatment ofluminal breast cancer and Her2 targeted therapy, such astrastuzumab for HER2þ/ER� tumors, have led to improvedsurvival for a subset of patients with breast cancer (2–4).However, the basal-like subtype, which comprises approxi-mately 15% of invasive breast cancers and is generally triple-negative [ER�, progesterone receptor–negative (PR�), andHER2�], lacks targeted therapy (5, 6). Currently, chemotherapyis the only option for the treatment of triple-negative breastcancers (TNBC), but its clinical benefit is limited to a subset ofpatients. Because of poor prognosis and a more aggressivephenotype, there is an urgent clinical need to identify noveltherapeutic targets for TNBCs.

Akt is a key regulator of numerous cellular phenotypesassociated with cancer, including cell survival, proliferation,

and metastasis (7). Hyperactivation of Akt due to mutations inthe PIK3CA, the catalytic subunit of the p110a subunit ofphosphoinositide 3-kinase, PTEN loss, INPP4B loss, or HER2amplification are common features of many tumors (8, 9). Thethree mammalian Akt isoforms (Akt1, Akt2, and Akt3) areencoded by distinct genes, have high sequence similarity andare activated by near-identical mechanisms (10, 11). Thecritical role of Akt in modulating cancer cell survival andgrowth has been well characterized (12). However, the roleplayed by individual Akt isoforms in different molecular sub-types of breast cancer has not been extensively evaluated. Inparticular, it is not known whether a specific Akt isoform playsa predominant role in TNBC. In the context of breast cancerinvasion and metastasis, Akt isoforms have nonredundantroles whereby Akt1 inhibits invasion and metastasis, yet Akt2promotes these phenotypes both in vitro and in mouse modelsof breast cancer progression (11–14). Akt3 is arguably the leaststudied isoform, and its function in breast cancer cell prolif-eration, survival, and migration is not known. Nevertheless,isoform-specific functions of Akt3 have been evaluated, espe-cially in knockoutmice inwhich the brain size of Akt3 nullmiceis reduced (15, 16). Akt3 has a putative oncogenic function issupported by the observation that it is overexpressed with highenzymatic activity in ER� breast cancer cells (17). This agreeswith the analysis by The Cancer Genome Atlas Project (TCGA)that has reported upregulation of AKT3 expression in 28% ofTNBCs (5). The recent identification of somatic mutations ofAKT3, including MAGI3–Akt3 and Akt3E17K, in different can-cers also points to an important role of this isoform intumorigenesis (18, 19). However, a causal role for Akt3 inbreast cancer initiation and growth has not been examined.

Here, we report that Akt3 is a critical regulator of the growthof TNBCs. Downregulation of Akt3 using short hairpin RNA(shRNA) inhibits tumor spheroid growth in three-dimensional(3D) as well as in xenografts. Akt3 depletion is accompanied by

Authors' Affiliations: 1Department of Pathology, Beth Israel DeaconessMedical Center; 2Department of Medicine, Harvard Medical School;3Department of Medical Oncology, Dana-Farber Cancer Institute; and4Department of Medicine, Brigham and Women's Hospital, Boston,Massachusetts

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Alex Toker, Beth Israel Deaconess MedicalCenter, Harvard Medical School, 330 Brookline Avenue, EC/CLS-633A,Boston, MA 02215. Phone: 617-735-2482; Fax: 617-735-2480; E-mail:[email protected]; and Kornelia Polyak, Department of MedicalOncology, Dana-Farber Cancer Institute. Phone: 617-632-2106; Fax: 617-582-8490; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2175

�2013 American Association for Cancer Research.

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robust upregulation of the cell-cycle inhibitor p27. Silencingp27 rescues spheroid growth inhibition mediated by Akt3depletion, indicating that Akt3 modulates tumor growth, atleast in part, via p27. These findings point to a previouslyunderappreciated isoform-selective role for Akt3 in the tumor-igenesis of TNBC, and demonstrate that inhibition of Akt3-specific signaling might be exploited for therapeutic purposes.

Materials and MethodsCell cultureMCF7, MDA-MB-231, MDA-MB-468, T47D, Hs578T, and

HEK293T cells were obtained from American Type CultureCollection (ATCC) and maintained in Dulbecco's ModifiedEagle Medium (DMEM; Cellgro) supplemented with 10% FBS(HyClone). SKBR3 andMDA-MB-453 cells obtained fromATCCwere cultured in McCoy's 5A medium (Cambrex) supplemen-ted with 10% FBS. BT-549 cells obtained from ATCC weregrown in RPMI-1640 medium supplemented with 10% FBS.SUM-159-PT cells were cultured in Ham's F12 medium (Cell-gro) supplemented with 5% FBS, 1 mg/mL hydrocortisone(Sigma-Aldrich), and 5 mg/mL insulin (Sigma-Aldrich). ZR-75-30 and BT474 cells obtained from ATCC were cultured inRPMI-1640 medium (Cambrex) supplemented with 10% FBSand 10 mg/mL insulin. MCF10DCIS.com (20) were grown inDMEM/Ham's F12 medium supplemented with 5% equineserum (Gibco-BRL), 10 mg/mL insulin, 500 ng/mL hydrocorti-sone (Sigma-Aldrich), 20 ng/mL EGF (R&D Systems), and 100ng/mL cholera toxin (Sigma-Aldrich). All cell lines obtainedfrom the cell banks listed above are tested for authenticationusing short tandem repeat profiling and passaged for fewerthan 6 months, and routinely assayed for mycoplasmacontamination.

3D cultures3D cultures were prepared as previously described (21).

Briefly, chamber slides were coated with growth factor–reduced Matrigel (BD Biosciences) and allowed to solidify for30 minutes. About 4,000 MCF10DCIS or MDA-MB-231 cells inassay medium were seeded on coated chamber slides. Assaymedium for MCF10DCIS contains DMEM/Ham's F12 supple-mented with 2% equine serum, 10 mg/mL insulin, 500 ng/mLhydrocortisone, 5 ng/mL EGF, 100 ng/mL cholera toxin,2 mg/mL puromycin, and 2% Matrigel. MDA-MB-231 assaymedium contains DMEM supplemented with 2% tet systemapproved FBS (Clontech) and 5% Matrigel. The assay mediumwas replaced every 4 days. About 100 ng/mL doxycycline wasadded every 2 or 3 days.

AntibodiesAnti-Akt1monoclonal antibody (mAb), anti-Akt2mAb, anti-

Akt3 mAb, anti-phospho-Akt S473 (pAkt) mAb, and anti-p27mAb were obtained from Cell Signaling Technology. Anti-b-actin mAb was purchased from Sigma-Aldrich. Anti-bromo-deoxyuridine (BrdUrd) mAb was obtained from Roche. Anti-p85 polyclonal antibody was generated in house and has beendescribed previously (22). Horseradish peroxidase (HRP)–con-jugated anti-mouse and anti-rabbit immunoglobulin G anti-body were purchased from Chemicon.

RNA interferenceFor doxycycline-inducible shRNA-mediated knockdown of

Akt isoforms, a set of single-stranded oligonucleotides encod-ing the Akt1, Akt2, and Akt3 target shRNA and its complementwere synthesized. The hairpin sequences have been validatedpreviously (11). Akt1, sense, 50-CCGGGAGTTTGAGTACCT-GAAGCTGCTCGAGCAGCTTCAGGTACTCAAACTCTTTTTG-30; Akt2, sense, 50-CCGGGCGTGGTGAATACATCAAGACCT-CGAGGTCTTGATGTATTCACCACGCTTTTTG-30; and Akt3,sense, 50-CCGGCTGCCTTGGACTATCTACATTCTCGAGAAT-GTAGATAGTCCAAGGCAGTTTTTG-30. The oligonucleotidesense and antisense pair was annealed and inserted into tet-on pLKO. To produce lentiviral supernatants, 293T cells werecotransfected with control or shRNA-containing tet-on pLKOvectors, vesicular stomatitis virus G glycoprotein, and psPAX2for 48 hours. p27 shRNA sequence (sense, 50-CCGGAAGT-ACGAGTGGCAAGAGGTGCTCGAGCACCTCTTGCCACTCG-TACTTTTTTTG-30) previously validated (23) was cloned intothe tet-onpLKO lentiviral expression systemasdescribed above.Cells stably expressing doxycycline-inducible shRNA were cul-tured in medium containing puromycin (0.5–2 mg/mL) Geneknockdown was induced by incubating cells with 100 ng/mLdoxycycline for 48 to 72 hours.

PlasmidsFor doxycycline-inducible overexpression of Akt3, HA–

Akt3/pTRIPZ was constructed. HA–Akt3 cDNA was amplifiedby PCR from HA–Akt3/pcDNA3. The resulting PCR productwas digested with restriction enzymes AgeI and ClaI, followedby insertion into pTRIPZ lentiviral vector (Thermo Scientific).

shRNA screenScreening was performed as described previously (24).

Briefly, cells were infected with a library of lentiviral shRNAsdirected against 26 human kinases commonly gained andoverexpressed in breast cancer. Infected cells were selectedfor puromycin resistance, and cultured for 6 days. Cell viability/proliferation was assessed using CellTiter-Glo (Promega). Sup-pression of growth was defined as the capacity for at least twoclones of shRNA targeting the same gene to decreased prolif-eration by �30% compared with control.

Xenograft studiesFemale nude mice (6–8-week-old) were purchased from

Taconic and maintained and treated under specific pathogen-free conditions. All procedures were approved by the Institu-tional Animal Care and Use Committee at Dana-Farber CancerInstitute (Boston, Massachusetts) and conform to the federalguidelines for the care and maintenance of laboratory animals.Themicewere injectedsubcutaneouslywith1�105MCF10DCISor 2 � 106 MDA-MB-468 cells in media with 50% Matrigel. Themice were randomly divided into control and treatment groupsand treatedwithwater supplementedwith1mg/mLdoxycyclineand 2% sucrose or standardwater supply. Tumor formationwasexamined every 2 to 3 days for the whole duration of theexperiment. One hour before tumor harvesting, BrdUrd wasinjected intraperitoneally to assess cell proliferation. Tumorswere harvested and weighed at the experimental endpoint.

Akt3 in Triple-Negative Breast Cancer

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Immunohistochemistry of formalin-fixed paraffin-embedded xenograft tumors

Xenograft tumors were deparaffinized in xylene and hydrat-ed in a series of ethanol. After heat-induced antigen retrieval incitrate buffer (pH 6), the samples were blocked with donkeyserum. Slides were then incubated with primary antibodies forovernight at 4�C. Samples were washed three times with TBSfollowed by incubation with biotinylated secondary antibodiesfor 1 hour. The slides were washed and developed using the3,30-Diaminobenzidine Metal Enhanced Kit (Vector Labs) andcounter-stained with hematoxylin.

Transwell migration assaysA total of 1 � 105 cells in serum-free medium containing

0.1% bovine serum albuminwere added to the upper chambersin triplicate. NIH 3T3 cell–conditioned medium was added tothe lower chambers. After 2 to 16 hours of incubation at 37�C,nonmigrated cells on Transwell filters (8 mm pore size; Corn-ing)were removed. Cells that hadmigrated to the bottomof thefilters were fixed and stained using the Hema-3 stain set(Protocol).

Cell viability assaysMDA-MB-231 and SKBR3 cells were seeded 24 hours before

inhibitor treatment into 96-well plates at density of 2,000 to4,000 cells per well in 100 mL medium. Cell viability wasmeasured 72 hours after inhibitor treatment using the watersoluble tetrazolium salt (WST)-1 assay (Clontech) according tothe manufacturer's protocol.

Quantitative real-time PCRTotal RNA was isolated with TRizol (Invitrogen) according

to the manufacturer's protocol. Reverse transcription wasperformed using random hexamers and multiscribe reversetranscriptase (Applied Biosystems). Quantitative real-timePCR was performed using an ABI Prism 7700 sequence detec-tor. Akt isoform–specific primers have been validated previ-ously (25). Akt1 primer: sense, 50-CAAGCCCAAGCACCGC-30;Akt2 primer: sense, 50-GCAAGGCACGGGCTAAAG-30; andAkt3 primer: sense, 50-GAAGAGGAGAGAATGAATTGTAGT-CCA-30. PCR reactions were carried out in triplicate. Quanti-fication of mRNA expression was calculated by the dCTmethod with glyceraldehyde-3-phosphate dehydrogenase asthe reference gene.

ImmunoblotsCells were washed with PBS at 4�C and lysed in radioimmu-

noprecipitation assay buffer (1% NP-40, 0.5% sodium deoxycho-late, 0.1% SDS, 150 mmol/L NaCl, 50 mmol/L Tris–HCl(pH 7.5), proteinase inhibitor cocktail, 50 nmol/L calyculin, 1mmol/L sodium pyrophosphate, and 20 mmol/L sodium fluo-ride) for 15 minutes at 4�C. Cell extracts were precleared bycentrifugation at 13,000 rpm for 10 minutes at 4�C and proteinconcentrationwasmeasuredwithBio-Radprotein assay reagentusing a Beckman Coulter DU-800 machine. Lysates were thenresolved on 10% acrylamide gels by SDS–PAGE and transferredelectrophoretically to nitrocellulose membrane (Bio-Rad) at100 V for 60 minutes. The blots were blocked in Tris-bufferedsaline (TBST) buffer (10 mmol/L Tris–HCl, pH 8, 150 mmol/L

NaCl, and 0.2% Tween 20) containing 5% (w/v) nonfat dry milkfor 30 minutes, and then incubated with the specific primaryantibodydiluted inblockingbuffer at 4�Covernight.Membraneswere washed three times in TBST, and incubated with HRP-conjugated secondary antibody for 1 hour at room temperature.Membranes were washed three times and developed usingenhanced chemiluminescence substrate (Pierce).

Survival analyses in clinical cohorts of TNBCTo determine the association of AKT3 mRNA expression

with patient survival, we used two publicly available clinicallyannotated breast cancer gene expression datasets. The first is apublished gene expression microarray (GEM) meta-datasetthat integrates gene expression and survival data from a totalof 19 published studies, including a total of 2,731 patients, asdescribed in ref. 26. Of note, 527 of these patients weremolecularly subtyped using the three-gene model of (27) asER�/HER2�. Given that true PR expression is not observed inER� breast cancer (28), this group is representative of TNBC.To assess the association of AKT3 expression with survivalamong these patients with ER�/HER2� breast cancer, weperformed a Cox proportional hazards analysis. To assess theassociation of a binary AKT3 expression score with survival, wethresholded the AKT3 expression at the TNBC populationmedian and plotted Kaplan–Meier curves in AKT3-highexpressing versus ATK3-low expressing tumors. We assessedthe significance in difference between survival curves byperforming a log-rank test. We performed a similar set ofanalyses on the METABRIC dataset, which is composed of atotal of approximately 2,000 breast cancer cases with clinicalfollow-up, as described in ref. 29. Pathologic data for ER, PR,and HER2 expression were available for this cohort, and basedon these data we identified 319 patients with TNBC. TheMETABRIC dataset contained four probes for theAKT3mRNA.For each probe, we performed Cox proportional hazardsanalysis to overall survival. To assess the association of abinary AKT3 expression score with survival, we computed themean AKT3 expression for each patient and thresholded themean AKT3 expression score at the TNBC population median.We plotted Kaplan–Meier curves in AKT3-high expressingversus AKT3-low expressing tumors and performed a log-ranktest to assess differences in the curves.

ResultsOverexpression of Akt3 in TNBC

To identify genes that are necessary for growth of TNBC, wescreened a panel of 17 commonly studied breast cancer lines aswell as two triple-negative nontumorigenic, immortalizedmammary epithelial lines (MCF-10A and MCF-12A) with alibrary of lentiviral shRNAs targeting 26 protein kinases ampli-fied in breast tumors. Akt3 was found to be one candidate genethat was preferentially required for TNBC growth. In 10 of 10TNBC lines tested (including bothmesenchymal and basal-likesubtypes), Akt3 depletion leads to growth inhibition (Fig. 1A).In contrast, proliferation is inhibited in only 3 of 9 luminalcancer lines transduced with Akt3 shRNA (P for difference inproportions ¼ 0.009). To investigate whether there is a corre-lation between Akt3 expression and breast tumor subtypes, we

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analyzed a dataset of breast cancer from TCGA (n ¼ 825).SampleswithAkt3 amplification have an overrepresentation inthe TNBC subtype (14%) and an underrepresentation in lumi-nal tumors (3%; Fig. 1B). In addition, whereas Akt3 mRNA isupregulated in 2% luminal breast tumors, its expression issignificantly higher in 21% of TNBCs. To determine whetherthis is also observed in breast cancer cell lines, we quantifiedmRNA levels for individual Akt isoforms. Although all cell linestested express Akt1 and Akt2, Akt3 is exclusively expressed inTNBC lines (Fig. 1C). Correspondingly, 5 of 5 triple-negativelines express Akt3 at the protein level as observed by immu-noblot analysis, whereas only one of the tested luminal linesshows detectable Akt3 expression (T47D; Fig. 1D).

To evaluate the association of Akt3 mRNA expression withpatient survival in TNBC, we used two of the largest sets ofpublicly available clinically annotated breast cancer geneexpression data. The first is a published GEM meta-datasetthat integrates gene expression and survival data from a totalof 19 published studies, including a total of 2,731 patients, asdescribed previously (26). The second analysis was performedon the full METABRIC dataset, which is composed of a total ofapproximately 2,000 breast cancer cases with clinical follow-up, as described previously (29). Analysis of both datasetsprovides no evidence to suggest that AKT3 mRNA expressionlevels are an important predictor of survival in TNBC (Sup-plementary Fig. S1). However, mRNA expression levels may

Figure 1. Preferential amplification andoverexpression of Akt3 in TNBCs. A, plot showinggrowth inhibition byAkt3 shRNAs in apanel of breast cell lines using aloss-of-function screen. Inhibition was scored when growth was inhibited by �30%, with at least two clones of Akt3 shRNA. B, bar graph depictingthe percentage of Akt3 amplification and mRNA upregulation in the indicated tumors, using a dataset from The Cancer Genome Atlas. C, analysis ofAkt isoform mRNA expression in various breast cancer cell lines by real-time RT-PCR. The levels of Akt isoform mRNAs are expressed as a ratio relative tothe Akt1 mRNA in each cell line. D, immunoblotting showing expression of Akt isoforms in a panel of breast cancer lines. Actin was used as loading control.M, mesenchymal-like.






show poor correlation with levels of activated Akt3, and thusmay be poor markers of pathway activation. Thus, to definitelydetermine the association of Akt3 signaling with patientsurvival, it will be important to perform analyses assessingthe relationship between phosphorylated/activated Akt3 andpatient survival on large clinical cohorts. These data arecurrently unavailable from public databases of clinically anno-tated molecular profiling data in breast cancer.

Akt3 depletion potently inhibits breast tumor spheroidgrowth and sensitizes TNBC to an Akt inhibitor

To explore the function of Akt3 in TNBC, we generated apanel of breast cancer lines with tet-on doxycycline-inducibleAkt1, Akt2, or Akt3 shRNA. Upon doxycycline administration,Akt isoforms are depleted specifically and quantitatively(Fig. 2A). We investigated the consequence of Akt3 silencingon tumor spheroid growth using a 3D culture system in vitrothat more accurately recapitulates phenotypes that govern

tumor growth in vivo. Depletion of Akt3 in MDA-MB-231 cellspotently inhibits spheroid growth (Fig. 2B), resulting in a 57%reduction of spheroid size (Fig. 2C). In contrast, the effect ofAkt1 or Akt2 depletion on spheroid growth is much moremodest. A 26% reduction in spheroid size is observed in Akt2-depleted cells, whereas there is no significant change inspheroid size when Akt1 is silenced. Knockdown of Akt3 inanother triple-negative line, MCF10DCIS, cloned from a xeno-graft lesion formed by premalignant MCF-10AT cells and hasthe oncogenicmutation H1047R in the p110a catalytic subunit(PIK3CA; ref. 30), has similar effect on the inhibition of spheroidgrowth (56% reduction; Fig. 2D). Conversely, depletion of Akt1or Akt2 has minimal effect on the growth of spheroids. Thesedata suggest that Akt3 has a selective role in promoting tumorspheroid growth of TNBCs.

Data from a recent study have demonstrated that themajority of TNBC are resistant to the pan-Akt inhibitorGDC-0068 (31). Because most of the TNBC lines tested express

Figure 2. An essential role of Akt3 for the growth of triple-negative breast tumor spheroids. A, MDA-MB-231 cells expressing tet-on Akt isoform shRNA weretreated with doxycycline (Dox; 100 ng/mL) for 3 days. Whole-cell lysates were subjected to immunoblotting. B, MDA-MB-231 cells were grown in 3D culturesfor 7 days in the presence or absence of doxycycline (100 ng/mL). Representative phase-contrast images are shown. C, the size of spheroids (n� 50) grown inB was quantified and depicted in bar graph. Error bars, mean � SEM. D, immunoblotting showing knockdown of Akt isoforms in tet-on MCF10DCIScells when treated with doxycycline (100 ng/mL) for 3 days. p85 was used as a loading control. Bar graph depicts spheroid size of MCF10DCIS cells grown in3D culture for 7 days in the presence or absence of doxycycline (100 ng/mL). �, P < 0.05; ��, P < 0.01; ��, P < 0.001. E, MDA-MB-231 cellsexpressing tet-on Akt3 shRNA were treated with or without doxycycline (100 ng/mL) for 2 days. Cells were then seeded to 96-well plates, followed byGSK690693 treatment for 3 days. Cell viability was assessed by WST assays. Error bars, SEM between replicates (n ¼ 3). F, SKBR3 cells expressingtet-on Akt3/pTRIPZwere treatedwith or without doxycycline (100 ng/mL) for 3 days. Cells were treatedwith inhibitor and assessed for viability as described inE; whole-cell lysates were subjected to immunoblotting.

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Akt3, we next determined whether Akt3 loss in TNBC mightresult in increased sensitivity to Akt inhibition. AlthoughMDA-MB-231 cells are resistant to the ATP-competitive inhibitorGSK690693 (IC50: 24.2 mmol/L), loss of Akt3 results in a 6-folddecrease in IC50 (3.8 mmol/L; P < 0.0001; Fig. 2E). Similarly, Akt3depletion in MCF10DCIS cells leads to a 28-fold decrease inIC50 (0.54 vs. 15.4 mmol/L in control cells; P < 0.0001; data notshown). To further determine the role of Akt3 in drug sensi-tivity, we overexpressed Akt3 in a luminal breast cancer line,SKBR3, which does not express this Akt isoform. Overexpres-sion of Akt3 results in a 2.7-fold increase in IC50 for GSK690693compared with control cells (13.4 vs. 5 mmol/L in control cells;P < 0.05; Fig. 2F). Together, these data indicate that Akt3 playsan important role in conferring resistance to pan-Akt inhibi-tors in TNBCs.p27 is a key regulator of cell-cycle progression. Studies have

shown that Akt inhibits p27 expression via the transcriptionfactor forkhead box O (32). Moreover, phosphorylation by Akthas been shown to promote the proteasomal degradation ofp27 (33), thereby regulating its expression at both the tran-scriptional and posttranslational level. However, it is notknown whether p27 is regulated redundantly by all Akt iso-forms, or whether specificity exists. In this context, inMCF10DCIS cells Akt1 silencing results in increased p27protein expression (Fig. 3A), whereas p27 levels are diminishedupon Akt2 silencing. Interestingly, p27 is significantly elevatedin Akt3-depleted cells. Similar results are observed in two other

TNBC lines, MDA-MB-231 and MDA-MB-468 (Fig. 3A). Todetermine whether Akt3 promotes tumor cell proliferation bymodulating p27 expression levels, we performed a rescueexperiment by silencing p27. As shown in Fig. 3B, approxi-mately 50% of p27 is depleted by specific shRNA. Downregula-tion of p27 alone has minimal effect on 3D spheroid growth(Fig. 3C). In contrast, Akt3 shRNA–mediated spheroid growthinhibition is completely rescued by p27 silencing, indicatingthat the effect of Akt3 on tumor spheroid growth is mediated,at least in part, by p27.

RoleofAkt isoforms inmodulating invasiveness of tumorspheroids

We and others have shown that Akt1 and Akt2 have oppos-ing functions in regulating breast cancer cell invasion andmetastasis (12). However, a functional role for Akt3 in mod-ulating cancer cell migration has not been evaluated. To assesschemotactic cell migration, Transwell migration assays wereperformed. In three TNBC lines (MCF10DCIS, MDA-MB-468,and BT-549), Akt1 and Akt2 silencing leads to increased anddecreased cell migration, respectively (Fig. 4A), in agreementwith published studies. A statistically significant but modestenhancement of migration is observed upon Akt3 silencing inall three lines. Furthermore, Akt1 silencing promotes anabnormal branching morphology with cells protruding intoMatrigel (Fig. 4B). In contrast, spheroids remain round anduniform when Akt2 or Akt3 is depleted. These results indicate

Figure 3. p27 is a downstreameffector of Akt3 in regulating tumorspheroid growth. A, MCF10DCISandMDA-MB-468 cells expressingtet-on Akt isoform shRNA weretreated with doxycycline(Dox; 100 ng/mL) for 3 days,followed by immunoblot analysis.MDA-MB-231 cells were infectedwith Akt isoform shRNA/pLKOlentiviral vectors for 3 days,followed by immunoblot analysis.B, MCF10DCIS cells were infectedwith tet-on p27 shRNA lentiviralvector. Three days afterdoxycycline (100 ng/mL)treatment, cell lysates wereimmunoblotted with anti-p27 andanti-p85. C, MCF10DCIS cellsexpressing tet-on p27 shRNA and/or Akt3 shRNA were seeded to 3Dculture for 24 hours, followed bytreatment with dox (100 ng/mL) foran additional 7 days. Spheroid size(n ¼ 100) was quantified.��, P < 0.001.






that Akt3 plays a critical role in modulating tumor spheroidgrowth in the absence of significant effects on breast cancercell invasive migration.

Akt3 downregulation attenuates breast tumor growthin vivo

Next, we evaluated the contribution of Akt3 to tumorigen-esis in vivo. Because Akt3 selectively inhibits tumor spheroidgrowth of TNBC lines, MDA-MB-468 cells stably expressing tet-on Akt1, Akt2, and Akt3 shRNA were injected subcutaneouslyinto nude mice. When Akt3 is depleted by inducing shRNAexpression via doxycycline in drinking water, there is a signif-icant reduction in tumor growth (Fig. 5A). In striking contrast,silencing Akt1 or Akt2 has no significant effect on tumorgrowth. To confirm the knockdown of Akt isoforms and toassess their effect on cell proliferation as well as p27 expres-sion, immunohistochemistry (IHC) was performed on xeno-grafts harvested at the end of the study. Akt protein levels aredramatically reduced in doxycycline-treated tumors whencompared with their vehicle-treated counterparts (Fig. 5B).Depletion of Akt3, but not Akt1 or Akt2, is concomitant with asignificant reduction in BrdUrd staining, consistent with the

notion that Akt3 plays a critical role in tumor cell proliferationin vivo. Similar to what is observed with the in vitro analysis,depletion of Akt3, but not Akt1 or Akt2, leads to a markedelevation of p27 expression. Interestingly, silencing of eitherAkt1 or Akt3 markedly reduces tumor growth in a distincttriple-negative line, MCF10DCIS (Fig. 5C). The more markedeffect of Akt1 on MCF10DCIS tumor growth in vivo comparedwith in vitro could be due to paracrine function of Akt1 in thetumor-associated stroma and microenvironment (12). Doxy-cycline itself has no effect on tumor growth because the growthof tumors containing empty vector in the absence of doxycy-cline is indistinguishable from doxycycline-treated tumors(Fig. 5C). Similar to what is observed in MDA-MB-468 cells,silencing Akt3, but not Akt1 or Akt2, leads to robust upregula-tion of p27 expression (Fig. 5D). Taken together, these datademonstrate that TNBC growth can be inhibited by silencingAkt3 alone.

DiscussionTNBCs are especially aggressive and have a relatively poor

prognosis compared with other breast tumor subtypes. In thepresent study, we set out to identify molecular targets of TNBC

Figure 4. Isoform-specific functionsof Akt isoforms on breast cancercell migration and invasion. A,MCF10DCIS, MDA-MB-468, andBT-549 cells expressing tet-on Aktisoform shRNA were treated withdoxycycline (Dox; 100 ng/mL).Three days after treatment, cellswere subjected to Transwellmigration assays. Relativemigration (y-axis), ratio of thenumber of migrated cells in testversus control. Error bars, mean �SEM. �, P < 0.05; ��, P < 0.01;��,P < 0.001. B, MCF10DCIS cellsexpressing tet-on Akt isoformshRNA were seeded to 3D culturefor 2 days, followed by treatmentwith doxycycline (100 ng/mL) for anadditional 4 days.

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with therapeutic potential using an RNAi functional geneticscreen. We find that Akt3 silencing preferentially inhibits theproliferation of TNBC cell lines both in vitro and in xenografts.Consistent with this, Akt3 is overexpressed in 21% and 2% oftriple-negative and luminal breast tumors, respectively. Todetermine the contribution of individual Akt isoforms intriple-negative tumor growth, we used an inducible shRNAstrategy. Silencing Akt3, but not Akt1 or Akt2, dramaticallyreduces tumor spheroid growth in 3D. The selective effect ofAkt3 in triple-negative tumor is noteworthy because it has beenlargely assumed that Akt isoforms play redundant roles inregulating tumor proliferation and growth. Furthermore, anisoform-specific function of Akt3 in any cancer context has notyet demonstrated. In breast cancer, published studies havefocused on the opposing functions of Akt1 and Akt2 on cellmigration and invasion. Several downstream Akt targets havebeen shown to contribute to the differential functions of these

two isoforms in motility, including palladin, nuclear factor ofactivated T cells, tuberous sclerosis complex 2, andb1 integrins(34–37). In this study, we find that althoughAkt3 is required forTNBC proliferation and tumor growth, unlike Akt1 it does notpromote an invasive phenotype. These findings have signifi-cant implications for targeted therapeutics, because currentlyall Akt small molecule inhibitors in phase I and II clinical trialsare not isoform selective. One prediction is that pan Aktinhibitors could have undesired clinical effects by actuallypromoting invasion and even metastasis, because Akt1 hasbeen shown to have both and antiinvasive and antimetastaticactivity in vivo. In contrast, targeting Akt3 exclusively in TNBCsmay have the advantage of inhibiting tumor growth withoutany significant impact on metastatic dissemination. Further-more, due to the critical roles of Akt isoforms in tissuehomeostasis, pan-Akt inhibitors are expected to be doselimiting. Instead, we would argue that isoform-specific

Figure 5. Akt3 depletion attenuates tumor growth in vivo. A, doxycycline (Dox; 1 mg/mL) was administered to mice in drinking water started 1 day aftersubcutaneous injection of MDA-MB-468 cells expressing Akt isoform shRNA (n ¼ 10). Tumor volume was measured on days 22, 31, 40, 48, and 64 afterinjection. Bar graphs, tumor xenograft weights 64 days after injection. ��, P < 0.001. B, Akt isoforms, BrdUrd, and p27 IHC staining for xenografts inA. C, MCF10DCIS cells expressing Akt isoform shRNA or empty vector were injected subcutaneously into nude mice (n ¼ 10). Doxycycline (1 mg/mL) wasadministered tomice in drinking water started 1 day after injection. Plots depict tumor volumesmeasured on days 11, 14, 18, 22, and 26 after injection. D, Aktisoforms and p27 IHC staining for xenografts in C.






inhibitors, including Akt3-specific drugs, would likely have lessassociated toxicity and provide a better therapeutic window.

Further support for the need to develop Akt3-specific inhi-bitors for the therapeutic targeting of TNBC tumors comesfrom a recent study on the sensitivities of different breastcancer lines to the pan-Akt inhibitor GDC-0068 (31). Of allluminal lines tested in this study, 5 of 6 lines are sensitive toGDC-0068 with an IC50 < 2 mmol/L. In agreement with ourexpression analysis, most of these lines have undetectablelevels of Akt3. In contrast, 5 of 5 TNBC lines tested expressAkt3, and 3 of themare insensitive toGDC-0068 (MDA-MB-468,MDA-MB-231, andMCF10A: IC50 > 10 mmol/L; BT-549: IC50 5.6mmol/L; Hs578T IC50 2.2 mmol/L). Consistent with this, weshow that MDA-MB-231 and MCF10DCIS cells are resistant tothe pan-Akt inhibitor GSK690693. Importantly, Akt3 depletionin both lines resulted in robust sensitization to the drug,suggesting a critical role of Akt3 in conferring resistance topan-Akt inhibitor in TNBCs. It will be interesting to determinewhether there is a correlation between Akt3 expression andsensitivity to pan-Akt inhibitors in clinical samples.

On the basis of the gene expression signatures, sevendistinct subtypes of TNBC have been proposed, with aclassification that comprises two basal-like, immunomodu-latory, mesenchymal, mesenchymal stem-like (MSL), andluminal androgen receptor subtypes (38). Recent reportsindicated that different subtypes of TNBCs may responddifferently to targeted and chemotherapies (39). Our datapoint to a distinct function of Akt3 in MDA-MB-468 andMDA-MB-231 breast cancer lines, which belong to the basal-like 1 and MSL subtype, respectively. Future studies warrantevaluating the impact of Akt isoform–specific signaling inthe various proposed TNBC subtypes.

Our study has also uncovered distinct roles of Akt iso-forms in the regulation of p27. It has been shown that Akt isa negative regulator of p27. Here, we demonstrate that Akt2,in contrast with Akt1, positively regulates p27 expression.Surprisingly, silencing Akt3 results in a robust elevation ofp27 expression concomitant with inhibition of the growth ofTNBC lines in vitro as well as in xenografts. Using an shRNArescue strategy, we show that p27 plays a critical function inthe regulation of tumor spheroid growth mediated by Akt3.These findings not only lend further support to the notion ofnonredundant functions of Akt isoforms at the molecularlevel, they also suggest the potential use of p27 as a bio-marker of Akt3 activity.

Depletion of Akt3 alone inhibits MCF10DCIS andMDA-MB-468 xenograft tumor growth. Interestingly, although Akt1 hasminimal effect in modulating spheroid growth, silencing Akt1in MCF10DCIS in xenografts leads to a reduction of tumorgrowth comparable with that observed with Akt3 depletion.This is likely due to the functional importance of Akt1 signalingin the tumor-associated stroma because this isoform plays animportant role in promoting VEGF-mediated angiogenesis(40). Whether angiogenesis contributes to the effect of Akt1onMCF10DCIS growth in ourmodel remains to be determined.

In conclusion, our studies demonstrate that targeting Akt3and downstream signaling may be an effective approach toinhibit growth of TNBC. Because Akt3-specific inhibitors haveyet to be developed, we propose that the development of suchinhibitors could constitute the basis for Akt3 as a novel targetfor single agent or combination therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y.R. Chin, K. Polyak, A. TokerDevelopment of methodology: Y.R. ChinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.R. Chin, T. Yoshida, A. MarusykAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.R. Chin, T. Yoshida, A. Marusyk, A.H. Beck, K.Polyak, A. TokerWriting, review, and/or revisionof themanuscript:Y.R. Chin, A.Marusyk, A.H. Beck, K. Polyak, A. TokerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A.H. Beck, A. TokerStudy supervision: Y.R. Chin, K. Polyak, A. Toker

AcknowledgmentsThe authors thank the confocal imaging core and histology core at BIDMC for

their technical support with IHC; Casey Stottrup for technical assistance with 3Dcultures, migration assays, and immunoblotting; Evan Lien for providingpTRIPZ-Akt3 plasmid; and members of the Toker laboratory for discussions.

Grant SupportThis study was supported in part by grants from the NIH (K99CA157945 to

Y.R. Chin; CA092644 and T32 CA081156–09 to A. Toker), the Susan G. KomenFoundation (K. Polyak), from a sponsored research grant from ImClone/Eli Lilly(A. Toker and Y.R. Chin), and from an award from the Klarman FamilyFoundation (A.H. Beck).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 2, 2013; revised November 4, 2013; accepted November 20,2013; published OnlineFirst December 12, 2013.

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2014;74:964-973. Published OnlineFirst December 12, 2013.Cancer Res Y. Rebecca Chin, Taku Yoshida, Andriy Marusyk, et al. Targeting Akt3 Signaling in Triple-Negative Breast Cancer

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