Key Survival Factor, Mcl-1, Correlates with …...Cell Death and Survival Key Survival Factor, Mcl-1, Correlates with Sensitivity to Combined Bcl-2/Bcl-xL Blockade Michelle M.Williams1,

Cell Death and Survival

Key Survival Factor, Mcl-1, Correlates withSensitivity to Combined Bcl-2/Bcl-xL BlockadeMichelle M.Williams1, Linus Lee2, Donna J. Hicks1, Meghan M. Joly1, David Elion1,Bushra Rahman1, Courtney McKernan1, Violeta Sanchez3, Justin M. Balko1,3,Thomas Stricker4,5, Monica Valeria Estrada4, and Rebecca S. Cook1,2,5

Abstract

An estimated 40,000 deaths will be attributed to breast cancer in2016, underscoring the need for improved therapies. Evading celldeath is amajor hallmark of cancer, driving tumor progression andtherapeutic resistance. To evade apoptosis, cancers use antiapop-totic Bcl-2 proteins to bind to and neutralize apoptotic activators,suchasBim. Investigationof antiapoptoticBcl-2 familymembers inclinical breast cancer datasets revealed greater expression andmorefrequent gene amplification of MCL1 as compared with BCL2 orBCL2L1 (Bcl-xL) across three major molecular breast cancer sub-types, Luminal (A and B), HER2-enriched, and Basal-like. WhileMcl-1protein expressionwas elevated inestrogen receptora (ERa)-positive andERa-negative tumors as comparedwithnormal breast,Mcl-1 stainingwashigher inERaþ tumors. TargetedMcl-1blockadeusing RNAi increased caspase-mediated cell death in ERaþ breastcancer cells, resulting in sustained growth inhibition. In contrast,combined blockade of Bcl-2 and Bcl-xL only transiently induced

apoptosis, as cells rapidly acclimated through Mcl-1 upregulationand enhanced Mcl-1 activity, as measured in situ using Mcl-1/Bimproximity ligation assays. Importantly, MCL1 gene expressionlevels correlated inversely with sensitivity to pharmacologic Bcl-2/Bcl-xL inhibition in luminal breast cancer cells, whereas norelationship was seen between the gene expression of BCL2 orBCL2L1 and sensitivity to Bcl-2/Bcl-xL inhibition. These resultsdemonstrate that breast cancers rapidly deploy Mcl-1 to promotecell survival, particularly when challenged with blockade of otherBcl-2 family members, warranting the continued development ofMcl-1–selective inhibitors for targeted tumor cell killing.

Implications: Mcl-1 levels predict breast cancer response toinhibitors targeting other Bcl-2 family members, and demon-strate the key role played by Mcl-1 in resistance to this drugclass. Mol Cancer Res; 15(3); 259–68. �2016 AACR.

IntroductionThe intrinsic apoptotic pathway is tightly regulated by Bcl-2

family members to support developmental processes and properphysiologic function (1). Apoptosis dysregulation often producespathologic consequences, including cancer formation, progres-sion, and therapeutic resistance (2). At the center of the intrinsicapoptotic pathway are the "effectors" Bax and Bak, which oligo-merize at the outer mitochondrial membrane (OMM) by bindingto "activators" (Bim, Bid, and Puma; ref. 3). Bak/Bax oligomer-ization promotes pore formation in the OMM, resulting in

mitochondrial depolarization, disruption of oxidative phosphor-ylation, mitochondrial cytochrome-c release into the cytoplasm,and apoptosome activation, thus initiating caspase-dependentapoptosis (4–6). Antiapoptotic Bcl-2 family proteins (Bcl-2,Bcl-A1, Bcl-xL, Bcl-w, and Mcl-1) restrain the intrinsic apoptoticpathway by binding and sequestering effectors (7) and/or acti-vators (8), thus favoring cell survival. Proapoptotic "sensitizers"(Bad, Hrk, and Noxa) bind and saturate antiapoptotic Bcl-2proteins, thus favoring apoptosis (9–11). A delicate balance inthe relative ratio of antiapoptotic Bcl-2 proteins to apoptoticactivators or sensitizers is necessary for cell survival regulation.Cancers can exploit this pathway to evade apoptosis, oftenthrough increased levels of antiapoptotic Bcl-2 factors (12–14).

Nearly 250,000newbreast cancer caseswill be diagnosed in theUnited States in 2016 (15). Despite advances in detection andtreatment of breast cancers, it is estimated that up to 40,000patients will die from breast cancer in the United States each year,oftendue to recurrentmetastatic disease, highlighting the need forimproved treatments that promote tumor cell killing. Severalstudies suggest that antiapoptotic Bcl-2 family proteins may beparticularly attractive therapeutic targets in breast cancers. Forexample, Bcl-2 expression was observed in up to 70% of ERaþ

breast cancers (16). While Bcl-xL remains less studied in primarybreast tumors, Bcl-xL expression was increased in ductal carcino-ma in situ (DCIS) compared with normal breast in some breastcancer subtypes (17). Interestingly, many breast cancers increaselevels of Bcl-2 family proteins following treatmentwith tamoxifen(18), fulvestrant (19), and neoadjuvant chemotherapy (NAC;

1Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.2Department of Biomedical Engineering, Vanderbilt University, Nashville, Ten-nessee. 3Department ofMedicine, Vanderbilt UniversityMedical Center, Nashville,Tennessee. 4DepartmentofPathology,Microbiologyand Immunology,VanderbiltUniversity Medical Center, Nashville, Tennessee. 5The Vanderbilt-Ingram CancerCenter, Vanderbilt University Medical Center, Nashville, Tennessee.

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

Current address for M.M. Joly: Department of Molecular and Medical Genetics,OregonHealth and ScienceUniversity, 3181 S.W. SamJackson ParkRd., Portland,Oregon.

Corresponding Author: Rebecca S. Cook, Vanderbilt University Medical Center,759 Preston Research Building, 2220 Pierce Ave., Nashville, TN 37232. Phone:615-936-3813; Fax: 615-936-2911; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0280-T

�2016 American Association for Cancer Research.

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ref. 20). Bcl-2 andBcl-xL levels reportedly predict poor response totaxanes (21), adriamycin (22), and the HER2 monoclonal anti-body Herceptin (23). In addition, Bcl-2 family proteins are oftenupregulated in endocrine-resistant cancers (24, 25). These find-ings support an intense interest in research strategies to block theactivity of Bcl-2 family proteins as a means to enhance tumor cellkilling.

Pharmacologic inhibition of Bcl-2 family proteins has beenachieved using compounds that bind to the BH3-domain bindingpocket of Bcl-2 family members. These BH3 "mimetics" block theinteraction of Bcl-2 family proteins with BH3-domain containingproapoptotic factors (i.e., Bcl-2 activators or sensitizers), includ-ing Bim (26). ABT-199/venetoclax, which specifically blocks Bcl-2from interacting with BH3-domain–containing proapoptotic fac-tors, is currently approved for clinical use in chronic lymphocyticleukemia (CLL; ref. 27), and is in clinical trial in several additionalcancers, including breast cancers (28). ABT-263/navitoclax bindsto and blocks the activity of Bcl-2 and Bcl-xL, and is showingefficacy in early-phase clinical trials in hematologic malignancies(29–31). Preclinical studies in luminal breast cancers show thatABT-263 (targeting Bcl-2 and Bcl-xL) or ABT-199 (targeting Bcl-2)may effectively increase tumor killingwhen cells arefirst "primed"with tamoxifen (32, 33).

These advances in Bcl-2 family targeting warrant a greater under-standing of the molecular characteristics of breast cancers thatmight benefit from this treatment strategy. Examination of largeclinical datasets identified expression and gene amplification ofBCL2 and BCL2L1 (Bcl-xL). However, we found that MCL1 geneexpression occurred more frequently in breast cancers than otherBcl-2 family members. Disruption of Mcl-1 activity increasedcaspase-activated apoptosis and impaired cell growth to a greaterextent than combined disruption of Bcl-2 and Bcl-xL. Importantly,expression levels of MCL1 predicted sensitivity to ABT-263 in apanel of breast cancer cell lines, which may inform results inongoing clinical trials, or guide patient selection for future trials.

Materials and MethodsExpression analysis of publicly available cancer cell line andbreast cancer datasets

mRNA expressions of MCL1, BCL2, and BCL2L1 (Bcl-xL) werecurated using cBio Portal (www.cbio.org) for cancer cell lines(CCLE) and breast tumor specimens (TCGA). Breast cancer speci-mens were stratified on the basis of PAM50 molecular markers(TCGA), and comparative genomic hybridization (CGH) analysiswas used to observe alterations at the genetic level (amplifica-tions). mRNA expressions of MCL1, BCL2, and BCL2L1 in breastcancer cell lines (CCLE) were correlated to the IC50 of ABT-263 asdetermined by the Sanger Institute (http://www.cancerrxgene.org/); data were fit to linear regression.

Western blottingCells and tumor tissue were homogenized in ice-cold lysis

buffer [50 mmol/L Tris pH 7.4, 100 mmol/L NaF, 120 mmol/LNaCl, 0.5% NP-40, 100 mmol/L Na3VO4, 1� protease inhibitorcocktail (Roche), 0.5 mmol/L proteasome inhibitor (Santa CruzBiotechnology)]. Proteins were resolved on 4%–12% SDS-PAGEgels and transferred to nitrocellulose membranes, which wereblocked in 3% gelatin in TBS-T (Tris-buffered saline, 0.1% Tween-20), incubated in primary antibody [Mcl-1 S19, Bim, Bcl-2, Bcl-xL(Santa Cruz Biotechnology 1:500), b-actin, E-Cadherin (Cell

Signaling Technology, 1:10,000)], secondary antibodies [rabbit,goat, mouse (Santa Cruz Biotechnology, 1:5,000–10,000)], anddeveloped with ECL substrate (Thermo Scientific).

Proximity ligation assayCells cultured in 96-well plates were fixed with methanol,

stained with the Duolink (Sigma) PLA protocol according tomanufacturer's directions using Mcl-1 (Santa Cruz Biotechnolo-gy, 1:25) and Bim (Santa Cruz Biotechnology, 1:25) antibodies,counterstained with Hoechst, and scanned by ImageXpress MicroXL Automated Microscope. Proximity ligation assay (PLA) fluo-rescent puncta and Hoechst-stained cells were enumerated usingImageJ software.

Caspase activity assayA total of 5� 103 cells/well or 1� 104 cells/well were seeded in

96-well plates in growth media and were treated with ABT-263 orDMSO for 4 to 48 hours. Caspase-Glo 3/7 Assay (Promega) wasused according to the manufacturer's directions. Luminescencewas measured on a Glomax MutliþDetection System (Promega)luminometer and was standardized to protein values.

Cell cultureCell lines were purchased directly from ATCC (Homo sapiens

ATCC CRL 2327; HTB-22; HTB-133; CRL-1500), and cultured ingrowth media (DMEM, 10% FBS, 1� antibiotics/antimycotics).Cells were transduced with lentiviral particles expressing threedistinct shControl or shMCL1 sequences (Santa Cruz Biotechnol-ogy) and kept under constant puromycin selection (1 mg/mL, LifeTechnologies). For cell growth analyses, 2.5 � 103 cells/well[growth 3D Matrigel (BD Biosciences)] or 5 � 103 cells/well(growth monolayer) were seeded in a 96-well or 12-well plate,respectively. Media, antibiotic, and/or drug were changed every 3days. For analysis, three-dimensional (3D) colonies were imagedafter 14 days (Motic AE3, ProRes CapturePro v2.8.0) and enu-merated using ImageJ software. Colonies in monolayer werestained with 0.01% w/v crystal violet (Sigma Life Sciences) andmeasured using ImageJ. Trypan blue–excluding cells werecounted after seeding 5 � 104 cells/well in 12-well plates andtreating with drug for 48 hours.

Statistical analysisStatistical significance (P < 0.05)was determined using Student

unpaired two-tailed t test or ANOVAwith Bonferroni post hoc testsfollowed by Student unpaired two-tailed t test using GraphPadPrism5 software.

ResultsMcl-1 is highly expressed in breast cancers

Antiapoptotic Bcl-2 familymember transcripts were assessed inCancer Cell Line Encyclopedia (CCLE) tumor cell line expressiondatasets (34). BCL2 transcripts were high in tumors of hemato-logic origin, but were relatively low in epithelial tumor cells,including breast, whereas BCL2L1 (Bcl-xL) transcripts were higherin tumors of epithelial origin (Supplementary Fig. S1). MCL1levels were relatively high across several cancers of epithelial(lung, breast, ovary, pancreas, prostate, and stomach) and hema-tologic (B-cell lymphomas,myelomas) origin, and inmelanomas(Fig. 1A). Focusing specifically on breast cancer, we assessed

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MCL1, BCL2, and BCL2L1 in cell lines stratified by PAM50molecular subtypes (35–37). MCL1 was detected at higher levelsthanBCL2 andBCL2L1 across all breast cancermolecular subtypes(Fig. 1B). This observation was confirmed by Western blot anal-ysis in a smaller panel of breast cancer lines, showing abundantMcl-1 expression across most lines, and variable levels of Bcl-2and Bcl-xL (Supplementary Fig. S2).

Next, we queried RNA-Seq data from The Cancer GenomeAtlas(TCGA)-curated clinical breast cancer datasets for total MCL1,BCL2, and BCL2L1 transcript counts, finding more MCL1 inLuminal A (N ¼ 333), Luminal B (N ¼ 325), HER2-enriched(N ¼ 150), and Basal-like (N ¼ 211) samples than BCL2 andBCL2L1 (Fig. 1C). Basal-like tumors harbored highest MCL1transcripts, followed by Luminal A, HER2-enriched, and finallyLuminal B (Fig. 1D). CGH analysis of TCGA Luminal (A and B)breast cancers (N ¼ 324) demonstrated that MCL1 gene ampli-ficationswere found in 21 of 324 tumor samples (7%), whichwasmore frequent than amplifications in BCL2 (2/324) and BCL2L1(1/324; Fig. 1E). Gene amplifications in MCL1 also occurred inHER2-enriched and Basal-like breast cancer specimens at rateshigher than what was seen for other Bcl-2 family-encoding genes.Immunohistochemical analysis of clinical breast tumor speci-

mens (N ¼ 266) showed little Mcl-1 staining in normal breastepithelium, but substantial Mcl-1 upregulation in breast tumorspecimens, with the highest expression seen in ERaþ tumors(Fig. 1F). These results suggest a role for Mcl-1 in breast cancerbiology,motivating our continued investigation ofMcl-1 in ERaþ

breast cancers.

Mcl-1 inhibition decreases luminal breast cancer tumor cellgrowth

We usedMCL1 shRNA sequences (shMCL1; 2–3 sequences percell line) in the luminal breast cancer cell lines HCC1428, MCF7,T47D, and ZR75-1 to knock downMcl-1 expression. Western blotanalysis of polyclonal puromycin-selected cells demonstrateddecreased Mcl-1 protein expression in each cell line, with nochange in Bcl-2 and Bcl-xL expression (Fig. 2A). PLA (38) showeda decreased association betweenMcl-1 and Bim in cells expressingshMCL1 as compared with shControl nontargeting sequences(Fig. 2B). Mcl-1 knockdown increased caspase-3/7 activity inthree of four cells lines (Fig. 2C), and decreased cell growth inthree of four lines grown inmonolayer (Fig. 2D) or in 3DMatrigel(Fig. 2E). These results confirm that specific Mcl-1 inhibitionincreases cell death in some, but not all ERaþ breast cancer cells.

Figure 1.

Mcl-1 expression is highest in ERþ

breast cancers. A, CCLE-curatedcancer cells were grouped accordingto tumor type and assessed for mRNAexpression of MCL1 using cBio Portal(www.cbio.org). B, CCLE-curatedbreast cancer cells were groupedaccording to PAM50 molecularsubtype and assessed for mRNAexpression ofMCL1, BCL2, and BCL2L1using cBio Portal (www.cbio.org).C and D, Clinical breast tumorspecimens curated by TCGA andsubjected to RNA-Seq analysis weregrouped according to PAM50molecular subtype, then assessed forabsolute read for transcripts encodingMCL1, BCL2, and BCL2L1 using cBioPortal (www.cbio.org). Unpaired two-tailed Student t test. E, Clinical breasttumor specimens curated by TCGAand subjected to CGH analysis weregrouped according to PAM50molecular subtype, and then assessedfor amplifications of genes encodinganti-apoptotic Bcl-2 family members.F,Breast tumor tissuemicroarray (N¼266) was stained for Mcl-1 and scoredfor percent of tumor cells that are Mcl-1–positive by a breast pathologist.Samples were stratified by ER status,ERþ, n¼132; ER–, n¼134. The average% Mcl-1–positive cells (�SE) is shownas the midline. Individual samples areshown as data points (Studentunpaired two-tailed t test).

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ERaþbreast cancer cell lines have limited sensitivity to ABT-263To determine how blockade of other Bcl-2 family members

impacts the growth and survival of ERaþ breast cancer cells, we

treated HCC1428, MCF7, T47D, and ZR75-1 with ABT-263, acompound that inhibits two Bcl-2 family members, Bcl-2 andBcl-xL. Although each cell line showed increased caspase-3/7

Figure 2.

Decreased activity and expression of Mcl-1 induces tumor cell killing. A–E, Polyclonal pools of cells stably expressing shCtrl (shControl) or shMcl-1 sequenceswere assessed. A, Western blot analysis of whole-cell lysates. B, Cells were assessed by PLA to detect Mcl-1/Bim interactions. Right, representative images:red, Mcl-1/Bim proximity; blue, Hoechst. Left, average cytosolic puncta/cell (�SE) was quantitated for 3 cells/sample, n ¼ 3 (assessed in triplicate), Studentunpaired two-tailed t test. C, Cells were assessed for caspase activity by Caspase-3/7 Glo assay. Average RLU (�SE) is shown, n ¼ 6–9, Student unpairedtwo-tailed t test. D, Cells were grown for 7 days in monolayer. Average cell area (�SE) was determined using crystal violet, setting the number of shCtrl coloniesequal to 1 as a common reference of comparison for each cell line (n ¼ 6–9 per group). Student unpaired two-tailed t test. E, Cells embedded in Matrigelwere cultured for 14 days. Number of colonies/well was measured, setting the number of shCtrl colonies equal to 1 as a common reference of comparisonfor each cell line (n ¼ 6–9 per group). Student unpaired two-tailed t test.

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activity at 4-hour exposure to ABT-263 (1.0 mmol/L), caspaseactivation was not sustained through 48 hours in three of the fourcell lines (Fig. 3A), suggesting a rapid loss of sensitivity to ABT-263. Consistent with these findings, ABT-263 did not affect thetotal number ofMCF7, T47D, or ZR75-1 cells grown inmonolayerfor 48 hours (Fig. 3B), or in 3D Matrigel for 14 days (Fig. 3C;Supplementary Fig. S3). PLA (38) confirmed that ABT-263 dis-rupted interaction of its target protein Bcl-2with Bim (Fig. 3D andE), confirming on-target activity of ABT-263 at 4- and 24-hourtreatment, despite the lack of caspase activation at distal timepoints in three of the four cells tested.

ABT-263 induces Mcl-1 expression and activityCells were treated with a time course of ABT-263 to determine

whether rapid upregulation of Bcl-2 family proteins could desen-

sitize cells to ABT-263. Although Bcl-xL expression remainedrelatively unchanged after 24-hour treatment with ABT-263 (Sup-plementary Fig. S4A), Bcl-2 expression was elevated at this timepoint. Despite an increase in Bcl-2 levels, Bcl-2/Bim interactionsremain inhibited at 24-hour treatment (Fig. 3D and E), suggestingthat elevated Bcl-2 expression does not enhance the ability forBcl-2 to sequester Bim in the presence of ABT-263. Alternatively,Mcl-1 expression was promptly increased within 4- to 8-hourtreatment with ABT-263 (Fig. 4A). This rapid upregulation wasalso sustained, as Mcl-1 levels were increased at 7-day treatmentwith ABT-263 in three of four cell lines (Fig. 4A and B). IncreasedMcl-1/Bim interactions were observed in each cell line upontreatment with ABT-263 (Fig. 4C and D). While Mcl-1/Biminteraction increased in three of four cell lines at 4-hour treatment,these interactions decreased or returned to baseline at 24 hours in

Figure 3.

Luminal breast cancer cell lines have limited sensitivity to ABT-263. A, Caspase activity was measured in cells treated for 4 or 48 hours with 1.0 mmol/LABT-263 using a luminescent Cleaved Caspase-3/7-Glo assay. Average (�SE) relative luminescence is shown, Student unpaired two-tailed t test, n ¼ 3. B, Cellswere treated 48 hours with 1.0 mmol/L ABT-263, and live cells (Trypan blue–negative) were counted. Values shown are average cell number (�SE), Studentunpaired two-tailed t test, n ¼ 3. C, Cells were Matrigel embedded and cultured for 14 days. Average number of colonies/well (�SE) is shown. t test, n ¼ 3. D andE, PLA of cells treated 4 or 24 hours �1.0 mmol/L ABT-263. Representative images are shown. Red, Bcl-2/Bim proximity; blue, Hoechst (D). Values shown areaverage cytosolic puncta/nuclei (�SE), n ¼ 3 (E).






MCF7 and ZR75-1 cells, possibly due to decreased Bim expressionat this time point (Supplementary Fig. S4B). In addition, thedelayedupregulation ofMcl-1/Bim interactions inHCC1428 cells(24 hours) relative to the other three cell lines (4 hours) mayexplain the relatively increased sensitivity of HCC1428 cells toABT-263 (as shown in Fig. 3), suggesting that complex interac-tions between Bcl-2 family proteins may determine sensitivity toABT-263.

We assessed MCF7 xenografts treated in vivo with ABT-263(20 mg/kg) for 16 days. Coprecipitation of Bim with Bcl-2 wasseen in control-treated tumors, but was absent in MCF7 xeno-

grafts treated with ABT-263 (Fig. 4E), confirming on-targetactivity of ABT-263 within tumors. Increased Mcl-1 was seenin ABT-263–treated MCF7 whole tumor lysates (Fig. 4F), andPLA measured increased Mcl-1/Bim interactions in tumors trea-ted with ABT-263 as compared with vehicle-treated tumors (Fig.4G). Although decreased recovery of signal was achieved informalin fixed paraffin embedded as compared with what wasseen in methanol-fixed MCF7 cell cultures (comparing Fig. 4Cto G), these results are consistent with the idea that tumorsin vivo upregulate Mcl-1 activity in response to blockade of otherBcl-2 family members similar to what was seen in cell culture.

Figure 4.

Increased Mcl-1 expression and activity in luminal breast cancer cells treated with ABT-263. A and B, Western blot analysis of whole-cell lysates after treatmentwith 1.0 mmol/L ABT-263 or DMSO for 0–48 hours (A) or 0, 3, or 7 days (B). C and D, PLA of cells treated 4 or 24 hours �1.0 mmol/L ABT-263. Representativeimages are shown. Red, Bim/Mcl-1 proximity; blue, Hoechst (C). Average cytosolic puncta/cell (�SE) was quantitated for 3 cells/sample, n ¼ 3 (assessed intriplicate), Student unpaired two-tailed t test (D). E–G, MCF7 xenografts were treated with daily ABT-263 (20 mg/kg) for 16 days by oral gavage. E,Immunoprecipitation (IP) of Bim or IgG control was conducted on whole tumor lysates. F, Whole tumor lysates were assessed by Western blot analysis.G, PLA of MCF7 xenografts treated daily with ABT-263 for 16 days. Average puncta/cell was quantitated for three fields/tumor, n ¼ 4.

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MCL1 expression correlates inversely with sensitivity toBcl-2/Bcl-xL inhibition

Bcl-2 and/or Bcl-xL inhibition is currently under clinical inves-tigation for the treatment of breast cancers. Given the high Mcl-1expression levels inbreast cancers, and the capacity for rapidMcl-1upregulation following Bcl-2/Bcl-xL using ABT-263, we assessedthe relationship betweenMCL1 and sensitivity to ABT-263 acrossa panel of CCLE-curated ERaþ (n¼ 16),HER2-amplified (n¼ 9),

and triple-negative (N ¼ 18) breast cancer cell lines (usingdatasets published in refs. 34 and 39). MCL1 transcript levelscorrelated with the ABT-263 IC50 in ERaþ and HER2-amplifiedcells, but not in triple negative breast cancer (TNBC) cells(Fig. 5A). Interestingly, BCL2 and BCL2L1 transcript levels didnot correlate directly or inverselywith the ABT-263 IC50 in any celltype. These findings support the notion that Mcl-1 may be for amarker of de novo resistance of breast cancers to ABT-263. To test

Figure 5.

Mcl-1 expression correlates with reduced sensitivity to ABT-263. A, MCL1, BCL2, and BCL2L1(Bcl-xL) mRNA expression in luminal breast cancer cell lines(CCLE) was compared with the ABT-263 IC50 (Sanger Institute). Data were fit to a linear regression. R2 and P values shown for MCL1. R2 and P values for BCL2 are0.05, 0.37 (ERaþ), 0.05, 0.55 (HER2), and 0.07, 0.28 (TNBC), respectively; and for BCL2L1 are 0.00, 0.97 (ERaþ), 0.03, 0.52 (HER2), and 0.05, 0.58 (TNBC),respectively. B–D, Human Mcl-1 was overexpressed in cells by adenoviral expression. B, Whole-cell lysates after 48-hour treatment with adenovirus. C, PLA inHCC1428 and T47D cells treated with an his-Mcl-1 adenovirus for 48 hours, then treated with ABT-263 (1.0 mmol/L) for 4 hours, n ¼ 3 (assessed in triplicate),Student unpaired two-tailed t test. D, Relative caspase activity of cells treated ABT-263 (1.0mmol/L) for 4 hours. Average (�SE) relative luminescence is shown,n ¼ 3, Bonferroni post hoc test followed by Student unpaired two-tailed t test. E, Model for Mcl-1–mediated resistance to ABT-263.






this idea directly, we overexpressed Mcl-1 in HCC1428, MCF7,and T47D cells (Fig. 5B). Although Bim interactions with Mcl-1were upregulated by 20%–45% in cells transduced with the his-Mcl-1 adenovirus, we found ABT-263 more potently upregulatedMcl-1/Bim interactions in transduced cells (Fig. 5C), demonstrat-ing that Mcl-1 acts as a sink for Bim upon ABT-263 treatmentand that ABT-263 promotes maximal Mcl-1/Bim interactions.Consistent with the idea that Mcl-1 levels contribute to de novoABT-263 resistance, we found that Mcl-1 overexpression substan-tially decreased caspase-3/7 activation in ABT-263-treated cells(Fig. 5D), further suggesting that ERaþ breast cancer cells useupregulation of Mcl-1 to evade cell death when challenged withABT-263 (Fig. 5E).

DiscussionNearly 40,000 breast cancer–related deaths occur annually in

the United States, often in the context of recurrent and/or ther-apeutically resistant disease (15). Additionalmolecularly targetedtreatment strategies that effectively induce tumor cell killing couldpotentially decrease tumor recurrences, and would increase ther-apeutic options for patients with resistant disease. Tumors oftenrely on antiapoptotic Bcl-2 family proteins to evade tumor celldeath. Given the clinical success of ABT-199 in CLL (27), andongoing clinical investigations of ABT-263 and ABT-199 in breastcancers (28), we were motivated to understand which breastcancers express high levels of Bcl-2 family members, as this mightindicatewhichbreast cancerswouldbenefit fromBcl-2 and/or Bcl-xL inhibition. We also investigated the Bcl-2 family member Mcl-1, given recently developed compounds that target Mcl-1 activitythat have transitioned into early-phase clinical trials for hemato-logic malignancies (40). Data shown herein suggest that Mcl-1may be a key survival factor across all breast cancer subtypes.

In previous studies, a dependency screening approach identi-fied Mcl-1 as a key survival factor in TNBCs (41), consistent withour observations presented herein that among Bcl-2 family mem-bers, MCL1 mRNA expression was frequently higher than BCL2and BCL2L1mRNA in breast cancers, including TNBCs (Fig. 1C).At the protein level, immunohistochemical Mcl-1 staining ofbreast cancer tissue microarrays demonstrated profoundly elevat-ed Mcl-1 in breast cancers as compared with normal breastepithelium (Fig. 1F). Furthermore, Mcl-1 levels were elevated inbreast tissue containing ERa expression, suggesting that Mcl-1may confer a selective survival advantage to breast cancer cells,particularly luminal breast cancer cells, but may also represent avulnerability that can be exploited therapeutically. Mcl-1 knock-down in luminal breast cancer cells decreased Mcl-1 activity, asmeasured in situ by Mcl-1/Bim interactions (Fig. 2B). Impor-tantly, Mcl-1 knockdown decreased tumor cell survival andtumor cell growth (Fig. 2C–E). These studies complementearlier studies from other groups indicating that Mcl-1 knock-down in some triple-negative (20, 42) and/or HER2-amplifiedbreast cancer cells (43, 44) increases caspase activity. In con-trast, combined Bcl-2 and Bcl-xL inhibition using ABT-263 hadonly an acute impact on caspase activation (Fig. 3A) and didnot affect tumor cell growth (Fig. 3B and C). These findings arein agreement with studies from other groups demonstratingthat ABT-263 was insufficient as a single agent to induce tumorcell killing in luminal breast cancer cells, but only once cellswere primed with tamoxifen did Bcl-2/Bcl-xL or selective Bcl-2inhibition with ABT-199 induce cell death (32).

Several studies show Mcl-1 production and stability is respon-sive to cellular cues, including inhibition of other Bcl-2 familymembers (45–49), making Mcl-1 ideally suited for rapidly evad-ing therapeutically induced tumor cell death [reviewed in ref. 2],and emphasizing the value of Mcl-1 as a therapeutic target.Interestingly, we found that rapid and sustained Mcl-1 inductionoccurred in response to ABT-263 in luminal breast cancer cells(Fig. 4), althoughMcl-1 depletion did not result uniformly in Bcl-2 or Bcl-xL upregulation in luminal breast cancer cells (Fig. 2A).This suggests that Mcl-1 may be a dominant antiapoptotic signalin luminal breast cancers. Previous studies in hematologic malig-nancies, colorectal cancers, and small-cell lung cancers show thatMcl-1 drives innate resistance to ABT-263, while Mcl-1 suppres-sion could restore sensitivity to ABT-263 (45, 49, 50). Althoughthis idea needs to be tested further in luminal breast cancers, it ispossible that maximal tumor cell killing may only be achievedwhen Bcl-2, Bcl-xL, and Mcl-1 are inhibited, suggesting that thetoxicities associated with targeting all three family members needto be explored. In addition, we show herein that MCL1 geneexpression levels correlated inversely with sensitivity to ABT-263(Fig. 5A), suggesting that MCL1 might be used as predictor ofpatient response to ABT-263 or ABT-199, a hypothesis that couldbe tested in ongoing and future clinical trials.

In summary, we find that MCL1 gene expression and amplifi-cation are frequent occurrences in breast cancers, and that geneticMcl-1 inhibition increased apoptosis in luminal breast cancercells, resulting in increased growth inhibition. In contrast, Bcl-2/Bcl-xL inhibition using ABT-263 did not sustain tumor cell killingor growth inhibition. Cells rapidly responded to ABT-263 byincreasing Mcl-1 expression, but Mcl-1 depletion did not induceBcl-2 or Bcl-xL upregulation. Together, these findings support arole for Mcl-1 in survival of breast cancer cells, warranting con-sideration of Mcl-1 in the design and interpretation of clinicaltrials investigating Bcl-2 family inhibitors.

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

Authors' ContributionsConception and design: M. Williams, R.S. CookDevelopment of methodology: M. Williams, D. Elion, V. Sanchez, R.S. CookAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Williams, L. Lee, D.J. Hicks, M.M. Joly, D. Elion,C. McKernan, M.V. EstradaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Williams, D. Elion, B. Rahman, J.M. Balko,T. StrickerWriting, review, and/or revision of the manuscript: M. Williams, M.M. Joly,J.M. Balko, T. Stricker, R.S. CookAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Williams, D.J. Hicks, B. RahmanStudy supervision: R.S. Cook

AcknowledgmentsWe thank Dr. Stephen Fesik for critical review of this manuscript. We are

grateful to Dr. Carlos Arteaga for thoughtful discussion of this manuscript andJoshua Bauer from the Vanderbilt High Throughput Screening Facility for hisassistance with imaging and analysis of the Proximity Ligation Assay.

Grant SupportThis work was supported by Specialized Program of Research Excellence

(SPORE) grant NIHP50 CA098131 (Vanderbilt-Ingram Cancer Center), CancerCenter Support grant NIH P30 CA68485 (Vanderbilt-Ingram Cancer Center),NIH F31 CA195989-01 (M.M. Williams) and CTSA UL1TR000445 from the

Williams et al.





National Center for Advancing Translational Sciences. R.S. Cook received NIHaward R01CA143126 and Susan G. Komen for the Cure grant KG100677.M.M.Joly received NRSA F31 predoctoral award CA186329-01. M. Williams receivedNRSA F31 predoctoral award CA195989-01 and an award from the VanderbiltInstitute for Clinical and Translational Research (CTSA UL1TR000445 from theNational Center for Advancing Translational Sciences).

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

Received August 19, 2016; revised November 16, 2016; accepted December12, 2016; published OnlineFirst December 30, 2016.

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2017;15:259-268. Published OnlineFirst December 30, 2016.Mol Cancer Res Michelle M. Williams, Linus Lee, Donna J. Hicks, et al. Bcl-2/Bcl-xL BlockadeKey Survival Factor, Mcl-1, Correlates with Sensitivity to Combined

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Key Survival Factor, Mcl-1, Correlates with …...Cell Death and Survival Key Survival Factor, Mcl-1, Correlates with Sensitivity to Combined Bcl-2/Bcl-xL Blockade Michelle M.Williams1,