V-ATPase Inhibition Regulates Anoikis Resistance and Metastasis … · Cancer Biology and Signal Transduction V-ATPase Inhibition Regulates Anoikis Resistance and Metastasis of Cancer

Cancer Biology and Signal Transduction

V-ATPase Inhibition Regulates Anoikis Resistance andMetastasis of Cancer Cells

Christina M. Schempp1, Karin von Schwarzenberg1, Laura Schreiner2, Rebekka Kubisch2,Rolf M€uller3, Ernst Wagner2, and Angelika M. Vollmar1

AbstractFightingmetastasis is amajor challenge in cancer therapy andnovel therapeutic targets anddrugs are highly

appreciated. Resistance of invasive cells to anoikis, a particular type of apoptosis induced by loss of cell–matrix

contact, is a major prerequisite for their metastatic spread. Inducing anoikis in metastatic cancer cells is

therefore a promising therapeutic approach. The vacuolar-ATPase (V-ATPase), a proton pump located at the

membrane of acidic organelles, has recently come to focus as an antimetastatic cancer target. As V-ATPase

inhibitors have shown to prevent invasion of tumor cells and are able to induce apoptosis, we proposed that V-

ATPase inhibition induces anoikis-related pathways in invasive cancer cells. We used the V-ATPase inhibitor

archazolid to investigate themechanismof anoikis induction in variousmetastatic cancer cells (T24,MDA-MB-

231, 4T1, 5637) in vitro. Anoikis induction by archazolidwas characterized by decreased c-FLIP expression and

caspase-8 activation as well as reduction of active integrin-b1 and an early increase of the proapoptotic protein

BIM.However,we observed that archazolid also inducesmechanisms opposing anoikis such as degradation of

BIM mediated by extracellular signal-regulated kinase (ERK), Akt and Src kinases at later time points and

induction of reactive oxygen species. Still, intravenous injection of archazolid-treated 4T1-Luc2 mouse breast

cancer cells resulted in reducedmetastasis inmouse lungs. Thus, V-ATPase inhibition is not only an interesting

option to reduce cancer metastasis, but also to better understand anoikis resistance and to find choices to fight

against it. Mol Cancer Ther; 13(4); 926–37. �2014 AACR.

IntroductionThe cause of death by cancer is mainly not the primary

tumor but the development ofmetastases in distinct organs(1). Metastasis is a highly complex process following seq-uential steps that comprise dissociation of cancer cells fromthe site of origin, their survival and travel in the circulationas well as migration and proliferation in distinct targetorgans (2–4). Becauseof the clinical importance, there is stillthe need to better understand the major determinants ofmetastasis and to identify therapeutical targets and path-ways suitable for fighting the metastatic process.

We recently introduced the vacuolar-ATPase (V-ATPase)as a promising new antimetastatic target showing that

the V-ATPase inhibitor archazolid inhibits cancer cellmigration (5).

V-ATPases are ATP-dependent proton pumps ubiqui-tously expressed, regulating the pH in endomembranesystems thus affecting receptor-mediated endocytosisand intracellular trafficking (6). Archazolid is a myxobac-terial second metabolite, first isolated from Archangiumgephyra (7), which binds the subunit c in the V0 domain ofthe V-ATPase and thereby inhibits its activity (8).

Besides affecting motility of invasive cancer cells, arch-azolid also induces apoptotic cell death. We thereforehypothesized that archazolid might also affect the initialsteps of metastatic dissemination, which is characterizedby the ability of invasive tumor cells to survive in a state ofdetachment from the extracellular matrix (9).

Although nontumoral cells respond to loss of cell–matrixcontact by a particular type of apoptosis, which has beentermed anoikis (10, 11), metastatic cancer cells are insensi-tive to anoikis. This allows their survival after detachmentfrom the primary tumor and their travel through the blood-stream to distant organs (3, 11). In other words, anoikisresistance is a precondition and a hallmark for the meta-static spread of tumor cells (12). Inducing anoikis by drugsis a promising option for the treatment ofmetastatic cancerbut calls for a profound understanding of the mechanismsunderlying anoikis resistance in invasive cancer.

Anoikis is initiated by disruption of integrin ligationof cells to the ECM. Integrin binding activates distinct

Authors' Affiliations: 1Department of Pharmacy, Pharmaceutical Biologyand 2Department ofPharmacy,PharmaceuticalBiotechnology,University ofMunich, Munich; and 3Helmholtz Institute for Pharmaceutical ResearchSaarland, Helmholtz Centre for Infection Research and Department ofPharmaceuticalBiotechnology, SaarlandUniversity,Saarbr€ucken,Germany

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

C.M. SchemppandK. vonSchwarzenberg contributed equally to thiswork.

Corresponding Author: Angelika M. Vollmar, Department of Pharmacy,Pharmaceutical Biology,University ofMunich,Germany, Butenandtstrasse5-13, 81377 Munich, Germany. Phone: 0049-89-2180-77172; Fax: 0049-89-2180-77170; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0484

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(4) April 2014926

on November 17, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst January 30, 2014; DOI: 10.1158/1535-7163.MCT-13-0484

http://mct.aacrjournals.org/

cell survival signaling cascades comprising downstreamplayers such as focal adhesion kinase (FAK), Src kinase,PI3K/Akt, and extracellular signal-regulated kinase(ERK). Detachment of cells, that is, loss of integrin signal-ing not only inhibits survival signals, but also activatesspecific apoptotic processes (13–15).Anoikis in line with classical apoptosis can follow both

the extrinsic pathway triggered by cell surface deathreceptors and the intrinsic pathway driven by mitochon-dria (12, 16). Activation of caspase-8 is considered as amajor player of the extrinsic pathway, and the activationof proapoptoticmembers of the Bcl-2 family, in particular,the protein BIM and mitochondrial cytochrome C releaseas features of the intrinsic pathway of anoikis. However, across-talk between intrinsic and extrinsic pathways fre-quently occurs (3, 17, 18).Detachment can also induce autophagy as a survival

mechanism preventing anoikis. It has been shown thatinhibition of autophagy increased caspase-3 activity indetached cells (19). BIM was also suggested to play a rolein autophagy by interacting with beclin-1 and inhibitingautophagosome formation (20).V-ATPase inhibitionon theother hand is known to block late-stage autophagy due toalkalization of lysosomes and therefore inhibition of pro-tein degradation in autolysosomes. V-ATPase inhibitionmight also affect integrin signaling as integrin activities aredependent on fast endocytosis rates and V-ATPase regu-lates receptor recycling via acidification of endosomes andlysosomes (21–23). That is why it has been hypothesizedthat V-ATPase inhibition affects anoikis induction.In fact, we report here, that archazolid induces anoikis in

invasive urinary and breast cancer cells. Anoikis is pro-voked by a reduction of active integrin-b1, an activationof caspase-8, an early translocation of BIM to themitochon-dria followed by release of cytochrome C. Of note, arch-azolid-treatedbreast tumor cells injected (intravenously) inmice showed a reduced formation of lung metastasis. Ourstudy further identified counter mechanisms induced byarchazolid treatment like a strong proteasomal downregu-lation of BIM provoked by generation of reactive oxygenspecies (ROS) and activation of prosurvival kinase Akt.

Materials and MethodsCompoundsArchazolid (Supplementary Fig. S1) was purified

and isolated as described previously (7). The protea-some-inhibitors MG-132 and bortezomib as well as theAkt-inhibitor LY 294002, ERK-inhibitor PD 98059, and c-Src-inhibitor saracatinib, all dissolved in dimethyl sulf-oxide (Sigma-Aldrich)were purchased fromSelleckchem.N-Acetyl-L-Cystein (NAC; Sigma-Aldrich)was dissolvedin PBS (pH 7.4). The following antibodies were used forimmunoblotting: Actin (Millipore), pFAK/FAK (SantaCruz Biotechnology), integrin-b1, c-FLIP, BIM, CytC,COX IV, pAkt/Akt, pERK/ERK, pSrc/Src (Cell SignalingTechnology), integrin-b1 active form 12G10 (Abcam),goat-anti-mouse IRDye 800cw (Li-COR GmbH), goat-anti-rabbit 680 (AlexaFluor, Life Technologies), horserad-

ish peroxidase (HRP) goat-anti-mouse (Santa CruzBiotechnology), and HRP goat-anti-rabbit (Bio-Rad).

Cell cultureThehumanurinarycarcinomacell lineT24wasprovided

by Dr. B. Mayer (Surgical Clinic, LMU,Munich, Germany)in 2009 and authenticated in April 2013 by the DSMZ byDNA profiling of eight highly polymorph short tandemrepeats (STR) regions. Cells were cultured in McCoy’smedium containing 10% heat-inactivated fetal calf serum(FCS, Biochrom AG), 1% glutamine (1.5 mmol/L), and 1%penicillin-streptomycin at 37�C and 5% CO2. MDA-MB-231 and 5637 cells were purchased from CLS cell linesservice GmbH in May 2011 and April 2013. CLS authen-ticates all cell lines by DNA profiling via STR analysis.The mouse breast cancer cell line 4T1-Luc2 was pur-chased from Caliper Life Science in January 2012. Cal-iper analyzed the cells by IMPACT 1 PCR profiling. 4T1-Luc2 and 5637 cells were maintained in RPMI-1640medium with 10% FCS. MDA-MB-231 cells were culti-vated in Dulbecco’s Modified Eagle Medium (HighGlucose) with 10% FCS. All media were purchased fromPAA laboratories.

In vivo experimentsTwenty, 4- to 6-week old female BALB/cByJRj mice

(Janvier) were housed in individually ventilated cageswith a 12-hour day/night cycle and food and water adlibitum. Mice were injected with archazolid pretreated (10nmol/L, 24hours) or untreated4T1-Luc2 cells (1� 105) viathe tail vein. Bioluminescence of metastasized cells wasmonitored at day 8 after cell injection under anesthesia(2% isoflurane in oxygen) using the IVIS Lumina systemwith Living Image software 3.2 (Caliper Life Sciences) 15minutes after intraperitoneal injection of 6 mg Na-lucif-erin (Promega). Thereafter, mice were sacrificed by cer-vical dislocation, their lungs harvested, imaged, andweighted. The total flux/area of the defined region ofinterest was calculated as photon/second/cm2. All in vivoexperiments were performed according to the guidelinesof the German law for protection of animal life andapproved by the local ethics committee.

Colony formation assayArchazolid-treated T24 cells (5 � 103, 24 hours) were

suspended in a 0.4% agarose-medium mix (low-meltingtemperature agarose, LONZA), seeded on 6-well platesprecoated with 1% agarose, and incubated for 9 days at37�C. Evolved colonies were stained with MTT (Sigma-Aldrich) dye, photographed, and analyzed by ImageJ1.46r software.

Detachment-induced anoikis assayT24,MDA-MB-231, 4T1, or 5637 cells (7� 104)were kept

in suspensionbyusingpoly-HEMA[poly(2-hydroxyethylmethacrylate); Sigma-Aldrich] coated culture dishes toprevent adhesion (24, 25). The culture medium was sup-plemented with 1% methylcellulose (Sigma-Aldrich) to

Archazolid Induces Anoikis in Metastatic Cancer Cells

www.aacrjournals.org Mol Cancer Ther; 13(4) April 2014 927




increase viscosity to prevent cell clumping. Apoptoticdeath was analyzed as described (26): permeabilized cellsstainedwith propidium iodide (50 mg/mL) were analyzedfor their subdiploid DNA content by flow cytometry(FACSCanto II, BD Biosciences). Cell death was furtheranalyzed by the propidium iodide exclusion assay count-ing propidium iodide (5 mg/mL) positive, nonpermeabi-lized cells by flow cytometry.

Western blot analysis and cytosol-mitochondriafractionation

Floating cells were treated with archazolid and thelisted inhibitors for the indicated timeframes and subse-quently lysed. Equal amounts of the proteins were sep-arated by SDS-PAGE and transferred onto nitrocellulosemembranes by tank blotting. For detection of specific

proteins, the ECL detection system (Amersham Pharma-cia Biotech) or the Odyssey Infrared Imaging systemversion 2.1 (LI-COR Biosciences) was used.

For cytosol-mitochondria fractionation, cells were har-vested, incubated with a permeabilization buffer (man-nitol 210 mmol/L, sucrose 200 mmol/L, HEPES, pH 7.2,10 mmol/L, Na2EGTA 0.2 mmol/L, succinate 5 mmol/L,bovine serum albumin 0.15%, digitonin 80 mg/mL; 20minutes, on ice), and centrifuged (10 minutes, 1,300 rpm,4�C). The supernatant was collected (cytosolic fraction)and the pellet permeabilized with 0.1% TritonX-100 (15minutes, on ice; mitochondrial fraction).

Flow cytometry analysis of cell surface integrinActive integrin-b1 on the cell surface was examined

using the conformation specific integrin-b1 active form

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Figure 1. Reduction of anchorage-independent colony formation andinduction of anoikis by archazolid(Arch.). A, left, T24 cells werepretreated with archazolid (1 and10 nmol/L, 24 hours) or leftuntreated (Co) and subsequentlycultured in a soft agar layer for 9days. Anchorage-independentgrowth was analyzed by countingstained colonies (MTT). Bars, thepercentage of colony formationcompared with control.��, P < 0.001, n ¼ 3. Right,representative colonies of treatedand untreated T24 cells are shown.B, detached T24, 5637, and4T1cellswere treatedwith differentconcentrations of archazolidfor 48 hours and analyzedfor apoptotic cell death.��, P < 0.001, n ¼ 3. DetachedMDA-MB-231 cells were treatedwith archazolid (10 nmol/L,72 hours) and apoptotic cells werequantified by flow cytometry.�, P < 0.05, n¼ 3. Bars, the relativeinduction of apoptosis comparedwith control cells. Bars alwaysrepresent themean�SEMof threeindependent experiments done intriplicate.

Schempp et al.

Mol Cancer Ther; 13(4) April 2014 Molecular Cancer Therapeutics928




12G10 antibody (Abcam). Floating and adherent cellswere treated with archazolid (10 nmol/L, 24 hours),harvested on ice (adherent cells were trypsinized withtrypsin/EDTA first), washed once with PBS, and incu-bated with the active integrin-b1 antibody (45 minutes,4�C). After washingwith PBS, cells were incubatedwith afluorescent secondary antibody (45 minutes, 4�C) andanalyzed by flow cytometry.

Caspase activityAfter treating cells with archazolid (1 nmol/L, 10

nmol/L, 48 hours), the activity of caspase-8 was mea-sured using a commercial caspase-8 activity assay (Cal-biochem) based on the cleavage of a caspase-8–specific

7-amino-4-trifluoromethyl coumarin labeled peptidesequence. The fluorometric shift over 5 hours was mon-itored by a fluorescent plate reader (SpectraFluorPlus,Tecan) calculating the relative enzyme activity dis-played as the relative fluorescence signal (RFU).

Intracellular ROS levelROSwasmeasuredbyusing the 20,70-dichlorofluorescin

diacetate dye (DCFDA; Sigma-Aldrich). Cells were har-vested after incubation with the indicated substances andstained with 20 mmol/L DCFDA for 30 minutes at 37�C,washed once with PBS, and measured by flow cytometry(excitation 488 nm, emission, excitation 535 nm, FACS-Canto II, BD Biosciences).

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Figure 2. Pretreatment of invasivebreast cancer cells with archazolidreduced metastasis in mouselungs. 4T1-Luc2 cells werepretreated with archazolid (Arch.;10 nmol/L, 24 hours) or leftuntreated and subsequentlyinjected (1 � 105) in the tail vain of10 BALB/cByJRj mice per group.A, at day 8 after inoculation, lungmetastasis were monitored bybioluminescence read out bydorsoventral and ventrodorsalmiceimaging. B, bioluminescencesignals were calculated as totalflux/area. Bars, mean � SEM.�, P < 0.05, n ¼ 10.






Statistical analysisAll experiments were performed at least three times

in triplicate. All statistic analyses were performed usingGraphPad Prism 5.0 software.

One-way ANOVA with Tukey posttest and for twocolumn comparison unpaired Students t-test was per-formed as significance analysis. Error bars indicate SEM.

ResultsArchazolid impairs anchorage-independent growthand induces anoikis in invasive cancer cells

To start with, anoikis resistance of the invasive cancercell lines T24, 5637 (urinary), MDA-MB-231 (mammary),and 4T1-Luc2 (mouse mammary) was shown by theirability to grow and proliferate in an anchorage-indepen-dent state (Fig. 1A and B).

Interestingly, pretreatment of T24 cells with archazolid(Supplementary Fig. S1A) for 24 hours impaired anchor-age-independent growth as shown by a reduction ofviable colonies in a soft agar colony formation assay (Fig.1A). To confirm that archazolid in fact inhibits V-ATPase,

endolysosomal pH was monitored by staining cells withLysotracker dye (Supplementary Fig. S1B). To excludeapoptosis responsible for the shown results cells werestained with propidium iodide before seeded to the softagar (Supplementary Fig. S2A). Inhibition of colony for-mation could also be observed by the V-ATPase inhibitorconcanamycin (Supplementary Fig. S2B), suggesting a V-ATPase–dependent effect.

To further analyze effects on anoikis, cells were kept in adetached status using poly-HEMA–coated culture dishes.Treatment with archazolid (10 nmol/L) induced anoikis indetached cancer cell lines T24, 5637, and 4T1 after 48 hoursand MDA-MB-231 after 72 hours, respectively (Fig. 1B).

In comparison, adherent T24 cells showed a highersensitivity toward archazolid after 48 hours (Supplemen-tary Fig. S3) pointing to death resistance mechanisms ofdetached cells.

Archazolid-treated tumor cells lose their metastaticpotential in vivo

The anoikis inducing effect of archazolid washypothesized to affect the invasive potential of tumor

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Figure 3. Archazolid (Arch.)treatment decreased activeintegrin-b1 and phosphorylation ofthe FAK, accompanied bycaspase-8 activation anddownregulation of c-FLIP. A, left,cell surface level of integrin-b1 wasanalyzed by an antibodyrecognizing only active integrin-b1and compared between adherentand detached (24 hours) T24 cells.Right, levels of total and activeintegrin-b1 on the cell surface ofarchazolid-treated (24 hours),floating T24 cells was investigatedby antibody staining and flowcytometry. ��, P < 0.001, n ¼ 3. B,the phosphorylation state of theFAK after archazolid treatment(24 and 48 hours, T24 cells) isshown by Western blot analysiswith the correspondingquantification of three independentexperiments. �, P < 0.05.(Continued on the following page.)

Schempp et al.





cells in vivo. To confirm this hypothesis, an in vivomodel based on the 4T1-Luc2 mouse mammary tumorcell line has been used. These cells are highly metastaticand disseminate quickly to the lungs when injectedintravenously and are therefore resistant to anoikis.4T1-Luc2 cells are further engineered to express aluciferase reporter to enable real-time monitoring ofdeveloping tumors by live imaging. As shown in Fig.2A, 4T1-Luc2–injected animals formed easily detect-able lung metastases; however, 4T1-Luc2 cells pre-treated with 10 nmol/L archazolid for 24 hours showeda significant reduction of lung metastases (Fig. 2B).Archazolid-treated cells did not show signs of apopto-sis at the time of the intravenous injection (Supplemen-tary Fig. S4).

Underlying mechanismsArchazolid treatment reduces active b1-integrin and

FAK activity in detached cells. Integrin-b1 is a majorplayer responsible for cell adhesion to the ECM.Activatedby attachment integrin-b1 signaling inhibits anoikis andpromotes cell survival (27).

Figure 3A shows that the level of active integrin-b1(analyzed by an integrin-b1 conformation specific anti-body) is similar between adherent and detached (24hours) T24 cells. However, archazolid-treated detachedcells (24 hours) display a reduction of active integrin-b1compared with detached control cells, whereas surfacelevel of total integrin-b1 was not affected by archazolidtreatment.

The FAK is recruited and activated by integrin bindingto their ECM ligands, leading to the activation of severaldownstream survival signals via the PI3K/Akt andthe Raf/MEK/ERK pathway (14). As shown in Fig. 3Barchazolid-treated floating cells showed a reduced phos-phorylation of FAK in the total cell lysate after 24 and 48hours of treatment.

Archazolid induces activation of caspase-8 anddownregulation of c-FLIP

As caspase-8 induction is a known hallmark of anoikis,its activity was measured in cell lysates using a fluoro-phore generating substrate for caspase-8. FloatingT24 and4T1 cells were stimulated for 48 hours with different

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Figure 3. (Continued. ) C, treated(48 hours) T24 and 4T1 cells wereharvested and the total cell lysatewas analysed for caspase-8activity. TRAIL, as a positivecontrol, was applied 1 hour beforeactivity measurement. Top,representative graph of caspase-8activity measurement over 5 hours(T24 cells) displayed as RFU.Bottom, evaluation of threeindependent experiments for T24and 4T1 cells presented as relativeRFU/minute. ��, P < 0.001;��,P<0.05;n¼3.D, left, the level ofcaspase inhibitor c-FLIP wasinvestigated by Western blotanalysis in T24 cells afterarchazolid treatment (48 hours).Right, quantification of threeindependent experiments. Barsalways represent the mean � SEMof three independent experimentsconducted in triplicate. All Westernblot experiments show arepresentative blot out of threeindependent experiments.�, P < 0.01.






archazolid concentrations, displaying a significant inc-rease in caspase-8 activity compared with control cells(Fig. 3C). TRAIL was used as a positive control.

Also, the FLICE-like inhibitory protein (c-FLIP), a well-described inhibitor of caspase-8 (28), which contributes toanoikis resistance by overexpression in malignant cells(3), is reduced after 48 hours of treatment in floating T24cells (Fig. 3D).

Archazolid treatment rapidly induced BIMtranslocation to the mitochondria leading tocytochrome C release

The Bcl-2 protein BIM is considered to be the majorplayer of the intrinsic-mediated anoikis pathway(29). Figure 4A indicates that cellular BIM expressionis not affected by archazolid treatment for 5 hours;however, BIM is translocated and enriched to mitochon-dria at early time points after archazolid exposure (i.e., 3and 5 hours). BIM localized at the mitochondrial outermembrane is responsible for Bax and Bak activationleading to the release of cytochrome C in the cytosol andsubsequently to apoptosis (30). A decrease of cyto-chrome C in the mitochondrial fraction was observedafter 24-hour treatment (Fig. 4B). Obviously, both theintrinsic and the extrinsic pathways are activated byarchazolid.

Archazolid triggers mechanisms opposing anoikisSustained exposure of detached cells to archazolid

results in a strong reduction of BIM (Fig. 5A and Supple-mentary Fig. S5), which is due to proteasomal degradationshown by the use of two proteasome inhibitors (MG-132and bortezomib; Fig. 5B and Supplementary Fig. S6).Interestingly, archazolid treatment increases ROS levelsignificantly after 16 to 48 hours (Fig. 6C and Supplemen-tary Fig. S7A) compared with floating control cells andinhibition of ROS generation by the antioxidant NACrescues BIM from proteasomal degradation induced byarchazolid alone (Fig. 5C), indicating counter mechanismsinduced by archazolid.

In addition, kinases involved in anoikis resistance(Akt, ERK, Src) were shown to be affected by detach-ment of T24 cells shown by an early increase of theirphosphorylation (Supplementary Fig. S8A and S8B).Treatment with archazolid even further increased thephosphorylation of Akt and did not downregulatephosphorylation of ERK and Src (Fig. 5D and Supple-mentary Fig. S8C), suggesting archazolid inducedcounter mechanisms. Of note, using specific inhibitors,that is, LY 294002 (Akt), PD 98059 (ERK), and Saraca-tinib (c-Src) leads to apoptosis (Supplementary Fig. S9)and prevents the decrease of BIM in archazolid-treatedcells (Fig. 5D).

Targeting regulators of BIM degradation increasesarchazolid-induced anoikis

Combination of archazolid with the proteasome inhi-bitors MG-132 (100 nmol/L) and bortezomib (5 ng/mL)

induced significant higher apoptosis rates (synergistic) inT24 cells and rescued BIM from degradation (Fig. 6A).NACaswellwas able to increase cell death in combinationwith archazolid (Fig. 6B) and rescued BIM (Fig. 5C),suggesting a role for ROS in anoikis induction. Themechanisms leading to archazolid induced anoikis andcounter mechanisms are further depicted in a cartoon(Supplementary Fig. S10).

DiscussionThiswork disclosed that pharmacologic inhibition of V-

ATPase by archazolid induces anoikis in invasive cancercells, which contributes to the distinct antimetastaticaction of archazolid in vivo. Thus we could add importantnew information about the role of V-ATPase in cancerdissemination.

Up to now, it has been reported that the abundance ofV-ATPase on the plasma membrane of tumor cellscorrelates with their invasiveness. Several well-knownV-ATPase inhibitors like concanamycin and bafilomy-cin were shown to lead to growth arrest and cell deathinduction in a variety of tumor cells (6, 31, 32). Also, thenewly developed V-ATPase inhibitors salicylihalamide(33) and NIK-12192 (34) have demonstrated antitumoractivity although the exact molecular mechanisms ofV-ATPase inhibitors leading to inhibition of tumor cellinvasion remain to be elucidated. Cell surface located V-ATPase is hypothesized to create a proton efflux leadingto an acidic pericellular microenvironment that pro-motes the activity of proinvasive proteases (35). How-ever, recently there is increasing evidence that theendolysosomal V-ATPase is important as antitumor-al/antimetastatic target. Recent own work showed thatV-ATPase inhibition by archazolid impairs endocytotictraffic of migratory signaling molecules such as Rac1and EGF receptor (EGFR, which is pivotal for directedand polarized cell movements; ref. 5). Abrogation ofendosomal trafficking by V-ATPase inhibition wasreported to also have impact on tumor growth andapoptosis induction suppressing activation of impor-tant signaling molecules such as Rab27B or activation ofcaspase-8 (36, 37).

Ourwork using archazolid as a tool to inhibit V-ATPaseprovides important information on regulation of anoikisand especially anoikis resistance in cancer.

From a physiologic point of view, anoikis is an impor-tant mechanism to remove cells that are currently not intheir correct location lacking a correct adhesion and thusto guarantee tissue homeostasis and prevent dysplasticgrowth. In other words, most cell types need proper cell–cell and cell–matrix interactions to survive. Cell to ECMinteractions occur mainly through specific integrins andtrigger a cascade of prosurvival and proliferative signals(12, 14).

Cancer cells in contrast, do not require adhesion to theECM to survive and proliferate, they are mostly insensi-tive to anoikis and in fact resistance to anoikis is a key

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regulator for tumor cell invasion and metastasis (12).Tumor cells use various strategies to acquire anoikisresistance such as the constitutively activation of survivalpathways (PI3K/Akt, MEK/ERK, and Src familykinases), alteration of the integrin expression pattern orgenerating oxidative stress, and inhibition of apoptoticpathways (3, 12, 38, 39).In experimental anoikismodels, cells are forced to grow

anchorage independent, which triggers the above-men-tioned stress and survival responses leading to a selec-tion of the "fittest," which are often resistant to che-motherapeutics. This is supported by our observationthat adherent cancer cells are significantlymore sensitiveto archazolid than detached cells (Supplementary Fig.S3). The clear effect of archazolid on anchorage-inde-pendent growth of cancer cells as well as the inductionof cell death in cells forced to stay detached is thusremarkable. Even more so having learned that archazo-

lid treatment itself potently induces anoikis resistancevia ROS generation and activation of Akt, which togetherwith constitutive active ERK and c-Src leads to thedegradation of BIM.

Obviously it is important to find out how a compoundlike archazolid finally achieves anoikis induction, mean-ing which pathways are involved.

Anoikis is characterized as an apoptotic cell deathexecuted by features of the intrinsic and extrinsic apo-ptotic pathway and the loss of survival signals byunligated anchorage proteins like integrins (4). Theextrinsic pathway is initiated by caspase-8 activatingeffector caspases or promoting mitochondrial cyto-chrome C release (4).

Archazolid evidently uses the extrinsic apoptotic path-way as shown by a downregulation of c-FLIP and adistinct caspase-8 activation. Recruitment of caspase-8and its activation have been shown to occur by loss of

T24

Whole lysate

BIM

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Arch. (nmol/L) Co 10

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/VD

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4T1, 5 h

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/CO

X I

V

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Co 10

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Figure 4. Archazolid (Arch.)treatment rapidly induced BIMtranslocation to the mitochondrialeading to release of cytochromeC. A, Western blot analysis of BIMexpression in whole-cell lysate ofuntreated and archazolid-treatedT24 cells after 5 hours. BIM levelwas analyzed in mitochondrialfraction of cells treated witharchazolid for early time points(3 and 5 hours, T24 and 4T1 cells).Immunoblotting for mitochondrialCOX IV and VDAC was used ascontrol for protein loading.Quantification of multipleindependent experiments wasperformed. �, P < 0.05. B, themitochondrial cytochrome C levelafter archazolid treatment (24 and48 hours) is shown byWestern blotanalysis ofmitochondrial protein ofT24 cells. COX IV immunoblottingserved as loading control. All blotsshow a representative blot out ofthree independent experiments.






anchorage to ECM and unligated integrins (17, 40).Integrins with the b1-subunit in common are the majorreceptors for ECM components responsible for cell–ECM

interactions (14). Interestingly cell surface b1-integrinin 24-hour floating cancer cells was still as active asin adherent cells, suggesting an inside-out integrin

BIM

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0.0

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RO

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vel

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old

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p-Akt

(Ser473)

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nitcAnitcA


LY-294002 (5 μmol/L)

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(Thr202/

Tyr204)

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nitcAnitcA


PD 98059 (5 μmol/L) - - + +

D

p-Src

(Tyr416)

MIBcrS

nitcAnitcA


Saracatinib (100 nmol/L)

- - + +

++--

++--

++--

++-- ++--

Figure 5. Counter mechanisminduced by archazolid (Arch.). A,BIM degradation. BIM protein levelin the whole-cell lysate (left) and themitochondrial fraction (right) afterprolonged treatment with archazolid(24, 48 hours) were analyzed byWestern blot analysis. B, BIMrescue. A combination of archazolid(10 nmol/L) with proteasomeinhibitor MG-132 (24 hours; left) orbortezomib (BOR, 48 hours; right)was used to investigate changes inBIM protein level by Western blotanalysis. The combination wasapplied simultaneously to the cellsat time of detachment. C, ROSgeneration. Archazolid inducesgeneration of ROS, which can beinhibited by the antioxidant NACafter 48 hours of treatment (left).ROS generation was measured asindicated in Materials and Methods.Bars, the mean � SEM of threeindependent experimentsconducted in triplicate.��, P < 0.001. Right, ROSgeneration is involved in BIMdegradation by archazolid. Westernblot analysis showed that ROSscavenger in combination witharchazolid (24 hours) rescued thedegradation of BIM. D, activation ofsurvival kinases. Left, activation ofAkt, ERK, and Src kinases wasinvestigated after 48 hours for Aktand ERK and 24 hours for Src ofarchazolid treatment by Westernblot analysis of their phosphorylatedsites. Right, application of inhibitors,i.e., LY 294002 (Akt), PD 98059(ERK), or saracatinib (c-Src), wereused to analyze changes in BIMprotein level. All Western blotexperiments show a representativeblot out of three independentexperiments. All experiments wereperformed with T24 cells.

Schempp et al.





activation supporting anoikis resistance of these cells (41).Of note, archazolid reduced the amount of active integrin-b1 on the cell surface leaving the total integrin levelconstant.In consequence downstream prosurvival signals like

the phosphorylation of one key player in anoikis protec-tion, the FAK (27) was affected by archazolid treatment.FAK together with c-Src then interact with numerousmolecules recruiting and activating other prosurvivalproteins like PI3K/Akt or ERK (16). We observed thatT24 cells shortly after detachment (5 hours) induce anoikisresistance by activating Akt and ERK, which decreaseswhen cells were detached for 24 hours, suggesting arestored balance between apoptotic and survival players(Supplementary Fig. S8A and S8B). C-Src kinase phos-phorylation is not changed despite detachment over 24hours (Supplementary Fig. S8B).

Unexpectedly, we did not observe inhibitory effects ofarchazolid on eitherAkt, ERK, or c-Src but an activation ofAkt, which is considered to be part of the counter pro-survival mechanism induced by archazolid.

Anoikis due to the intrinsic pathway is mainly ini-tiated by BIM (42). BIM, a member of the Bcl-2 familyactivates Bax and Bak, which leads to the permeabiliza-tion of the outer mitochondrial membrane and therelease of cytochrome C to the cytosol thereby activat-ing a caspase cascade inducing cell death (30, 43). Infact, BIM has been reviewed as a potential target fortumor therapy as BIM promotes anoikis in many tumorcell types and BIM suppression supports metastasisand chemoresistance (29). BIM regulation is dependenton cell surface molecules like integrins and the EGFR(18).

We found that BIM was rapidly translocated to themitochondria (Fig. 4A) by archazolid treatment followedby cytochrome C release (Fig. 4B) pointing to an involve-ment of the intrinsic pathway. Of note, BIM inhibitionseems to be also themajor factor used by T24 cells to induceresistance to anoikis as BIM gets strongly degraded at latertime points of archazolid exposure (Fig. 5A). The fact thatrescue of BIM degradation using proteasome inhibitorsMG-132 and bortezomib synergistically increase archazo-lid-inducedcelldeathunderscores the important roleof thisBH-3 only protein in the regulation of anoikis and its failure(Figs. 5B and 6A).

As kinases such as ERK, Akt, and c-Src are known toregulate BIMdegradation andexpression, itwas temptingto use specific kinase inhibitors to gain further insight inthe archazolid triggered BIM removal and thus chemore-sistance (29). The relevance of these kinases in survivalwas shown by the fact that kinase inhibitors alone at highconcentration induce anoikis (Supplementary Fig. S9).Costimulation of archazolid with moderate concentra-tions of kinase inhibitors rescuedBIMdecrease, indicatingthat cells actively block BIM activation by various path-ways in response to archazolid treatment (Fig. 5D). Thisfurther underlines the highly efficient survival and anoi-kis resistance mechanisms of invasive tumor cells andhighlights compounds such as archazolid still inducingcell death.

In addition, ROS are considered critical players inanoikis resistance. Integrin-mediated adhesion inducesa transient burst of high ROS levels transducing survivalsignals by activation of Src kinases. Activated Src kinasestrans-phosphorylate EGFR ligands, activating the ERKand PI3K/Akt pathways. Scavenging of ROS in adherentcells leads to BIM induction and cell death (38, 44). ROSare also recognized as second messenger in cell growth,proliferation, adhesion, and cell spreading in untrans-formed cells (45). Induction of ROS in tumor cells iscorrelated to tumor initiation and progression as well aswith tumor invasiveness (46). Anoikis-sensitive cellsshow decreased ROS levels after detachment correlatingwith cell death induction (38). Now, we found that arch-azolid treatment of floating cells resulted in elevated ROS

A

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tic r

ate

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ell

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Apopto

tic r

ate

X-f

old

of

contr

ol ***

***

Co Co10 10

- - + +

- - - + + +

- - + +

Figure 6. Inhibition of resistance pathways triggers archazolid (Arch.)-induced anoikis. A, floating T24 cells were either treated with archazolid(10 nmol/L) alone or in combination with the proteasome inhibitor MG-132 or bortezomib for 48 hours. Subsequently apoptosis induction wasdetermined as described in Materials and Methods. ��, P < 0.001. B,detached T24 cells were treated with archazolid (5 nmol/L) alone or incombination with NAC (10 mmol/L) for 48 hours. Induction of apoptosiswas measured by propidium iodide staining and flow cytometry.��,P < 0.01. Bars always represent themean�SEMof three independentexperiments conducted in triplicate.






levels compared with untreated cells (Fig. 5C and Sup-plementary Fig. S7A), but did not exceed the steady statelevel of ROS in untreated adherent cells (SupplementaryFig. S7B). Still, cell death was induced by archazolid. Thiselevated ROS could be a further explanation for theprominent removal of BIM protein. We showed in accor-dance to Giannoni and colleagues that cotreatment ofarchazolid with an ROS scavenger lead to increased celldeath and increased BIM levels (38). Therefore, ROSmustplay a critical role in resistance to archazolid-inducedanoikis.

In sum,wedemonstrate that archazolid induces anoikisin highly invasive tumor cells (Supplementary Fig. S10).Anoikis induction is accompanied by initiation of highlyproductive resistant mechanisms especially degradationof BIM and induction of ROS. Understanding themode ofactions leading to cell death by archazolid treatment andthe challenges of counter reactions can help gaining dee-per insight in anoikis resistance mechanisms, chemore-sistance and the metastatic transition of detached tumorcells.

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

Authors' ContributionsConception anddesign:C.M. Schempp,K. von Schwarzenberg, R.M€uller,A.M. VollmarDevelopment of methodology: C.M. Schempp, A.M. VollmarAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Kubisch, E. Wagner, A.M. VollmarAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): C.M. Schempp, K. von Schwarzenberg,R. M€uller, A.M. VollmarWriting, review, and/or revision of the manuscript: C.M. Schempp,K. von Schwarzenberg, R. M€uller, E. Wagner, A.M. VollmarAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. von Schwarzenberg, A.M.VollmarStudy supervision: K. von Schwarzenberg, E. Wagner, A.M. Vollmar

AcknowledgmentsThe authors thank Dr. Romina Wiedmann for her contribution to this

work.

Grant SupportThis work was supported by the German Research Foundation

(DFG-FOR 1406; to A.M. Vollmar, E. Wagner and R. M€uller) and Nano-systems Initiative Munich (NIM; to A.M. Vollmar, E. Wagner).

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 June 24, 2013; revised January 8, 2014; accepted January 22,2014; published OnlineFirst January 30, 2014.

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2014;13:926-937. Published OnlineFirst January 30, 2014.Mol Cancer Ther Christina M. Schempp, Karin von Schwarzenberg, Laura Schreiner, et al. of Cancer CellsV-ATPase Inhibition Regulates Anoikis Resistance and Metastasis

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