B8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance Sha Jin1,Wei-Chun Lee1, Daniela Aust2,3,4, Christian Pilarsky5, and Nils Cordes1,4,6,7,8

Published OnlineFirst July 23, 2019; DOI: 10.1158/1541-7786.MCR-18-1352

Signal Transduction and Functional Imaging Molecular Cancer Research b8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance Sha Jin1,Wei-Chun Lee1, Daniela Aust2,3,4, Christian Pilarsky5, and Nils Cordes1,4,6,7,8

Abstract

Pancreatic ductal adenocarcinoma (PDAC) stroma, com- microtubule-dependent perinuclear-to-cytoplasmic shift as posed of extracellular matrix (ECM) proteins, promotes well as strong changes in its proteomic interactome regard- therapy resistance and poor survival rate. Integrin-mediated ing the cell functions transport, catalysis, and binding. Parts cell/ECM interactions are well known to control cancer cell of this interactome link b8 integrin to autophagy, which is survival, proliferation, and therapy resistance. Here, we diminished in the absence of b8 integrin. Collectively, our identified b8 integrin in a high-throughput knockdown data reveal b8 integrin to critically coregulate PDAC cell screen in three-dimensional (3D), ECM-based cell cultures radiochemoresistance, intracellular vesicle trafficking, and for novel focal adhesion protein targets as a critical deter- autophagy upon irradiation. minant of PDAC cell radiochemoresistance. Intriguingly, b8 integrin localizes with the golgi apparatus perinuclearly in Implications: This study identified b8 integrin as an essen- PDAC cells and resection specimen from PDAC patients. tial determinant of PDAC cell radiochemosensitivity and as Upon radiogenic genotoxic injury, b8 integrin shows a a novel potential cancer target.

Introduction systematically evaluated in large clinical trials nor showed great advantages over standard of care yet (2, 3). Pancreatic ductal adenocarcinoma (PDAC) is one of the five One putative cancer target in PDAC is the PDAC cell/ most lethal malignancies in the world. Although the 5-year overall extracellular matrix (ECM) interaction as PDACs are stroma- survival rate is about 15% to 20% for resectable patients (1, 2), rich tumors (4). This stroma consists of numerous ECM compo- most patients presenting at late stage with a treatment-refractory nents such as collagens, laminins, fibronectin, and hyaluronic disease survive significantly less. Neoadjuvant chemotherapy acid (5). Cells interact with ECM components via cell adhesion [e.g., gemcitabine, nab-paclitaxel, FOLFIRINOX (folinic acid, receptors that coalesce with receptor tyrosine kinases, adapter and 5-fluorouracil, irinotecan, and oxaliplatin)] is administered to signaling molecules to form focal adhesion complexes. These patients whose tumors seem irresectable or borderline resectable. membranous multiprotein complexes are essentially coregulating Alternatives such as radiotherapy and biologicals were neither key cell functions such as survival, cell death, proliferation, meta- stasis, and therapeutic resistance (6, 7). In previous work, we and others have documented the radiochemosensitizing potential of 1OncoRay, National Center for Radiation Research in Oncology, Faculty of integrin and focal adhesion protein targeting, as the major family € Medicine and University Hospital Carl Gustav Carus, Technische Universitat facilitating cell/ECM interactions. Examples for preclinically iden- Dresden, Helmholtz-Zentrum Dresden, Rossendorf, Dresden, Germany. fi 2Institute for Pathology, University Hospital Carl Gustav Carus, Technische ti ed novel targets are b1 integrin (8), avb3 integrin (9), avb6 Universitat€ Dresden, Dresden, Germany. 3NCT Biobank Dresden, University integrin (10), FHL2 (11), APPL1 and 2 (12), Caveolin 1 (8, 13), as Hospital Carl Gustav Carus, Technische Universitat€ Dresden, Dresden, Germany. well as small integrin-binding ligand N-linked glycoproteins 4German Cancer Consortium (DKTK), Partner Site Dresden, Heidelberg, (called SIBLINGs) or secreted protein acidic and rich in cysteine Germany. 5Department of Surgery, Universitatsklinikum€ Erlangen, Friedrich- (called SPARC) families (14). Particularly, the anti-integrin € 6 Alexander Universitat Erlangen, Erlangen, Germany. Department of Radiother- approaches were exploited for molecular imaging (15), whereas apy and Radiation Oncology, Faculty of Medicine and University Hospital Carl biologicals inhibiting focal adhesion proteins (FAP) have not Gustav Carus, Technische Universitat€ Dresden, Dresden, Germany. 7Institute of Radiooncology, OncoRay, Helmholtz-Zentrum Dresden, Rossendorf, Dresden, found their way into the clinic. Owing to the critical function of Germany. 8German Cancer Research Center (DKFZ), Partner Site Dresden, focal adhesions for prosurvival signaling in normal and cancer Heidelberg, Germany. cells, we established a high-throughput esiRNA-based screening Note: Supplementary data for this article are available at Molecular Cancer for more physiologic three-dimensional (3D) cell cultures to Research Online (http://mcr.aacrjournals.org/). more systematically characterize the role of FAPs for PDAC fi S. Jin and W.-C. Lee contributed equally to this article. radiochemoresistance. Intriguingly, we identi ed b8 integrin as top druggable target eliciting radiosensitization of PDAC cells. Corresponding Author: Nils Cordes, Technische Universitat€ Dresden, Fetscher- b8 integrin, a 769-amino acid containing type I transmem- strasse 74/PF 41, Dresden 01307, Germany. Phone: 49-351-458-7401; Fax: 49- 351-458-7311; E-mail: [email protected] brane protein, consists of a large extracellular domain, a VWFA domain, four cysteine-rich repeats, and a short cytosolic Mol Cancer Res 2019;17:2126–38 domain (16, 17). Recent studies demonstrated that, unlike other doi: 10.1158/1541-7786.MCR-18-1352 b integrins, the cytosolic tail of b8 integrin shares no apparent 2019 American Association for Cancer Research. homology and does not directly influence cell adhesion. This

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suggests b8 integrin signaling to be distinct from other b integ- 101) and 1% nonessential amino acids (Gibco, 11140050). rins (16). A connection between b8 and aV integrin as well as the Patient-derived primary cell cultures (PacaDD119, PacaDD137, TGFb signaling cascade has been reported (18). Others exhibited and PacaDD159) and COLO357 cells were cultured in DMEM b8 integrin involvement in liver cancer resistance to gefitinib (19), with 33% K-SFM (Gibco, 17005034) and 13% FCS. All cells were interactions with EphB4 receptor (20), as well as dependency of maintained at 37 C with 8.5% CO2 at pH 7.4. In all experiments, differentiation and radiosensitivity on b8 integrin in glioma asynchronously growing cells were used. All cells were tested cells (21). negative for Mycoplasma by using a mycoplasma detection kit Based on the fact that PDAC is highly therapy refractory, there from Minerva biolabs (VenorGeM OneStep). appears an imbalance in survival and death mechanisms per se and under treatment. Partly, the therapy resistance found in PDAC Construct of b8 integrin arises from autophagy (22). Autophagy is a highly conserved Human b8 integrin generated by PCR-based amplification catabolic process involving the formation of double-membraned from cDNA ORF Clone, purchased from Sino Biological vesicles known as autophagosomes that engulf cellular proteins (HG10367-M) with specific primers (pAcGFPC1-ITGB8-XhoI-F and organelles for delivery to the lysosome (23). The impact of and pACGFPC1-ITGB8-KpnI-R). Constructs were flanked with autophagy seems tissue- and cancer type dependent. In general, XhoI and KpnI restriction sites and inserted into the XhoI and KpnI autophagy has two opposing functions. One is cytoprotective, sites of pAcGFP-C1 (Clontech). eliciting therapeutic resistance; the other one is cytotoxic, induc- ing autophagic cell death. Recent studies have shown autophagy Plasmid DNA transfection as a prosurvival and resistance mechanism against chemotherapy Cells were plated onto uncoated 35-mm dishes with a 0.17-mm treatment in PDAC (24, 25). glass bottom (MatTek) and allowed to reach 60% to 70% con- fl In the present study, we explored the function of b8 integrin in uency. pAcGFPC1-ITGB8 (b8/AcGFP) and pAcGFPC1 empty PDAC therapy resistance and unraveled parts of its contributing vector (control, data not shown) were introduced into the cells molecular mechanism. In PDAC cell lines and PDAC primary cell using Lipofectamine LTX (Invitrogen, 15338100) according to the fl cultures grown in 3D lrECM as well as in clinical samples from manufacture's protocol. Brie y, cells were incubated in 250 mL PDAC patients, we observed that b8 integrin inhibition sensitizes OptiMEM with a DNA-mix of 250 mL (containing plasmid DNA – PDAC cells to X-rays and gemcitabine and localizes perinuclearly of 1 2 mg/mL and 10 mL Lipofectamine LTX with 15 mL with PLUS with the golgi apparatus. Furthermore, our data demonstrate that Reagent in 240 mL OptiMEM). Transfection media were removed ionizing radiation induces a microtubule-dependent perinuclear- after 4 hours, and cells were further incubated in fresh medium. to-cytoplasmic shift of b8 integrin and changes in the composi- esiRNA transfection tion of the b8 integrin interactome to transport, catalysis, and Cells (250,000) were seeded per well of 6-well plate, esiRNAs binding upon irradiation. Parts of the protein interactome of b8 or nonspecificRLUCesiRNAof1mg/mL final concentrations integrin facilitate a connection to autophagy, which is diminished (Eupheria Biotech) were transfected by using oligofectamine in the absence of b8 integrin. (Invitrogen, 12252011) according to the manufacturer's protocol. Knockdown efficiency of b8 integrins was measured Materials and Methods by Western blotting. Antibodies and reagents The antibodies against b1 integrin (WB 1:1,000, Abcam, Radiation exposure ab179471), b8 integrin (IF 1:200, WB 1:1,000; Abcam, ab80673, Irradiation was delivered at room temperature using 2, 4, or Rb, recognizes aa 614-663 (this antibody was generally used 6 Gy single doses of 200-kV X-rays (Yxlon Y.TU 320; Yxlon; dose fi in this study); Abnova, H00003696-M01, Mo, recognizes aa rate 1.3 Gy/minute at 20 mA) ltered with 0.5-mm Cu as 392–503 (this antibody was used only where specifically indi- published (11). The absorbed dose was measured using a Duplex cated), GM130 (IF 1:100, WB 1:1,000, BD, 610822), MEK1/2 dosimeter (PTW). (WB 1:1,000, Cell Signaling Technology, 4694s), gH2AX (WB 3D high-throughput esiRNA-based screening (3DHT-esiRNAS) 1:1,000, Cell Signaling Technology, 9718s), aV integrin (IF against FAPs and data analysis 1:250, Novus, NB100-2618), APPL2 (IF 1:100, Sigma-Aldrich, 3DHT-esiRNAS was performed in MiaPaCa2 cells in 96-well SAB1400605), Caveolin 1 (IF 1:100, BD, 610407), LC3B plate. The esiRNAs of 10 ng/mL final concentration were trans- (IF 1:500, Sigma-Aldrich, SAB4200361), b-actin (WB 1:10,000, fected using oligofectamine. The cells were subcultured 24 hours Sigma-Aldrich, A5441), HRP-conjugated secondary antibody after transfection. Cell suspensions were mixed with laminin-rich (GE, Rb NXA931, Mo NXA931), Alexa Fluor 488– or Alexa Fluor extracellular matrix (IrECM, final conentration at 0.5 mg/mL) and 594–conjugated secondary antibodies (1:500, Life Technologies) seeded into a new 96-well plate precoated with 1% agarose. After were purchased and used as indicated. 1-day incubation, 3D IrECM cultured cells were irradiated with Cell culture 6 Gy X-rays. Grown tumoroids containing 50 cells or more were PDAC cell lines BxPC3, MiaPaCa2, Panc-1, and Patu8902 were counted 8 days after irradiation. The percentage of tumoroid purchased from ATCC; Capan-1, patient-derived primary cell formation (N) is given by: lines (PacaDD119, PacaDD137, PacaDD159) and COLO357 cells were a kind gift from Chr. Pilarsky (TU Dresden). Origin Xn X ¼ 1 ðÞx x and stability of the cells were routinely monitored by short- i 0 2 i¼1 tandem repeat analysis (microsatellites). Established cell lines were cultured in Dulbecco's modified Eagle medium (DMEM, where n is the number of tumoroids in each well treated with Gibco, 61965026) with 10% fetal calf serum (FCS, PAN, A15- target esiRNAs, n0 is average of control samples without

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irradiation or with 6 Gy. The average of six data sets was collected Coloc 2 plugin of Fiji. Images for translocation and expression of from three independent screens. b8 integrin were recorded by spinning disk confocal microscopy (Olympus IX83 microscope with Yokogawa CSU-X1 Confocal 3D tumoroid formation Spinning Disc unit, and iXon Ultra 897 EMCCD Camera) with In addition to the 3D high-throughput screen, as described 60/1.2 NA water immersion objective. Z stacks with a step of above, 3D tumoroid formation was determined under identical 1 mm were acquired per image. Maximum intensity projection of z growth conditions after irradiation (2–6 Gy X-rays) or gemcita- stacks was processed using Fiji. All the detected b8 integrin signals bine treatment (for 24 hours; PBS as control) as published from the whole cell were analyzed. The cell size and nucleus size previously (26, 27). For gemcitabine, media were removed, cells were measured on bright field images. Coordinates of each b8 repeatedly washed, and fresh media were added to allow tumor- integrin (Xn, Yn) were determined by the position of maximum oid growth over 8 days. Each point on the survival curves repre- intensity. Distance of each b8 integrin to nucleus center (D) is sents the mean surviving fraction from at least three independent given by experiments. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi D ¼ ðÞx x 2 þ ðÞy1 y0 2 Western blotting analysis 1 0 Cells were lysed with modified RIPA buffer consisting of where x0 and y0 are the local positions of the nucleus center. 50 mmol/L Tris-HCl (pH 7.4), 1% Nonidet-P40, 0.25% sodium Relative b8 integrin to nucleus center distance (rel. D), deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L NaVO4, 2 mmol/L NaF (all Sigma-Aldrich), complete protease inhibitor cocktail (Roche) as described previously (27). Total rel: D ¼ D Nucleus size protein amount was measured by BCA assay (Thermo Fisher Cell size Scientific). After SDS-PAGE and transfer of proteins onto nitro- cellulose membranes (GE Healthcare), probing of specific avg: value at p: IR time norm: distance ¼ proteins was accomplished using indicated primary antibodies avg: value of unirradiated sample and horseradish peroxidase–conjugated donkey anti-rabbit and sheep anti-mouse antibodies. Enhanced chemiluminescent reagent (GE Healthcare) was used for detection of proteins on Sulforhodamine B (SRB) assay X-ray films (GE Healthcare), and protein expression levels were BxPC3 cells grown in a 96-well plate were treated with colchi- measured by densitometry. cine, paclitaxel, chloroquine, and gemcitabine with serial dilu- tion. After 3 days, cells were fixed with trichloroacetic acid. After Protein fractionation assay washing with tap water, 50 mL of 0.04% (wt/vol) SRB solution The Subcellular Protein Fractionation Kit for Cultured Cells was added to each well and incubated for 1 hour. Then, plates (Calbiochem, ProteoExtract Subcellular Proteome Extraction Kit, were rinsed four times with 1% (vol/vol) acetic acid to remove 539790) was used according to the manufacturer's protocol. In unbound dye. Tris base solution (pH 10.5; 100 mL of a 10 mmol/L brief, cell lysis and fractionation were conducted after 24-hour solution) was added to solubilize the protein-bound dye. plating. Equal loading was ensured by total protein concentration Measurement of absorbance was accomplished at 510 nm in a measurement using the BCA assay (Pierce). Fractionation efficacy microplate reader (Tecan). was confirmed with detection of b1 integrin for the membrane fraction, MEK1/2 for the cytosol fraction and gH2AX for the Histology nuclear fraction. Patient material was examined according to the ethic approval (EK 378092017; dated February 1, 2018) provided by the ethic Immunofluorescence staining committee of the Technische Universit€at Dresden. The tissue used In brief, cells were grown on coverslip or 35-mm dishes with a was provided by the tissue bank for tumor and normal tissues of 0.17-mm glass bottom (MatTek), fixed with 3.7% PFA at room the UCC/NCT Dresden and used in accordance with the regula- temperature for 15 minutes, permeabilized with 0.25 % Triton tions of the tissue bank. For immunofluorescence analysis, par- X-100 for 10 minutes, and washed with PBS as published (27). affin sections were deparaffinized in xylene and rehydrated. Then, cells were incubated with blocking buffer (1% BSA in PBS) Antigen retrieval was performed in 10 mmol/L citric acid, at room temperature for 1 hour followed by incubation with pH 6.0 at 98C for 15 minutes, and sections were stained with primary and secondary antibodies in blocking buffer in the hematoxylin and eosin or antibodies against b8 integrin. For dark at room temperature for 1 hour. The cells were washed tyramide-based immunofluorescence detection, the TSA-kit with PBS three times between each step. Then, the cover slides (Thermo Fisher Scientific) was used according to the manufac- were mounted using ProLong Diamond Antifade Mountant with turer's instructions. Sections were mounted using ProLong DAPI. Samples were stored in dark until microscopy. Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) for nuclear counterstaining. Images were acquired using an Microscopy and image analysis Axioscope1 plus fluorescence microscope (Zeiss). To investigate the localization of b8 integrin and expression levels of LC3, samples were imaged by a Zeiss Axioscope1 epi- Immunoprecipitation fluorescence microscope using a 40/0.75 or 100/1.25 oil Immunoprecipitation was performed as previously pub- objective. Images for colocalization analysis were acquired by a lished (26). In brief, 1 mg of whole-cell lysates (Cell Lysis Buffer, Zeiss LSM 510 meta microscope using 63/1.2 water immersion Cell Signaling Technology) was incubated with specific antibo- objective and were analyzed by Pearson correlation analysis using dies at 4 C for 1 hour. Beads were washed with Cell Lysis Buffer

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(Cell Signaling Technology) and added to lysates. After overnight of The Cancer Genome Atlas database of the PDAC patient incubation at 4C, immunoprecipitates were washed, mixed with cohort suggested high b8 integrin expression levels result in sample buffer, and loaded on SDS gels. shorter survival of PDAC patient as compared with lower expression levels (Fig. 2B; ref. 29). Then, we determined the Sequential immunoprecipitation/mass spectrometry–based protein expression level of b8 integrin in six established proteomics (IP-MS) and data analysis PDAC cell lines (BxPC3, Capan1, Colo357, Mia PaCa2, Panc1, To identify b8 integrin interacting proteins, mass spectrometric and Patu8902) and three human PDAC patient primary cell analysis was carried out at the Max Planck Institute of Molecular cultures (PacaDD119, PacaDD137, and PacaDD159) grown in Cell Biology and Genetics Mass Spectrometry (MPI-CBG MS) IrECM 3D (Fig. 2C and D). The expression of b8integrinvaried Facility (Dresden, Germany) and performed as published (28). In in a cell line–dependent manner (Fig. 2C and D). To charac- brief, immunoprecipitates were separated by gel electrophoresis, terize the subcellular location of b8 integrin, immunofluores- in-gel digested with trypsin and peptides recovered from the gel cence staining was performed in a panel of human pancreatic matrix analyzed on a LTQ Orbitrap XL mass spectrometer cancer cell lines indicating b8 integrin to be located in the (Thermo Fisher Scientific) coupled on-line to the Ultima3000 perinuclear area in PDAC cells (Fig. 3A, B, and D). In contrast, a LC system (Dionex) via a TriVersa robotic ion source (Advion clear b8 integrin accumulation at the plasma membrane was BioScience). PANTHER (Protein ANalysis THrough Evolutionary observable in glioblastoma cell lines (Fig. 3C and E). Next, we Relationships) classification system (http://www.pantherdb. examined cytosol, membrane, and nucleus protein fractions org/) was used to categorize interactomes of b8 integrin accord- revealing b8 integrin localized in cytosol and membranes, in ing to their functions. Connections between the identified b8 contrast to our b1 integrin control found only in the membrane integrin interactome and autophagy were analyzed by the fraction (Supplementary Fig. S2A and S2B). Intriguingly, con- Autophagy Regulatory Network (http://arn.elte.hu/). firmatorydataforperinuclearlocalizationin situ are presented by b8 integrin staining in resection specimen of human PDAC Statistical analysis (Fig. 3F and G). To further address this abnormal localization, All results represent mean standard deviation (SD) of at least we screened the International Cancer Genome Consortium three independent experiments. Unpaired, two-sided Student t (ICGC; icgc.org) database for mutations in the ITGB8 gene test was performed with Microsoft Excel. A P value of less than (https://icgc.org/ZyH). In 52 of 472 donors, 83 mutations are 0.05 was considered statistically significant. found of which 67 are in introns and 9 in exons. The COSMIC database provided no ITGB8 gene mutations in any of the cell lines used in the presented study (https://cancer.sanger.ac. Results uk/cosmic/gene/samples?all_data¼&coords¼AA%3AAA&dr¼ High-throughput RNAi screen in 3D PDAC cells identifies &end¼770&gd¼&id¼5149&ln¼ITGB8&seqlen¼770&sn¼ potential focal adhesion protein targets involved in pancreas&src¼gene&start¼1#positive). These findings demon- radioresistance strate a b8 integrin expression in PDAC cells without func- The role of FAPs in PDAC therapy resistance has not been tionally critical gene mutations and, as compared with other comprehensively unraveled. By means of a 3D high-throughput integrin subunits, an unusual subcellular localization that RNA interference (3D-HTP-RNAi) screen (Fig. 1A; Supplementary warrants further investigation. Table S1), we sought to identify novel potential FAP candidates whose depletion potently diminishes PDAC tumoroid forming b8 integrin is a critical regulator of cellular sensitivity to ability under basal (Fig. 1B) or irradiation conditions (Fig. 1C). ionizing radiation and cytotoxic drugs We found basal tumoroid forming ability to be significantly To confirm that b8 integrin plays an essential role in PDAC (P < 0.05) compromised upon depletion of, for example, radiochemoresistance, we effectively silenced b8 integrin in a Synemin, JUB, ITGB5, CRK, GAB1, and Caveolin 1 relative to panel of PDAC cell lines (Fig. 4A and B), detected unaltered controls (Fig. 1B). Upon 6 Gy X-rays, we surprisingly found tumoroid growth (Fig. 4C and E) but observed a highly significant none of the FAP depletions to result in a significantly enhanced decrease in tumoroid growth upon irradiation relative to controls radiosensitivity of MiaPaCa2 cells (Fig. 1D). We proceeded (Fig. 4D and E; Supplementary Fig. S3A–S3D). In addition, b8 with the top 10 candidates for further evaluation based on the integrin–depleted PDAC cell lines (BxPC3 and Patu8902) following criteria (Supplementary Fig. S1): (i) novelty in the exhibited enhanced chemosensitivity to gemcitabine (Fig. 4F; context of radiosensitivity, (ii) current knowledge about the Supplementary Fig. S3E). Collectively, our data suggest a critical candidates and, (iii) higher RNA expression levels in PDAC function in PDAC cell survival after X-ray irradiation and gemci- compared with normal tissue according to the Oncomine data- tabine treatment. base. Our analyses favored b8 integrin as a novel and potent FAP candidate whose depletion enhanced, although not signi- Changes in the composition of the b8 integrin interactome to ficantly, PDAC cell radiosensitivity in our screen. transport, catalysis, and binding upon irradiation To define the interacting proteins likely to be causative for the b8 integrin is overexpressed and located in the perinuclear perinuclear localization of b8 integrin, sequential immunopre- region in PDAC cipitation-mass spectrometry was used. First, we found 133 Next, we performed an Oncomine data (https://www. proteins bound to b8 integrin in unirradiated cells (Fig. 5A; oncomine.org) analysis and found a significant, 3.1-fold Supplementary Table S2). Intriguingly, the interactions changed upregulation of b8 integrin mRNA expression level in PDAC dramatically at 2 hours after 6-Gy X-rays, revealing 637 interacting as compared with normal pancreas (Fig. 2A). Furthermore, an proteins (Fig. 5A), of which only 32 proteins were present under analysis using OncoLnc. (http://www.oncolnc.org) for analysis both untreated and irradiation conditions. This tremendous

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A 0 Gy

KIF11 C20orf42 CORO1B FHL2 FLNA GRB2 PXN LASP1 HAX1 PFN1 LDB3 SSH3BP LPP SYNM Tumoroid SORBS1 NCK2 CASS4 NF2 CRKL PARVA ZFYVE21 N = 100 × n/n0, ITGA5 esiRNA Perform FBLIM1 ACTN1 FERMT3 CTTN GNB2L1 PARVB ITGA4 ITGAM GRB7 ARPC2 JUB SHC1 LIMS2 SORBS3 ITGAL ITGB6 MSN BCAR1 NEDD9 CRK PALLD VCL ITGB5 SDCBP counting & analyzing RLUC PLEKHC1 CORO2A GAB1 KEAP1 ITGA3 RDX transfection 3D cell culture N, normalized average tumoroid formation in percentage TES VASP ITGB1BP1 SYNM LIMS1 IRS1 ITGAE TENC1 CD151 ACTB NDEL1 CAV1 OSTF1 VIL2 ITGB4 CEACAM1 ITGA6 ITGAV TLN1 ITGB2 TRPM7 NUDT16L1 ENG n, tumoroid number upon KD of target proteins ITGB7 ITGA10 LRP1 PLEKHC1 LPXN CFL1 6 Gy n0, average tumoroid number in control sample FAPs esiRNA library Incubation 24 h

Incubation 24 h Tumoroid formation

B Surviving upon KD of target proteins + 0 Gy (normalized on control)

n.s. * ** *** 100 formation/%

Avg. tumoroid 0 NF2 LPP JUB TES VCL ZYX PXN PVR VIL2 RDX CRK SVIL ENG IRS1 MSN TLN1 CFL1 FHL2 LDB3 LRP1 FLNA TNS1 THY1 PFN1 CD47 KTN1 LPXN HAX1 PKD1 CAV1 NRP2 VASP KIF11 CTTN NRP1 CALR GAB1 SHC1 SDC4 CRKL ACTB NCK2 RLUC GRB2 GRB7 ENAH NEXN SMPX TRIP6 ITGB5 ITGA3 ITGA1 ITGA7 LIMS2 ITGA4 ITGA6 ITGB6 ITGA8 ITGB1 ITGA2 ITGB7 SYNM LIMS1 ITGB4 ITGA5 ITGB2 SYNM ITGB8 ITGAL ITGB3 ITGA9 ITGAE ITGAV ITGAX ITGAD LASP1 CD151 PALLD SH2B1 ITGAM NDEL1 KEAP1 OSTF1 ACTN1 CASS4 TENC1 BCAR1 ARPC2 CSRP1 PARVB NEDD9 KCNH2 PARVA TRPM7 ITGA10 ITGA11 PPFIA1 SDCBP FBLIM1 SLC3A2 GNB2L1 SSH3BP FERMT3 SORBS1 TGFB1I1 SORBS2 SORBS3 C20orf42 CORO2A ITGB3BP CORO1B ZFYVE21 SH3KBP1 PLEKHC1 PLEKHC1 ITGB1BP1 CEACAM1 NUDT16L1

C Surviving upon KD of target proteins + 6 Gy (normalized on control)

n.s. * ** *** 100 formation/% Avg. tumoroid 0 NF2 JUB LPP ZYX TES VCL PVR PXN VIL2 RDX CRK SVIL ENG IRS1 MSN FHL2 TLN1 CFL1 LRP1 LDB3 PFN1 THY1 KTN1 TNS1 FLNA CD47 HAX1 PKD1 LPXN CAV1 CALR VASP ACTB NCK2 KIF11 NRP2 CRKL GAB1 SDC4 CTTN NRP1 SHC1 GRB2 RLUC GRB7 ENAH NEXN SMPX TRIP6 ITGB7 ITGA7 ITGB6 ITGA8 ITGB8 ITGA5 ITGA2 ITGA6 ITGA3 ITGB3 ITGB4 SYNM ITGAL ITGB5 ITGB1 ITGA9 ITGA1 ITGB2 ITGA4 LIMS1 LIMS2 SYNM ITGAE ITGAX ITGAV ITGAD LASP1 CD151 ITGAM PALLD SH2B1 NDEL1 OSTF1 KEAP1 TENC1 CASS4 ACTN1 ARPC2 BCAR1 CSRP1 TRPM7 PARVA KCNH2 PARVB NEDD9 PPFIA1 ITGA10 ITGA11 SDCBP FBLIM1 SLC3A2 GNB2L1 SSH3BP FERMT3 SORBS2 TGFB1I1 SORBS3 SORBS1 C20orf42 CORO2A ITGB3BP CORO1B ZFYVE21 SH3KBP1 PLEKHC1 PLEKHC1 ITGB1BP1 CEACAM1 NUDT16L1

D Enhancement ratio of radiosensitivity by KD (1 = no effect; >1 = radioprotection; <1 = radiosensitization) 2

6 Gy/untreated 0 NF2 LPP JUB TES ZYX VCL VIL2 PXN PVR RDX CRK ENG SVIL MSN IRS1 TLN1 CFL1 FHL2 LRP1 LDB3 PFN1 TNS1 FLNA THY1 KTN1 CD47 CAV1 HAX1 LPXN PKD1 CRKL SDC4 ACTB VASP NRP1 KIF11 SHC1 CALR NRP2 GAB1 CTTN NCK2 GRB7 GRB2 RLUC ENAH NEXN TRIP6 SMPX ITGAL LIMS1 SYNM ITGB2 ITGB5 ITGB4 ITGA1 ITGA6 ITGA4 ITGA8 ITGB7 ITGA3 ITGA9 SYNM ITGA7 ITGA2 LIMS2 ITGB6 ITGA5 ITGB1 ITGB3 ITGB8 ITGAE ITGAV ITGAX ITGAD LASP1 CD151 ITGAM SH2B1 PALLD KEAP1 OSTF1 NDEL1 TENC1 ACTN1 CASS4 CSRP1 BCAR1 ARPC2 PARVB NEDD9 TRPM7 KCNH2 PARVA ITGA10 PPFIA1 ITGA11 SDCBP FBLIM1 SLC3A2 GNB2L1 SSH3BP FERMT3 SORBS1 TGFB1I1 SORBS3 SORBS2 C20orf42 CORO1B CORO2A ITGB3BP ZFYVE21 SH3KBP1 PLEKHC1 PLEKHC1 ITGB1BP1 CEACAM1 NUDT16L1

Figure 1. High-throughput esiRNA knockdown screen of 117 FAPs in 3D PDAC cell culture. A, Workflow of esiRNA screen. B–D, 3D tumoroid formation in unirradiated (B) or 6 Gy X-ray–irradiated cells (C) upon knockdown (KD) of FAPs. D, Enhancement ratio of radiosensitivity upon knockdown (tumoroid number at 6 Gy)/ (tumoroid number at 0 Gy). Results for b8 integrin depletion are shown in red. Black columns indicate nonspecific esiRNA control. All results show mean SD (n ¼ 3; two-sided t test; , P < 0.05; , P < 0.01; , P < 0.005; n.s., not significant).

change in the interactome stimulated us to perform a GO ICGC database revealed 54 of 472 donors with mutations analysis based on the protein molecular functions according (https://icgc.org/Zyr). In total, 78 gene mutations can be to the PANTHER classification system (see the categorization of found, of which 60 are located in introns and 3 are categorized proteins in Supplementary Table S2). We calculated the increas- missense. Similar to ITGB8, no mutations were recognized in ed rate between the protein numbers in unirradiated versus any of the used PDAC cell lines using the COSMIC data- 6-Gy–irradiated cells. The number of interacting proteins in- base(https://cancer.sanger.ac.uk/cosmic/gene/samples?all_data¼ creased in different categories as follows: transporter activity by &coords¼AA%3AAA&dr¼&end¼1049&gd¼&id¼5530&ln¼ 95%, antioxidant activity by 100%, catalytic activity by approx- ITGAV&seqlen¼1049&sn¼pancreas&src¼gene&start¼1#positive). imately 84%, signal transducer activity by approximately 83%, OncoLnc. (http://www.oncolnc.org) based analysis of the PDAC structural molecule activity by approximately 84%, receptor patient cohort indicated high aV integrin expression to signifi- activity by approximately 64%, binding by approximately cantly correlate with shorter survival of PDAC patient as com- 68%, and translation regulator activity by approximately 86%, pared with lower expression levels (http://www.oncolnc.org/ respectively (Fig. 5B). Considering both factors, the absolute kaplan/?lower¼10&upper¼90&cancer¼PAAD&gene_id¼ number of proteins and their relative increase before and after 3685&raw¼ITGAV&species¼mRNA) (Supplementary Fig. S5). irradiation, we reckoned that the function of b8 integrin under- goes a critical shift toward transport/binding and catalysis. b8 integrin translocated from perinuclear area to cytosol Moreover, we checked a small selection of putative proteins upon irradiation related to transport (golgi apparatus reported by GM130; APPL2 To contextually connect the b8 integrin interactome data with and Caveolin 1) and stress (mitochondria) and published inter- b8 integrin behavior upon irradiation, we next conducted immu- action partners (aV integrins; ref. 18). In contrast to mitochon- nofluorescence staining followed by analysis of positive particle dria, the previously reported interacting integrin av, APPL2, and intensity and the distance from the cell center to the particles. We Caveolin 1, only GM130 colocalized to b8 integrin as indicative correlated this by multiplying with a correction factor, which was from the calculated Pearson correlation (0.55 0.04; Fig. 5C and determined by measuring the ratio of cell size to nucleus size. D; Supplementary Fig. S4). Under normal conditions, b8 integrin was located in the peri- As another possible explanation for the abnormal b8 integrin nuclear area. However, upon irradiation, the distance of b8 localization, genetic mutations were evaluated in ITGAV. The integrin particles increased from center to nucleus, indicating a

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A β8 integrin B mRNA expression β8 integrin P = 4.8E−8 Low (Fold change: 3.1) 100 High P n 4 = 0.04; = 52 50 Figure 2. PDAC

mRNA and protein expression of b8 integrin. A, median- 0

2 (n = 39) mRNA expression level of b8 integrin in healthy Overall survival/% 0

and in PDAC pancreatic tissue using the Log −4 0 1,000 2,000 3,000 Oncomine database (https://www.oncomine. centered intensity n.c. org). Box plot illustrates maximum, 75th n Days percentile, median, 25th percentile, and the ( = 39) minimum; study by Badea et al. B, Correlation of b8 integrin gene expression and PDAC patient survival rate analyzed using OncoLnc. (http:// C Patient-derived D www.oncolnc.org). C, Western blot analysis for PDAC b8 integrin expression from whole-cell lysates of 1 tumoroids derived from 3D lrECM-grown established PDAC cell cultures and patient- derived primary cell cultures. D, Densitometry analysis of B normalized to b-actin. All results 0.5 show mean SD (n ¼ 3). of actin/AU Rel. integrin β 8 BxPC3 Capan-1 COLO357 MiaPaca 2 Panc 1 Patu8902 PaCaDD119 PaCaDD137 PaCaDD159 β8 integrin 0

β-Actin BxPC3 Panc 1 Capan-1 Patu8902 COLO357 MiaPaca 2 PaCaDD119 PaCaDD137 PaCaDD159 movement of b8 integrin–positive particles from the perinuclear such as MAPK8, PRKAR1A, SAFB2, PRKAG1, DNAJB1, SMC1A, area to the cell membrane. This dynamic was accompanied by a MYH4, RHEB, FKBP1A, RPA2, HADHA, HACD3, and DIP2B significant, approximately 4-fold elevation of b8 integrin expres- categorized as proteins regulating autophagy according to the sion at already 2 hours as well as 24 hours after genotoxic stress PANTHER classification system. Collectively, our interactome induced by 6-Gy X-ray irradiation (Fig. 6A and B). Furthermore, findings suggest b8 integrin to contribute to the regulation of both average and maximum distances as measures of perinuclear- autophagy after exposure to X-rays in PDAC cells. to-cytoplasmic translocation were significant at 24 hours but not 2 hours after irradiation (Fig. 6A, C, and D). Depletion of b8 integrin reduces autophagy induction The association of the b8 integrin interactome proteins led us to Next, we investigated whether b8integrinper se or together speculate that the microtubule system is involved in the radio- with the microtubule system affects LC3B expression as key genic b8 integrin perinuclear-to-cytoplasmic shift. Therefore, we readout for autophagy. In addition to the microtubule assem- pretreated cells with the microtubule assembly inhibitor colchi- bly inhibitor colchicine, we pretreated cells with the microtu- cine and found a loss of the significance change in average and bule stabilizing agent paclitaxel and the autophagy inhibitor maximum distance of b8 integrin (Fig. 6E–G). This finding chloroquine (Supplementary Fig. S6A–S6C; ref. 30). Unirra- suggests microtubule dependence of the radiogenic perinuc- diated esiRNA control cells showed nonsignificant elevation in lear-to-cytoplasmic translocation of b8 integrin. LC3B intensity under colchicine and chloroquine (Fig. 7B and C; Supplementary Fig. S7), whereas paclitaxel led to a slight b8 integrin interactome connects to autophagy reductionofLC3Bintensity(Fig.7B).Incontrast,b8 integrin Based on our observations that PDAC cells are radiochemo- depletion elicited a significant decline in LC3B fluorescence sensitized by b8 integrin targeting and b8 integrin protein inter- intensity under exposure to all three agents, including PBS, action partners are associated not only with transporter activity relative to esiRNA controls (Fig. 7B). Upon irradiation, the and binding but also with catalytic activity, we hypothesized b8 intensity of LC3B was not significantly increased in esiRNA integrin to function, for example, in autophagy. Hence, candidate control cells independent of PBS or a specificagentrelative proteins involved in autophagy were identified in the list of to unirradiated esiRNA controls (Fig. 7B). Similar to nonirra- interacting proteins (Fig. 7A). Although electron transfer flavo- diated cells, b8integrin–depleted and irradiated cells showed protein alpha subunit (ETFA), thymopoietin (TMPO), and gly- asignificant reduction in LC3B intensity (Fig. 7B). coprotein P43 (RBMX) dispersed from b8 integrin at 2 hours after To determine whether the conditions tested for LC3B fluo- 6-Gy X-rays (Fig. 7A), DnaJ homolog subfamily C member 7 rescence intensity affect PDAC cell survival, cells were depleted (DNAJC7), aldehyde dehydrogenase 9 family members A1 of b8 integrin without and with colchicine, paclitaxel or (ALDH9A1) already present under nonirradiated conditions the autophagy inhibitor chloroquine. Intriguingly, 3D lrECM showed an enhanced binding to b8 integrin at 2 hours after PDAC tumoroid formation remained unaffected upon treat- irradiation. Intriguingly, we also found newly connecting proteins ment (Fig. 7D), whereas irradiated and treated PDAC cell

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A PDAC cell lines C Glioblastoma cell lines β8 integrin DAPI Phalloidin Overlay β8 integrin BF Overlay BxPC3 U-251 MG DD-T4 Capan-1

D β8/AcGFP Anti-β8 Overlay BF COLO357 BxPC3

β8/AcGFP1 vs. anti-β8 of whole cell area: Pearson R = 0.6 MiaPaCa2 E β8/AcGFP Anti-β8 Overlay BF Panc1 U-251 MG β8/AcGFP1 vs. anti-β8 of whole cell area: Pearson R = 0.8

Patu8902 F PDAC patient biopsy – H&E PaCaDD119 PaCaDD137 PaCaDD159 G PDAC patient biopsy – IF

Anti-β8 B Area 1 Antibody 1 Antibody 2 BF Pearson R (aa 614–663) (aa 392–503) (Ab.1 vs. Ab. 2):

0.9 BxPC3 Area 2

0.64 MiaPaCa2

β8 integrin Nucleus

Figure 3. Subcellular localization of b8 integrin in PDAC cells and tissue. A, Immunofluorescence staining of b8 integrin (green), actin stained with phalloidin/Alexa 594 (red), and nucleus with DAPI (blue). B, Endogenous b8 integrin detected using two different anti-b8 integrin antibodies for nearly membrane domain (aa 614– 663, in green) and the headpiece (aa 392–503, red) in BxPC3 and MiaPaCa2 cells visualized by Alexa 405 and Alexa 647, respectively. In this study, the antibody detecting the aa 614–663 sequence was generally used. C, Immunofluorescence staining of b8 integrin (green) in glioblastoma cells. Black arrows indicate membrane expression of b8 integrin. D and E, BxPC3 (D) and U-251 MG (E) cells were transfected with b8-AcGFP plasmid and then costained with anti-b8 antibody. White line represents plasma membrane; blue line represents nucleus based on BF images. F and G, hematoxylin and eosin (H&E; F) and immunofluorescence staining of integrin b8(G, plus DAPI) in human resection specimen from PDAC patients. White boxes indicate the same area. Scale bars, (E and F)100mm; zoomed areas 1 and 2 in F, 10 mm.

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A β8 integrin β-Actin B esi RNA control esi integrin β8:− + − + esi β8 integrin

BxPC3 1 Capan-1 COLO357 MiaPaca 2 0.5 Panc 1

Patu8902 Fold change PaCaDD119 0 PaCaDD137

BxPC3 Panc 1 Capan-1 Patu8902 COLO357MiaPaca 2 PaCaDD119PaCaDD137

C Unirradiated/ D 6 Gy X-rays/ plating efficiency surviving fraction esi RNA control esi RNA control esi β8 integrin esi β8 integrin Figure 4. 20 50 b8 integrin is critical for PDAC cell ** *** * ** *** *** * ** sensitivity to ionizing radiation and chemotherapy. A and B, esiRNA- mediated b8 integrin depletion 10 25 analysis by Western blot (A)and densitometry (B). C and D, 3D tumoroid forming ability in 0 0 Tumoroid formation/%

unirradiated (C) and in 6 Gy X-rays Tumoroid formation/% irradiated samples upon b8 integrin silencing. E, Representative phase BxPC3 Panc 1 BxPC3 Panc 1 Capan-1 Capan-1 contrast images of PDAC tumoroids Patu8902 Patu8902 COLO357MiaPaca 2 COLO357MiaPaca 2 (C and D). F, 3D tumoroid forming PaCaDD119PaCaDD137 PaCaDD119PaCaDD137 ability upon gemcitabine treatment in BxPC3 and Patu8902 cells. All results E Unirradiated 6 Gy X-rays show mean SD (n ¼ 3; two-sided t test; , P < 0.05; , P < 0.01; esi RNA control esi β8 integrin esi RNA control esi β8 integrin , P < 0.005).

F 100 BxPC3 100 Patu8902

50 50 formation/% Rel. tumoroid

0 0 1 10 100 1,000 10,000 1 10 100 1,000 10,000 Gemcitabine/nmol/L Gemcitabine/nmol/L esi β8 integrin esi RNA control cultures under removal of b8 integrin showed significant lower functionally effective for cell survival only upon stress as tumoroid formation. This effect was not significantly different induced here by X-ray irradiation. between the two microtubule-inhibiting agents, but significantly lower for chloroquine (Fig. 7E). The salient findings are that b8 integrin regulates LC3B expression in a microtubule- Discussion independent manner and that the interplay between b8integ- Integrin-mediated cell/ECM interactions fundamentally core- rin and autophagy measured by LC3B intensity becomes gulate cancer cell resistance to therapy. Owing to the massive

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A β8 integrin interactome 6 Gy X-rays

Unirradiated

n n n Number of proteins: = 101 =32 = 605

B β8 integrin involved in molecular functions

Transporter activity 0 Gy Unirradiated and 6 Gy X-rays 6 Gy X-rays Antioxidant activity Catalytic activity Signal transducer activity Structural molecule activity Receptor activity Binding Translation regulator activity 0 50 100 150 200 Number of proteins C

GM130 Mitochondria Integrin αV APPL2 Caveolin 1 β8 integrin Nucleus β8 integrin Nucleus β8 integrin Nucleus β8 integrin Nucleus β8 integrin Nucleus D 0.75

0.5

0.25

Pearson correlation 0 GM130 Mitochondria Integrin αV APPL2 Caveolin 1

Figure 5. b8 integrin interactome and subcellular colocalization. A, Sequential immunoprecipitation-mass spectrometry–based analysis of b8 integrin interactome was performed in unirradiated and 6-Gy X-rays irradiated Patu8902 cells. Time point of analysis after irradiation was 2 hours. B, Comparative changes in the b8 integrin interactome between control and irradiation upon categorization into different molecular functions using the PANTHER classiﬁcation system. C, Immunoﬂuorescence costaining of b8 integrin with GM130 (Golgi), mitochondria, aV integrin, APPL2, and Caveolin 1. Scale bars, 10 mm. D, Pearson correlation analysis of C. Data show mean SD (n ¼ 3).

stroma in PDAC, we conducted a high-throughput knockdown functions transport, catalysis, and binding upon radiogenic gen- screen in three-dimensional (3D), ECM-based cell cultures to otoxic injury, and (v) connects with parts of its interactome to identify novel focal adhesion protein targets. Here, we show that autophagy. b8 integrin (i) is overexpressed in PDAC relative to normal b8 integrin does not show an apparent homology to other b pancreas, (ii) is critically involved in radiochemoresistance in integrin subunits in its cytosolic tail, questioning its role in PDAC cells, (iii) colocalizes with the golgi apparatus perinuclearly adhesion to ECM and downstream signaling (16, 17). Nonethe- in PDAC cells and in resection specimen from PDAC patients, (iv) less, b8 integrin is overexpressed in various cancer types originat- shows a microtubule-dependent perinuclear-to-cytoplasmic shift ing from brain (21), lung (31), prostate (20), and gastrointestinal and strong changes in its proteomic interactome regarding the cell tract (19, 33) as well as pancreas as shown here. Intriguingly,

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β8 integrin β8 integrin + nucleus *** A B *** 8

4 n.c.

per cell 2 Rel. β 8 level 1

0 n.c. 2 h p. IR 24 h p. IR C *** 2 h p. IR 2 Figure 6. Translocation of b8 integrin from perinucleus area toward the plasma membrane upon stress. A, Representative 1 maximum intensity projection of z stacks shows b8 integrin subcellular localization Norm. avg. distance in 6-Gy X-ray–irradiated BxPC3 cells using 0 immunoﬂuorescence staining. Signals of 24 h p. IR n.c. 2 h p. IR 24 h p. IR b8 integrin were analyzed in the whole D cell. B, Image-based expression analysis *** of radiogenic induction of b8 integrin β8 integrin β8 integrin + nucleus 2 expression. C and D, Both analyses of A E for average (C) and maximum (D) distance between b8 integrin–positive particles and the center of the nucleus. E, 1

Representative maximum intensity n.c. projection of z stacks shows b8 integrin Norm. max. distance subcellular localization in 6-Gy X-ray– 0 irradiated cells pretreated with colchicine. n.c. 2 h p. IR 24 h p. IR F and G, Both analyses of E for average F (C) and maximum (D) distance between n.s. 8 integrin–positive particles and the b 1 center of the nucleus. Scale bars, 10 mm. b8 integrin, green; nucleus (stained with

DAPI), gray. Results show mean SD Colchicine (n ¼ 3; data were collected from 20 0.5 to 40 cells from three independent experiments; two-sided t test; Norm. avg. distance P ﬁ ; < 0.005; n.s., not signi cant). 0 n.c. Colchicine Colchicine G 2 h p. IR 24 h p. IR 2 h p. IR

Colchicine n.s. 1

0.5 Norm. max. distance 24 h p. IR Colchicine 0 n.c. Colchicine Colchicine 2 h p. IR 24 h p. IR although other b integrins are located in the cell membrane, functionality in the protein using the ICGC and COSMIC b8 integrin is found in the perinuclear region in 3D IrECM databases. Hence, the underlying mechanism possibly involv- grown PDAC cultures as well as in resection specimen from ing abnormal function of golgi and endoplasmic reticulum PDAC patients. Our characterization of this perinuclear local- proteins requires further exploration. ization revealed colocalization with the golgi apparatus In addition, b8 integrin is documented to impact on cells from marker GM130 but neither with the reported integrin aVto different tumor types. In HepG2 liver cancer cells, b8 integrin form one of the primary receptors for latent-TGFb1 and latent- contributes to resistance to geﬁtinib via multidrug transporters TGFb3 (18) nor mitochondria, APPL2, or Caveolin 1. Genetic and apoptosis regulators (19). It interacts with EphB4 receptor in mutations in ITGB8 as well as its reported binding partner prostate cancer cells (20) and controls differentiation and radio- ITGAV were not detected in regions inevitably entailing dys- sensitivity of glioma cells (21). Moreover, b8 integrin has central

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A B Unirradiated 6 Gy X-rays Unirradiated 6 Gy X-rays esi RNA control esi integrin β8 esi RNA control esi integrin β8 MAPK8 PRKAR1A SAFB2 PRKAG1

DNAJB1 Integrin β8 SMC1A

MYH4 n.c. RHEB FKBP1A RPA2

HADHA LC3 + DAPI DIP2B HACD3 DNAJC7 ETFA ALDH9A1

TMPO Integrin β8 RBMX

3 1.5 0 1.5 3

Intensity/AU Colchicine (Col) LC3 + DAPI

D Unirradiated esi RNA control esi integrin β8

n.s. Integrin β8

10 Paclitaxel (Pac)

0 LC3 + DAPI

Tumoroid formation/% n.c. Col Pac Chl

E 6 Gy x-rays esi RNA control esi integrin β8 Integrin β8

50 n.s. n.s. Chloroquin (Chl) 25 LC3 + DAPI

0 Tumoroid formation/% n.c. Col Pac Chl C Unirradiated 6 Gy X-rays esi RNA control esi integrin β8 esi RNA control esi integrin β8 2

1 Norm. avg. Flu. I. of LC3 0 Col Col Chl Col Chl Chl Col Chl n.c. n.c. n.c. n.c. Pac Pac Pac Pac

Figure 7. b8 integrin interaction with autophagy proteins upon irradiation and reduced LC3 expression under b8 integrin silencing. A, Autophagy-associated proteins identiﬁed using the (http://arn.elte.hu/) increasingly interact with b8 integrin upon irradiation. B and C, Representative images (B) and analyses (C)ofLC3 expression at 24 hours after 6-Gy X-rays irradiation in the presence of colchicine (5 nmol/L), paclitaxel (1 nmol/L) or chloroquine (25 mmol/L) in b8 integrin– depleted and control cells. D and E, 3D tumoroid formation upon b8 integrin depletion in BxPC cells in the presence of colchicine (5 nmol/L), paclitaxel (1 nmol/L), or chloroquine (25 mmol/L) plus/minus 6 Gy X-rays. Scale bars, 10 mm. LC3B, red; nucleus, gray. Results show mean SD (n ¼ 3; two-sided t test; , P < 0.05; , P < 0.005; n.s., not signiﬁcant).

roles in promoting glioma initiation in vitro and in vivo and its data demonstrating that b8 integrin targeting sensitizes PDAC inhibition reduces glioma cell self-renewal ability, stemness, and cells grown under 3D lrECM-based conditions to irradiation and migration ability (21, 33). These observations are in line with our chemotherapy.

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Little is known about the underlying mechanisms how b8 unchanged tumoroid forming capacity in b8 integrin– integrin controls cancer cell survival. We, therefore, characterized expressing cells that were pretreated with these three agents. In the b8 integrin proteomic interactome and found pronounced contrast, radiosensitization occurred in dependence of b8 integrin changes in its composition upon radiogenic genotoxic injury. The depletion without strong impact of microtubule or autophagy deduced categorization of interacting proteins from this analysis inhibitors. revealed that b8 integrin engages in transporter activity, antiox- Taken together, b8 integrin is overexpressed in PDAC and plays idant activity, catalytic activity, signal transducer activity, struc- an essential role in PDAC cell radiochemosensitivity. Based on its tural molecule activity, receptor activity, binding, and translation translocation and regulation of autophagy upon radiogenic gen- regulator activity in genotoxically injured cells. This wide spec- otoxic injury, b8 integrin seems to be critically involved in trum of functions stimulated us to focus on transport and prosurvival mechanisms. The b8 integrin–driven molecular cir- catalysis. cuitry and prosurvival signaling networks as well as its drugg- Upon genotoxic stress, we found b8 integrin to undergo a ability warrant further investigations to identify the potential of perinuclear-to-cytoplasmic translocation that seems cytoprotec- b8 integrin as a potential cancer target. tive, as a siRNA-mediated b8 integrin depletion resulted in elevated cell death and radiosensitization. Mechanistically, the Disclosure of Potential Conflicts of Interest cytoprotective process depended on functional microtubules. D. Aust is a consultant/advisory board member for Roche Pharma and Astra- Disturbing microtubule assembly by the microtubule inhibitor Zeneca. No potential conflicts of interest were disclosed by the other authors. colchicine strongly reduced the perinuclear-to-cytoplasmic translocation and induced radiosensitization. Which proteins from Authors' Contributions our b8 integrin interactome profile are responsible for these Conception and design: S. Jin, W.-C. Lee, N. Cordes Development of methodology: S. Jin, W.-C. Lee, N. Cordes actions require further investigation. Acquisition of data (provided animals, acquired and managed patients, The interactome also included proteins involved in autophagy. provided facilities, etc.): S. Jin, W.-C. Lee, D. Aust, C. Pilarsky Autophagy is a regulated catabolic pathway to degrade cellular Analysis and interpretation of data (e.g., statistical analysis, biostatistics, organelles and macromolecules. In malignant tumors, autophagy computational analysis): S. Jin, W.-C. Lee, D. Aust, C. Pilarsky, N. Cordes can either function as a prosurvival response to stress such as Writing, review, and/or revision of the manuscript: S. Jin, W.-C. Lee, D. Aust, starvation, hypoxia, and chemotherapy and radiotherapy that is N. Cordes Administrative, technical, or material support (i.e., reporting or organizing thought to mediate resistance to anticancer therapies or as an data, constructing databases): S. Jin, W.-C. Lee antisurvival response (23, 34). Intriguingly, b8 integrin seems to Study supervision: N. Cordes be a critical autophagy regulator, and autophagy is a cytoprotective mechanism in PDAC. These findings are in line with pub- Acknowledgments lished work describing high levels of basal autophagy and macro- The authors thank Inga Lange for excellent technical assistance and all the pinocytosis arising from intense metabolic rewiring (35). members of the Core Facility Cellular Imaging (CFCI) at Faculty of Medicine € Although the various proteins found in the b8 integrin interac- Carl Gustav Carus, Technische Universitat Dresden for technical assistance. The tome also require further attention to unravel the underlying tissue used was provided by the "Tumor- und Normalgewebebank des UCC Dresden" and used in accordance with the regulations of the tissue bank (and mechanism, some are already well-known determinants of the the vote of the ethics committee from the Technische Universit€at Dresden). This autophagy process such as RHEB (36, 37), DNAJB1 (38), project has received funding from the European Union's Horizon 2020 research FKBP1A (39), and HADHA (40). Importantly, we observed and innovation program under the Marie Skøodowska-Curie grant agreement increasing interactions of these autophagy-associated proteins No. 642623. with b8 integrin upon radiogenic genotoxic stress. Accordingly, the b8 integrin expression in PDAC cells seems connected with The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked autophagy detected by LC3 intensity. LC3 intensity was clearly advertisement fi in accordance with 18 U.S.C. Section 1734 solely to indicate dependent on b8 integrin expression but neither signi cantly this fact. altered by the two microtubule-inhibiting agents, colchicine and paclitaxel, nor the late-phase autophagy inhibitor chloroquine. Received December 21, 2018; revised March 26, 2019; accepted July 17, 2019; Concerning tumoroid formation, it was interesting to exhibit published first July 23, 2019.

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2138 Mol Cancer Res; 17(10) October 2019 Molecular Cancer Research

β8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance

Sha Jin, Wei-Chun Lee, Daniela Aust, et al.

Mol Cancer Res 2019;17:2126-2138. Published OnlineFirst July 23, 2019.

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