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Cancer Biology and Signal Transduction

Upregulation of AKT3 Confers Resistance to theAKT Inhibitor MK2206 in Breast CancerCasey Stottrup, Tiffany Tsang, and Y. Rebecca Chin

Abstract

Acquired resistance to molecular targeted therapy represents amajor challenge for the effective treatment of cancer. Hyperactiva-tion of the PI3K/AKT pathway is frequently observed in virtuallyall human malignancies, and numerous PI3K and AKT inhibitorsare currently under clinical evaluation. However, mechanisms ofacquired resistance to AKT inhibitors have yet to be described.Here, we use a breast cancer preclinical model to identify resis-tance mechanisms to a small molecule allosteric AKT inhibitor,MK2206. Using a step-wise and chronic high-dose exposure,breast cancer cell lines harboring oncogenic PI3K resistant toMK2206 were established. Using this model, we reveal that AKT3expression is markedly upregulated in AKT inhibitor–resistantcells. Induction of AKT3 is regulated epigenetically by the bro-modomain and extra terminal domain proteins. Importantly,

knockdown of AKT3, but not AKT1 or AKT2, in resistant cellsrestores sensitivity to MK2206. AKT inhibitor–resistant cells alsodisplay an epithelial to mesenchymal transition phenotype asassessed by alterations in the levels of E-Cadherin, N-Cadherin,and vimentin, as well as enhanced invasiveness of tumor spher-oids. Notably, the invasive morphology of resistant spheroids isdiminished uponAKT3 depletion.We also show that resistance toMK2206 is reversible because upon drug removal resistant cellsregain sensitivity to AKT inhibition, accompanied by reexpressionof epithelial markers and reduction of AKT3 expression, implyingthat epigenetic reprogramming contributes to acquisition ofresistance. These findings provide a rationale for developingtherapeutics targeting AKT3 to circumvent acquired resistance inbreast cancer. Mol Cancer Ther; 15(8); 1964–74. �2016 AACR.

IntroductionBreast cancer is the most frequently diagnosed malignancy in

women. Genetic and epigenetic deregulation of the phosphoi-nositide 3-kinase (PI3K)/AKT signaling pathway is highly prev-alent in breast cancer (1). Mutations in PIK3CA, the catalyticsubunit of the p110a subunit of PI3K, loss of PTEN, and ampli-fication of HER2 are common genomic abnormalities in tumorcells leading to hyperactivation of PI3K/AKT signaling and sub-sequent phenotypes associated with malignancy (2, 3). Based onthis knowledge, a number of small molecule inhibitors targetingvarious components of the PI3K/AKT pathway are in clinicaldevelopment and evaluation. For example, both allosteric andATP-competitive AKT inhibitors (MK2206: allosteric; GDC0068and GSK690693: ATP-competitive) are being assessed in clinicaltrials for various aggressive cancers as monotherapy or as com-bination strategies (4, 5). In cell-free assays using purified recom-binant AKT, these pan-AKT inhibitors inhibit all three AKT iso-forms with nanomolar potencies. Preliminary evidence of clinicalactivity is observed with combination of MK2206 and trastuzu-mab in patients with HER2-positive solid tumors in a phase I

clinical trial (6). In a separate phase Ib study, the combination ofthe PI3K inhibitor BKM120 and trastuzumab was evaluated inpatients with HER2-positive advanced/metastatic breast cancerresistant to trastuzumab-based therapy, and initial evidence oftherapeutic efficacy has been presented (7). However, the expe-rience from other molecular targeted therapies suggests that theclinical benefits of these PI3K/AKT inhibitors are likely to belimited by the development of acquired resistance in patients.

There are a few documented mechanisms of resistance totargeted therapy for inhibitors that target PI3K or AKT (8). Ofthese, overexpression and/or amplification of Myc, Notch,RSK3/4, HER3, and PI3K itself have been proposed to conferresistance to certain PI3K inhibitors in the context of breastcancer (9–14). In addition, RNA sequencing analysis revealedan induction of the receptor tyrosine kinase (RTK) AXL in headand neck squamous cell carcinomas (SCC) upon adaptation tothe PI3Ka inhibitor BLY719. AXL dimerizes with EGFR andresults in the activation of mTOR and resistance. Importantly,overexpression of AXL is observed in SCC tumors of patientstreated with BLY719 (15). With respect to AKT inhibitors,upregulation and activation of RTKs have been implicated inacquisition of resistance to targeted therapy. For example, acutetreatment of breast cancer cells with an allosteric AKT inhibitorinduces marked upregulation of insulin receptor, IGF1R andHER3, via a FOXO-dependent manner (16). The same studyalso showed that phosphorylation of multiple RTKs isenhanced upon AKT inhibition, by relieving mTORC1-medi-ated feedback inhibition. HER3 expression has also shown tobe induced in triple-negative breast cancer (TNBC) acutelytreated with the catalytic AKT inhibitor GDC0068 (17). Con-sistent with preclinical studies, compensatory feedback activa-tion of HER3 and ERK has been observed in tumor biopsiesfrom patients treated with GDC0068 (18).

Department of Pathology, Beth Israel Deaconess Medical Center,Harvard Medical School, Boston, Massachusetts.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Yuet Rebecca Chin, 330 Brookline Ave, E/CLS 633A,Department of Pathology, Beth Israel Deaconess Medical Center, HarvardMedical School, 3 Blackfan Circle, Boston, MA 02115. Phone: 617-735-2484; Fax:617-735-2480; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0748

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(8) August 20161964

on March 1, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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Delineating the spectrum of resistance mechanisms is criticalfor the development of strategies to prevent or treat resistanttumors. Because mechanisms of acquired resistance to AKT inhi-bitors have not been elucidated, in this study we used a breasttumor line harboring an activating mutation in PI3K and gener-ated cells resistant to a selective AKT inhibitor, MK2206, bychronic adaptation.We show that these cells display upregulationof AKT3 protein and function, accompanied by a phenotypicswitch in the epithelial to mesenchymal transition (EMT). Deple-tion of AKT3 in resistant cells restores sensitivity to MK2206,highlighting AKT3 as a candidate for conferring resistance tomolecular targeted therapy in breast cancer.

Materials and MethodsCell culture

T47D, HEK293T, and MDA-MB-468 cells were obtainedfrom ATCC and maintained in Dulbecco's modified Eaglemedium (DMEM; Cellgro) supplemented with 10% tet sys-tem-approved fetal bovine serum (FBS; Clontech). All cell linesobtained from the cell banks listed above are tested for authen-tication using short tandem repeat (STR) profiling and passagedfor fewer than 6 months, and routinely assayed for mycoplasmacontamination.

AKT inhibitor–resistant line generationTheMK2206-resistant cell line T47DMK0.2-5was generated by

gradual dose escalation of the AKT inhibitor MK2206 from 0.2mmol/L to 5 mmol/L for a period of 2 months, then the cells weremaintained in 5 mmol/L MK2206. T47D MK5-resistant line wasgenerated by chronic exposure of cells to 5 mmol/L MK2206 for 2months. T47D GDC0.2-5–resistant line was generated by cultur-ing cells in increasing concentration of the AKT inhibitorGDC0068 from 0.2 mmol/L to 5 mmol/L for 2 months. T47Dparental cells were maintained in growth medium containingdimethyl sulfoxide (DMSO).

Fresh AKT inhibitor or DMSO was replaced every 3 to 4 days.Cells were considered resistant when they could be culturedroutinely in growth medium containing 5 mmol/L MK2206 orGDC0068. MK2206 and GDC0068 have been described previ-ously (4, 19) and were obtained from Selleck Chemical.

3D cultures3D cultures were prepared as previously described (20).

Briefly, chamber slides were coated with growth factor-reduced Matrigel (BD Biosciences) and allowed to solidifyfor 30 minutes. Cells (1 � 104) in assay medium were seededon coated chamber slides. Assay medium contained DMEMsupplemented with 10% FBS and 2% Matrigel. The assaymedium was replaced every 4 days. Doxycycline (dox, 100ng/mL) was added every 2 or 3 days.

AntibodiesAll primary antibodies in this study except p85 were

obtained from Cell Signaling Technology. Anti-p85 polyclonalantibody was generated in-house and has been described (21).Horseradish peroxidase–conjugated anti-mouse and anti-rab-bit immunoglobulin G (IgG) antibody were purchased fromChemicon.

RNA interferenceFor dox-inducible shRNA-mediated knockdown of AKT iso-

forms, a tet-on shRNA/pLKO system was used. The hairpinsequences targeting AKT1, AKT2, and AKT3 have been validated,and the construction of tet-on AKT isoform shRNA/pLKO vectorshas been described previously (22). Akt1, sense, 50-CCGGGA-GTTTGAGTACCTGAAGCTGCTCGAGCAGCTTCAGGTACTCAA-ACTCTTTTTG-30; Akt2, sense, 50-CCGGGCGTGGTGAATACAT-CAAGACCTCGAGGTCTTGATGTATTCACCACGCTTTTTG-30;Akt3, sense, 50- CCGGCTGCCTTGGACTATCTACATTCTCGA-GAATGTAGATAGTCCAAGGCAGTTTTTG-30. To produce len-tiviral supernatants, 293T cells were cotransfected with con-trol or shRNA-containing tet-on pLKO vectors, VSVG andpsPAX2 for 48 hours. Cells stably expressing dox-inducibleshRNA were cultured in medium containing puromycin (2mg/ml). Gene knockdown was induced by incubating cellswith 100 ng/mL dox for 48 to 72 hours.

PlasmidsFor dox-inducible overexpression of AKT3, cells were infected

with HA-AKT3/pTRIPZ lentiviral vector. Construction of HA-AKT3/pTRIPZ has been described previously (23).

Cell viability assaysCells were seeded 24 hours before inhibitor treatment into 96-

well plates at density of 5,000 cells per well in 100 mL medium.Cell viability was measured 48 hours after inhibitor treatmentusing theWST-1 assay (Clontech) according to themanufacturer'sprotocol.

In vitro kinase assaysAkt3 was immunoprecipitated from cell extracts and incubated

with 100 ng of GSK3b peptide in the presence of 250 mmol/L coldATP in a kinase buffer for 1 hour at 30�C. The kinase reaction wasstopped by the addition of SDS-PAGE loading buffer, and thesamples were assayed by immunoblotting.

Transwell invasion assaysTranswellfilters (8-mmpore size; Corning)were coatedwith 1.5

mgMatrigel (BD Biosciences). A total of 1� 105 cells in serum-freemedium containing 0.1% BSA were added to upper Transwellchambers in triplicate. Conditioned medium from NIH 3T3 cellswas used as chemoattractant, and was added to the lower cham-bers. After 7 hours of incubation at 37�C, non-invaded cells onTranswell filters were removed. Cells that had invaded andmigrated to the bottom of the filters were fixed and stained usingthe Hema-3 stain set (Fisher HealthCare Protocol).

Quantitative real-time RT-PCRTotal RNA was isolated with an RNeasy Mini Kit (Qiagen)

according to the manufacturer's protocol. Reverse transcriptionwas performed using random hexamers and multiscribe reversetranscriptase (Applied Biosystems). Quantitative real-timePCR was performed using an ABI Prism 7700 sequence detector.AKT3 primer: sense, 50-GAAGAGGAGAGAATGAATTGTAGTCCA-30; anti-sense, 50-AGTAGTTTCAAATAGTCAAAATCATTCATTG-30

(24); IGF1R primer: sense, 50-TTCAGCGCTGCTGATGTG-30; anti-sense, 50-GGCTCATGGTGATCTTCTCC-30 (25). PCR reactionswere carried out in triplicate. Quantification of mRNA expression

AKT3 and Breast Tumor Resistance

www.aacrjournals.org Mol Cancer Ther; 15(8) August 2016 1965




was calculated by the dCT method with GAPDH as the referencegene.

Copy number analysis with quantitative real-time PCRGenomic DNA was isolated with the QIAamp DNA Mini Kit

(Qiagen) according to the manufacturer's protocol. Real-timePCR was performed using an ABI Prism 7700 sequence detector.AKT3 primer: sense, 50-CTGGACATCACCAGTCCTAGCTC-30;anti-sense, 50-ACCCTTGGCTGGTCTGGG-30 (26); CEP17 primer:sense, 50-GCTGATGATCATAAAGCCACAGGTA-30; anti-sense, 50-TGGTGCTCAGGCAGTGC-30 (27). PCR reactionswere carriedoutin triplicate. Quantification of copy number was calculated by thedCT method with CEP17 as the reference gene.

ImmunoblottingCells werewashedwith PBS at 4�Cand lysed inRIPAbuffer [1%

NP-40, 0.5% deoxycholic acid (SDC), 0.1% SDS,150 mmol/LNaCl, 50 mmol/L Tris-HCl (pH 7.5), proteinase inhibitor cock-tail, 50 nmol/L calyculin, 1 mmol/L sodium pyrophosphate, 20mmol/L sodiumfluoride] for 15minutes at 4�C.Cell extractswereprecleared by centrifugation at 13,000 rpm for 10minutes at 4�C,andprotein concentrationwasmeasuredwith theBio-Radproteinassay reagent using a Beckman Coulter DU-800 machine. Lysateswere then resolved on 10% acrylamide gels by SDS-PAGE andtransferred electrophoretically to nitrocellulose membrane(BioRad) at 100 V for 60 minutes. The blots were blocked inTBST buffer (10mmol/L Tris-HCl, pH 8, 150mmol/L NaCl, 0.2%Tween 20) containing 5% (w/v) nonfat dry milk for 30 minutes,and then incubated with the specific primary antibody diluted inblocking buffer at 4�C overnight. Membranes were washed threetimes in TBST and incubated with horseradish peroxidase–con-jugated secondary antibody for 1 hour at room temperature.Membranes were washed 3 times and developed using enhancedchemiluminescence substrate (Pierce).

ResultsEstablishment and characterization of AKT inhibitor–resistantbreast tumor lines

In vitro modeling of acquired resistance has successfully iden-tified a resistance mechanism observed in cancer patients treatedwith kinase inhibitors. To explore mechanisms that mediateresistance to AKT inhibitors, we set out to model resistance toan allosteric AKT inhibitor, MK2206, currently in phase II clinicaltrials, using the luminal breast tumor cell line T47D, whichharbors an activating PIK3CA mutation (H1047R). This cell linehas been shown to be sensitive to MK2206 (28). MK2206-resis-tant derivative T47D lines were cultured either in graduallyincreasing doses (starting at 0.2 mmol/L) or at a constant concen-tration of 5 mmol/L inhibitor, until pools of cells growing in thepresence of 5mmol/L drugwere established. The resulting pools ofcells were termed T47DR (MK0.2-5; step-wise fashion) and T47DR (MK5; chronic high-dose fashion), respectively. Both resistantlines show resistance toMK2206 in cell viability assays, evidencedby >10-fold shifts in IC50 to MK2206 compared with the DMSO-treated parental pools (Fig. 1A). The T47DR lines also show cross-resistance to the ATP-competitive AKT inhibitor GDC0068 (Fig.1A). In dose response studies, T47DR lines are broadly resistant tothe inhibitory effect of MK2206 and GDC0068 at multiple nodesof the PI3K/AKT pathway, including pAKT S473, pPRAS40 T246,pGSK3b S9, and p4EBP1 S65 (Fig. 1B).

Upregulation of AKT3 as a novel mechanism of acquiredresistance to AKT inhibitors

The three AKT isoforms have distinct functions in modulat-ing phenotypes commonly associated with cancer. For exam-ple, whereas AKT1 is a breast cancer metastasis suppressor (29–31), AKT2 promotes invasion and metastasis of breast cancer invitro and in vivo (32, 33). In addition, recent studies have alsorevealed a specific role of AKT3 in regulating the growth ofTNBC (23). Resistant lines were profiled with immunoblotanalysis to identify relative expression levels of AKT1, AKT2,and AKT3. Cells resistant to MK2206 markedly exhibitincreased AKT3 expression levels when compared with parentalcells (Fig. 2A). Conversely, expression of AKT1 and AKT2 isindistinguishable between parental and resistant cells. In addi-tion, AKT3 expression is increased in a T47D R line establishedby culturing cells in gradually increasing doses of GDC0068(R (GDC0.2-5); Fig. 2A), suggesting that AKT3 upregulation isnot specific to a particular class of the AKT inhibitor. Impor-tantly, upregulation of AKT3, but again neither AKT1 nor AKT2,is also observed in multiple resistant derivatives of the triple-negative cell line MDA-MB-468, including cells resistant to theATP-competitive AKT inhibitor GSK690693 (SupplementaryFig. S1). These data indicate that upregulation of AKT3 is ageneral feature of acquired resistance to AKT inhibitors in breastcancer cell lines.

It has been shown that IGF1R levels are enhanced in breastcancer cells treated with AKT inhibitors (16). Consistent withthis, we observe upregulation of IGF1R in the T47D R lines (Fig.2A). Upregulation of both AKT3 and IGF1R protein expressionin resistant cells correlates well with upregulation at the tran-scriptional level as assessed by real-time RT-PCR (Fig. 2B) andRNA-sequencing (RNA-seq; Supplementary Table S1). Toexamine if AKT3 is amplified in resistant cells, quantitativereal-time PCR were performed on 10 subclones of T47D paren-tal lines as well as the resistant lines. There is no significantdifference of AKT3 copy number in resistant cells as comparedwith parental cells, whereas AKT3 amplification is found in thetriple-negative MDA-MB-468 line (Fig. 2C). Because it hasrecently been demonstrated that members of the bromodo-main and extra terminal domain (BET) family of proteins actepigenetically to regulate various components of the PI3Kpathway, including IGF1R (34), we next evaluated the effectsof BET proteins inhibition on the expression of AKT3 andIGF1R in T47D cells using two different small-molecule inhi-bitors, JQ1 and iBET. In T47D parental cells, AKT3 and IGF1Rare induced after 48 hours of MK2206 treatment (Fig. 2D). Theupregulation of AKT3 and IGF1R is inhibited when cells werepretreated with JQ1 or iBET. Similarly, both JQ1 and iBET lowerthe expression of AKT3 and IGF1R in the resistant cells. Thesedata demonstrated that an epigenetic pathway regulates theinduction of AKT3 and IGF1R in T47D R cells. To examine if theintrinsic kinase activity of AKT3 is changed in the resistant cellsand if its activity can be inhibited by MK2206, we haveperformed in vitro kinase assays. MK2206 was removed fromcells for 48 hours before harvesting of cell lysates. When anequal amount of Akt3 in the parental and resistant cells wasimmunoprecipitated, AKT3 expressed in the resistant cellsexhibits lower ability to phosphorylate GSK3b peptides (Fig.2E). These data suggest that AKT3 in the resistant cells is nothyperactive, and that it could be bound to and inhibited byMK2206, which has a long half-life (60–90 hours; ref. 6).

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Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics1966




Knockdown of AKT3 in resistant cells restores sensitivity to theAKT inhibitor MK2206

To investigate the role of AKT3 in determining sensitivity toMK2206, AKT3 was expressed using a tetracycline-on (dox)-inducible system. Whereas T47D cells are sensitive to MK2206(IC50: 0.17 mmol/L), ectopic expression of AKT3 results in a 16-fold increase in IC50 (2.71 mmol/L, P < 0.0001; Fig. 3A). Bycontrast, AKT3 depletion in T47D cells using shRNA leads to a3.5-fold decrease in IC50 (0.06 mmol/L vs. 0.21 mmol/L incontrol cells; P < 0.0001; Fig. 3B). Dox itself has no effect ondrug sensitivity because the IC50 of tumor cells containingempty vectors in the absence of dox is indistinguishable fromdox-treated cells (Supplementary Fig. S2a). To further deter-mine the specific role of AKT3 in inhibitor sensitivity, dox-inducible shRNA to deplete AKT1, AKT2, or AKT3 in parentaland T47D R lines was used. Upon dox administration, AKTisoforms are depleted specifically and quantitatively (Fig. 3C).We have demonstrated a specific role for AKT3 in regulatingTNBC spheroid growth using a three-dimensional (3D) cell

culture system (23). In the luminal T47D model, depletion ofAKT3 in parental cells results in a small, but statisticallysignificant reduction in spheroid size (23% reduction; Fig.3C). Conversely, depletion of AKT1 or AKT2 has minimal effecton spheroid growth. In resistant lines, depletion of AKT3potently inhibits spheroid growth relative to parental cells(47% reduction; Fig. 3C), consistent with the notion thatproliferation of resistant cells is driven by increased expressionof AKT3. By contrast, the effect of AKT1 or AKT2 depletion onspheroid growth is much more modest. A 21% reduction inspheroid size is observed in AKT1-depleted cells, whereas thereis no significant difference in spheroid size when AKT2 isdepleted. We next assessed the contribution of AKT isoformsin determining sensitivity to MK2206. In parental lines, where-as AKT1 or AKT2 depletion has no effect on sensitivity toMK2206, loss of AKT3 results in a 4.8-fold decrease in IC50

(Fig. 3D). Importantly, depletion of AKT3 restores sensitivity ofT47D R (MK5) to MK2206 to levels equivalent to parental cells(Fig. 3D). In contrast, knockdown of AKT1 or AKT2 in the

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T47D AKT inhibitor–resistant cells showresistance to inhibition along the PI3K/AKT axis. A, T47D parental (black) andMK2206-resistant (green) cells wereseeded to 96-well plates in the absenceof MK2206 for 24 hours. Cells were thentreated with MK2206 or GDC0068 for48 hours. Cell viability was determinedbyWST assays and calculated relative tothe untreated cells. Error bars, mean �SEM, with n ¼ 3. B, T47D parental andMK2206-resistant cells were seeded toplates in the absence of MK2206 for48 hours. Cells were then treatedwith MK2206 or GDC0068 for 1 hour.Whole-cell lysates were subjected toimmunoblotting for AKT signalingpathway components. p85 wasimmunoblotted as a loading control.






resistant cells by using shRNAs targeting two distinct regions ofthe AKT1 and AKT2 transcripts did not affect MK2206 sensi-tivity (Fig. 3D; Supplementary Fig. S2b). Similarly, AKT3 deple-tion in another T47D R line (MK0.2-5) sensitizes cells toMK2206 in a dose-dependent manner (Supplementary Fig.S3). We also examined if the activation of AKT3 in resistantcells is mediated by IGF1R. Treatment of resistant cells with anIGF1R inhibitor, AEW541, greatly reduces the phosphorylationof AKT and PRAS40, as well as cell viability (Fig. 3E), suggestingthat the IGF1R pathway drives AKT3 activation in T47D R cells.Taken together, these findings demonstrate that AKT3 upregu-lation in breast cancer cells confers resistance to the AKTinhibitor MK2206.

Cellular reprogramming and reversibility ofMK2206-resistant T47D cells

Next, to determine if genetic or epigenetic mechanisms areresponsible for the establishment of AKT inhibitor resistance, we

examined if the resistance phenotype is reversible upon drugdiscontinuation. MK2206 was removed from resistant lines for3 weeks prior to analysis. Compared with cells continuouslytreated with MK2206, resistant cells that had drug removedpartially reacquire AKT inhibitor sensitivity (Fig. 4A). Further-more, when the cells of drug removal were rechallenged withMK2206, they regain resistance rapidly after 2 weeks of drugexposure (Fig. 4B). The AKT signaling profile of MK2206-with-drawn cells is comparable with parental cells (Fig. 4C). Whereasphosphorylation of PRAS40 and 4EBP1 is largely refractory to theinhibitory effects of MK2206 in T47D R lines, upon drug removalfor 9 days, MK2206 inhibits PRAS40 and 4EBP1 phosphorylationto levels similar to those seen in parental cells. The reversibility ofboth signaling and resistance phenotypes is indicative of anepigenetic mechanism.

Because increasing evidence indicates an important role for theEMT in drug resistance (35), and there is clear evidence forepigenetic reprogramming in epithelial–mesenchymal plasticity

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Upregulation of AKT3 in AKT inhibitor–resistant breast tumor cells. A,Western blot analysis of lysates from T47D parental and resistant cells. It has been shown thatthe ATP-competitive inhibitor GDC0068 locks AKT in a nonfunctional yet hyperphosphorylated state (19). The slower migrating protein bands of AKT1-3 inGDC0068-resistant cells represent the hyperphosphorylated form of AKTs. Protein levels were quantified with ImageJ from NIH software. The levels of protein areexpressed as a ratio relative to the p85 protein in each sample (n ¼ 3). B, mRNA levels of AKT3 and IGF1R in T47D parental and resistant cells were analyzed byquantitative real-time RT-PCR. The levels of mRNA are expressed as a ratio relative to the GAPDH mRNA (n ¼ 3). C, AKT3 gene copy levels in MDA-MB-468 cells,T47D parental line (10 subclones), as well as resistant cells were analyzed by quantitative PCR. The copy number of AKT3 is expressed as a ratio relativeto the CEP17 reference gene in each sample (n¼ 3).D, T47D parental and resistant cellswere seeded to plates in the absence of MK2206 for 24 hours. Cellswere thentreated with JQ1 (0.3 mmol/L; Cayman Chemical) or iBET151 (1 mmol/L; Cayman Chemical) for 30 minutes, followed by MK2206 (1 mmol/L) for 48 hours. Whole-celllysates were immunoblotted for the indicated antibodies. E, T47D parental and resistant cells were seeded to plates in the absence of MK2206 for 48 hours.AKT3was immunoprecipitated from the cell lysate. In vitro kinase assaywas thenperformedusingGSK3bpeptides as substrates. The kinase reactionwas terminated,and samples were immunoblotted.

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AKT3 expression determines sensitivity of breast cancer cells to the AKT inhibitor.A, T47D cells infectedwith tet-on AKT3/pTRIPZ lentiviral vector were treatedwithdox for 3 days. Cell viability was assessed byWST assays. Data, mean� SEM, n¼ 2. Cell lysates were analyzed by immunoblotting. B, T47D cells expressing tet-onAKT3 shRNA were treated with dox for 3 days. Cell viability was calculated relative to the untreated cells. Data, mean � SEM; n ¼ 3. Knockdown ofAKT3 was confirmed byWestern blot analysis. C, T47D parental and resistant cells containing tet-on AKT1 (#1), AKT2 (#1), or AKT3 shRNAwere grown in 3D culturefor 5 to 8 days in the presence or absence of dox. Spheroid size was quantified in pixel area using ImageJ and depicted in the bar graph. Error bars, mean � SEM.� , P < 0.05; �� , P < 0.01; �� , P < 0.001 (Student t test, n � 40). Knockdown of AKT isoforms was confirmed by treating cells with dox for 72 hours, followedby immunoblotting. D, T47D parental and resistant cells containing tet-on AKT1 (#1), AKT2 (#1), or AKT3 shRNA were treated with dox for 3 days, followed bypreformingWST cell viability assays. Data, mean� SEM; n¼ 3. E, T47D-resistant cells were seeded to plates in the absence of MK2206 for 48 hours. Cells were thentreated with AEW541 (1 mmol/L) for 1 hour, followed by MK2206 (0.1 mmol/L) for 1 hour. Whole-cell lysates were analyzed by immunoblotting. To assess cellviability, T47D-resistant cells were seeded to 96-well plates in the absence of MK2206 for 24 hours. Cells were then treated with AEW541 (1 mmol/L) and/or MK2206(0.15 mmol/L) for 48 hours, followed by WST assays. Data, mean � SEM. � , P < 0.05; �� , P < 0.01; ns, not significant (Student t test, n ¼ 3).






(36), we next investigated if the resistant cells show hallmarks ofEMT. Expression of the epithelial gene E-cadherin is decreasedwhereas the mesenchymal markers N-cadherin and vimentin areincreased in T47D R lines, relative to parental cells (Fig. 4C). InBoyden chamber invasion assays, T47D R lines also exhibitenhanced invasiveness (Fig. 5A), consistent with the acquisitionof EMT. In addition, expression of E-cadherin is inversely corre-lated with the expression levels of AKT3, whereby E-cadherinexpression is decreased and AKT3 expression is increased in T47DR lines, again compared with parental cells. Conversely, upregula-tion and downregulation of E-cadherin and AKT3, respectively, is

observed upon drug removal in resistant cells (Fig. 4C). Thesefindings indicate that acquisition of AKT inhibitor resistance inT47D tumor cells is accompanied by an EMT program that isassociated with upregulation of AKT3.

AKT3 depletion promotes epithelial phenotype in MK2206-resistant breast tumor spheroids

We next explored whether AKT3 regulates epithelial character-istics of AKT inhibitor–resistant cells. T47D R (MK5) and parentalcells containing tet-on AKT3 shRNA were cultured in 3D. Inagreement with published findings (37), T47D cells form normal

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Induction of EMT in AKT inhibitor–resistant cells is associated withupregulation of AKT3. A, to assess thereversibility of the effects of chronicAKT inhibition, a fraction ofT47D-resistant cells was split from theMK2206 culture and maintained inDMSO for 3 weeks. Cells were thentreated with MK2206 (2.5 mmol/L) for48 hours, followed by WST cellviability assays. Data, mean � SEM.� , P < 0.05; �� , P < 0.01; �� , P < 0.001;ns, not significant (Student t test,n¼ 3).B, to assess the ability of cells toregain resistance after drug removal, afraction of cells that have beencultured in the absence of drug for2 weeks were rechallenged withMK2206 for 1 or 2 weeks, followed byWST assays. wk, weeks. C, T47Dparental and resistant cells, as well asresistant cells that have been culturedin the absence of MK2206 for 9 dayswere seeded to plates in the absenceof MK2206 for 48 hours. Cells werethen treated with MK2206 (0.1 or 1mmol/L) for 1 hour. Whole-cell lysateswere immunoblotted for the indicatedantibodies.

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round 3D spheroids (Fig. 5B). Depletion of AKT3 has minimaleffect on spheroid morphology. Consistent with changes in EMTmarkers, MK2206-resistant spheroids display invasive morpho-genesis, and AKT3 depletion reverses the resistant spheroid phe-notype to organized round structures (Fig. 5B).

DiscussionDespite the initial response of tumors to targeted therapeutic

agents, most patients relapse and develop resistance, leading tolimited clinical benefit. Various resistance mechanisms havebeen identified for inhibitors targeting the EGFR, BRAF, andMAPK pathways (38, 39). By contrast, relatively few mechan-isms have been identified and implicated in resistance to PI3Kpathway inhibitors (8–14). Importantly, despite over 80 cur-rent clinical trials for various AKT inhibitors, resistancemechanisms in cells and patients treated chronically with thesecompounds have yet to be identified. In the present study, weset out to identify and delineate resistance mechanisms to anAKT inhibitor, MK2206, using the T47D breast cancer cell linethat harbors a PIK3CA mutation. We show that AKT3, but notAKT1 or AKT2, is upregulated at the mRNA and protein level inresistant cells. Functional studies show that acquisition ofresistance is specifically due to the increased expression ofAKT3, as evidenced by the restoration of sensitivity to MK2206in resistant cells upon AKT3 depletion. To our knowledge, thisis the first report of a chronic resistance model for an AKTinhibitor in cancer.

In our breast tumor model of resistance, we observe AKT3upregulation not only with the allosteric AKT inhibitorMK2206, but also with the ATP-competitive inhibitorsGDC0068 and GSK690693. Whereas MK2206 has �5-foldlower IC50 toward recombinant AKT1 and AKT2 than AKT3(AKT1: 8 nmol/L; AKT2: 12 nmol/L; AKT3: 65 nmol/L; ref. 4),the ATP-competitive inhibitors have similar potency toward allthree AKT isoforms (GDC0068: AKT1: 5 nmol/L; AKT2: 18nmol/L; AKT3: 8 nmol/L, GSK690693: AKT1: 2 nmol/L; AKT2:13 nmol/L; AKT3: 9 nmol/L; refs. 40, 41). In addition to theluminal subtype of breast cancer, AKT3 is also overexpressed inresistant cells of the triple-negative subtype. In this context, aspecific role of AKT3 in regulating TNBC growth has been

demonstrated in vitro and in vivo, and the cell cycle inhibitorp27 appears to be critical for the ability of AKT3 in modulatingproliferation (23). The underlying molecular mechanism(s) forAKT3-mediated drug resistance in breast cancer is yet to bedefined. In human glioblastoma, AKT3 is highly expressed andits expression is significantly correlated with DNA repair genes(42). Moreover, in a mouse model of glioma, AKT3 overexpres-sion enhances DNA repair pathways and confers resistance oftumor cells to radiation and temozolomide. It will be inter-esting to determine if DNA repair is involved in conferringresistance to AKT inhibitors. It has also been reported that inresponse to targeted agents, cancer cells develop resistance byupregulating both the levels of the targeted kinase and increas-ing intrinsic kinase activity (43). To examine this, we performedin vitro kinase assays, and our data indicated that AKT3 expres-sing in the MK2206-resistant cells is not hyperactive and couldstill be inhibited by MK2206. Although upregulation of AKT3as a resistance mechanism has yet to be verified in matchedpatient biopsies of pre- and posttreatment of AKT inhibitors,copy number alteration analysis of TNBC clinical samplesshows that AKT3 is amplified in �15% of chemotherapy-resistant tumors (44). In addition, in a systematic functionalscreen performed in breast cancer cell lines, AKT3 is one of thegenes shown to support proliferation and survival of tumorcells upon PI3K inhibition (13). In a separate study usingHER2þ mammary tumor cells from Balb-neuT mice, AKT3depletion upregulates estrogen receptor alpha and sensitizestumor cells to the estrogen receptor modulator tamoxifen (45).In addition to breast cancer and glioblastoma, AKT3 has alsobeen implicated in resistance of other aggressive tumors. Inmetastatic melanoma, AKT3 plays a critical role in mediatingresistance to an inhibitor targeting mutant BRAF (46). BRAFinhibitor–induced upregulation of BH3-only proapoptotic pro-teins Bim-EL and Bmf is attenuated by ectopic expression ofAKT3. Our current work focuses primarily on the T47D breasttumor line, which has a PIK3CA mutation, whether the iso-form-specific function of AKT3 plays a critical role in acquiredresistance in other breast tumor lines, contexts, and tumor typesawaits further studies.

To explore genes that may be involved in the regulation ofAKT3 and IGF1R, as well as to examine global changes of gene

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AKT3 regulates invasiveness of AKT inhibitor–resistant breast tumor cells.A, T47D parental and resistant cells were subjected to a Transwell invasion assay. Relativeinvasion (y axis) ¼ ratio of the number of invaded cells in test versus control. � , P < 0.05 (Student t test, n ¼ 3). B, T47D parental and resistant cellsexpressing tet-on AKT isoform shRNA were grown in 3D culture for 8 to 15 days in the presence or absence of dox. Morphology of spheroids is shown in therepresentative phase-contrast images.






expression, we analyzed the transcriptome of T47D parental andMK2206-resistant cells using RNA-seq.We identified 525 and 402protein-coding genes that are upregulated (log of change > 0.5) ordownregulated (log of change < 0.5), respectively, in MK2206-resistant lines (Supplementary Table S1). In addition to AKT3 andIGF1R, a fewother genes in the PI3K/AKTpathway are found to beupregulated in the resistant lines, including HER3, insulin recep-tor substrate 2 (IRS2), and discoidin domain receptor 1 (DDR1).Agreeing with previous studies that implicated a role for HER3 inAKT inhibitor resistance (16–18), HER3 expression is increased inour resistant lines. Anupregulation of IRS2, a cytoplasmic adaptorprotein that mediates the activation of PI3K (47), suggests afeedforward loop activating AKT signaling in the resistant cells.In addition, DDR1, which has been shown to interact with andpositively regulate IGF1R expression, is upregulated. This collagenreceptor tyrosine kinase has also been demonstrated to promoteEMT and cancer progression (48). Two other upregulated genesthat warrant further studies are the transcription factor FOXD3,where its consensus binding sequence is found on the AKT3promoter, as well as BCL2, an antiapoptotic protein that drivescell survival.

The reversibility of drug resistance in the context of increasedAKT3 expression suggests that epigenetic alterations are respon-sible. Indeed, induction of AKT3 in MK2206-resistant cells isregulated epigenetically by BET proteins. Our RNA-seq dataalso showed that one of the BET proteins, BRD1, is upregulatedin the MK2206-resistant lines (Supplementary Table S1). Itwould be interesting to examine if BRD1 is the major proteinmediating the epigenetic regulation of AKT3. The EMT pheno-type is associated with both intrinsic and acquired resistance tovarious kinase inhibitors (49, 50), and is subject to epigeneticregulation. In our resistant lines, we observe changes in theexpression of multiple EMT-associated genes, accompanied byaltered AKT3 expression. Consistent with this, expression ofAKT3 is highly correlated with EMT activators such as ZEB1 inclinical breast tumors (Spearman's correlation: 0.61; cBiopor-tal.org; refs. 51, 52). Our resistant lines also show mesenchy-mal-associated characteristics, including the enhancement ofinvasiveness. We observe that MK2206-resistant spheroids aremore invasive than spheroids from parental cells. Notably,AKT3 depletion reduces invasiveness of cells in resistant tumorspheroids. It has been shown that AKT1 and AKT2 have oppos-ing roles in breast cancer cell invasion and metastasis (53). Bycontrast, the role of AKT3 in this phenotype has not beenexamined in detail, although the present findings are consistentwith previous studies showing that unlike AKT1 depletion,silencing AKT3 does not result in an invasive morphology ofMCF10A spheroids (23). It will also be interesting to examine ifinhibition of AKT3 reduces metastasis of resistant cells in an invivo setting.

These findings have important clinical implications becauseAKT inhibitors in clinical development target all three isoforms,and an AKT3-selective inhibitor has yet to be developed. Giventhe undesired metastatic phenotype of breast cancer cells uponinhibition of AKT1, and the critical role of AKT2 in regulatingglucose homeostasis, combined with studies highlighting a rolefor AKT3 in TNBC growth, these findings advocate for treatingTNBC and other breast tumors overexpressing AKT3 with anAKT3-selective inhibitor to curb toxicities (23). Our data alsoreveal an isoform-selective role of AKT3 in an in vitro resistancemodel. Based on the concept that it is preferable to prevent theemergence of resistance, rather than treating resistance once itdevelops, if AKT3, but not AKT1 or AKT2, induction is observedin patients that develop resistance, these findings provide arationale for the development of potent AKT3-selective smallmolecule inhibitors for treating breast tumors in which AKT3 isa driver for growth.

Disclosure of Potential Conflicts of InterestY.R. Chin reports receiving a commercial research grant from Pfizer CTI. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Y.R. ChinDevelopment of methodology: Y.R. ChinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Stottrup, T. Tsang, Y.R. ChinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Stottrup, T. Tsang, Y.R. ChinWriting, review, and/or revision of the manuscript: Y.R. ChinStudy supervision: Y.R. Chin

AcknowledgmentsThe thank Samuel Klempner for advice in generating drug-resistant tumor

lines, Ruslan Sadreyev and Fei Ji from the Nextgen Sequencing Core of Mas-sachusetts General Hospital for their assistance in analyzing the RNA sequenc-ing data, members of the Chin laboratory for discussions, andmembers of AlexToker laboratory for advice.

Grant SupportThis study was supported in part by a grant from the NIH National Cancer

Institute (Y.R. Chin and C. Stottrup; R00CA157945), a grant from the VFoundation for Cancer Research (Y.R. Chin), and a sponsored research grantfrom Pfizer CTI (Y.R. Chin and T. Tsang).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 16, 2015; revised May 24, 2016; accepted May 27, 2016;published OnlineFirst June 13, 2016.

References1. EllisMJ, PerouCM. The genomic landscape of breast cancer as a therapeutic

roadmap. Cancer Discov 2013;3:27–34.2. Thorpe LM, Yuzugullu H, Zhao JJ. PI3K in cancer: divergent roles of

isoforms, modes of activation and therapeutic targeting. Nat Rev Cancer2015;15:7–24.

3. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol3-kinases as regulators of growth and metabolism. Nat Rev 2006;7:606–19.

4. Yap TA, Yan L, Patnaik A, Fearen I,OlmosD, Papadopoulos K, et al. First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients withadvanced solid tumors. J Clin Oncol 2011;29:4688–95.

5. Rodon J, Dienstmann R, Serra V, Tabernero J. Development of PI3Kinhibitors: lessons learned from early clinical trials. Nat Rev Clin Oncol2013;10:143–53.

6. Hudis C, Swanton C, Janjigian YY, Lee R, Sutherland S, Lehman R, et al. Aphase 1 study evaluating the combination of an allosteric AKT inhibitor


Stottrup et al.




(MK-2206) and trastuzumab in patients with HER2-positive solid tumors.Breast Cancer Res 2013;15:R110.

7. Saura C, Bendell J, JerusalemG, Su S, Ru Q, De Buck S, et al. Phase Ib studyof buparlisib plus trastuzumab in patients withHER2-positive advanced ormetastatic breast cancer that has progressed on Trastuzumab-based ther-apy. Clin Cancer Res 2014;20:1935–45.

8. Brown KK, Toker A. The phosphoinositide 3-kinase pathway and therapyresistance in cancer. F1000Prime Rep 2015;7:13.

9. Liu P, Cheng H, Santiago S, Raeder M, Zhang F, Isabella A, et al. OncogenicPIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms. Nat Med2011;17:1116–20.

10. Ilic N, Utermark T, Widlund HR, Roberts TM. PI3K-targeted therapy canbe evaded by gene amplification along the MYC-eukaryotic translationinitiation factor 4E (eIF4E) axis. Proc Natl Acad Sci U S A 2011;108:E699–708.

11. Muellner MK, Uras IZ, Gapp BV, Kerzendorfer C, Smida M, Lech-termann H, et al. A chemical-genetic screen reveals a mechanism ofresistance to PI3K inhibitors in cancer. Nat Chem Biol 2011;7:787–93.

12. Serra V, Scaltriti M, Prudkin L, Eichhorn PJ, Ibrahim YH, Chandarlapaty S,et al. PI3K inhibition results in enhanced HER signaling and acquired ERKdependency in HER2-overexpressing breast cancer. Oncogene 2011;30:2547–57.

13. Serra V, Eichhorn PJ, Garcia-Garcia C, Ibrahim YH, Prudkin L, Sanchez G,et al. RSK3/4 mediate resistance to PI3K pathway inhibitors in breastcancer. J Clin Invest 2013;123:2551–63.

14. Huw LY, O'Brien C, Pandita A, Mohan S, Spoerke JM, Lu S, et al.Acquired PIK3CA amplification causes resistance to selective phos-phoinositide 3-kinase inhibitors in breast cancer. Oncogenesis 2013;2:e83.

15. ElkabetsM, Pazarentzos E, JuricD, ShengQ, Pelossof RA, Brook S, et al. AXLmediates resistance to PI3Kalpha inhibition by activating the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell carcinomas.Cancer Cell 2015;27:533–46.

16. Chandarlapaty S, Sawai A, Scaltriti M, Rodrik-Outmezguine V, Grbovic-Huezo O, Serra V, et al. AKT inhibition relieves feedback suppression ofreceptor tyrosine kinase expression and activity. Cancer Cell 2011;19:58–71.

17. Tao JJ, Castel P, Radosevic-Robin N, Elkabets M, Auricchio N, Aceto N,et al. Antagonism of EGFR and HER3 enhances the response to inhi-bitors of the PI3K-Akt pathway in triple-negative breast cancer. SciSignal 2014;7:ra29.

18. Yan Y, Serra V, Prudkin L, Scaltriti M,Murli S, RodriguezO, et al. Evaluationand clinical analyses of downstream targets of theAkt inhibitorGDC-0068.Clin Cancer Res 2013;19:6976–86.

19. Lin J, Sampath D, Nannini MA, Lee BB, Degtyarev M, Oeh J, et al.Targeting activated Akt with GDC-0068, a novel selective Akt inhibitorthat is efficacious in multiple tumor models. Clin Cancer Res 2013;19:1760–72.

20. Debnath J,Muthuswamy SK, Brugge JS.Morphogenesis andoncogenesis ofMCF-10Amammary epithelial acini grown in three-dimensional basementmembrane cultures. Methods 2003;30:256–68.

21. Kapeller R, Toker A, Cantley LC, Carpenter CL. Phosphoinositide 3-kinasebinds constitutively to alpha/beta-tubulin and binds to gamma-tubulin inresponse to insulin. J Biol Chem 1995;270:25985–91.

22. Chin YR, YuanX, Balk SP, Toker A. PTEN-deficient tumors depend onAKT2for maintenance and survival. Cancer Discov 2014;4:942–55.

23. Chin YR, Yoshida T, Marusyk A, Beck AH, Polyak K, Toker A. TargetingAkt3 signaling in triple-negative breast cancer. Cancer Res 2014;74:964–73.

24. Ringel MD, Hayre N, Saito J, Saunier B, Schuppert F, Burch H, et al.Overexpression and overactivation of Akt in thyroid carcinoma. CancerRes 2001;61:6105–11.

25. Yuen JS, Cockman ME, Sullivan M, Protheroe A, Turner GD, Roberts IS,et al. The VHL tumor suppressor inhibits expression of the IGF1R and itsloss induces IGF1R upregulation in human clear cell renal carcinoma.Oncogene 2007;26:6499–508.

26. Moore DA, Saldanha G, Ehdode A, Mughal MZ, Potter L, Dyall L, et al.Duplex ratio tests as diagnostic biomarkers inmalignant melanoma. J MolDiagn 2015;17:616–22.

27. Heredia NJ, Belgrader P, Wang S, Koehler R, Regan J, Cosman AM, et al.Droplet Digital PCRquantitation ofHER2 expression in FFPE breast cancersamples. Methods 2013;59:S20–3.

28. Hofmann I,Weiss A, ElainG, SchwaederleM, SterkerD, Romanet V, et al. K-RASmutant pancreatic tumors showhigher sensitivity toMEK than to PI3Kinhibition in vivo. PLoS One 2012;7:e44146.

29. Maroulakou IG, Oemler W, Naber SP, Tsichlis PN. Akt1 ablation inhibits,whereas Akt2 ablation accelerates, the development of mammary adeno-carcinomas in mouse mammary tumor virus (MMTV)-ErbB2/neu andMMTV-polyoma middle T transgenic mice. Cancer Res 2007;67:167–77.

30. Hutchinson JN, Jin J, Cardiff RD,Woodgett JR,MullerWJ. ActivationofAkt-1 (PKB-alpha) can accelerate ErbB-2-mediated mammary tumorigenesisbut suppresses tumor invasion. Cancer Res 2004;64:3171–8.

31. Yoeli-Lerner M, Yiu GK, Rabinovitz I, Erhardt P, Jauliac S, Toker A. Aktblocks breast cancer cell motility and invasion through the transcriptionfactor NFAT. Mol Cell 2005;20:539–50.

32. Arboleda MJ, Lyons JF, Kabbinavar FF, Bray MR, Snow BE, Ayala R, et al.Overexpression of AKT2/protein kinase Bbeta leads to up-regulation ofbeta1 integrins, increased invasion, and metastasis of human breast andovarian cancer cells. Cancer Res 2003;63:196–206.

33. Dillon RL, Marcotte R, Hennessy BT, Woodgett JR, Mills GB, Muller WJ.Akt1 and akt2 play distinct roles in the initiation and metastatic phases ofmammary tumor progression. Cancer Res 2009;69:5057–64.

34. Stratikopoulos EE, Dendy M, Szabolcs M, Khaykin AJ, Lefebvre C, ZhouMM, et al. Kinase and BET inhibitors together clamp inhibition of PI3Ksignaling and overcome resistance to therapy. Cancer Cell 2015;27:837–51.

35. Wang Z, Li Y, Ahmad A, Azmi AS, Kong D, Banerjee S, et al. TargetingmiRNAs involved in cancer stem cell and EMT regulation: an emergingconcept in overcoming drug resistance. Drug Resist Updat 2010;13:109–18.

36. Tam WL, Weinberg RA. The epigenetics of epithelial-mesenchymal plas-ticity in cancer. Nat Med 2013;19:1438–49.

37. Han J, Chang H, Giricz O, Lee GY, Baehner FL, Gray JW, et al. Molecularpredictors of 3D morphogenesis by breast cancer cell lines in 3D culture.PLoS Comput Biol 2010;6:e1000684.

38. Ercan D, Xu C, Yanagita M, Monast CS, Pratilas CA, Montero J, et al.Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors.Cancer Discov 2012;2:934–47.

39. Villanueva J, Infante JR, Krepler C, Reyes-Uribe P, Samanta M, Chen HY,et al. ConcurrentMEK2mutation andBRAF amplification confer resistanceto BRAF and MEK inhibitors in melanoma. Cell Rep 2013;4:1090–9.

40. Blake JF, Xu R, Bencsik JR, Xiao D, Kallan NC, Schlachter S, et al. Discoveryand preclinical pharmacology of a selective ATP-competitive Akt inhibitor(GDC-0068) for the treatment of human tumors. J Med Chem 2012;55:8110–27.

41. Rhodes N, Heerding DA, Duckett DR, Eberwein DJ, Knick VB, Lansing TJ,et al. Characterization of an Akt kinase inhibitor with potent pharmaco-dynamic and antitumor activity. Cancer Res 2008;68:2366–74.

42. Turner KM, Sun Y, Ji P, Granberg KJ, Bernard B, Hu L, et al. Genomicallyamplified Akt3 activates DNA repair pathway and promotes glioma pro-gression. Proc Natl Acad Sci U S A 2015;112:3421–6.

43. Lackner MR, Wilson TR, Settleman J. Mechanisms of acquired resistance totargeted cancer therapies. Future Oncol 2012;8:999–1014.

44. Balko JM, Giltnane JM, Wang K, Schwarz LJ, Young CD, Cook RS, et al.Molecular profiling of the residual disease of triple-negative breast cancersafter neoadjuvant chemotherapy identifies actionable therapeutic targets.Cancer Discov 2014;4:232–45.

45. Grabinski N, Mollmann K, Milde-Langosch K, Muller V, Schumacher U,Brandt B, et al. AKT3 regulates ErbB2, ErbB3 and estrogen receptor alphaexpression and contributes to endocrine therapy resistance of ErbB2(þ)breast tumor cells from Balb-neuT mice. Cell Signal 2014;26:1021–9.

46. Shao Y, Aplin AE. Akt3-mediated resistance to apoptosis in B-RAF-targetedmelanoma cells. Cancer Res 2010;70:6670–81.

47. Landis J, Shaw LM. Insulin receptor substrate 2-mediated phosphatidyli-nositol 3-kinase signaling selectively inhibits glycogen synthase kinase3beta to regulate aerobic glycolysis. J Biol Chem 2014;289:18603–13.

48. Mata R, Palladino C, Nicolosi ML, Lo Presti AR,Malaguarnera R, RagusaM,et al. IGF-I induces upregulation ofDDR1 collagen receptor in breast cancercells by suppressing MIR-199a-5p through the PI3K/AKT pathway. Onco-target 2016;7:7683–700.






49. Frederick BA, Helfrich BA, Coldren CD, Zheng D, ChanD, Bunn PAJr, et al.Epithelial to mesenchymal transition predicts gefitinib resistance in celllines of head and neck squamous cell carcinoma and non-small cell lungcarcinoma. Mol Cancer Ther 2007;6:1683–91.

50. Suda K, Tomizawa K, Fujii M, Murakami H, Osada H, Maehara Y, et al.Epithelial to mesenchymal transition in an epidermal growth factorreceptor-mutant lung cancer cell line with acquired resistance to erlotinib.J Thorac Oncol 2011;6:1152–61.

51. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al.Integrative analysis of complex cancer genomics and clinical profiles usingthe cBioPortal. Sci Signal 2013;6:pl1.

52. Cerami E,Gao J,DogrusozU,Gross BE, Sumer SO, Aksoy BA, et al. The cBiocancer genomics portal: an open platform for exploring multidimensionalcancer genomics data. Cancer Discov 2012;2:401–4.

53. Chin YR, Toker A. Function of Akt/PKB signaling to cell motility, invasionand the tumor stroma in cancer. Cell Signal 2009;21:470–6.


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2016;15:1964-1974. Published OnlineFirst June 13, 2016.Mol Cancer Ther Casey Stottrup, Tiffany Tsang and Y. Rebecca Chin MK2206 in Breast CancerUpregulation of AKT3 Confers Resistance to the AKT Inhibitor

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