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GX15-070 (Obatoclax) Induces Apoptosis and InhibitsCathepsin D- and L–Mediated Autophagosomal Lysis inAntiestrogen-Resistant Breast Cancer Cells

Jessica L. Schwartz-Roberts1,2, Ayesha N. Shajahan1, Katherine L. Cook1, Anni W€arri1,Mones Abu-Asab3, and Robert Clarke1,2

AbstractIn estrogen receptor–positive (ERþ) breast cancer cells, BCL2 overexpression contributes to antiestrogen

resistance. Direct targeting of the antiapoptotic BCL2 members with GX15-070 (obatoclax), a BH3-mimetic

currently in clinical development, is an attractive strategy to overcome antiestrogen resistance in some breast

cancers. Recently, GX15-070 has been shown to induce both apoptosis and autophagy, yet the underlying cell

deathmechanisms have yet to be elucidated.Here,we show thatGX15-070 ismore effective in reducing the cell

density of antiestrogen-resistant breast cancer cells versus sensitive cells and that this increased sensitivity of

resistant cells to GX15-070 correlates with an accumulation of autophagic vacuoles. Formation of autophago-

somes in GX15-070-treated cells was verified by changes in expression of the lipidation of microtubule-

associated protein-1 light chain-3 and both confocal and transmission electron microscopy. While GX15-070

treatment promotes autophagic vacuole and autolysosome formation, p62/SQSTM1, a marker for autophagic

degradation, levels accumulate. Moreover, GX15-070 exposure leads to a reduction in cathepsin D (CTSD) and

L (CTSL1) protein expression that would otherwise digest autolysosome cargo. Thus, GX15-070 has dual roles

inpromoting cell death: (i) directly inhibiting antiapoptotic BCL2 familymembers, thereby inducingapoptosis;

and (ii) inhibiting downstreamCTSD andCTSL1 protein expression to limit the ability of cells to use degraded

material to fuel cellular metabolism and restore homeostasis. Our data highlight a new mechanism of GX15-

070-induced cell death that could be used to design novel therapeutic interventions for antiestrogen resistant

breast cancer. Mol Cancer Ther; 12(4); 448–59. �2013 AACR.

IntroductionApproximately two thirds of newly diagnosed inva-

sive breast tumors express the estrogen receptor-a (ER)protein (ER-positive; ERþ; ref. 1) and most will betreated with an endocrine therapy such as an antiestro-gen or aromatase inhibitor. Antiestrogens can inhibit ERfunction and/or expression, blocking the ER-regulatedsignaling that induces breast cancer cell survival andproliferation (2). Tamoxifen (TAM), a selective ER mod-ulator (SERM), is the most frequently prescribed anti-estrogen and is effective in increasing overall survival

and reducing the incidence of ERþ disease in high-riskwomen (3). The selective ER downregulator (SERD)fulvestrant [Faslodex, ICI182780 (ICI)], does not exhibitthe partial agonist activities of some SERMs and is oftenan effective treatment option following relapse ontamoxifen or an aromatase inhibitor (4, 5). Despite thewidespread clinical efficacy of antiestrogens in thetreatment of ERþ breast cancers, approximately half ofthese women will exhibit de novo or acquired resistanceto endocrine therapies (6).

Breast cancer cells can acquire resistance to antiestro-gens through changes in molecular signaling that affectcell proliferation and death. The B-cell lymphoma 2(BCL2) gene family encodes central regulatory proteinswith both antiapoptotic (BCL2, BCLW, BCL-xL, MCL1,and A1) and proapoptotic functions (BAX, BAK, andBH3-only proteins; ref. 7). BH3-only members interactwith the core antiapoptotic BCL2 proteins to promoteapoptosis by activating BAX and/or BAK, which leadsto downstream cytochrome c release (8). BCL2 alsointeracts with beclin-1 (BECN1), a critical regulator ofautophagy that facilitates autophagosome production(9). Before systemic therapy, BCL2 overexpression oftencorrelates with ER-a and is usually a favorable prog-nostic indicator (10). However, BCL2 levels decrease in

Authors' Affiliations: 1Department of Oncology, Lombardi Comprehen-sive Cancer Center; 2Department of Physiology and Biophysics, George-town University School of Medicine,Washington, District of Columbia; and3Sectionof ImmunopathologyandLaboratory of Immunology,National EyeInstitute, NIH, Bethesda, Maryland

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

Corresponding Author:Robert Clarke, Department of Oncology, George-town University School of Medicine, Research Building W405A, 3970Reservoir Road NW, Washington, DC 20057. Phone: 202-687-3755; Fax:202-687-7505; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0617

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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tumors that respond to 3 months of tamoxifen therapy,whereas BCL2 expression is high in tumors that remain3 months after tamoxifen (11, 12). Thus, targeting theantiapoptotic BCL2 family members may be a usefulstrategy to overcome antiestrogen resistance in somebreast cancers.Recently, a new series of small molecules that mimic

BH3-only proteins have been generated, constituting anew class of potentially useful drugs. Bymimicking BH3-only proteins (such as NOXA, PUMA, BID, BAD, andBIM), antiapoptotic BCL2 members can be sequestered,thus, allowing BAK and BAX to activate the intrinsicapoptotic pathway. Among the BH3-mimetics, GX15-070 (GX; obatoclax) is an indole bipyrrole compound thatcan inhibit all known prosurvival BCL2 family members(13, 14). GX15-070 is currently under investigation inphase II clinical trials for the treatment of leukemia,lymphoma, myelofibrosis, and mastocytosis (15, 16). Pre-vious studies report that GX15-070 overcomes resistanceto lapatinib, a tyrosine kinase inhibitor often used inHER2-amplified breast cancer (17). While GX15-070 caninduce mitochondrial apoptosis (18), the precise molecu-lar mechanism(s) of cell death by GX15-070 is unclear.Several reports suggest that GX15-070 may induce autop-hagy and other forms of death as an alternate mechanismto caspase-dependent apoptosis (17, 19, 20).We examined the effects of GX15-070 and an antiestro-

gen using two separate models of antiestrogen resistancein breast cancer (21). Comparisons were made betweenMCF7 (ERþ, estrogen-dependent, antiestrogen-sensitive)and MCF7/RR [ERþ, estrogen-independent, tamoxifen-resistant; ICI182780-sensitive, derived from MCF7 cellsselected against tamoxifen (22, 23)] cells and betweenestrogen-independent MCF7/LCC1 [ERþ, antiestrogen-sensitive, derived by in vivo selection of MCF7 cells(24)] and MCF7/LCC9 (ERþ, estrogen-independent,ICI182780-resistant; tamoxifen cross-resistant, derivedfrom MCF7/LCC1 cells selected against ICI182780; ref.21) cells. In this report, we show that inhibiting antiapop-totic BCL2 family expression with GX15-070 induces celldeath in antiestrogen-resistant breast cancer cell linesthrough the completion of apoptosis and a cathepsin-mediated inhibition of autophagy. These findings haveimportant clinical implications and provide amechanisticrationale for the use of GX15-070 in combination with anantiestrogen for the treatment of ERþ breast cancers.

Materials and MethodsCell culture and reagentsMCF7 human breast cancer cells were provided by Dr.

Marvin Rich (Karmanos Cancer Institute, Detroit, MI);MCF7/RR, LCC1, and LCC9 cells were established aspreviously described (21, 22, 24). MCF7 and MCF7/RRcells were cultured in improvedMinimal Essential Media(IMEM; Invitrogen) with phenol red and supplementedwith 5% FBS. LCC1 and LCC9 cells were routinely grownin phenol red–free IMEM supplemented with 5% char-

coal-stripped calf serum. Cells were authenticated byDNA fingerprinting and tested regularly for Mycoplasmainfection. GX15-070 was purchased from Selleck Chemi-cals; ICI182780 (Faslodex; fulvestrant) and Z-VAD-FMKfrom Tocris Bioscience; Bafilomycin A1 (BAF) from EMDBiosciences; 4-hydroxytamoxifen (tamoxifen), hydroxy-chloroquine (HCQ), BAPTA-AM, and 3-methyladenine(3-MA) were from Sigma-Aldrich. The cathepsin L(CTSL1; 1-napthalenesulfonyl-Ile-Trp-aldehyde) and D(Ac-Leu-Val-Phe-aldehyde) inhibitors were from EnzoLife Sciences and Bachem, respectively.

Cell proliferationCells were seeded at a density of 5� 103 per well in 96-

well plates and, 24 hours later, treated with the indicatedconcentration of GX15-070 or vehicle control. Cells wereincubated with drug for 48 hours or 6 days, with mediacontaining either drug or vehicle being replaced every 3days. Following treatment, cells were stained with acrystal violet staining solution as previously described(25). Sodiumcitrate bufferwasused to extract thedye, andabsorbance was measured at 550 nmol/L using a micro-plate reader (Bio-Rad). Cell density was calculated fromthe crystal violet assay.

RNA interferenceATG7 and BECN1 siRNAwere fromCell Signaling and

Origene, respectively. Cells were transfected using Lipo-fectamine RNAiMAX (Invitrogen) according to the man-ufacturer’s instructions.

Western blot analysisLCC1, LCC9,MCF7, andMCF7/RR cells were plated in

6-well dishes and the following day treated with 100nmol/L GX � 100 nmol/L ICI/TAM, 100 nmol/L ICI/TAM, or vehicle control. Lysates were harvested 48 hourslater for protein analysis as previously described (3).Protein expression was measured by probing proteinswith the following antibodies overnight at 4�C: ATG7,BECN1, CTSB, CTSD, LC3B, PARP (Cell Signaling); p62(BD Transduction Labs); BCL2 (Enzo Life Sciences); andCTSL1 (eBioscience). To confirm equal loading of thegels, membranes were reprobed for b-actin (Santa CruzBiotechnology).

Reverse transcription PCRRNA was extracted using TRIzol (Invitrogen). Two

microgramRNAwasused fromeach sample as a templatefor cDNA synthesis with the High Capacity RNA-to-cDNAKit (Invitrogen). PCR amplificationwas conductedusing the following primers (purchased from IntegratedDNA Technologies): cathepsin B (CTSB): 50-GCCGCC-GAGCTCATGTGGCAGCTCTGGGCCTCC-30 (forward)and 50-ATTATTCCCGGGTTAGATCTTTTCCCAGTAC-TG-30 (reverse), cathepsin D (CTSD): 50-GTGCCTG-CCAGTCAGCGTCGTCAG-30 (forward) and 50-CCTGC-TCAGGTAGAAGGAGAAGATG-30 (reverse), andCTSL1:

Obatoclax Inhibits Autolysosome Degradation

www.aacrjournals.org Mol Cancer Ther; 12(4) April 2013 449




50-GACTCTGAGGAATCCTATCCA-30 (forward) and 50-AAGGACTCATGACCTGCATCAA-30 (reverse).

Apoptosis and autophagosome formation assaysCells were plated in 6-well tissue culture plates 1 day

before treatment with 100 nmol/L GX � 100 nmol/LICI/TAM, 100 nmol/L ICI/TAM, or vehicle control.Forty-eight hours posttreatment, cells were harvestedand stained as described in the Enzo Life SciencesAnnexin V–FITC Apoptosis Detection Kit for flowcytometry. Accumulation of autophagic vesicles wasmeasured using a modified monodansylcadaverineaccording to the manufacturer’s instructions (Cyto-IDAutophagy Detection Kit; Enzo Life Sciences). Stainedcells were detected and appropriate signals measuredby fluorescence-activated cell sorting (LCCC FACSShared Resource).

Autophagosome maturationLCC9 cells (1 � 105) were seeded onto 18 mm � 18

mm glass coverslips and 24 hours later were transfectedwith LC3 tagged with a GFP and/or a p62 cDNA taggedwith a red fluorescent protein (RFP). An mRFP-GFPtandem fluorescent-tagged LC3 vector (Addgene) wasused to assess whether GX15-070 inhibited autophagicflux (26). The following day, cells were treated with 500nmol/L GX15-070, 100 nmol/L ICI182780, or 5 nmol/LBAF in CCS-IMEM. The pH of autolysosomes wasmeasured in LCC9 cells using the LysoSensor Greendye (Invitrogen) as indicated by the supplier. Twenty-four hours posttreatment, cells were fixed, mounted oncoverslips, and visualized as previously described (27).

Orthotopic xenografts in athymic nude miceLCC1orLCC9cellswere injectedorthotopically into the

mammary fat pads of 5-week-old ovariectomized athymicnude mice as previously described (28). Mice were sacri-ficed after 9 weeks; tumors were removed at necropsy,fixed in neutral buffered formalin, and processed usingroutine histologic methods.

ImmunohistochemistryFive-micrometer sections from LCC1 and LCC9 paraf-

fin-embedded tissueswere stainedwithmouse anti-BCL2(Dako, 1:150) antibody as previously described (28). Acomputer-assisted counting technique with a grid filter toselect cells was used to quantify the immunohistochem-ical staining of BCL2 (29).

Electron microscopyFollowing 24 hours of treatment with 500 nmol/L

GX15-070 or vehicle control, LCC9 cells were harvestedand fixed in a glutaradehyde/paraformaldehyde solu-tion. Embedding sectioning and stainingwere carried outas previously described (30). After fixation, cells weredouble stained with uranyl acetate and lead citrate. Elec-tron micrographs of ultrathin sections (90 nm) were

viewed at a magnification of �10,000/�15,000 with aJEOL JM1010 transmission electron microscope.

Cathepsin activityCathepsin activity was determined using the commer-

cial assay provided by Biovision according to the manu-facturer’s protocol. Cells were seeded in 10 cm2 dishes24 hours before treatment with 100 nmol/L GX � 100nmol/L ICI, 100 nmol/L ICI182780, or vehicle control.Forty-eight hours posttreatment, cathepsin activity wasmeasured using 10 mmol/L CTSB (Ac-RR-AFC) or Lsubstrate (Ac-FR-AFC). A fluorometer (Tecan) was usedto quantify the cleavage of synthetic substrate of CTSBand CTSL1. Cathepsin activity was expressed as relativefluorescence units (RFU) per microgram protein.

Statistical analysisThe statistical significance of differences between 2

groups was analyzed by 2-tailed Student t tests. Formultiple group comparisons, Bonferroni multiple com-parison test was applied following one-way ANOVA.Results were considered to be significantly different atP < 0.05. Statistical analysis was carried out using thePrism version 5.0 software.

ResultsGX15-070 alone and in combination with anantiestrogen inhibits breast cancer cell density

Endogenous BCL2 protein expressionwasmeasured inMCF7, MCF7/RR, LCC1, and LCC9 breast cancer cells(Fig. 1A). Increased BCL2 expression was observed inestrogen-regulated MCF7 cells and estrogen-indepen-dent, antiestrogen-resistant LCC9 cells (Fig. 1A). We alsomeasured total BCL2 protein by immunohistochemistryin LCC1 and LCC9 mammary tumor xenografts. Datafrom these studies revealed that LCC9 tumors had sig-nificantly higher BCL2 expression compared with LCC1tumors (Supplementary Fig. S1A and S1B; P ¼ 0.0005).Given that GX15-070 is a BH3 mimetic that inhibits allknown antiapoptotic BCL2 members (structure shownin Fig. 1B), we sought to determine the effect of GX15-070 on antiestrogen-sensitive and -resistant breast cancercells. Increasing concentrations of GX15-070 (0, 0.1, 0.5, 1,and 10 mmol/L) inhibited both antiestrogen-sensitive(LCC1 and MCF7) and -resistant (LCC9 and MCF7/RR)cell density after 48 hours (Fig. 1C). When combined withthe antiestrogen tamoxifen over the course of 6 days,GX15-070 had an additive effect on inhibiting the relativecell density ofMCF7 andMCF7/RR cells (Fig. 1D),where-as no significant additive effect was observed in the LCC1and LCC9 cells with GXþICI (Fig. 1D). We also measuredAnnexin V–stained cells following GX15-070 exposureand observed that GX15-070 enhanced apoptosis consis-tent with its on-target effects (Supplementary Fig. S2Aand S2B). These data suggested that GX15-070 enhancedapoptosis-mediated cell death in antiestrogen-sensitiveand -resistant breast cancer cells.

Schwartz-Roberts et al.

Mol Cancer Ther; 12(4) April 2013 Molecular Cancer Therapeutics450




GX15-070 induces BECN1-dependentautophagosome formation, but autophagy is notrequired for GX15-070 toxicityWhile GX15-070 enhanced apoptosis, the pan-caspase

inhibitor Z-VAD-FMK failed to abrogate killing by GX15-070 � an antiestrogen (Supplementary Fig. S3). Further-more, Z-VAD-FMK failed to reduce the number of apo-ptotic cells following GX15-070 exposure, suggesting thatGX15-070-mediated apoptosis occurred via a caspase-independent mechanism (Supplementary Fig. S3).Because cell viability was not affected by a pan-caspaseinhibitor and because others have linked autophagic cell

death to GX15-070, we determined whether GX15-070either alone or in combination with an antiestrogen couldinduce autophagosome formation in breast cancer cells.Wemeasured the initial phaseof autophagy in response toGX15-070� antiestrogen treatment by selectively labelingautophagosomes with a modified monodansylcadaver-ine. GX15-070 alone and in combination with an anties-trogen significantly increased the number of autophagicvacuoles in antiestrogen-sensitive and -resistant cell lines(Fig. 2A; P ¼ 0.0001).

We next explored the effect of GX15-070 on autophagyregulation by measuring the formation of LC3-II, which

Figure 1. GX15-070 and anantiestrogen inhibit breast cancercell density. A, MCF7, MCF7/RR,LCC1, andLCC9 cells were grown in6-well plates under basal conditionsfor 48 hours. Whole cell lysates wereprepared and immunoblottedagainst BCL2; b-actin serves as theloading control. Densitometricanalysis from 3 or more separateexperiments. B, chemical structureofGX15-070.C, LCC1, LCC9,MCF7,and MCF7/RR cells were seeded in96-well tissue culture dishes 24hours before treatment with GX15-070 at the indicated concentration.Forty-eight hours posttreatment,cells were stained with crystal violetand cell density was determined witha plate reader at wavelength 550 nm.D, LCC1, LCC9, MCF7, and MCF7/RR cells were plated in 96-well tissueculture plates and treated with theindicated drug(s) for 6 days (refedwith medium containing vehicle ordrug on day 3) before measuring cellproliferation as in C. Data arepresented as relative cell density andrepresent the mean � SEM for 3 ormore independent experiments;�, P < 0.05; ��, P < 0.001 versusvehicle/control.






participates in elongation of the autophagosome mem-brane (31). In LCC9 cells, 48 hours of GX15-070 � anantiestrogen significantly induced the formation ofLC3-II (Fig. 2B;P¼ 0.0005).When autophagywas blockedby RNA interference (RNAi)–targeting BECN1, a criticalregulator of autophagy (31), LC3-II cleavage was signif-icantly reduced (Fig. 2B; P ¼ 0.0005). In contrast,the increase in autophagosome formation observed afterGX15-070 exposurewas not suppressed by the addition ofRNAi-targeting ATG7, a protein necessary for the forma-

tion of the preautophagosomal structure (ref. 31; Fig. 2B).To assess whether GX15-070-induced autophagosomeformationwas calcium-dependent, LCC9 cellswere trans-fected with a GFP-tagged version of LC3, and the follow-ingday treatedwith 500nmol/LGX15-070 and10mmol/Lof the calcium chelator BAPTA-AM. Formation of LC3-GFP aggregates increased with GX15-070 and BAPTA þGX treatment, suggesting that GX15-070-mediated LC3punctae formation was largely independent of calciumsignaling (Fig. 2C).

Figure 2. GX15-070–mediated autophagosome formation is dependent on BECN1. A, LCC1, LCC9, MCF7, and MCF7/RR cells were seeded in 6-well tissueculture plates 24 hours before treatment with the indicated drug(s). Forty-eight hours posttreatment, autophagosome formationwas detected as described inMaterials and Methods. Data are presented as percentage of total cells positive for green fluorescence and represent the mean � SEM for 3 or moreindependent experiments; �,P <0.05 versus control/vehicle experiment. B, LCC9 cellswere transfectedwithBECN1 siRNA (siBECN1), ATG7 siRNA (siATG7),or control siRNA (siCtrl) 24 hours before treatment with GX� ICI, ICI182780, or vehicle control. Forty-eight hours posttreatment, cells were lysed and proteinexpressionwasmeasuredby probing proteinswith the indicated antibodies; b-actin served as the loading control. Representative images from3 independentexperiments. C, LCC9 cells transfectedwith LC3-GFPwere treatedwith 500 nmol/LGX�10 mmol/L BAPTA-AM for 24 hours. Cells were fixed, permeabilized,stained with DAPI, and visualized by confocal microscopy. D, LCC1 and LCC9 cells were treated with GX15-070, ICI182780, and the indicated autophagyinhibitor for 48 hours. Cell viability was assessed using a crystal violet assay. Data are presented as relative density and represent the mean � SEM for 3 ormore independent experiments, �, P < 0.05; ��, P < 0.001 between indicated experimental groups.






To determine whether inhibition of autophagy couldsuppress GX15-070-mediated cell death, we used severalautophagy inhibitors in combinationwithGX� ICI. LCC1and LCC9 cells were treated with 500 nmol/L GX � 100nmol/L ICI, 100 nmol/L ICI182780, or a combination ofICI182780,GX15-070, and one of the autophagy inhibitors,3-MA(5mmol/L; early-stage inhibitor),HCQ(10mmol/L;late-stage inhibitor), or BAF (5 nmol/L; late-stageinhibitor). 3-MA slightly suppressed activity of the drugcombination in LCC1 (P ¼ 0.04) but not LCC9 cells (Fig.2D). Because 3-MA inhibits autophagy by blocking autop-hagosome formation via inhibition of type III phosphoi-nositide 3-kinase (PI3K), these data suggested that GX15-070-induced autophagy was independent of PI3K. BAF,which results in the accumulation of autophagosomes,promoted drug combination lethality in LCC1 and LCC9cells (Fig. 2D; P¼ 0.0006 and 0.003, respectively), whereasHCQþGXþICI reduced cell viability equally to GXþICI(Fig. 2D). Despite BECN1-dependent LC3-II processingfollowing GX15-070 exposure, GX15-070 reduced equallythe cell density of LCC9 cells transfected with control orBECN1 siRNA (Supplementary Fig. S4).

GX15-070 induces autophagic vacuole and lysosomeformation in antiestrogen resistant LCC9 breastcancer cellsTo confirm our observation that GX15-070 induces

autophagosome formation, we used electron microscopyto examine LCC9 cells treated with GX15-070. Untreatedcontrol cells seemed to have a normal cytoplasmwith fewautophagic vacuoles (Fig. 3; arrows). Twenty-four hoursof GX15-070 exposure resulted in an accumulation oflysosomes (marked "L") and autophagic vacuoles (Fig.3B). Under higher magnification, autophagic vesicleswere observed to have typical double-membrane bound-aries containing electron dense material (Fig. 3B). Mito-chondria (marked "M") were also enlarged in the GX15-070–treated cells (Fig. 3B), suggesting that the cell was in

the later stages of apoptosis (32). The electron micro-graphs revealed that GX15-070 increased lysosome andautophagic vacuole formation, leading us to examineproteins involved in autolysosome formation anddegradation.

GX15-070 treatment results in the accumulation ofLC3-II and p62 proteins

We next investigated the role of GX15-070 on laterevents of the autophagy process. In mammalian cells,p62/sequestosome-1 (SQSTM1) is implicated in autopha-gic cargo recognition and is lost in the final stages ofautophagy during autolysosome degradation (31). UsingWestern blot analysis, we found an accumulation of p62protein in antiestrogen-sensitive and -resistant breastcancer cells following treatment with GX15-070 (Fig.4A). By immunofluorescence, we measured LC3 punctaeand p62 protein expression in LCC9 cells treated with 100nmol/L ICI182780, 500 nmol/LGX15-070, ICIþGX, or thelate-stage autophagy inhibitor, 5 nmol/L BAF. 40,6-Dia-midino-2-phenylindole (DAPI) staining shows the loca-tion of the nuclei; when merged with LC3-GFP and redfluorescent p62 cDNA, the levels of LC3 punctae and p62were elevated in GX15-070- and BAF-treated cells com-pared with both vehicle- and ICI182780-treated cells (Fig.4B). Accumulation of LC3 punctae andp62 suggested thatGX15-070 functioned as a downstream autophagyinhibitor.

GX15-070 blocks autophagic degradation throughattenuation of cathepsin activity

In the final stages of autophagy, mature autolysosomesare subjected to proteolytic degradation, leading to areduced level of autophagic contents and substrates suchas p62 (33). Therefore, we focused on the lysosomalhydrolases CTSB, CTSD, and CTSL1, as potential targetsof GX15-070. Forty-eight hours of GX15-070 exposureresulted in a 5-fold inhibition of CTSD and CTSL1 protein

Figure 3. GX15-070 inducesautophagosome and lysosomeformation in antiestrogen-resistantLCC9 cells. A, electron microscopyimages of vehicle (control) LCC9-treated breast cancer cells. Thecytoplasm seems normal andcontains few autophagic vacuoles.B, following 24 hours of 500 nmol/LGX15-070 exposure, the number ofautophagic vacuoles in LCC9 cellsincreased. Swollen mitochondria (M)and an increase in the number oflysosomes (L) were specificallyobserved in cells treated with GX15-070.






expression in LCC1, LCC9, and MCF7 cells (Fig. 5A).While GX15-070 reduced CTSB modestly in LCC1 cells,ICI182780 alone stimulated expression of the 3 cathepsinsin antiestrogen-sensitive cells (Fig. 5A). We further exam-ined the mechanism of GX15-070 bymeasuring cathepsinmRNA expression in LCC1 and LCC9 cells. CTSB, CTSD,and CTSL1 mRNA levels remained unchanged afterGX�ICI exposure (Supplementary Fig. S5).

We next assessed whether cathepsin activity was atten-uated by GX15-070 by measuring activity of B and L inLCC1 and LCC9 cells exposed to 100 nmol/L GX � 100nmol/L ICI, 100 nmol/L ICI182780, or vehicle control. GX� ICI significantly inhibited CTSL1 activity in LCC1 andLCC9 cells after 48 hours (Fig. 5B; P ¼ 0.0001 and 0.0001,respectively). While GX15-070 slightly reduced CTSBactivity in LCC1 cells (P ¼ 0.004), CTSB activity wasunaffected by GX15-070 in LCC9 cells (Fig. 5C). Withouta specific substrate, activity for CTSD could not be deter-mined. To further elucidate how GX15-070 affects autop-hagosome maturation, we transfected LCC9 with anmRFP-GFP tandem-tagged LC3. Typically, GFP-LC3 isdegraded by hydrolases following autophagosome–lyso-some fusion (26); however, GX15-070 induced an accu-mulation ofmRFPandGFP, suggesting that a reduction incathepsin protein expression inhibited the degradation ofGFP (Supplementary Fig. S6). Acidic pH is required forcathepsin activity (34), so we determined whether GX15-070 affected lysosomal pH. Merged fluorescence imagesof LCC9 cells stained with a red fluorescent dye to mea-sure acidic organelles and a green dye to measure pHshow as yellow and indicate an acidic environment (Fig.5D). GX15-070 clearly induced an acidic pH suitable for

cathepsin activity when compared with vehicle-treatedcontrol cells (Fig. 5D). These results indicate that inhibi-tion of cathepsin protein expression by GX15-070 inhibitsautophagic flux, and these changes were not mediated bychanges in lysosomal pH.

Inhibition of cathepsin L and D results in breastcancer cell death by blocking autophagosomaldegradation

Wenext sought to determinewhether known inhibitorsof CTSL1 and CTSD would also reduce breast cancer cellgrowth through the inhibition of autophagosomal lysis.Relative cell proliferation of LCC1 and LCC9 cells wasinhibited by 20 mmol/L of the CTSL1 and 50 mmol/L ofthe CTSD inhibitor alone and in combination with 100nmol/L ICI182780 after 48 and 24 hours, respectively (Fig.6A; P ¼ 0.0001). Cell death was accompanied by anaccumulation of p62 in antiestrogen-sensitive and -resis-tant cells, which is indicative of inhibition of autophagicflux (Fig. 6B). CTSL1 and CTSD inhibition also increasedautophagosome formation in both LCC1 and LCC9 cells(Fig. 6C; P ¼ 0.0001). Taken together, these data providestrong evidence that GX15-070 inhibits autophagic deg-radation through a CTSL1- and CTSD-dependent mech-anism, which causes cancer cells to lose their ability torecycle subcellular components through autophagy andrestore metabolic homeostasis.

DiscussionIncreased expression of BCL2 and/or BCLW plays a

role in antiestrogen resistance by allowing cells to evade

Figure 4. GX15-070 results in LC3and p62 accumulation. A, LCC1,LCC9, MCF7, and MCF7/RR cellswere seeded in 6-well plates 24hours before treatment with theindicated drug(s). Forty-eighthours posttreatment, cells werelysed and the indicated proteinswere detected by immunoblot;b-actin served as the loadingcontrol. Representative imagesfrom 3 or more independentexperiments. B, LCC9 cellstransfected with LC3-GFP and ap62 cDNA tagged with a RFP weretreatedwith 100 nmol/L ICI182780,500 nmol/L GX15-070, ICI þ GX, 5nmol/L BAF, or vehicle control.Twenty-four hours posttreatment,the cells were fixed, permeabilized,stained for DAPI, and visualized byconfocal microscopy.






apoptosis (35). Using multiple cell lines and differentendocrine therapies, we show that GX15-070, a small-molecule pan-inhibitor of antiapoptotic BCL2 familymembers, potentiates cell death in both antiestrogen-sen-sitive and -resistant human breast cancer cells. Whilereports describe apoptosis and autophagy as the majorforms of death induced by GX15-070 (14, 18, 19), thedownstream mediators of GX15-070-induced autophagyhave yet to be elucidated. We show that GX15-070’santicancer efficacy is due to the blockade of antiapoptoticBCL2 family members and an increase in autophagy

initiation without the complete digestion of autolyso-somes; perhaps one form of apparent autophagic celldeath. Furthermore, GX15-070 inhibits the protein expres-sion of CTSD and CTSL1 that would ultimately limit cellsfrom effectively recycling cargo that could be used to fuelcell metabolism and restore metabolic homeostasis.

Using multiple cell lines affected differently by estro-gen, we were able to establish a role for GX15-070 inantiestrogen-resistant breast cancer. BCL2 and CTSD areboth estrogen-regulated genes (36, 37), so assays wereconductedwith orwithout estrogen to determinewhether

Figure 5. GX15-070 inhibits autophagic degradation through attenuation of cathepsin activity. A, LCC1, LCC9, MCF7, and MCF7/RR cells were treatedwith the indicated amount of GX � ICI/TAM, ICI/TAM, or vehicle control for 48 hours and expression of the indicated proteins were detected byimmunoblot; b-actin served as the loading control; n¼ 3. B and C, LCC1 and LCC9 cells were assayed for CTSL1 andCTSB activity as described inMaterialsand Methods. Data represent the mean fluorescence unit relative to the vehicle control for 3 or more independent experiments; �, P < 0.05; ��, P <0.01;��,P <0.001 versus control/vehicle treatment. D, LCC9cellswere incubatedwithCellLight LysoTracker Red (30 particles per cell)�500nmol/LGX15-070 for24 hours 15 minutes before fixation; cells were incubated with a LysoSensor Green dye. Merged (yellow) images are indicative of an acidic pH.






the effects of GX15-070 were mediated by ER signaling.We hypothesized that GX15-070 would inhibit the prolif-eration of cells that express high levels of antiapoptoticBCL2. While BCL2 members are known to regulate apo-ptosis (35), several antiapoptotic BCL2 members alsoaffect autophagy through their interaction with BECN1(9). In the antiestrogen-resistant LCC9 cells,we observed astrong induction of apoptosis and autophagy comparedwith their LCC1 parental cells following GX15-070 treat-ment alone.Wenoted a similar induction of apoptosis andautophagy inMCF7 cells compared with their tamoxifen-resistant MCF7/RR–derived cells. Thus, ERþ breasttumors that have high BCL2 expression seem to be goodcandidates for GX15-070 � antiestrogen treatment.

GX15-070 seems to induce apoptosis as measured byAnnexin V localization to the outer leaflet of the plasmamembrane (Supplementary Fig. S3). To determinewheth-er GX15-070-mediated cell deathwas caspase-dependent,we treated cells with GX15-070 in combination with thepan-caspase inhibitor Z-VAD-FMK. While Z-VAD-FMKinhibited PARP cleavage, it did not influence the effects ofGX15-070 on cell density and Annexin V translocation(Supplementary Fig. S3). Thus, we hypothesized thatGX15-070 killed breast cancer cells by a caspase-indepen-dent cell death mechanism. Z-VAD-FMK can potentiatenecrotic cell death in certain cell types (38), suggestingthat the drug combination killing could have resultedfrom off-target effects of Z-VAD-FMK. Further studies

Figure 6. CTSL1 and CTSD mediate autolysosomal membrane component degradation. A, LCC1 and LCC9 cells were incubated with 20 mmol/L ofCTSL1 inhibitor or 50 mmol/L of CTSD inhibitor � 100 nmol/L ICI182780, 100 nmol/L ICI182780, or vehicle control for 48 or 24 hours, respectively. Cellproliferation was assessed as changes in cell density measured using crystal violet staining. Data are presented as relative density and represent themean � SEM for 3 or more independent experiments; ��, P < 0.001 versus control/vehicle experiment. B, indicated proteins were detected by immunoblot;b-actin served as the loading control; representative images from 3 or more independent experiments. C, LCC1 and LCC9 cells were treated as in A.Cells were detected for autophagosome formation by measuring a modified monodansylcadaverine probe by flow cytometry. Data are presented aspercentage of total cells positive for green fluorescence and represent the mean � SEM for 3 or more independent experiments; ��, P < 0.01;��, P < 0.001 versus control/vehicle experiment.






investigating the effects of Z-VAD-FMKþGX onmetabol-ic regulators and necroptosis are in progress.Recent studies have associated increased autophagy

with endocrine resistance and imply that autophagy pro-vides one mean for cells to delay an apoptotic cell death(35, 39–41). However, when autophagy persists at highlevels, it is often associated with cell death (31). The BH3mimetic GX15-070 can induce both apoptosis and autop-hagy (18, 19). Therefore,we expected to see both apoptosisand autophagy implicated in GX15-070-induced lethalityin antiestrogen-sensitive and -resistant breast cancer cells.Indeed,GX15-070promotedautophagic vacuole and lyso-some formation in LCC9 cells (Fig. 3B). However, this wasaccompanied by an accumulation of p62 (Fig. 4A), whichsuggests an impaired ability to degrade contents of autop-hagic vesicles. We also observed swollen mitochondria inGX15-070–treated cells (Fig. 3B). Previous reports charac-terizemitochondria swellingwith release of cytochrome cin the later stages of apoptosis (32).GX15-070–induced LC3 processing has been suggested

to depend on ATG7 (18). However, we show that LC3-IIformation following GX15-070 exposure is dependent onBECN1 and independent of ATG7 (Fig. 2B). BECN1 con-tains a BH3 domain and its ability to initiate autophagy isinhibited by antiapoptotic BCL2 family members (9). It ispossible that inhibition of antiapoptotic BCL2members byGX15-070 releases free BECN1, allowing for BECN1-dependent autophagosome formation. However, GX15-070 reduces cell density in the presence of an autophagyinhibitor (Fig. 2D) and BECN1 siRNA (SupplementaryFig. S4), suggesting that GX15-070 toxicity promoted celldeath through an unknown, PI3K-, BECN1-, and ATG7-independent mechanism. These different cell deathmechanisms show the plasticity of breast cancer cellsignaling to regulate cell fate in response to endocrine-based stress.To determine the precise role of GX15-070 in autop-

hagy maturation, we systematically measured thedownstream events of autophagy. Previous reportssuggest that defective autophagic degradation, reflectedby the accumulation of undigested autophagosomesand p62 protein, may contribute to cell death inducedby a combination of GX15-070 and lapatinib (42). How-ever, the mechanism by which GX15-070 inhibits autop-hagy is unclear. In the present study, we showed thatdegradation of the autophagy substrate, p62, and clear-ance of autolysosomes is blunted in GX15-070–treatedcells. This is further supported by data illustrating thatGX15-070 prevents the degradation of GFP and mRFP-tagged LC3, suggesting GX15-070 inhibits autophagicflux (Supplementary Fig. S6). This led us to focus onstudying CTSB and CTSL1, which are known to degradeautolysosome contents (43), and CTSD that is tightlyregulated by estrogen (37) and often overexpressed inERþ breast cancers (44).Our data show that expression of CTSD, CTSL1, and

to a lesser extent CTSB, protein was suppressed inGX15-070–treated cells (Fig. 5A). Importantly, CTSB,

CTSD, and CTSL1 have each been implicated in tumorinvasion, and metastasis (37, 45). Consistent with theprotein expression data, we also reported reduced pro-teolytic activity of CTSB and CTSL1 (Fig. 5B). Despitedecreases in protein expression, we did not observe anychanges in CTSB, CTSD, or CTSL1 mRNA expressionfollowing GX15-070 exposure (Supplementary Fig. S5).Increased secretion of CTSD and CTSL1 could contrib-ute to reduced intracellular levels; however, we mea-sured cytosolic cathepsin expression following GX15-070 treatment and found no difference in cathepsinprotein expression when compared with vehicle-treatedcells (Unpublished Data). Thus, it is possible that GX15-070 modulated the posttranslational modification ofcathepsins, which, in turn, attributed to the increase inLC3-II and p62 protein levels. To mimic the effects ofGX15-070, we suppressed CTSD and CTSL1 with achemical inhibitor and treated cells with ICI182780. Aswith GX15-070 treatment, we detected an accumulationof autophagosomes in cells with depressed CTSL1 andCTSD activity (Fig. 6C). This observation is consistentwith the ability of CTSL1 to degrade lysosomal mem-brane components (43). Because CTSD and CTSL1 areimplicated in breast cancer progression, inhibition ofcathepsin activity using GX15-070 or other strategiesmay be clinically beneficial.

In summary, our results show that inhibition ofantiapoptotic BCL2 expression by GX15-070 effectivelyreduces breast cancer cell growth alone and additivelywith an antiestrogen. Mechanistically, GX15-070induces apoptosis by freeing up BH3-only BCL2 mem-bers and increasing autophagic vacuole formation inbreast cancer cells. Despite the increase in autophago-some formation with GX15-070 exposure, the decisionfor the cell to undergo cell death is driven by GX15-070’s ability to impair cathepsin protein expression,which leads to the accumulation of autophagosomesand autolysosomes but interrupts completion of deg-radation of autophagic vacuoles. This study providesstrong data supporting the potential use of GX15-070and an endocrine therapy for the treatment of ERþbreast cancer cells with detectable BCL2, CTSD, andCTSL1 expression.

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

DisclaimerThe content of this article is solely the responsibility of the authors and

does not necessarily represent the official views of the National CancerInstitute or the NIH.

Authors' ContributionsConception and design: J.L. Schwartz-Roberts, A.N. Shajahan, K.L. Cook,R. ClarkeDevelopment of methodology: J.L. Schwartz-Roberts, A.N. Shajahan, R.ClarkeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.L. Schwartz-Roberts, A.N. Shajahan, A. W€arri,M. Abu-Asab






Analysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): J.L. Schwartz-Roberts, M. Abu-Asab, R.ClarkeWriting, review, and/or revision of the manuscript: J.L. Schwartz-Roberts, A.N. Shajahan, K.L. Cook, A. W€arri, M. Abu-Asab, R. ClarkeAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.L. Schwartz-Roberts, R. ClarkeStudy supervision: A.N. Shajahan, R. Clarke

AcknowledgmentsThe authors thank technical services provided by the Microscopy and

Imaging, Flow Cytometry, Histopathology, and Tissue Culture SharedResources. The authors also thank Margaret Axelrod, and Drs. M. Liu, T.Sherman, and P. Furth.

Grant SupportJ.L. Schwartz-Roberts is the recipient of a NIH training grant (grant no.

F31CA165514-01A1). This research was supported by Public Health Ser-vice Awards U54-CA149147, R01-CA131465, and Susan G. Komen GrantKG090245 (to R. Clarke). The project described earlier was supported byAward Number P30CA051008 from the National Cancer Institute.

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 July 13, 2012; revised January 28, 2013; accepted January 28,2013; published OnlineFirst February 8, 2013.

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2013;12:448-459. Published OnlineFirst February 8, 2013.Mol Cancer Ther Jessica L. Schwartz-Roberts, Ayesha N. Shajahan, Katherine L. Cook, et al. Breast Cancer Cells

Mediated Autophagosomal Lysis in Antiestrogen-Resistant−and L GX15-070 (Obatoclax) Induces Apoptosis and Inhibits Cathepsin D-

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