Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer Casey Stottrup, Tiffany Tsang, and Y

Published OnlineFirst June 13, 2016; DOI: 10.1158/1535-7163.MCT-15-0748

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer Casey Stottrup, Tiffany Tsang, and Y. Rebecca Chin

Abstract

Acquired resistance to molecular targeted therapy represents a knockdown of AKT3, but not AKT1 or AKT2, in resistant cells major challenge for the effective treatment of cancer. Hyperactiva- restores sensitivity to MK2206. AKT inhibitor–resistant cells also tion of the PI3K/AKT pathway is frequently observed in virtually display an epithelial to mesenchymal transition phenotype as all human malignancies, and numerous PI3K and AKT inhibitors assessed by alterations in the levels of E-Cadherin, N-Cadherin, are currently under clinical evaluation. However, mechanisms of and vimentin, as well as enhanced invasiveness of tumor spher- acquired resistance to AKT inhibitors have yet to be described. oids. Notably, the invasive morphology of resistant spheroids is Here, we use a breast cancer preclinical model to identify resis- diminished upon AKT3 depletion. We also show that resistance to tance mechanisms to a small molecule allosteric AKT inhibitor, MK2206 is reversible because upon drug removal resistant cells MK2206. Using a step-wise and chronic high-dose exposure, regain sensitivity to AKT inhibition, accompanied by reexpression breast cancer cell lines harboring oncogenic PI3K resistant to of epithelial markers and reduction of AKT3 expression, implying MK2206 were established. Using this model, we reveal that AKT3 that epigenetic reprogramming contributes to acquisition of expression is markedly upregulated in AKT inhibitor–resistant resistance. These ﬁndings provide a rationale for developing cells. Induction of AKT3 is regulated epigenetically by the bro- therapeutics targeting AKT3 to circumvent acquired resistance in modomain and extra terminal domain proteins. Importantly, breast cancer. Mol Cancer Ther; 15(8); 1964–74. 2016 AACR.

Introduction clinical trial (6). In a separate phase Ib study, the combination of the PI3K inhibitor BKM120 and trastuzumab was evaluated in Breast cancer is the most frequently diagnosed malignancy in patients with HER2-positive advanced/metastatic breast cancer women. Genetic and epigenetic deregulation of the phosphoi- resistant to trastuzumab-based therapy, and initial evidence of nositide 3-kinase (PI3K)/AKT signaling pathway is highly prev- therapeutic efficacy has been presented (7). However, the expe- alent in breast cancer (1). Mutations in PIK3CA, the catalytic rience from other molecular targeted therapies suggests that the subunit of the p110a subunit of PI3K, loss of PTEN, and ampli- clinical benefits of these PI3K/AKT inhibitors are likely to be fication of HER2 are common genomic abnormalities in tumor limited by the development of acquired resistance in patients. cells leading to hyperactivation of PI3K/AKT signaling and sub- There are a few documented mechanisms of resistance to sequent phenotypes associated with malignancy (2, 3). Based on targeted therapy for inhibitors that target PI3K or AKT (8). Of this knowledge, a number of small molecule inhibitors targeting these, overexpression and/or amplification of Myc, Notch, various components of the PI3K/AKT pathway are in clinical RSK3/4, HER3, and PI3K itself have been proposed to confer development and evaluation. For example, both allosteric and resistance to certain PI3K inhibitors in the context of breast ATP-competitive AKT inhibitors (MK2206: allosteric; GDC0068 cancer (9–14). In addition, RNA sequencing analysis revealed and GSK690693: ATP-competitive) are being assessed in clinical an induction of the receptor tyrosine kinase (RTK) AXL in head trials for various aggressive cancers as monotherapy or as com- and neck squamous cell carcinomas (SCC) upon adaptation to bination strategies (4, 5). In cell-free assays using purified recom- the PI3Ka inhibitor BLY719. AXL dimerizes with EGFR and binant AKT, these pan-AKT inhibitors inhibit all three AKT iso- results in the activation of mTOR and resistance. Importantly, forms with nanomolar potencies. Preliminary evidence of clinical overexpression of AXL is observed in SCC tumors of patients activity is observed with combination of MK2206 and trastuzu- treated with BLY719 (15). With respect to AKT inhibitors, mab in patients with HER2-positive solid tumors in a phase I upregulation and activation of RTKs have been implicated in acquisition of resistance to targeted therapy. For example, acute treatment of breast cancer cells with an allosteric AKT inhibitor Department of Pathology, Beth Israel Deaconess Medical Center, induces marked upregulation of insulin receptor, IGF1R and Harvard Medical School, Boston, Massachusetts. HER3, via a FOXO-dependent manner (16). The same study Note: Supplementary data for this article are available at Molecular Cancer also showed that phosphorylation of multiple RTKs is Therapeutics Online (http://mct.aacrjournals.org/). enhanced upon AKT inhibition, by relieving mTORC1-medi- Corresponding Author: Yuet Rebecca Chin, 330 Brookline Ave, E/CLS 633A, ated feedback inhibition. HER3 expression has also shown to Department of Pathology, Beth Israel Deaconess Medical Center, Harvard be induced in triple-negative breast cancer (TNBC) acutely Medical School, 3 Blackfan Circle, Boston, MA 02115. Phone: 617-735-2484; Fax: treated with the catalytic AKT inhibitor GDC0068 (17). Con- 617-735-2480; E-mail: [email protected] sistent with preclinical studies, compensatory feedback activa- doi: 10.1158/1535-7163.MCT-15-0748 tion of HER3 and ERK has been observed in tumor biopsies 2016 American Association for Cancer Research. from patients treated with GDC0068 (18).

1964 Mol Cancer Ther; 15(8) August 2016

AKT3 and Breast Tumor Resistance

Delineating the spectrum of resistance mechanisms is critical RNA interference for the development of strategies to prevent or treat resistant For dox-inducible shRNA-mediated knockdown of AKT iso- tumors. Because mechanisms of acquired resistance to AKT inhi- forms, a tet-on shRNA/pLKO system was used. The hairpin bitors have not been elucidated, in this study we used a breast sequences targeting AKT1, AKT2, and AKT3 have been validated, tumor line harboring an activating mutation in PI3K and gener- and the construction of tet-on AKT isoform shRNA/pLKO vectors ated cells resistant to a selective AKT inhibitor, MK2206, by has been described previously (22). Akt1, sense, 50-CCGGGA- chronic adaptation. We show that these cells display upregulation GTTTGAGTACCTGAAGCTGCTCGAGCAGCTTCAGGTACTCAA- of AKT3 protein and function, accompanied by a phenotypic ACTCTTTTTG-30; Akt2, sense, 50-CCGGGCGTGGTGAATACAT- switch in the epithelial to mesenchymal transition (EMT). Deple- CAAGACCTCGAGGTCTTGATGTATTCACCACGCTTTTTG-30; tion of AKT3 in resistant cells restores sensitivity to MK2206, Akt3,sense,50- CCGGCTGCCTTGGACTATCTACATTCTCGA- highlighting AKT3 as a candidate for conferring resistance to GAATGTAGATAGTCCAAGGCAGTTTTTG-30. To produce len- molecular targeted therapy in breast cancer. tiviral supernatants, 293T cells were cotransfected with control or shRNA-containing tet-on pLKO vectors, VSVG and Materials and Methods psPAX2 for 48 hours. Cells stably expressing dox-inducible shRNA were cultured in medium containing puromycin (2 Cell culture mg/ml). Gene knockdown was induced by incubating cells T47D, HEK293T, and MDA-MB-468 cells were obtained with 100 ng/mL dox for 48 to 72 hours. from ATCC and maintained in Dulbecco's modified Eagle medium (DMEM; Cellgro) supplemented with 10% tet system-approved fetal bovine serum (FBS; Clontech). All cell lines Plasmids obtained from the cell banks listed above are tested for authen- For dox-inducible overexpression of AKT3, cells were infected tication using short tandem repeat (STR) profiling and passaged with HA-AKT3/pTRIPZ lentiviral vector. Construction of HA- for fewer than 6 months, and routinely assayed for mycoplasma AKT3/pTRIPZ has been described previously (23). contamination. Cell viability assays Cells were seeded 24 hours before inhibitor treatment into 96- AKT inhibitor–resistant line generation well plates at density of 5,000 cells per well in 100 mL medium. The MK2206-resistant cell line T47D MK0.2-5 was generated by Cell viability was measured 48 hours after inhibitor treatment gradual dose escalation of the AKT inhibitor MK2206 from 0.2 using the WST-1 assay (Clontech) according to the manufacturer's mmol/L to 5 mmol/L for a period of 2 months, then the cells were protocol. maintained in 5 mmol/L MK2206. T47D MK5-resistant line was generated by chronic exposure of cells to 5 mmol/L MK2206 for 2 months. T47D GDC0.2-5–resistant line was generated by cultur- In vitro kinase assays ing cells in increasing concentration of the AKT inhibitor Akt3 was immunoprecipitated from cell extracts and incubated GDC0068 from 0.2 mmol/L to 5 mmol/L for 2 months. T47D with 100 ng of GSK3b peptide in the presence of 250 mmol/L cold parental cells were maintained in growth medium containing ATP in a kinase buffer for 1 hour at 30 C. The kinase reaction was dimethyl sulfoxide (DMSO). stopped by the addition of SDS-PAGE loading buffer, and the Fresh AKT inhibitor or DMSO was replaced every 3 to 4 days. samples were assayed by immunoblotting. Cells were considered resistant when they could be cultured m routinely in growth medium containing 5 mol/L MK2206 or Transwell invasion assays GDC0068. MK2206 and GDC0068 have been described previ- Transwell filters (8-mm pore size; Corning) were coated with 1.5 ously (4, 19) and were obtained from Selleck Chemical. mg Matrigel (BD Biosciences). A total of 1 105 cells in serum-free medium containing 0.1% BSA were added to upper Transwell chambers in triplicate. Conditioned medium from NIH 3T3 cells 3D cultures was used as chemoattractant, and was added to the lower cham- 3D cultures were prepared as previously described (20). bers. After 7 hours of incubation at 37 C, non-invaded cells on Briefly, chamber slides were coated with growth factor- Transwell filters were removed. Cells that had invaded and reduced Matrigel (BD Biosciences) and allowed to solidify migrated to the bottom of the filters were fixed and stained using for 30 minutes. Cells (1 104) in assay medium were seeded the Hema-3 stain set (Fisher HealthCare Protocol). on coated chamber slides. Assay medium contained DMEM supplemented with 10% FBS and 2% Matrigel. The assay medium was replaced every 4 days. Doxycycline (dox, 100 Quantitative real-time RT-PCR ng/mL) was added every 2 or 3 days. Total RNA was isolated with an RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription was performed using random hexamers and multiscribe reverse Antibodies transcriptase (Applied Biosystems). Quantitative real-time All primary antibodies in this study except p85 were PCR was performed using an ABI Prism 7700 sequence detector. obtained from Cell Signaling Technology. Anti-p85 polyclonal AKT3 primer: sense, 50-GAAGAGGAGAGAATGAATTGTAGTCCA- antibody was generated in-house and has been described (21). 30; anti-sense, 50-AGTAGTTTCAAATAGTCAAAATCATTCATTG-30 Horseradish peroxidase–conjugated anti-mouse and anti-rab- (24); IGF1R primer: sense, 50-TTCAGCGCTGCTGATGTG-30; anti- bit immunoglobulin G (IgG) antibody were purchased from sense, 50-GGCTCATGGTGATCTTCTCC-30 (25). PCR reactions Chemicon. were carried out in triplicate. Quantification of mRNA expression

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was calculated by the dCT method with GAPDH as the reference Upregulation of AKT3 as a novel mechanism of acquired gene. resistance to AKT inhibitors The three AKT isoforms have distinct functions in modulat- Copy number analysis with quantitative real-time PCR ing phenotypes commonly associated with cancer. For exam- – Genomic DNA was isolated with the QIAamp DNA Mini Kit ple, whereas AKT1 is a breast cancer metastasis suppressor (29 in (Qiagen) according to the manufacturer's protocol. Real-time 31), AKT2 promotes invasion and metastasis of breast cancer vitro in vivo PCR was performed using an ABI Prism 7700 sequence detector. and (32, 33). In addition, recent studies have also 0 0 fi AKT3 primer: sense, 5 -CTGGACATCACCAGTCCTAGCTC-3 ; revealed a speci c role of AKT3 in regulating the growth of 0 0 fi anti-sense, 5 -ACCCTTGGCTGGTCTGGG-3 (26); CEP17 primer: TNBC (23). Resistant lines were pro led with immunoblot sense, 50-GCTGATGATCATAAAGCCACAGGTA-30; anti-sense, 50- analysis to identify relative expression levels of AKT1, AKT2, TGGTGCTCAGGCAGTGC-30 (27). PCR reactions were carried out and AKT3. Cells resistant to MK2206 markedly exhibit in triplicate. Quantification of copy number was calculated by the increased AKT3 expression levels when compared with parental dCT method with CEP17 as the reference gene. cells (Fig. 2A). Conversely, expression of AKT1 and AKT2 is indistinguishable between parental and resistant cells. In addition, AKT3 expression is increased in a T47D R line established Immunoblotting by culturing cells in gradually increasing doses of GDC0068 Cells were washed with PBS at 4C and lysed in RIPA buffer [1% (R (GDC0.2-5); Fig. 2A), suggesting that AKT3 upregulation is NP-40, 0.5% deoxycholic acid (SDC), 0.1% SDS,150 mmol/L not specific to a particular class of the AKT inhibitor. Impor- NaCl, 50 mmol/L Tris-HCl (pH 7.5), proteinase inhibitor cock- tantly, upregulation of AKT3, but again neither AKT1 nor AKT2, tail, 50 nmol/L calyculin, 1 mmol/L sodium pyrophosphate, 20 is also observed in multiple resistant derivatives of the triple- mmol/L sodium fluoride] for 15 minutes at 4C. Cell extracts were negative cell line MDA-MB-468, including cells resistant to the precleared by centrifugation at 13,000 rpm for 10 minutes at 4C, ATP-competitive AKT inhibitor GSK690693 (Supplementary and protein concentration was measured with the Bio-Rad protein Fig. S1). These data indicate that upregulation of AKT3 is a assay reagent using a Beckman Coulter DU-800 machine. Lysates general feature of acquired resistance to AKT inhibitors in breast were then resolved on 10% acrylamide gels by SDS-PAGE and cancer cell lines. transferred electrophoretically to nitrocellulose membrane It has been shown that IGF1R levels are enhanced in breast (BioRad) at 100 V for 60 minutes. The blots were blocked in cancer cells treated with AKT inhibitors (16). Consistent with TBST buffer (10 mmol/L Tris-HCl, pH 8, 150 mmol/L NaCl, 0.2% this, we observe upregulation of IGF1R in the T47D R lines (Fig. Tween 20) containing 5% (w/v) nonfat dry milk for 30 minutes, 2A). Upregulation of both AKT3 and IGF1R protein expression and then incubated with the specific primary antibody diluted in in resistant cells correlates well with upregulation at the tran- blocking buffer at 4C overnight. Membranes were washed three scriptional level as assessed by real-time RT-PCR (Fig. 2B) and times in TBST and incubated with horseradish peroxidase–con- RNA-sequencing (RNA-seq; Supplementary Table S1). To jugated secondary antibody for 1 hour at room temperature. examine if AKT3 is amplified in resistant cells, quantitative Membranes were washed 3 times and developed using enhanced real-time PCR were performed on 10 subclones of T47D paren- chemiluminescence substrate (Pierce). tal lines as well as the resistant lines. There is no significant difference of AKT3 copy number in resistant cells as compared Results with parental cells, whereas AKT3 amplification is found in the Establishment and characterization of AKT inhibitor–resistant triple-negative MDA-MB-468 line (Fig. 2C). Because it has breast tumor lines recently been demonstrated that members of the bromodo- In vitro modeling of acquired resistance has successfully iden- main and extra terminal domain (BET) family of proteins act tified a resistance mechanism observed in cancer patients treated epigenetically to regulate various components of the PI3K with kinase inhibitors. To explore mechanisms that mediate pathway, including IGF1R (34), we next evaluated the effects resistance to AKT inhibitors, we set out to model resistance to of BET proteins inhibition on the expression of AKT3 and an allosteric AKT inhibitor, MK2206, currently in phase II clinical IGF1R in T47D cells using two different small-molecule inhi- trials, using the luminal breast tumor cell line T47D, which bitors, JQ1 and iBET. In T47D parental cells, AKT3 and IGF1R harbors an activating PIK3CA mutation (H1047R). This cell line are induced after 48 hours of MK2206 treatment (Fig. 2D). The has been shown to be sensitive to MK2206 (28). MK2206-resis- upregulation of AKT3 and IGF1R is inhibited when cells were tant derivative T47D lines were cultured either in gradually pretreated with JQ1 or iBET. Similarly, both JQ1 and iBET lower increasing doses (starting at 0.2 mmol/L) or at a constant concen- the expression of AKT3 and IGF1R in the resistant cells. These tration of 5 mmol/L inhibitor, until pools of cells growing in the data demonstrated that an epigenetic pathway regulates the presence of 5 mmol/L drug were established. The resulting pools of induction of AKT3 and IGF1R in T47D R cells. To examine if the cells were termed T47D R (MK0.2-5; step-wise fashion) and T47D intrinsic kinase activity of AKT3 is changed in the resistant cells R (MK5; chronic high-dose fashion), respectively. Both resistant and if its activity can be inhibited by MK2206, we have lines show resistance to MK2206 in cell viability assays, evidenced performed in vitro kinase assays. MK2206 was removed from by >10-fold shifts in IC50 to MK2206 compared with the DMSO- cells for 48 hours before harvesting of cell lysates. When an treated parental pools (Fig. 1A). The T47D R lines also show cross- equal amount of Akt3 in the parental and resistant cells was resistance to the ATP-competitive AKT inhibitor GDC0068 (Fig. immunoprecipitated, AKT3 expressed in the resistant cells 1A). In dose response studies, T47D R lines are broadly resistant to exhibits lower ability to phosphorylate GSK3b peptides (Fig. the inhibitory effect of MK2206 and GDC0068 at multiple nodes 2E). These data suggest that AKT3 in the resistant cells is not of the PI3K/AKT pathway, including pAKT S473, pPRAS40 T246, hyperactive, and that it could be bound to and inhibited by pGSK3b S9, and p4EBP1 S65 (Fig. 1B). MK2206, which has a long half-life (60–90 hours; ref. 6).

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A Parental Parental MK0.2-5 125 MK5 125

100 100

IC : 3.58 IC50: 6.95 75 50 75 IC : 0.31 50 50 50 IC50: 0.22 25 25 Cell viability (%) Cell viability (%) 0 0 0 0.01 0.1 1 10 100 0 0.01 10.1 10 100 MK2206 (µmol/L) MK2206 (µmol/L)

Figure 1. T47D AKT inhibitor–resistant cells show Parental Parental resistance to inhibition along the PI3K/ MK5 MK0.2-5 125 125 AKT axis. A, T47D parental (black) and MK2206-resistant (green) cells were 100 100 seeded to 96-well plates in the absence IC : 2.21 of MK2206 for 24 hours. Cells were then 75 75 50 IC50: 0.53 treated with MK2206 or GDC0068 for 50 50 48 hours. Cell viability was determined IC : 0.06 IC : 0.06 by WST assays and calculated relative to 25 50 25 50 Cell viability (%) the untreated cells. Error bars, mean Cell viability (%) SEM, with n ¼ 3. B, T47D parental and 0 0 MK2206-resistant cells were seeded to 0 0.01 0.1 1 10 0 0.01 0.1 1 10 plates in the absence of MK2206 for GDC0068 (µmol/L) GDC0068 (µmol/L) 48 hours. Cells were then treated with MK2206 or GDC0068 for 1 hour. Whole-cell lysates were subjected to B T47D T47D T47D T47D T47D T47D immunoblotting for AKT signaling P R (MK0.2-5) R (MK5) P R (MK0.2-5) R (MK5) pathway components. p85 was µ µ MK2206 ( mol/L) – 1 5 – 1 5 – 1 5 GDC0068 ( mol/L) – 0.1 0.5 – 0.1 0.5 – 0.1 0.5 immunoblotted as a loading control. pAkt S473 pAkt S473

Pan Akt Pan Akt

pPRAS40 T246 pPRAS40 T246

pGSK3β S9 pGSK3β S9

p4EBP1 S65 p4EBP1 S65

p85 p85

Knockdown of AKT3 in resistant cells restores sensitivity to the culture system (23). In the luminal T47D model, depletion of AKT inhibitor MK2206 AKT3 in parental cells results in a small, but statistically To investigate the role of AKT3 in determining sensitivity to significant reduction in spheroid size (23% reduction; Fig. MK2206, AKT3 was expressed using a tetracycline-on (dox)- 3C). Conversely, depletion of AKT1 or AKT2 has minimal effect inducible system. Whereas T47D cells are sensitive to MK2206 on spheroid growth. In resistant lines, depletion of AKT3 (IC50:0.17mmol/L), ectopic expression of AKT3 results in a 16- potently inhibits spheroid growth relative to parental cells fold increase in IC50 (2.71 mmol/L, P < 0.0001; Fig. 3A). By (47% reduction; Fig. 3C), consistent with the notion that contrast, AKT3 depletion in T47D cells using shRNA leads to a proliferation of resistant cells is driven by increased expression 3.5-fold decrease in IC50 (0.06 mmol/L vs. 0.21 mmol/L in of AKT3. By contrast, the effect of AKT1 or AKT2 depletion on control cells; P < 0.0001; Fig. 3B). Dox itself has no effect on spheroid growth is much more modest. A 21% reduction in drug sensitivity because the IC50 of tumor cells containing spheroid size is observed in AKT1-depleted cells, whereas there empty vectors in the absence of dox is indistinguishable from is no significant difference in spheroid size when AKT2 is dox-treated cells (Supplementary Fig. S2a). To further deter- depleted. We next assessed the contribution of AKT isoforms mine the specific role of AKT3 in inhibitor sensitivity, dox- in determining sensitivity to MK2206. In parental lines, where- inducible shRNA to deplete AKT1, AKT2, or AKT3 in parental as AKT1 or AKT2 depletion has no effect on sensitivity to and T47D R lines was used. Upon dox administration, AKT MK2206, loss of AKT3 results in a 4.8-fold decrease in IC50 isoforms are depleted specifically and quantitatively (Fig. 3C). (Fig.3D).Importantly,depletionofAKT3restoressensitivityof We have demonstrated a specific role for AKT3 in regulating T47D R (MK5) to MK2206 to levels equivalent to parental cells TNBC spheroid growth using a three-dimensional (3D) cell (Fig.3D).Incontrast,knockdownofAKT1orAKT2inthe

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A B

3 2.5

2.5 2 2 1.5 1.5 1 1 (Akt3/GAPDH) 0.5 (IGF1R/GAPDH) 0.5 Akt3 mRNA levels Akt3 mRNA IGF1R mRNA levels IGF1R mRNA 0 0 ) ) P 5 P 5 K K (M (M R R

CD 1.4 **

1.2

0.8

0.6 ) -5 .2 0.4 E 0 K (M P R

Akt3 relative copy number 0.2 Input for IP (mg) 4 2

0 Akt3 IP: Akt3 pGSK3β S9 P (C1) P (C2) P (C3) P (C4) P (C5) P (C6) P (C7) P (C8) P (C9) P (C10) R (MK5)

R (MK0.2-5) MDA-MB-468

Figure 2. 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 that the ATP-competitive inhibitor GDC0068 locks AKT in a nonfunctional yet hyperphosphorylated state (19). The slower migrating protein bands of AKT1-3in GDC0068-resistant cells represent the hyperphosphorylated form of AKTs. Protein levels were quantiﬁed with ImageJ from NIH software. The levels of protein are expressed 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 by quantitative 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 relative to the CEP17 reference gene in each sample (n ¼ 3). D, T47D parental and resistant cells were seeded to plates in the absence of MK2206 for 24 hours. Cells were then treated 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-cell lysates were immunoblotted for the indicated antibodies. E, T47D parental and resistant cells were seeded to plates in the absence of MK2206 for 48 hours. AKT3 was immunoprecipitated from the cell lysate. In vitro kinase assay was then performed using GSK3b peptides as substrates. The kinase reaction was terminated, and samples were immunoblotted.

resistant cells by using shRNAs targeting two distinct regions of examined if the resistance phenotype is reversible upon drug theAKT1andAKT2transcriptsdidnotaffectMK2206sensi- discontinuation. MK2206 was removed from resistant lines for tivity (Fig. 3D; Supplementary Fig. S2b). Similarly, AKT3 deple- 3 weeks prior to analysis. Compared with cells continuously tion in another T47D R line (MK0.2-5) sensitizes cells to treated with MK2206, resistant cells that had drug removed MK2206 in a dose-dependent manner (Supplementary Fig. partially reacquire AKT inhibitor sensitivity (Fig. 4A). Further- S3). We also examined if the activation of AKT3 in resistant more, when the cells of drug removal were rechallenged with cells is mediated by IGF1R. Treatment of resistant cells with an MK2206, they regain resistance rapidly after 2 weeks of drug IGF1R inhibitor, AEW541, greatly reduces the phosphorylation exposure (Fig. 4B). The AKT signaling proﬁle of MK2206-with- of AKT and PRAS40, as well as cell viability (Fig. 3E), suggesting drawn cells is comparable with parental cells (Fig. 4C). Whereas that the IGF1R pathway drives AKT3 activation in T47D R cells. phosphorylation of PRAS40 and 4EBP1 is largely refractory to the Taken together, these ﬁndings demonstrate that AKT3 upregu- inhibitory effects of MK2206 in T47D R lines, upon drug removal lation in breast cancer cells confers resistance to the AKT for 9 days, MK2206 inhibits PRAS40 and 4EBP1 phosphorylation inhibitor MK2206. to levels similar to those seen in parental cells. The reversibility of both signaling and resistance phenotypes is indicative of an Cellular reprogramming and reversibility of epigenetic mechanism. MK2206-resistant T47D cells Because increasing evidence indicates an important role for the Next, to determine if genetic or epigenetic mechanisms are EMT in drug resistance (35), and there is clear evidence for responsible for the establishment of AKT inhibitor resistance, we epigenetic reprogramming in epithelial–mesenchymal plasticity

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A C T47D P T47D P T47D R (MK5) Tet-on shRNA Akt1 Akt2 Akt3 –Dox Dox –––+++ + Dox 5,000 3,000 125 4,500 Akt3 / 4,000 2,500 Akt1 pTRIPZ 3,500 2,000 100 Tet-on 3,000 Dox – + 2,500 1,500 Akt2 IC : 2.72 2,000 75 50 Akt1 shRNA 1,500 1,000

HA 1,000 (pixel area)

(pixel area) 500 500 Spheroid size Spheroid size Akt3 Akt3 50 0 0 IC50: 0.17 Dox – + Dox – + 25 p85 Cell viability (%) p85 0 4,000 3,000 0 0.01 0.1 1 10 3,500 2,500 3,000 2,000 MK2206 (µmol/L) 2,500 Tet-on 2,000 1,500 B 1,500 1,000 –Dox Akt2 shRNA 1,000 T47D R (MK5) (pixel area) (pixel area) 500 Spheroid size + Dox 500 Spheroid size 125 0 0 Dox – + Dox – + Tet-on shRNA Akt1 Akt2 Akt3 100 Tet-on IC50: 0.21 Dox –––+++ Akt3 shRNA 4,000 3,000 75 3,500 2,500 Dox – + 3,000 Akt1 50 2,500 2,000 Akt3 Tet-on 2,000 1,500 IC50: 0.06 1,500 Akt2 25 Akt3 shRNA 1,000 p85 Cell viability (%) 1,000 (pixel area) (pixel area) 500 Spheroid size Spheroid size 500 0 0 0 Akt3 0.01 0.1 1 10 0 Dox ––++Dox MK2206 (µmol/L) p85

D T47D P E T47D T47D T47D R (MK5 ) R (MK5) R (MK0.2-5)

–Dox –Dox MK2206 – + – + – + – + 125 + Dox 125 + Dox AEW541 – – + + – – + + 100 100 IC : >10 50 pAkt S473 75 75 Tet-on IC50: 0.55 IC50: >10 246 Akt1 shRNA 50 50 pPRAS40 T IC50: 0.46 25 25 Cell viability (%)

Cell viability (%) p85 0 0 0 0.01 0.1 1 10 0 0.01 0.1 1 10 MK2206 (µmol/L) MK2206 (µmol/L) * 100 ns

–Dox –Dox 80 125 125 + Dox + Dox 60 100 100 40 IC50: 6.79 Tet-on 75 75 IC : 0.51 50 IC : 4.74 20 Akt2 shRNA 50 Cell viability (%) 50 50 0 IC50: 0.57 25 25 MK2206 ++- Cell viability (%) Cell viability (%) - + 0 0 AEW541 + 0 0.01 0.1 1 10 0 0.01 0.1 1 10 T47D R (MK5) MK2206 (µmol/L) MK2206 (µmol/L) ** –Dox –Dox 100 ns 125 + Dox 125 + Dox 80 100 100 IC : 5.83 IC : 0.86 50 60 Tet-on 75 50 75 Akt3 shRNA 40 50 IC : 0.18 50 50 20

25 25 IC : 0.57 Cell viability (%) Cell viability (%) Cell Viability (%) 50 0 0 0 MK2206 ++- 0 0.01 0.1 1 10 0 0.01 0.1 1 10 AEW541 - + + MK2206 (µmol/L) MK2206 (µmol/L) T47D R (MK0.2-5)

Figure 3. AKT3 expression determines sensitivity of breast cancer cells to the AKT inhibitor. A, T47D cells infected with tet-on AKT3/pTRIPZ lentiviral vector were treated with dox for 3 days. Cell viability was assessed by WST assays. Data, mean SEM, n ¼ 2. Cell lysates were analyzed by immunoblotting. B, T47D cells expressing tet-on AKT3 shRNA were treated with dox for 3 days. Cell viability was calculated relative to the untreated cells. Data, mean SEM; n ¼ 3. Knockdown of AKT3 was confirmed by Western blot analysis. C, T47D parental and resistant cells containing tet-on AKT1 (#1), AKT2 (#1), or AKT3 shRNA were grown in 3D culture for 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, followed by 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 by preforming WST cell viability assays. Data, mean SEM; n ¼ 3. E, T47D-resistant cells were seeded to plates in the absence of MK2206 for 48 hours. Cells were then treated 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 cell viability, 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). www.aacrjournals.org Mol Cancer Ther; 15(8) August 2016 1969

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A ** B *** *** * ns 100 100 *** 120

80 80 100 80 60 60 60 40 40 40 20 20 20 Cell viability (%) Cell viability (%) Cell viability (%) 0 0 0 ) ) ) P 5 -5 P P 5 K .2 K (M 0 (M R K R (MK5) R (M R removal 3 wk removal 3 wk removal 4 wk ) ) ) 5 -5 5 Figure 4. K .2 K (M 0 (M R K R (MK5) removal 3 wk R Induction of EMT in AKT inhibitor– (M R resistant cells is associated with upregulation of AKT3. A, to assess the R (MK5) removal ; Drug again 1 wk R (MK5) removal ; Drug again 2 wk reversibility of the effects of chronic MK MK AKT inhibition, a fraction of T47D-resistant cells was split from the MK MK MK2206 culture and maintained in MK MK DMSO for 3 weeks. Cells were then treated with MK2206 (2.5 mmol/L) for 48 hours, followed by WST cell Week 0 4 7 0 4 8 viability assays. Data, mean SEM. , P < 0.05; , P < 0.01; , P < 0.001; ns, not signiﬁcant (Student t test, n ¼ 3). B, to assess the ability of cells to C T47D T47D T47D T47D T47D T47D regain resistance after drug removal, a P R (MK5) R (MK5) P R (MK0.2-5) R (MK0.2-5) fraction of cells that have been Removal 9 days Removal 9 days cultured in the absence of drug for MK2206 (µmol/L) – 0.1 1 – 0.1 1 – 0.1 1 MK2206 (µmol/L) – 0.1 1 – 0.1 1 – 0.1 1 2 weeks were rechallenged with MK2206 for 1 or 2 weeks, followed by Akt1 Akt1 WST assays. wk, weeks. C, T47D parental and resistant cells, as well as Akt2 Akt2 resistant cells that have been cultured in the absence of MK2206 for 9 days Akt3 Akt3 were seeded to plates in the absence of MK2206 for 48 hours. Cells were then treated with MK2206 (0.1 or 1 pAktS 473 473 pAktS mmol/L) for 1 hour. Whole-cell lysates were immunoblotted for the indicated 246 pPRAS40 T pPRAS40 T246 antibodies.

65 p4EBP1 S p4EBP1 S65

E Cadherin E Cadherin

N Cadherin N Cadherin

Vimentin Vimentin

p85 p85

(36), we next investigated if the resistant cells show hallmarks of observed upon drug removal in resistant cells (Fig. 4C). These EMT. Expression of the epithelial gene E-cadherin is decreased ﬁndings indicate that acquisition of AKT inhibitor resistance in whereas the mesenchymal markers N-cadherin and vimentin are T47D tumor cells is accompanied by an EMT program that is increased in T47D R lines, relative to parental cells (Fig. 4C). In associated with upregulation of AKT3. Boyden chamber invasion assays, T47D R lines also exhibit enhanced invasiveness (Fig. 5A), consistent with the acquisition AKT3 depletion promotes epithelial phenotype in MK2206- of EMT. In addition, expression of E-cadherin is inversely corre- resistant breast tumor spheroids lated with the expression levels of AKT3, whereby E-cadherin We next explored whether AKT3 regulates epithelial character- expression is decreased and AKT3 expression is increased in T47D istics of AKT inhibitor–resistant cells. T47D R (MK5) and parental R lines, again compared with parental cells. Conversely, upregula- cells containing tet-on AKT3 shRNA were cultured in 3D. In tion and downregulation of E-cadherin and AKT3, respectively, is agreement with published ﬁndings (37), T47D cells form normal

1970 Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics

AKT3 and Breast Tumor Resistance

A * B * 2 T47D P T47D R (MK5 )

1.5 No Dox + Dox No Dox + Dox

1 Tet-on Akt3 shRNA 0.5 Relative invasion

0 1 2 3 ) ) P 5 -5 K .2 M 0 ( K R (M R

Figure 5. AKT3 regulates invasiveness of AKT inhibitor–resistant breast tumor cells. A, T47D parental and resistant cells were subjected to a Transwell invasion assay. Relative invasion (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 cells expressing 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 the representative phase-contrast images. round 3D spheroids (Fig. 5B). Depletion of AKT3 has minimal demonstrated in vitro and in vivo, and the cell cycle inhibitor effect on spheroid morphology. Consistent with changes in EMT p27 appears to be critical for the ability of AKT3 in modulating markers, MK2206-resistant spheroids display invasive morpho- proliferation (23). The underlying molecular mechanism(s) for genesis, and AKT3 depletion reverses the resistant spheroid phe- AKT3-mediated drug resistance in breast cancer is yet to be notype to organized round structures (Fig. 5B). defined. In human glioblastoma, AKT3 is highly expressed and its expression is significantly correlated with DNA repair genes (42). Moreover, in a mouse model of glioma, AKT3 overexpres- Discussion sion enhances DNA repair pathways and confers resistance of Despite the initial response of tumors to targeted therapeutic tumor cells to radiation and temozolomide. It will be inter- agents, most patients relapse and develop resistance, leading to esting to determine if DNA repair is involved in conferring limited clinical benefit. Various resistance mechanisms have resistance to AKT inhibitors. It has also been reported that in been identified for inhibitors targeting the EGFR, BRAF, and response to targeted agents, cancer cells develop resistance by MAPK pathways (38, 39). By contrast, relatively few mechan- upregulating both the levels of the targeted kinase and increas- isms have been identifiedandimplicatedinresistancetoPI3K ing intrinsic kinase activity (43). To examine this, we performed pathway inhibitors (8–14). Importantly, despite over 80 cur- in vitro kinase assays, and our data indicated that AKT3 expres- rent clinical trials for various AKT inhibitors, resistance sing in the MK2206-resistant cells is not hyperactive and could mechanisms in cells and patients treated chronically with these still be inhibited by MK2206. Although upregulation of AKT3 compounds have yet to be identified. In the present study, we as a resistance mechanism has yet to be verified in matched set out to identify and delineate resistance mechanisms to an patientbiopsiesofpre-andposttreatmentofAKTinhibitors, AKT inhibitor, MK2206, using the T47D breast cancer cell line copy number alteration analysis of TNBC clinical samples that harbors a PIK3CA mutation. We show that AKT3, but not shows that AKT3 is amplified in 15% of chemotherapy- AKT1 or AKT2, is upregulated at the mRNA and protein level in resistant tumors (44). In addition, in a systematic functional resistant cells. Functional studies show that acquisition of screen performed in breast cancer cell lines, AKT3 is one of the resistance is specifically due to the increased expression of genes shown to support proliferation and survival of tumor AKT3, as evidenced by the restoration of sensitivity to MK2206 cells upon PI3K inhibition (13). In a separate study using þ in resistant cells upon AKT3 depletion. To our knowledge, this HER2 mammary tumor cells from Balb-neuT mice, AKT3 is the firstreportofachronicresistancemodelforanAKT depletion upregulates estrogen receptor alpha and sensitizes inhibitor in cancer. tumor cells to the estrogen receptor modulator tamoxifen (45). In our breast tumor model of resistance, we observe AKT3 In addition to breast cancer and glioblastoma, AKT3 has also upregulation not only with the allosteric AKT inhibitor been implicated in resistance of other aggressive tumors. In MK2206, but also with the ATP-competitive inhibitors metastatic melanoma, AKT3 plays a critical role in mediating GDC0068 and GSK690693. Whereas MK2206 has 5-fold resistance to an inhibitor targeting mutant BRAF (46). BRAF lower IC50 toward recombinant AKT1 and AKT2 than AKT3 inhibitor–induced upregulation of BH3-only proapoptotic pro- (AKT1: 8 nmol/L; AKT2: 12 nmol/L; AKT3: 65 nmol/L; ref. 4), teins Bim-EL and Bmf is attenuated by ectopic expression of the ATP-competitive inhibitors have similar potency toward all AKT3. Our current work focuses primarily on the T47D breast three AKT isoforms (GDC0068: AKT1: 5 nmol/L; AKT2: 18 tumor line, which has a PIK3CA mutation, whether the iso- nmol/L; AKT3: 8 nmol/L, GSK690693: AKT1: 2 nmol/L; AKT2: form-specific function of AKT3 plays a critical role in acquired 13 nmol/L; AKT3: 9 nmol/L; refs. 40, 41). In addition to the resistance in other breast tumor lines, contexts, and tumor types luminal subtype of breast cancer, AKT3 is also overexpressed in awaits further studies. resistant cells of the triple-negative subtype. In this context, a To explore genes that may be involved in the regulation of specific role of AKT3 in regulating TNBC growth has been AKT3 and IGF1R, as well as to examine global changes of gene

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Stottrup et al.

expression, we analyzed the transcriptome of T47D parental and These findings have important clinical implications because MK2206-resistant cells using RNA-seq. We identified 525 and 402 AKT inhibitors in clinical development target all three isoforms, protein-coding genes that are upregulated (log of change > 0.5) or and an AKT3-selective inhibitor has yet to be developed. Given downregulated (log of change < 0.5), respectively, in MK2206- the undesired metastatic phenotype of breast cancer cells upon resistant lines (Supplementary Table S1). In addition to AKT3 and inhibition of AKT1, and the critical role of AKT2 in regulating IGF1R, a few other genes in the PI3K/AKT pathway are found to be glucose homeostasis, combined with studies highlighting a role upregulated in the resistant lines, including HER3, insulin recep- for AKT3 in TNBC growth, these findings advocate for treating tor substrate 2 (IRS2), and discoidin domain receptor 1 (DDR1). TNBC and other breast tumors overexpressing AKT3 with an Agreeing with previous studies that implicated a role for HER3 in AKT3-selective inhibitor to curb toxicities (23). Our data also AKT inhibitor resistance (16–18), HER3 expression is increased in reveal an isoform-selective role of AKT3 in an in vitro resistance our resistant lines. An upregulation of IRS2, a cytoplasmic adaptor model. Based on the concept that it is preferable to prevent the protein that mediates the activation of PI3K (47), suggests a emergence of resistance, rather than treating resistance once it feedforward loop activating AKT signaling in the resistant cells. develops, if AKT3, but not AKT1 or AKT2, induction is observed In addition, DDR1, which has been shown to interact with and in patients that develop resistance, these findings provide a positively regulate IGF1R expression, is upregulated. This collagen rationale for the development of potent AKT3-selective small receptor tyrosine kinase has also been demonstrated to promote molecule inhibitors for treating breast tumors in which AKT3 is EMT and cancer progression (48). Two other upregulated genes a driver for growth. that warrant further studies are the transcription factor FOXD3, where its consensus binding sequence is found on the AKT3 Disclosure of Potential Conflicts of Interest promoter, as well as BCL2, an antiapoptotic protein that drives Y.R. Chin reports receiving a commercial research grant from Pfizer CTI. No cell survival. potential conflicts of interest were disclosed by the other authors. The reversibility of drug resistance in the context of increased AKT3 expression suggests that epigenetic alterations are responsible. Indeed, induction of AKT3 in MK2206-resistant cells is Authors' Contributions regulated epigenetically by BET proteins. Our RNA-seq data Conception and design: Y.R. Chin also showed that one of the BET proteins, BRD1, is upregulated Development of methodology: Y.R. Chin in the MK2206-resistant lines (Supplementary Table S1). It Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Stottrup, T. Tsang, Y.R. Chin would be interesting to examine if BRD1 is the major protein Analysis and interpretation of data (e.g., statistical analysis, biostatistics, mediating the epigenetic regulation of AKT3. The EMT pheno- computational analysis): C. Stottrup, T. Tsang, Y.R. Chin type is associated with both intrinsic and acquired resistance to Writing, review, and/or revision of the manuscript: Y.R. Chin various kinase inhibitors (49, 50), and is subject to epigenetic Study supervision: Y.R. Chin regulation. In our resistant lines, we observe changes in the expression of multiple EMT-associated genes, accompanied by Acknowledgments altered AKT3 expression. Consistent with this, expression of The thank Samuel Klempner for advice in generating drug-resistant tumor AKT3 is highly correlated with EMT activators such as ZEB1 in lines, Ruslan Sadreyev and Fei Ji from the Nextgen Sequencing Core of Mas- clinical breast tumors (Spearman's correlation: 0.61; cBiopor- sachusetts General Hospital for their assistance in analyzing the RNA sequenc- tal.org; refs. 51, 52). Our resistant lines also show mesenchy- ing data, members of the Chin laboratory for discussions, and members of Alex mal-associated characteristics, including the enhancement of Toker laboratory for advice. invasiveness. We observe that MK2206-resistant spheroids are more invasive than spheroids from parental cells. Notably, Grant Support AKT3 depletion reduces invasiveness of cells in resistant tumor This study was supported in part by a grant from the NIH National Cancer spheroids. It has been shown that AKT1 and AKT2 have oppos- Institute (Y.R. Chin and C. Stottrup; R00CA157945), a grant from the V ing roles in breast cancer cell invasion and metastasis (53). By Foundation for Cancer Research (Y.R. Chin), and a sponsored research grant contrast, the role of AKT3 in this phenotype has not been from Pfizer CTI (Y.R. Chin and T. Tsang). examined in detail, although the present findings are consistent The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked with previous studies showing that unlike AKT1 depletion, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate silencing AKT3 does not result in an invasive morphology of this fact. MCF10A spheroids (23). It will also be interesting to examine if inhibition of AKT3 reduces metastasis of resistant cells in an in Received September 16, 2015; revised May 24, 2016; accepted May 27, 2016; vivo setting. published OnlineFirst June 13, 2016.

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1974 Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics

Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer

Casey Stottrup, Tiffany Tsang and Y. Rebecca Chin

Mol Cancer Ther 2016;15:1964-1974. Published OnlineFirst June 13, 2016.

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