Autophagy Inhibition Improves Sunitinib Efﬁcacy in ... · Mol Cancer Ther; 16(11); 2502–15. 2017 AACR. Introduction Pancreatic neuroendocrine tumors (PanNET) show a neuro-endocrine

Cancer Biology and Translational Studies

Autophagy Inhibition Improves Sunitinib Efficacyin Pancreatic Neuroendocrine Tumors via aLysosome-dependent MechanismTabea Wiedmer1,2, Annika Blank1, Sophia Pantasis1, Lea Normand1, Ruben Bill3,Philippe Krebs1, Mario P. Tschan1,2, Ilaria Marinoni1, and Aurel Perren1

Abstract

Increasing the efficacy of approved systemic treatments inmetastasized pancreatic neuroendocrine tumors (PanNET) is anunmetmedical need. The antiangiogenic tyrosine kinase inhibitorsunitinib is approved for PanNET treatment. In addition, suniti-nib is a lysosomotropic drug and such drugs can induce lysosomalmembrane permeabilization as well as autophagy. We investi-gated sunitinib-induced autophagy as a possible mechanism ofPanNET therapy resistance. Sunitinib accumulated in lysosomesand induced autophagy in PanNET cell lines. Adding the autop-hagy inhibitor chloroquine reduced cell viability in cell lines andin primary cells isolated from PanNET patients. The same treat-ment combination reduced tumor burden in the Rip1Tag2 trans-genic PanNET mouse model. The combination of sunitinib and

chloroquine reduced recovery and induced apoptosis in vitro,whereas single treatments did not. Knockdown of key autophagyproteins in combination with sunitinib showed similar effect aschloroquine. Sunitinib also induced lysosomal membrane per-meabilization, which further increased in the presence of chlo-roquine or knockdown of lysosome-associated membrane pro-tein (LAMP2). Both combinations led to cell death. Our dataindicate that chloroquine increases sunitinib efficacy in PanNETtreatment via autophagy inhibition and lysosomal membranepermeabilization. We suggest that adding chloroquine to suniti-nib treatment will increase efficacy of PanNET treatment and thatsuch patients should be included in respective ongoing clinicaltrials. Mol Cancer Ther; 16(11); 2502–15. �2017 AACR.

IntroductionPancreatic neuroendocrine tumors (PanNET) show a neuro-

endocrine differentiation analogous to the cells of islets of Lan-gerhans and represent 3% of pancreatic tumors. PanNETs havesignificant malignant potential and surgery is the only curativeoption. Despite increasing availability of therapy options, stabledisease is achieved in only 30% of stage IV PanNET patients andpartial remission is observed only in rare cases (1–4). Sunitinib isa multi-tyrosine kinase inhibitor targeting mainly VEGF andplatelet-derived growth factor (PDGF) receptors leading to inhi-bition of angiogenesis and destabilization of existing vasculature(5). Sunitinib has a dual antitumor effect in PanNETs targeting thevasculature as well as targeting tyrosine kinases expressed bytumor cells (6, 7). Sunitinib is approved for treatment of advancedPanNETs and has been shown to prolong progression-free

survival (4). However, its efficacy is limited by intrinsic andacquired resistance. In Rip1Tag2 mice, a transgenic mouse modelfor PanNETs, mechanisms of acquired resistance towards suniti-nib have been described (8–10). Sunitinib is a lysosomotropicdrug, which freely diffuses the lysosomal membrane. In thelysosome, it is protonated due to the low pH, and thus accumu-lates due to the inability to diffuse through the membrane (11).Ellegaard and colleagues have shown that sunitinib inhibits acidsphingomyelinase (ASM), which influences lysosome stability(12). Sunitinib further leads to lysosomal leakage and later tomitochondrialmembrane permeabilization andultimately to celldeath (12). Interestingly, cancer cell lysosomes are less stable thannormal lysosomes due to transformation-associated changes inlysosome-associated membrane protein 1 (LAMP1) and LAMP2levels as well as in sphingolipid metabolism (13–15). Lysosomo-tropic drugs were shown to trigger lysosomal cell death even inapoptosis- and multidrug-resistant cancer cells (15). Therefore,targeting lysosomes is considered a promising yet rather unex-plored therapeutic strategy. More recently, it has been proposedthat leaky lysosomes do not necessarily lead to cell death. Instead,the release of lysosomal enzymes might affect physiologicalfunctions, for example influencing invasion by degradation offocal adhesions (16). In addition, lysosomes undergoing lyso-somalmembrane permeabilization (LMP) induce autophagy andare subsequently cleared by lysophagy (17–19).

Macroautophagy, hereafter named autophagy, is mainly a cellsurvivalmechanismactivatedupon cellular stress to sequester anddegrade long-lived proteins and damaged organelles and torecycle nutrients (20). Autophagy can promote tumorigenesis byincreasing cell survival in unfavorable environment such as hyp-oxia or anticancer treatments (21). Many anticancer treatments

1Institute of Pathology, University of Bern, Bern, Switzerland. 2Graduate Schoolfor Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.3Department of Internal Medicine, Regional Hospital Emmental Burgdorf, Burg-dorf, Switzerland.

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

I. Marinoni and A. Perren contributed equally to this article.

CorrespondingAuthors:Aurel Perren, Institute of Pathology, University of Bern,Murtenstrasse 31, Bern 3008, Switzerland. Phone: 413-1632-3223; Fax: 413-1632-4995; E-mail: [email protected]; and Ilaria Marinoni,[email protected]

doi: 10.1158/1535-7163.MCT-17-0136

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have been shown to affect autophagy in vitro and in vivo. Previ-ously, it was found that sunitinib modulates autophagy in othercellular systems in vitro (22–25). However, whether the modula-tion of autophagy by sunitinib is due to a block or an induction ofautophagy is controversial. As autophagy can promote tumori-genesis and resistance toward anticancer treatments, it has beenexploited as therapeutic target in vitro, in vivo and in early clinicaltrials. In clinical trials, the most commonly used treatments toinhibit autophagy are the antimalaria drugs chloroquine (CQ)and hydroxychloroquine (HCQ; ref. 26). Both are lysosomo-tropic compounds, which inhibit autophagy via blocking lyso-somal function. The role of autophagy and response toward itsinhibition depends on tumor stage. Indeed autophagy functionsare initially often tumor suppressing and at later stages tumorpromoting. In addition, the role of autophagy differs betweentumor types (21). The role of autophagy in mediating sunitinibresponse has not been studied in PanNET yet.

In this study, we investigated whether sunitinib upregulatesautophagy in PanNETs and whether this is important in mediat-ing therapy response or resistance. We assessed whether combi-nation with the autophagy inhibitor chloroquine enhanced theantitumor effect of sunitinib. Moreover, we questioned howsunitinib accumulation in lysosomes and LMP affect autophagyand treatment response.

Materials and MethodsDrugs, inhibitors, and antibodies

Bafilomycin A1 (B-1080) was purchased from LC Laboratories,chloroquine (C6628) from Sigma, and sunitinib (S1042) fromSelleckchem.

Antibodies against ATG5 (#2630), cleaved caspase-3 (#9661and #9664), cleaved PARP (#9541) were purchased from CellSignaling Technology, ATG7 (ab52472), and CD31 (ab28364)from Abcam, galectin 3 (#556904) from BD Biosciences, GAPDH(#MAB374, clone 6C5) from Millipore, LAMP2 (sc-18822) andSV40 (sc-20800) from Santa Cruz Biotechnology, Ki67(#RM-9106) from Thermo Fisher Scientific, and LC3B (NB600-1384) from Novus Biologicals.

Cell linesBON1 cell line was provided by E.J. Speel, Maastricht, Nether-

lands in 2011. QGP1 cell line was purchased from the JapaneseHealth Sciences Foundation, Osaka, Japan in 2011. Short tandemrepeat (STR) analysis by PCR was performed for both cell lines(QGP1 in 2011 and 2016, BON1 in 2014 and 2016). QGP1 cellswere authenticated. A BON1 profile does not exist yet but theprofile of these cells didnotmatch anyknownprofile of cancer celllines thus excluding contamination from other lines. In addition,expression of the specific neuroendocrine markers chromograninA and synaptophysin were routinely tested by IHC. BON1 cellswere cultured in DMEM/F12 medium (Sigma), QGP1 cells inRPMI1640 medium (Sigma). For both cell lines medium wassupplemented with 10% FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin and cells were kept in a humidified incubator at5% CO2 and 37�C. After thawing, cells were cultured for approx-imately two months.

Generation of knockdown cell linesShort hairpin RNA (shRNA) against ATG5 (NM_004849.1-

420s1c1, NM_004849.1-915s1c1, NM_004849.1-1170s1c1),

ATG7 (NM_006395.1-268s1c1, NM_006395.1-491s1c1,NM_006395.1-2173s1c1), and LAMP2 (NM_002294.1-291and NM_002294.1-561) as well as a nontargeting shRNAcontrol (SHC002) were delivered with a lentivirus expressingvector pLKO.1 (all from Sigma, MISSION shRNA). Lentivirusproduction and transduction were performed as describedpreviously (27). Cells were selected with 1.5 mg/mL puromy-cin for 3–4 days. Knockdown efficiency was validated byimmunoblotting of respective proteins.

Primary cell cultureFresh human PanNET tissue was obtained from patients who

underwent surgery at the Inselspital Bern (Bern, Switzerland) andhave signed an institutional informed consent. Ethical approvalwas given by the cantonal authorities (Kantonale Ethikkommis-sion Bern, Ref.-Nr. KEK-BE 105/2015). Fresh tissue was digestedwith collagenase IV (Worthington) and trypsin for 1 hour at 37�Cfollowed by red blood lysis with ACK buffer (Gibco). Single-cellsuspension was plated in 96-well plates and viability was mea-sured after 48-hour treatment with ATPlite assay kit (PerkinElmer) according to manufacturer's instructions. Protein extractswere collected from96-well plates using urea lysis buffer (8mol/Lurea, 0.5% Triton X-100) and immunoblotting was performed asdescribed previously. Primary tumor was from a male stage IIpatient (age 62, patient I) with a well-differentiated grade G2tumor showing no hormone secretion. Liver metastasis was froma male stage IV patient (age 65, patient II), with a well-differen-tiated grade G3 tumor showing no hormone secretion. Despitesunitinib is currently only approved for treatment of G1 and G2tumors, patient II was included in this study due to the well-differentiated state of the tumor. The coefficient of drug interac-tion (CDI) was calculated as CDI¼ AB/(A� B). AB is the relativeviability after the combination treatment of drugs A andB,while Aand B are the relative viability after single treatment with drug Aand B, respectively. CDI < 1 indicates a synergistic effect, CDI¼ 1an additive effect and CDI > 1 an antagonistic effect (28).

MTT and clonogenic assayCells were cultured and treated in 96-well plates. To perform

the MTT assay, cells were incubated with medium plus MTT(0.5 mg/mL) at 37�C for 30–45 minutes. Medium was removedand cells were lysed in DMSO. Sorensen solution (0.1 mol/Lglycine, 0.1 mol/L NaCl, pH 10.5 in water) was added andabsorbance measured at 570 nm. For the clonogenic assay, cellswere trypsinized after 72 hours of treatment and replated at equalnumbers in 6-well plates. Colonies were allowed to grow for twoweeks in normal growth medium followed by fixation andstaining of the colonies with 0.05% crystal violet in 30% ethanol.Colony numbers were measured with ColCount (OxfordOptronix).

Long-lived protein degradation assayCells were seeded in 24-well plates, radiolabeled with 0.1 mCi

14C-valine per mL (L-[U-14-C] valine; code CFB.75, Amersham)for 48 hours and treated with sunitinib for 24 hours. Remaining14C-valine was removed and cells were incubated for 1 hour inmedium supplemented with 10 mmol/L L-valine. During thisincubation time, short-lived proteins were degraded. After wash-ing, cells were incubated with medium supplemented with10 mmol/L L-valine and with or without BafA1 for 5.5 hours, inwhich long-lived proteins were degraded. Supernatant and

Autophagy Inhibition Improves Sunitinib Efficacy in PanNET

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trypsinized cells were collected and acid precipitation and cen-trifugation were performed to separate supernatant and pellet.Fractions were mixed with Microscint40 and radioactivity wasmeasured by liquid scintillation counting. Percentage of prote-olysis of long-lived proteins was calculated from supernatant andpellet fractions.

Immunoblotting and immunofluorescenceCells were lysed in urea buffer (8 mol/L urea, 0.5% Triton

X-100) supplemented with protease inhibitors (Roche). Forty to60 micrograms of protein was loaded on a 12% SDS-PAGE gel(Bio-Rad) and then transferred to a PVDF membrane (Bio-Rad).Incubation with primary antibodies was performed overnight at4�C, followed by washing steps and incubation with secondaryantibodies (DyLight 650 conjugate goat anti-rabbit and DyLight550 conjugate goat anti-mouse, ImmunoReagents andperoxidase-conjugated AffiniPure donkey anti-rabbit and donkeyanti-mouse, Jackson ImmunoResearch) for 1 hour at room tem-perature. Chemiluminescent or fluorescent signal was detectedusing ChemiDoc MP System (Bio-Rad). Total protein expressionfor quantification of specific protein expression was measured byuse of the stain-free gel technology and imaged with the Chemi-doc MP System (29).

For immunofluorescence, cells were plated on poly-L-lysinecoated coverslips and treated for 24–48 hours. Fixation andimmunofluorescence for galectin 3 was performed as describedpreviously (30). As secondary antibody, goat anti-mouse AlexaFluor 568 conjugate (Invitrogen) was used. Analysis was per-formed at a microscope Zeiss Axioplan 2 at �40 magnification.Samples were blinded and percentage of positive cells was cal-culated from500–600 counted cells on randomly chosen fields inthe DAPI channel.

Lysotracker stainingCells were incubated in coated glass-bottom plates and treated

with sunitinib for 72 hours. Medium was replaced by mediumcontaining 100 nmol/L Lysotracker Red DND-99 (L7528, Molec-ular Probes) and cells were incubated for 1 hour at 37�C.Mediumwas exchanged to normal medium and cells were imaged at aconfocal microscope Olympus FluoView-1000 at �63magnification.

In vivo experimentsRip1Tag2 (C57BL/6) mice were kindly provided by G. Chris-

tofori (Basel, Switzerland). All experimental protocols werereviewed and approved by the Cantonal Veterinary Office of Bern

(Bern, Switzerland). Mice were fed with food enriched in glucosestarting from 10 weeks of age. Rip1Tag2 mice were treated dailyfrom week 10 of age with 40 mg/kg sunitinib orally, 50 mg/kgchloroquine intraperitoneally or the respective vehicle control forthree weeks. Animals were monitored daily. At 13 weeks of age,animals were euthanized, tumor number (>1 mm) and size wasmeasured, and tissues were fixed in formalin overnight andembedded in paraffin. Tumor volume was calculated using theformulaV¼ 0.52� (width2)� length to estimate the volume of asphere (31). Tumor volume per mouse was calculated as the sumof single tumors.

IHCFor IHC and TUNEL (TdT-mediated dUTP nick end labeling)

on mouse pancreas, tissues were cut in 4-mm thick serial sectionsfollowed by deparaffinization, rehydration, and antigen retrievalusing an automated immunostainer (BondRX, Leica Biosystems).Antigen retrieval was performed for LC3Bwith Tris for 30minutesat 95�C, for Ki67, SV40, and CD31 with citrate for 30 minutes at95�C. Antibodies were diluted as follows: LC3B 1:4000, Ki671:400, SV40 1:100, CD31 1:100. For TUNEL, slides were incu-bated with TdT enzyme (Promega) for 20 minutes. Slides werecounterstained with hematoxylin and eosin (H&E). For livermetastasis assessment, four step sections of 75 mmwere cut fromliver tissue and stained with SV40 as described above. Number ofmetastasis was counted and summarized from all sections.Micro-vessel density (MVD) was evaluated by a staff pathologist(A. Blank) based on IHC for CD31 and classified as high or low.Classification of tumors was performed based on H&E stainingand according to classification criteria defined by Lopez andHanahan (32). Necrosis was evaluated by a staff pathologist(A. Blank) based on H&E staining, as percentage of area showingprenecrotic condensed nuclei (regional cell death) and necrosis(geographic necrosis) compared with total tumor volume. Rep-resentative pictures of analyses for classification and necrosis onH&E staining are shown in Supplementary Fig. S1A and S1B,respectively. Proliferation was measured as percentage of Ki67-positive cells compared with tumor volume in all tumors >1 mmand average permouse is shown.Number of TUNEL-positive cellswas counted in one high-power field chosen in the area of highestlabeling. Autophagy was assessed by IHC for LC3B, classifiedaccording to the abundance and number of LC3B punctae, rep-resentative of autophagosomes (33).Micewere classified as eitherLC3B punctae low or LC3B punctae high. IHC for LC3B on 30matched samples of human primary PanNET and liver metastasis(patient collective II as reported in ref. 34) was performed as

Figure 1.Sunitinib modulates autophagy in PanNET cell lines and primary cells and combination with autophagy inhibition reduces viability and recovery.A, Representative immunoblot for LC3B in BON1 and QGP1 cells upon 24-hour sunitinib (10 mmol/L), 24-hour CQ (20 mmol/L in BON1, 50 mmol/L in QGP1),5.5-hour BafA1 (200 nmol/L). LC3B-II levels were normalized to GAPDH or total protein, n � 4. B, Autophagic flux in BON1 and QGP1 cells calculatedfrom percentage of proteolysis in long-lived protein degradation assay (treatment with BafA1–treatment without BafA1), n ¼ 4. C, Immunoblots for LC3Bin primary cells isolated from PanNET patients I and II upon 7-hour sunitinib (10 mmol/L), 7-hour CQ (20 mmol/L), 2-hour BafA1 (200 nmol/L). LC3B-II levelswere normalized to total protein and are indicated below the corresponding LC3B-II immunoblot,n¼ 1.D,Viability of BON1 andQGP1 cells upon treatment. MTT assaywas performed after 72-hour treatment with sunitinib (10 mmol/L), CQ (BON1 20 mmol/L, QGP1 50 mmol/L), BafA1 (20 nmol/L), and valueswere normalized to control, n � 2. E, Viability of primary cells isolated from PanNET patients I and II upon treatment. ATPlite assay was performedafter 48-hour treatments with indicated concentrations of CQ and sunitinib and values were normalized with control, n ¼ 1, error bars show technical replicates.F, Representative picture and quantification of clonogenic assay in BON1 cells. The image shows triplicates for each treatment of one representativeexperiment. Colonies were fixed and stained after two weeks without treatment and colony numbers were normalized to control, n � 3. Statistical analysiswas performed using unpaired t test, � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.






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described above. Tumors were classified on the basis of theabundance and number of LC3B punctae in absent, low, or highLC3B punctae.

Statistical analysisStatistical analyses were performed with GraphPad Software.

Unpaired or paired t test was used to compare groups. Contin-gency tables were analyzed using Fisher exact test. Sample size (n)refers to biological replicates unless otherwise stated. �, P < 0.05;��, P < 0.01; ��, P < 0.001; ��, P < 0.0001.

ResultsSunitinib upregulates autophagy in PanNET cell lines andprimary cells

Weassessed sunitinib effects on autophagy in PanNET cell linesby immunoblotting for microtubule associated protein 1 lightchain 3B (MAP1LC3B/LC3B). The cytosolic form LC3B-I is lipi-dated to LC3B-II, which is incorporated into autophagosomesand represents autophagosome numbers. Sunitinib treatmentalone increased LC3B-II levels in both BON1 and QGP1 cells.To measure the autophagic flux we cotreated the cells with thelysosomal autophagy inhibitors chloroquine (CQ) or bafilomy-cin A1 (BafA1; ref. 35), which block the fusion of autophago-somes with lysosomes via elevation of lysosomal pH. In both celllines, LC3B-II levels further increased upon combination treat-ment of suntinib and autophagy inhibitors compared with singletreatments alone, indicating increased autophagic flux. The effectwas more prominent with CQ (Fig. 1A). Autophagy was alsomonitored by long-lived protein degradation assay (LLPDA;ref. 20). Sunitinib increased autophagy-specific proteolysis inboth cell lines (Fig. 1B; Supplementary Fig. S2A). We assessedbasal levels of autophagy in PanNET human tissue by IHC forLC3B. LC3B punctae, representative for autophagosomes, signif-icantly increased in liver metastasis compared with matchedprimary tumors, potentially indicating high levels of autophagyin stage IV patients (Supplementary Fig. S2B). Importantly, also inprimary cells freshly isolated from PanNET patients, sunitinibincreased LC3B-II levels, which were further elevated with autop-hagy inhibition (Fig. 1C). In conclusion, sunitinib upregulatesautophagy in PanNET cell lines and primary cells as measured byLC3B-II and proteolysis of long-lived proteins.

Reduced viability and recovery upon combination withautophagy inhibition

We hypothesized that sunitinib-induced autophagy may pro-vide a prosurvival mechanism limiting sunitinib antitumoraleffect and that combination with autophagy inhibitors might

have a beneficial effect in reducing cell viability. To address thishypothesis, we combined increasing doses of sunitinib with fixedconcentrations of CQ and BafA1, the latter two not affecting cellviability (Fig. 1D). Sunitinib decreased BON1 and QGP1 cellviability in a dose-dependent manner. The effect on viability wassimilar in both cell lines and seemed to be independent of targetreceptor expression levels (Supplementary Fig. S3A and S3B). CQfurther reduced cell viability when combined with 10 mmol/Lsunitinib (Fig. 1D; Supplementary Fig. S3A). Further experimentsin the cell lines were performed with these concentrations ofsunitinib and CQ. In primary cells from two PanNET patients,both sunitinib and CQ decreased viability in a dose-dependentmanner. Also, in primary PanNET cells, their combination led toan increased effect (Fig. 1E). Calculation of the coefficient of druginteraction (CDI) as described previously (28) indicated an addi-tive effect of sunitinib and CQ treatment in patient I (CDI¼ 1.01)and a trend towards a synergistic effect in patient II (CDI¼ 0.83).Cell-cycle analysis by FACS revealed that sunitinib induced a G1

cell-cycle arrest in PanNET cell lines, while the cell cycle was notaffected by CQ (Supplementary Fig. S3C). To assess the long-termeffect and recovery from treatments, a colony formation assay wasused. Sunitinib combined with autophagy inhibition led to asignificant 50% reduction in colony numbers (Fig. 1F).

Reduced tumor burden in vivo upon combination treatmentWe next assessed whether autophagy inhibition increases the

antitumoral effect of sunitinib in vivo in Rip1Tag2 mice, a trans-genic mouse model of PanNET. We started treatment of mice at10weeks of age,when tumors havedeveloped and canbe detectedmacroscopically (Fig. 2A). Mice were treated for three weeks witheither vehicle, CQ (50 mg/kg, daily i.p.), sunitinib (40 mg/kg,daily orally), or a combination thereof (Fig. 2A). A dose of50 mg/kg CQ corresponds to the human equivalent doseof 250 mg, which is the starting dose of phase I trials of CQ andHCQ in cancer (36, 37). Fifty mg/kg CQ led to an increase of LC3in a melanoma xenograft model and to intratumoral concentra-tions of CQ, which blocked autophagy efficiently in vitro (38).Tumor number was significantly reduced after combination treat-ment compared with single treatments (sunitinib P¼ 0.0045, CQP ¼ 0.0480). Tumor volume was reduced by 50% after combi-nation treatment compared with single treatments (sunitinib P¼0.0527, CQ P¼ 0.0050; Fig. 2A). Importantly, tumor number andvolume significantly increase during sunitinib treatment com-pared with the start at 10 weeks of age, but not during thecombination treatment (Fig. 2A). Microvessel density was clearlyreduced upon sunitinib and combination treatment andunchanged upon CQ treatment (Supplementary Fig. S4A). Pro-liferation did not change upon CQ treatment but decreased

Figure 2.Significant antitumor effect in vivo upon combination treatment. A, Graphic representation of the mouse treatment and macroscopically assessed tumorvolume and number in Rip1Tag2 mice at 10 weeks of age and after three weeks of daily treatment with vehicle, CQ, sunitinib, or sunitinib þ CQ (10 weeks n ¼ 10,vehicle n ¼ 13, CQ n ¼ 12, sunitinib n ¼ 12, sunitinib þ CQ n ¼ 12), unpaired t test. B, IHC staining for the proliferation marker Ki67. Percentage of Ki67-positivecells was estimated in 3–14 tumors >1 mm per mouse as shown in representative pictures with corresponding percentage of positive cells. C, Quantificationof Ki67 staining. The graph shows the mean percentage of positive cells per mouse (vehicle n ¼ 13, CQ n ¼ 12, sunitinib n ¼ 12, sunitinib þ CQ n ¼ 12),unpaired t test. D, Quantification of percentage of necrosis determined on H&E staining (vehicle n ¼ 13, CQ n ¼ 12, sunitinib n ¼ 12, sunitinib þ CQ n ¼ 12),unpaired t-test. E,Quantification of number of TUNEL-positive cells/mm2 as determined in one high-power field permouse (vehicle n¼ 13, CQ n¼ 12, sunitinib n¼ 12,sunitinib þ CQ n ¼ 12). F, IHC staining for LC3B. Classification of each mouse in LC3B punctae high or low based on abundance of dot staining as shown inrepresentative pictures (LC3B punctae low: no dots in most of the tumors, LC3B punctae high: dots in most of the tumors). The graph presents thepercentage of mice with LC3B high/low punctate staining (vehicle n¼ 13, CQ n¼ 12, sunitinib n¼ 12, sunitinibþ CQ n¼ 12), Fisher exact test. � , P < 0.05; ��, P < 0.01;�� , P < 0.001; �� , P < 0.0001.






significantly with sunitinib (P ¼ 0.0224) and the combinationtreatment (P¼ 0.0017) compared with vehicle treatment (Fig. 2BandC). In addition to reduced proliferation, necrosis was elevatedin tumors treatedwith sunitinib (P¼0.0005) or sunitinib andCQ

(P¼0.0003) comparedwith vehicle-treated tumors (Fig. 2D). Thenumber of TUNEL-positive cells increased significantly uponsunitinib (P ¼ 0.0222) and the combination (P ¼ 0.0163)treatment (Fig. 2E; Supplementary Fig. S4B). Tumor classification

Figure 3.

Sunitinib treatment of shATG5 and shATG7 cells only partially phenocopies combination with chloroquine. A, Viability of BON1 shControl, shATG5 andshATG7 cells upon treatment. MTT assay was performed after 72-hour treatment with sunitinib (10 mmol/L), CQ (20 mmol/L), and values were normalizedto the respective DMSO control, n � 3. B, Representative pictures of clonogenic assay in BON1 shControl, shATG5, and shATG7 cells. Colonies were fixedand stained after two weeks without treatment. Triplicates of one representative experiment are shown for each treatment. C, Clonogenic assay in BON1shControl, shATG5 and shATG7 cells. Colony numbers were normalized to the respective DMSO control, n � 3 D, Representative immunoblot for ATG5, ATG7,cleaved caspase-3 and cleaved PARP in BON1 shControl, shATG5 and shATG7 cells after 24-hour treatment with sunitinib (10 mmol/L), CQ (20 mmol/L),BafA1 (20 nmol/L), n ¼ 3. E, Representative immunoblot for ATG5, ATG7, cleaved caspase-3, and cleaved PARP in QGP1 shControl, shATG5 and shATG7 cellsafter 24-hour treatment with sunitinib (10 mmol/L), CQ (50 mmol/L), BafA1 (50 nmol/L), n ¼ 3.Statistical analysis was performed using unpaired t test, � , P < 0.05;�� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.

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Figure 4.

Sunitinib treatment of shLAMP2 cells phenocopies combination with chloroquine. A, Viability of BON1 shControl, shLAMP2-291 and shLAMP2-561 cells upontreatment.MTT assaywas performedafter 72-hour treatmentwith sunitinib (10mmol/L), CQ (20mmol/L) and valueswere normalized to the respectiveDMSOcontrol,n � 3. B, Representative pictures of clonogenic assay in BON1 shControl, shLAMP2-291 and shLAMP2-561 cells. Colonies were fixed and stained aftertwoweeks without treatment. Triplicates of one representative experiment are shown for each treatment. C, Clonogenic assay in BON1 shControl, shLAMP2-291 andshLAMP2-561 cells. Colony numbers were normalized to the respective DMSO control, n � 3 D, Representative immunoblot for LAMP2, cleavedcaspase-3 and cleaved PARP in BON1 shControl, shLAMP2-291, and shLAMP2-561 after 24-hour treatment with sunitinib (10 mmol/L), CQ (20 mmol/L),BafA1 (20 nmol/L), n ¼ 3. E, Representative immunoblot for LAMP2, cleaved caspase-3, and cleaved PARP in QGP1 shControl, shLAMP2-291 andshLAMP2-561 after 24-hour treatment with sunitinib (10 mmol/L), CQ (50 mmol/L), n ¼ 3. Statistical analysis was performed using unpaired t test, � , P < 0.05;�� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.






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revealed that invasiveness decreased upon sunitinib and com-bination treatment (Supplementary Fig. S4C), while incidenceof metastasis and number of metastases per mouse did notchange (Supplementary Fig. S4D). Punctate LC3B positivityby IHC was classified into low and high. The distribution ofLC3B punctae low versus LC3B punctae high increased towardLC3B punctae high in single and combination treatmentscompared with vehicle (Fig. 2F), indicating an upregulationof autophagy by sunitinib also in vivo. In summary, combi-nation treatment of suntinib and CQ leads to an increasedantitumor effect compared with single treatments in a pre-clinical setting.

Beneficial effect of combination treatment is partially due toautophagy inhibition

To prove a direct role of autophagy in limiting sunitinibantitumoral effect, we knocked down key autophagy proteins,autophagy-related protein 5 (ATG5), autophagy-related protein 7(ATG7), and lysosome-associated membrane protein 2 (LAMP2)by lentiviral delivery of shRNA. ATG5 and ATG7 are involved invesicle formation and lipidation of LC3, while LAMP2 is aglycoprotein localized on the lysosome membrane, which isinvolved in the fusion between autophagosome and lysosomeas well as in chaperone-mediated autophagy. Upon knockdown,protein levels and starvation-induced autophagic flux were bothreduced (Supplementary Fig. S5A–S5D). Proliferation and colonyformation were slightly reduced in cells with shATG5, shATG7, orshLAMP2 compared with control-transduced cells (Figs. 3Band 4B). Cells with shATG5 and shATG7 had reduced viabilityupon sunitinib treatment comparedwith control-transduced cells(Fig. 3A). In addition, recovery of sunitinib-treated shATG5 andshATG7 cells was significantly reduced compared with control-transduced cells to a similar extent as observed with the combi-nation of sunitinib and CQ (Fig. 3B and C). While neithersunitinib nor the autophagy inhibitors CQ and BafA1 aloneinduced apoptosis in BON1 cells, their combination led tocleavage of caspase 3 and PARP (Fig. 3D, Supplementary Fig.S6A and S6B). Sunitinib treatment did not induce apoptosis inBON1 ATG5 knockdown cells, whereas in QGP1 cells knockingdown ATG5 already induced apoptosis, which was furtherincreased by sunitinib treatment. In both cell lines, sunitinibtreatment of ATG7 knockdown cells induced apoptosis, but atlower levels compared with pharmacologic inhibition of autop-hagy (Fig. 3D and E). In summary, knockdown of early autophagygenes ATG5 and ATG7 only partially phenocopied the effectobserved with pharmacologic inhibitors.

Interestingly, knockdown of the late autophagy gene LAMP2better recapitulated the effect of pharmacologic inhibition. Suni-tinib treatment reduced viability of shLAMP2 cells comparedwithcontrol-transduced cells (Fig. 4A) as well as cell recovery similarlyto the combination treatmentof sunitinib andCQ(Fig. 4B andC).Sunitinib treatment of shLAMP2 BON1 and QGP1 cells inducedapoptosis to a level comparable or even stronger than combina-tion with autophagy inhibitors (Fig. 4D and E). The strong effectobserved on apoptosis upon sunitinib and LAMP2 knockdowncompared with ATG5 and ATG7 knockdown prompted us toinvestigate closer the involvement of lysosomes in sunitinib-mediated autophagy induction.

Effect of combination treatment depends on lysosome stabilityand is mediated by lysosomal membrane permeabilization

LAMP2 has been shown to contribute to lysosomal stability(14). Knockdownof LAMP2 sensitized BON1andQGP1 cell linestoward CQ (Fig. 5A–C) and the sensitivity toward CQ seemed tobe dependent on LAMP2 levels. BON1 cells with lower levels ofLAMP2weremore sensitive toCQ thanQGP1 (Fig. 5A–C).On thebasis of these results, we hypothesized that lysosomal stability isan important factor in the response towards sunitinib and chlo-roquine treatment. Sunitinib and chloroquine are both lysoso-motropic drugs, which due to their structure accumulate in lyso-somes (11). We confirmed the lysosomotropic property of suni-tinib by demonstrating that it colocalized with Lysotracker Red inBON1 and QGP1 cells (Fig. 5D). Lysosomotropic drugs caninduce lysosomal membrane permeabilization (LMP) leading torelease of lysosomal content in the cytoplasm, which in turn canactivate apoptosis (39–42), and autophagy (17–19). Immuno-fluorescence for galectin 1 or 3 is considered the gold standard fordetecting LMP (19). Upon permeabilization of lysosomes, galec-tins translocates from the cytoplasm and bind to the glycocalyx inlysosomes, which can be detected by immunofluorescence as adot-like staining (Fig. 5E). CQ alone did not induce LMP, whilesunitinib alone significantly induced LMP after 48-hour treat-ment, quantified as percentage of galectin 3–positive cells. Com-bination treatment of BON1 cells with sunitinib and CQ signif-icantly increased LMPafter 24 and48hours (Fig. 5F). Accordingly,sunitinib-treated shLAMP2 cells showed a significant increase ofLMP and to the same degree as observed in control-transducedcells treated with sunitinib and CQ (Fig. 5G). Together, our dataindicates that reduction of lysosomal stability, as in shLAMP2cells or upon combination of the lysosomotropic drugs sunitiniband CQ, promotes LMP-induced damage, which then triggersapoptosis.

Figure 5.Effect of combination treatment depends on lysosome stability and is mediated by lysosomal membrane permeabilization. A, Representative immunoblotsfor LAMP2 in BON1 and QGP1 shControl, shLAMP2-291 and shLAMP2-561 cells, n > 3. B, Viability of BON1 shControl, shLAMP2-291 and shLAMP2-561 cells upontreatment. MTT assay was performed after 72-hour treatment with increasing concentrations of CQ (20 mmol/L, 50 mmol/L, 100 mmol/L) and values werenormalized to the respective vehicle control, n¼ 3. C, Viability of QGP1 shControl, shLAMP2-291 and shLAMP2-561 cells upon treatment. MTT assay was performedafter 72-hour treatment with increasing concentrations of CQ (20 mmol/L, 50 mmol/L, 100 mmol/L) and values were normalized to the respective vehicle control,n ¼ 3. D, Representative images showing the colocalization of the sunitinib autofluorescence (72 hours, 10 mmol/L) in the FITC channel and LysotrackerRed staining lysosomes (1 hour, 100nmol/L) in theCy3 channel. Scalebar, 10mm, applies to all images.E,Representative immunofluorescent picture of a LMP-positiveBON1 cell showing galectin 3 dotted staining and a negative cell without dotted galectin 3 staining upon 48-hour sunitinib þ CQ treatment. Nuclei werestained with DAPI. Scale bar, 20 mm. F, LMP in BON1 cells upon treatment. Percentage of galectin 3–positive cells upon 24- and 48-hour treatment withsunitinib (10 mmol/L) and CQ (20 mmol/L) was calculated from 500–600 counted cells in randomly chosen fields, n ¼ 5. G, LMP in BON1 shControl, shLAMP2-291,and shLAMP2-561 cells upon treatment. Percentage of galectin 3–positive cells upon 24- and 48-hour treatment with sunitinib (10 mmol/L) and CQ (20 mmol/L)was calculated from 500–600 counted cells in randomly chosen fields, n � 5. Statistical analysis was performed using unpaired t test, � , P < 0.05; �� , P < 0.01;�� , P < 0.001; �� , P < 0.0001.






DiscussionOptions for anticancer therapy are limited for patients with

stage IV PanNETs. Since 2011, sunitinib has been approved fortreatment of advanced PanNETs but it has a limited efficacy (4).Here we investigated whether autophagy is involved in resis-tance to suntinib and therefore could be a potential therapeutictarget. We evaluated our hypothesis on the only two PanNETcell lines available, although these cell lines only partiallyrepresent PanNET biology (43, 44). However, no better in vitroPanNET model is currently available. Because of this limitation,we studied our hypothesis additionally in primary cells fromtwo PanNET patients and in vivo in a well-studied transgenicmouse model of PanNET. In PanNET cell lines and a transgenicPanNET mouse model, we demonstrated that sunitinibcombined with the autophagy inhibitor CQ has a significantlyenhanced antitumor effect compared with single treatmentalone. In addition, the beneficial effect of the combinationtreatment was confirmed in human primary cell culture,suggesting that these findings might be relevant for PanNETpatients.

First, we showed that sunitinib increases autophagy in vitro inPanNET cell lines, in cells isolated from human PanNET tumorsand in vivo in the Rip1Tag2 transgenic PanNET mouse model.Sunitinib alone led to an upregulation of LC3B-II levels corre-sponding to an increased number of autophagosomes in PanNETcell lines and primary cells. LC3B-II levels further elevated whenautophagy was blocked with lysosomal inhibitors indicatingincreased autophagic flux. In vivo, the number of mice with highLC3B punctate staining, representative for autophagosomes,increased upon sunitinib. Previous studies on other tumor typesin vivo and in patient samples also reported modulation ofautophagy upon sunitinib treatment (45, 46). In tumors, autop-hagy might be influenced by additional factors, which may alsoresult from sunitinib's antiangiogenic activity, such as hypoxia(47), and inflammation or acidosis (48, 49). These can be onlypartially mimicked in vitro. To our knowledge autophagy has notbeen assessed in human PanNET yet. We showed by immuno-histochemistry for LC3B that autophagy is present in PanNETswith increased levels in liver metastasis compared to primarytumors, suggesting autophagy as a potential target in stage IVpatients.

We observed reduced cell recovery and induction of apopto-sis in cell lines treated with the combination of sunitinib andCQ. Similarly, Eng and colleagues have shown a synergisticeffect of sunitinib and CQ in reducing cell proliferation in alung adenocarcinoma cell line (50). Combination of sunitinibwith CQ in the Rip1Tag2 model led to an increased antitumoraleffect compared with single treatments alone, as measured byreduced tumor burden, reduced proliferation, and increasednecrosis. Importantly, tumor burden increased during sunitinibtreatment compared with the start of the treatment, but notduring combination treatment, indicating that the combina-tion treatment strongly impairs tumor development. All thesunitinib-treated tumors were less vascularized; however, therewas no difference in CD31 staining upon combination withCQ. This suggests that the increased antitumoral effect by thecombination therapy is not explained by an effect on angio-genesis, but rather due to a direct effect on the cancer cells.Indeed, sunitinib has been shown to reduce tumor cell prolif-eration in vitro (23, 24, 50).

Sunitinib treatment reduced histologic signs of tumor inva-siveness but the incidence and number of liver metastasisremained unchanged. Reported data on sunitinib-inducedinvasiveness and metastasis in the Rip1Tag2 model are how-ever inconsistent. Sunitinib has been shown to lead toincreased invasiveness and incidence of metastasis (8, 9),while a recent publication reports no change in invasivenessand number of metastasis per mouse upon sunitinib treatment(51). One important difference among these studies includingours is the age of the mice and the duration of the treatment,which might affect invasiveness and metastasis formation. Apossible genetic drift of the Rip1Tag2 mice after breeding indifferent laboratories for a long time might be anotherreason for the different results (51, 52). In summary, ourfindings show an antitumor effect of the combination treat-ment in vivo.

Sunitinib combined with ATG5 or ATG7 knockdown resultedin little or almost no apoptosis activation, while sunitinib com-bined with lysosome-targeting autophagy inhibitors such as CQ,BafA1, or LAMP2 knockdown, induced apoptosis. Our datasuggest that lysosomes are centrally involved in the combinationeffect of sunitinib and CQ. This is in agreement with other studiesshowing that sunitinib as well as CQ and LAMP2 knockdowndestabilize lysosomes (12, 14, 40). Destabilization of lysosomescan lead to lysosomal membrane permeabilization (LMP), whichin turn can trigger cell death or autophagy (17–19). In BON1 cells,sunitinib alone induced LMP, which was further increased byaddition ofCQor upon knockdownof LAMP2. InQGP1, only thecombinationof sunitinib andahigh concentration ofCQ inducedLMP, while single treatments did not (Supplementary Fig. S7A).This different susceptibility toward LMP seems to correlate withautophagic flux. Indeed, when autophagic flux upon sunitinibtreatment is assessed by calculating the difference with andwithout CQ or BafA1, the two cell lines showed different results.In both cell lines, autophagic flux upon sunitinib increased ifLC3B-II levels were measured upon CQ treatment, while the fluxwas reduced whenmeasured upon BafA1 treatment (Supplemen-tary Fig. S7B). Interestingly, in QGP1 cells, a higher concentrationof CQ than the one used in BON1 increased flux, while a lowerconcentration of CQ also reduced the flux. (Supplementary Fig.S7C). These data suggest that BON1 cells are more susceptiblethan QGP1 toward LMP and show sunitinib-induced autophagy,while in QGP1 cells, sunitinib rather blocked autophagy. Indeed,a recent study showed that sunitinib has a dual, concentration-dependent effect on autophagy in BON1 cells and that thethreshold concentration differs between cell lines (53). Thisdifference between BON1 and QGP1 cell lines might be due tothe different levels of LAMP2 which influence lysosomal stabilityand lysosomal membrane permeabilitation (14). Indeed, ourdata showed that LAMP2 knockdown sensitized both cell linestoward sunitinib or CQ treatment. Similarly, LAMP1 or LAMP2knockdown sensitized osteosarcoma cells toward siramesine, alysosomotropic drug (14).

Our in vitro data suggest that sunitinib has a dual effect onautophagy by both blocking and inducing autophagy. We pro-pose that thedetermining factor for thenet effect of inhibition andinduction is lysosome stability, with lysosome instability favoringcell death. Of note, in this context CQ and LAMP2 knockdown,which block autophagy, also contribute to autophagy inductionvia their destabilizing effect on lysosomes. The effect of BafA1 onLMP seems to be controversial in our results as well as in the

Wiedmer et al.





literature and is probably context-dependent. In contrast to CQ,BafA1 does not accumulate in lysosomes but elevates the pH viainhibitionof vacuolar ATPase (20): BafA1 treatmentwas shown toinduce a release of cathepsin D from lysosomes followed byactivation of caspase-3 (54). On the other hand, pretreatmentwith BafA1 can limit LMP induced by another drug (40, 42, 55).Interestingly, BafA1 could revert sunitinib resistance resultingfrom lysosomal sequestration in renal and colon cancer celllines (11).

On the basis of our data, we propose that sunitinib accumulatesin lysosomes and induces LMP. Autophagy is upregulated forclearance of damaged lysosomes, leading to recovery of the cells,therefore playing a prosurvival role. Combination of sunitinibwith CQ or shLAMP2 leads to a higher destabilization of lyso-somes and increased LMP, which is associated with apoptosis-dependent cell death (Fig. 6).

Lysosomes are an attractive therapeutic target because cancercell lysosomes are less stable andmore prone to permeabilization(13). Many drugs approved for other diseases target lysosomes.Because of this property, they might be exploited also as antican-cer therapeutics (15). CQ, approved for treatment of malaria, is

already tested in several clinical trials in combinationwith variousanticancer treatments. In phase I clinical trials, hydroxychloro-quine (HCQ) led to a significant antitumor effect in combinationwith other therapeutics (36, 37). A clinical phase I trial is ongoingfor the combination of sunitinib and HCQ in advanced solidtumors that did not respond to chemotherapy (NCT00813423).Our study strongly supports the rationale of this combinationfor treatment of PanNET patients and their inclusion in suchclinical trials.

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

Authors' ContributionsConception and design: T. Wiedmer, M.P. Tschan, I. Marinoni, A. PerrenDevelopment of methodology: T. Wiedmer, P. Krebs, A. PerrenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Wiedmer, S. Pantasis, A. PerrenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Wiedmer, A. Blank, R. Bill, A. PerrenWriting, review, and/or revision of the manuscript: T. Wiedmer, A. Blank,S. Pantasis, P. Krebs, M.P. Tschan, I. Marinoni, A. Perren

Figure 6.

Proposed model of sunitinib and combination effect. Sunitinib accumulates in lysosomes and induces autophagy. Autophagy enables recovery of the cells,therefore playing a prosurvival role. If autophagy is blocked via knockdown of ATG5 or ATG7 in combination with sunitinib, reduced recovery is observed.Upon combination of sunitinib with CQ or LAMP2 knockdown, both combinations collaborate to a higher destabilization of lysosomes in addition toreduced recovery, leading to apoptosis-dependent cell death.






Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Pantasis, P. KrebsStudy supervision: M.P. Tschan, I. Marinoni, A. PerrenOther (laboratory technician; assistance of treatment of animals, laboratorywork): L. Normand

AcknowledgmentsWe would like to thank PD Dr. Deborah Stroka for critical comments on the

manuscript, Dr. Mario Noti for help and advice with animal experiments, andthe Translational Research Unit for cutting the slides and IHC. Tissues wereprovided by the Tissue Bank Bern.

Grant SupportThis work was supported by the Swiss Cancer League under Grant KFS-3360-

02-2014 (to A. Perren, M. Tschan, and I. Marinoni). Desir�ee and Niels YdeFoundation, grant no. 466-10 to I. Marinoni. I. Marinoni was supported by aSwiss National Science Foundation career grant (SNF-PMPDP3_164484).

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 February 10, 2017; revised June 8, 2017; accepted July 5, 2017;published OnlineFirst July 20, 2017.

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2017;16:2502-2515. Published OnlineFirst July 20, 2017.Mol Cancer Ther Tabea Wiedmer, Annika Blank, Sophia Pantasis, et al. Neuroendocrine Tumors via a Lysosome-dependent MechanismAutophagy Inhibition Improves Sunitinib Efficacy in Pancreatic

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