Secretome protein signature of human gastrointestinal stromal tumor cells

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Available online at www.sciencedirect.com

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Research Article

Secretome protein signature of human gastrointestinal stromal tumor cells Erik Berglunda,b,n, Elisabetta Daréc, Rui M.M. Brancad, Pinar Akcakayae, Robin Fröboma, Per-Olof Berggrenc, Weng-Onn Luie, Catharina Larssone, Jan Zedeniusa,b, Lukas Orred, Janne Lehtiöd, Jaeyoon Kimc, Robert Bränströma,b a

Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden Department of Breast and Endocrine Surgery, Karolinska University Hospital, Stockholm, Sweden c The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden d Cancer Proteomics Mass Spectrometry, Department of Oncology-Pathology, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden e Department of Oncology-Pathology, Karolinska Institutet, University Hospital, Cancer Center Karolinska, Stockholm, Sweden b

article information

abstract

Article Chronology:

Strategies for correct diagnosis, treatment evaluation and recurrence prediction are important for

Received 3 March 2015

the prognosis and mortality rates among cancer patients. In spite of major improvements in

Received in revised form

clinical management, gastrointestinal stromal tumors (GISTs) can still be deadly due to metastasis

4 May 2015

and recurrences, which confirms the unmet need of reliable follow-up modalities. Tumor-specific

Accepted 5 May 2015

secreted, shed or leaked proteins (collectively known as secretome) are considered promising sources for biomarkers, and suitable for detection in biofluids. Herein, we stimulated cell

Keywords: Sarcoma GIST Cell culture Conditioned media Secretome Proteomics

secretion in the imatinib-sensitive GIST882 cell line and profiled the secretome, collected as conditioned media, by using a shotgun proteomics approach. We identified 764 proteins from all conditions combined, 51.3% being predicted as classically/non-classically secreted. The protein subsets found were dependent on the stimulatory condition. The significant increase in protein release by the classical pathway was strongly associated with markers already found in other cancer types. Furthermore, most of the released proteins were non-classically released and overlapped to a high degree with proteins of exosomal origin. Imatinib pre-treatment radically changed these secretory patterns, which can have clinical implications when investigating biomarkers in imatinib-treated versus non-treated GIST patients. Our results show, for the first time, that GISTs contain a secretome signature. In the search for suitable biomarkers in the more complex GIST patient samples, this study aids in the understanding of basic GIST secretome characteristics. & 2015 Published by Elsevier Inc.

Abbreviations: BRAF, v-raf murine sarcoma viral oncogene homolog B1; CM, conditioned media; GIST, gastrointestinal stromal tumor; NF1, neurofibromin 1; TKI, tyrosine kinase inhibitor; PDGFR, platelet-derived growth factor receptor; SDH, succinate dehydrogenase n Correspondence to: Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, L1:03, SE-171 76 Stockholm, Sweden. E-mail address: [email protected] (E. Berglund).

http://dx.doi.org/10.1016/j.yexcr.2015.05.004 0014-4827/& 2015 Published by Elsevier Inc.

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

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Introduction

Materials and methods

Sarcomas constitute a heterogeneous group of tumors originating from non-epithelial tissues such as the mesenchyme, smooth muscle and bone [1]. Gastrointestinal stromal tumor (GIST) is the most common sarcoma in the alimentary tract and may be successfully targeted by the tyrosine kinase inhibitor (TKI) imatinib [2]. The unique gene alterations responsible for the development of the majority of GISTs are gain-of-function mutations in KIT [3] or platelet-derived growth factor receptor alpha (PDGFRA) [4], while a subset of KIT and PDGFRA negative GISTs harbor defects in BRAF, NF1, RAS or SDHx complex [5]. GISTs range from indolent micro-tumors to highly malignant disease. Although many operable GISTs can be cured by surgical resection solely, highly malignant and inoperable GISTs commonly recur or acquire TKI resistance [6]. These patients have a poor prognosis partly due to the lack of suitable follow-up strategies. One way of improving the clinical management is the identification of disease-specific biomarkers that can be applied for detection of recurrences, therapy responses and disease progression. GISTs have been proposed to have a neuroendocrine phenotype, and there are multiple reasons to believe that GISTs indeed are secretory: GIST cells arise from the interstitial cells of Cajal that have several characteristics similar to neuroendocrine cells. GIST cells exhibit the machinery necessary for cell secretion [7,8], and we have recently described an intracellular Ca2þ-dependent ATP release from GIST cells, which is an indirect indication of functional cell secretion [9]. Furthermore, the key oncogenic mutations of KIT and PDGFRA cause dysregulation of the PI3K-AKT, MEK-MAPK, and STAT signaling pathways [10–12], involved in cell cycle, protein translation, metabolism, apoptosis [13], and possibly cellular secretion [14,15]. However, the existence of a GIST secretome has not yet been identified. Bearing in mind that soft tissue sarcomas account for less than 1% of all malignancies [16], and GIST is a subtype with an annual incidence of 10–15 cases per million [17–19], biomarker validation is more complex in these malignancies than others. In addition, biomarker discovery in body fluids has proven to be challenging due to large and dynamic protein concentration ranges. A useful strategy to find biomarker candidates or groups of proteins is therefore to analyze proteins released into conditioned media (CM, the cell secretome) from cultured cells grown in serum-free media. The analysis of CM greatly enhances the chances of identifying a tumor secretome compared to complex clinical specimens by lowering the protein dynamic ranges and protein diversity. Proteomics approaches have been used previously in GIST, however those studies were designed to identify proteins associated with the malignant potential [20–23]. To our knowledge no studies exist that address secreted proteins from GIST cells. In this report, we have, for the first time, determined the existence and composition of a protein secretome from an imatinib-sensitive GIST cell line in vitro by applying a shotgun proteomics approach. The methodological approach was established and validated using a pancreatic β-cell line with an already known secretory signature.

Cell cultures and static incubations The murine insulin-secreting pancreatic β-cell line MIN6m9 was used for validation of the shotgun proteomics approach, and maintained in DMEM medium containing 1 mM sodium pyruvate and 11 mM glucose, supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 mg/ml streptomycin-sulfate and 5 nl/ml β-mercaptoethanol in a CO2 incubator at 37 1C. For proteomics studies cells were seeded at a density of 60,000 cells/cm2 in 24-well tissue culture plates and cultured for 48 h. Cells were washed twice with pre-warmed Hanks buffered salt solution (HBSS), containing (mM): 1.26 CaCl2, 0.49 MgCl2  6H20, 0.41 MgSO4  7H20, 5.33 KCl, 0.44 KH2PO4, 4.17 NaHCO3, 137.93 NaCl, 0.34 Na2HPO4, 0.1 D-glucose, pH 7.4, and maintained in the same buffer for 1 h at 37 1C. After the buffer was removed, the cells were stimulated with HBSS containing 25 mM glucose for 30 min using biological triplicates. The conditioned buffers as well as non-stimulated controls were collected on ice and centrifuged (5000g) at 4 1C for 10 min. The supernatants were stored at 80 1C for subsequent analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and for determination of insulin concentration. The imatinib-sensitive cell line GIST882 harboring a homozygous exon 13 missense KIT mutation [24] was used as a model system to examine if active protein secretion exists in GIST. GIST882 was kindly provided by Professor Jonathan Fletcher at Brigham and Women's Hospital, Boston, MA, USA, and verification of KIT mutation status has been previously published [9]. The cells were seeded in 175 cm2 flasks, cultured for three days until sub-confluence as previously described [25], and then carefully washed thrice. Following the third wash the cells were statically incubated for 5 min with 15 ml pre-warmed serum-free RPMI-1640 media supplemented with 5.5 mM glucose, 1.26 mM CaCl2, 100 units/ml penicillin G, 100 mg/ml streptomycin-sulfate and 0.21 mg/ml amphotericin B (control). Serum-free media was chosen to avoid masking effects of the major serum proteins such as bovine serum albumin (BSA). In addition to controls (biological triplicates) secretion stimulation was carried out by adding either 60 mM KCl (biological triplicates) or 2 mM thapsigargin (biological quadruplicates) for 5 min, which we have shown previously to be strong stimulators of ATP release in GIST and not to affect cell viability [9]. Furthermore, to see if the TKI imatinib affected the secretion pattern, cells were exposed to the therapeutic dose 1100 ng/ml imatinib during two hours before thapsigargin stimulation (biological quadruplicates). The conditioned medium was collected while holding the flasks upside down, kept on ice, and centrifuged to obtain cell free samples. The supernatant was then centrifuged at 4 ºC in Amicon Ultra-15 3 kD centrifugal tubes (cat no UFC900324, Billerica, MA, USA), thereby concentrating the samples approximately 30 times to final volumes of 500 ml per sample. To further clarify the protein-rich conditioned media, the samples were passed through 4 mm Millexs HV non-sterile syringe filters (cat no SLHV004NL, Millipore, Japan) according to the manufacturer's instructions. To decrease the risk of degradation of low-abundant proteins the supernatant was snapfrozen until LC-MS/MS and Western blot analysis.

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

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Insulin secretion measurements The ArcDia2-photon fluorescence excitation microparticle fluorometry (TPX) assay for insulin content (Arc-Dia Group, Turku, Finland) was used to measure insulin released by the MIN6m9 cells upon glucose stimulation. The assay was performed according to the manufacturer's instructions, using a recombinant human insulin standard to determine the insulin concentration, according to previously described methodology [26].

Sample preparation and LC-MS/MS configuration The collected supernatants ( 500 ml solution each) were reduced and denatured by addition of 2 ml of 50 mM DTT (dithiothreitol) and incubation at 95 1C for 20 min in a thermomixer. After cooling the samples, TEAB (triethylammonium bicarbonate) buffer was added to 50 mM (final concentration), followed by the addition of iodoacetamide (8 ml of a 50 mM solution) and incubation in the dark for 30 min to alkylate cysteins. Trypsin was added for overnight digestion at 37 ºC. Prior to MS analysis, the samples were cleaned up with strong cation exchange solid phase extraction with Strata-X-C cartridges (Phenomenex, P/N 8B-S029-TAK). Each GIST sample was run in LC–MS (Liquid Chromatography– Mass Spectrometry) technical triplicates to account for instrumental variations. MIN6m9 was used merely for method validation purposes and every biological sample (n¼ 3) was therefore analyzed once. In each LC–MS run, the LC auto sampler (HPLC 1200 system, Agilent Technologies) injected 4 ml into a C18 guard desalting column (Zorbax 300SB-C18, 5  0.3 mm, 5 mm bead size, Agilent). We then used a 15 cm long C18 picofrit column (100 mm internal diameter, 5 mm bead size, Nikkyo Technos Co., Tokyo, Japan) installed onto the nano-electrospray ionization (NSI) source. Solvent A was 97% water, 3% acetonitrile (ACN), 0.1% formic acid (FA); and solvent B was 5% water, 95% ACN, 0.1% FA. At a constant flow of 0.4 ml/min, the linear gradient went from 2% B up to 40% B in 15 min, followed by a steep increase to 100% B in 5 min. Online LC–MS was performed using a hybrid Q-Exactive mass spectrometer (Thermo Scientific). FTMS master scans with 70,000 resolution (and mass range 300–1700 m/z) were followed by data-dependent MS/MS (17,500 resolution) on the top 5 ions using higher energy collision dissociation (HCD) at 30% normalized collision energy. Precursors were isolated with a 2 m/z window. Automatic gain control (AGC) targets were 1e6 for MS1 and 1e5 for MS2. Maximum injection times were 100 ms for MS1 and 500 ms for MS2. The entire duty cycle lasted at most 2.5 s. Dynamic exclusion was used with 60 s duration. Precursors with unassigned charge state or charge state 1 were excluded. An underfill ratio of 1% was used.

Identification and quantification of secreted proteins All MS/MS spectra were searched by Sequest under the software platform Proteome Discoverer (v1.3.0.339, Thermo Scientific) against a fasta file containing the human protein subset of the Swissprot database (2012-10-08) using a 1% FDR at peptide level. Precursor mass tolerance of 10 ppm and product mass tolerance of 0.02 Da were used. Oxidation of methionine was used as variable modification and carbamidomethylation of cysteine used as fixed modification. Precursor peak areas were quantified using

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the “precursor ions area detector” module of Proteome Discoverer. This module averages the top three precursor ion areas to obtain the protein area value. To express quantitative changes of proteins identified in experimental groups from LC–MS runs, every protein area was normalized to the total sum of areas of all identified proteins. Only proteins present in at least two biological replicates were included in the quantitative analysis, and expressed as Log2 ratio, e.g, Log2(KCl/Con) [27]. To avoid taking logarithms for zero values, we set the arbitrary value of 46,171 and 16,750 for MIN6m9 and GIST882 respectively if no peptide was identified in an experimental group, which was half the smallest value among all normalized protein areas. Statistical significance in GIST882 was determined using one-way ANOVA, in MIN6m9 using Student's t-test. p-Values o0.05 were considered significant.

Assessment of secretory property of proteins Classical secretory proteins (SPþ, endoplasmic reticulum/Golgidependent pathway) with a N-terminal signal sequence were predicted using SignalP 4.1 software [28], accessed from http:// www.cbs.dtu.dk/services/SignalP/. SignalP predicts the existence and localization of signal peptide cleavage sites by using a combination of several artificial neural networks. Proteins were considered classically secreted if they had a signal peptide probability (D-cutoff score) Z0.45. Non-classically secreted pro teins (SP  ) were predicted by SecretomeP 2.0 [29] (http://www. cbs.dtu.dk/services/SecretomeP/), that obtains a combination of post-translational and locational protein characteristics to deter mine if a protein is non-classically secreted. Additionally, proteins with Gene Ontology (GO) annotation “extracellular region” were also considered secretory proteins (SP  ) [30]. To see if the identified proteins clustered differently between the cultured conditions, they were classified according to biological processes by using Database for Annotation, Visualization and Integrated Discovery (DAVID). This software utilizes the Fisher exact test to assess a protein's enrichment according to annotation terms [31]. Furthermore, cancer cells commonly secrete or shed exosomal proteins extracellularly as a mean to convey information between cells [32]. We therefore also compared the proteins found against the exosomal proteins reported in Exocarta (http://www.exocarta. org/) [33].

Western blot analysis Western blot analysis was carried out on the conditioned serumfree media (40 mg) from the GIST882 cells to validate the results. The GIST882 cells were homogenized in NP-40 Lysis buffer (Life Technologies) with the addition of protease inhibitor (Complete protease inhibitor cocktail; Roche) and 1 mM of phenylmethanesulfonyl fluoride (Sigma-Aldrich) to verify the existence of the proteins also within the GIST882 cells. Quantification was done using a Pierce™ BCA Protein Assay Kit (Thermo Scientific). The proteins was separated by Novexs NuPAGEs 12% Bis-Tris gels and transferred to nitrocellulose membranes (Life Technologies). After blocking with 5% non-fat milk diluted in TBS, membranes were incubated with the following antibodies: anti-PEA15 (#2780, Cell Signaling Technology) at 1:250 dilution, anti-HSP60 (#12165, Cell Signaling Technology) at 1:500 dilution, followed by anti-rabbit IgG-HRP (#170-6515; Bio-Rad Laboratories) at dilution 1:3000, as

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

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secondary antibody. Alternatively, anti-HSP27 (#2402, Cell Signaling Technology) at 1:500 dilution, followed by anti-mouse IgGHRP (A9044; Sigma) at dilution 1:10,000, as secondary antibody. Detection was carried out with Novex ECL HRP chemiluminescent substrate reagent (#WP20005; Life Technologies). Precision Plus Protein All Blue Standards (#161-0373; Bio-Rad) was used to determine relative molecular weights.

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MIN6m9 cells

GIST882 cells

Static incubation1 Collection of conditioned buffer/media Sample preparation

Results in solution tryptic digestion The methodological approach to profile secretome proteins by mass spectrometry after stimulation was first established and validated using a murine insulin-secreting pancreatic β-cell line (MIN6m9) containing a known secretory signature in response to glucose stimulation. The GIST882 secretome was subsequently analyzed following similar principles. The experimental strategy used to analyze the secretomes is outlined in Fig. 1.

Validation of secretome in MIN6m9 cells Proteins released into the extracellular buffer by MIN6m9 cells at basal (0.1 mM) and stimulatory (25 mM) glucose conditions (the latter of which is well known to increase insulin secretion) were compared. Following static incubation for 30 min, a total of 531 proteins were identified by mass spectrometry in at least two biological replicates (Supplementary Table 1S). We have classified proteins whose levels were increased or decreased according to quantitative changes and statistical significance (Log2 RZ 1.0 or Log2 Rr 1.0, p-Valueo0.05). Stimulation with glucose increased specifically the release of a subset of 19 proteins, mostly characterized by a signal peptide (73.7%) and by the annotation in Gene Ontology (GO) as “extracellular region” (73.7%) (Table 1). At the same time there was a significant decrease in the level of six proteins (Supplementary Table 2S). As expected, the majority of the proteins that were elevated by glucose stimulation have been reported to be localized in the insulin granules and to be cosecreted with insulin [26,34–36]. To confirm these results we measured the insulin concentration with the ArcDia TPX insulin assay. Stimulation with glucose for 30 min induced a 4-fold increase in the extracellular insulin level. The released insulin was 0.47270.078 ng/104 cells and 1.89270.216 ng/104 cells at 0.1 mM and 25 mM glucose, respectively (***po0.001, n ¼3).

strong cation exchange solid phase extraction LC-MS/MS analysis Validation by insulin determination

Informatics

Western blot validation

Lists of secreted proteins Fig. 1 – Workflow applied to evaluate the secretome from MIN6m9 and GIST882 cells. Conditioned media from the two cell lines were collected, centrifuged, concentrated and clarified as described in the Material and Methods section. The processed supernatant was then reduced, denatured, trypsinized and cleaned up with strong cation exchange solid phase extraction before subjecting the fractions to LC-MS/MS analysis in technical triplicates using a hybrid Q-Exactive mass spectrometer. The data were searched using the Proteome Discoverer software, normalized and quantitatively expressed as Log2 ratio. Data collected from MIN6m9 cells was validated using the ArcDia TPX insulin assay for insulin concentration determination, while the data from GIST882 cells was validated through Western blot verification of some of the quantitatively most expressed proteins detected by proteomics. 1MIN6m9 cells were stimulated for 30 min by 25 mM glucose, preceded by 1 h starvation in 0.1 mM glucose (n¼3). GIST882 cells were statically stimulated by adding 60 mM KCl (n¼ 3) or 2 lM thapsigargin for 5 min (n¼ 4). To evaluate if imatinib possessed modulatory effects on cell secretion GIST882 cells were subjected to 1100 ng/ml imatinib for 2 h before adding 2 lM thapsigargin for 5 min (n¼ 4).

Secretome of the GIST882 cell line GIST882 cells represent a primary human imatinib-sensitive GIST in vitro model for evaluation of the existence of a putative GIST cell secretome. In total, LC-MS/MS analysis identified 764 proteins in the conditioned media from all groups combined (Fig. 2). After excluding proteins not found in at least two biological replicates, 555 proteins were left for quantitative analysis. Among these 555 proteins, 55 (9.9%) were classically secreted proteins (SPþ), 221 (39.8%) were non-classically released (SP  ), 88 (15.9%) were nonsecretory, and 360 (64.9%) were of exosomal origin. Among the non-secretory, SPþ, and SP  proteins there were overlaps of 68.5%, 69.1%, and 59.3% with exosomal proteins, respectively. Under basal conditions (culture medium) 375 proteins could be detected, of which 50.3% were classically (9.4%) and nonclassically (40.9%) secreted. The secretory proteins in basal

conditions clustered according to biological processes by DAVID into functions responsible for e.g. regulation of apoptosis, cell growth, MAP kinase activity, immune response, development and response to wounding, organic substance, hormone stimulus, oxidative stress, endogenous and external stimuli (Table 4). Next, to define the proteins with increased secretion upon activation we compared proteins at basal (culture medium) and stimulatory conditions (stimulated by KCl or thapsigargin) known to prompt a rise in free cytoplasmic Ca2þ concentration ([Ca2þ]i) and ATP release from GIST882 cells [9]. Complete lists of quantitated proteins are provided in Supplementary Tables 3–6. We found 398 proteins in the KCl stimulated samples (Supplementary Table 3S). Among these, 200 were found to be secretory, 39 SPþ and the remaining 161 proteins were SP  . Based on quantitative changes and statistical significance (Log2 RZ1.0 or Log2 Rr  1.0, p-Valueo0.05)

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

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Table 1 – Proteins with significantly increased release from MIN6m9 cells after glucose stimulation. Accession number

Glucose stimulation 25 mM vs. 0.1 mMa

Protein name

(Uniprot)

Log2 Rb

p-Valuec

Secretoryd

Insulin-like growth factor II Neuroendocrine convertase 2 Pro-neuropeptide Y ProSAAS Neuroendocrine protein 7B2 GDP-L-fucose synthase Neuroendocrine convertase 1 26S proteasome non-ATPase regulatory subunit 13 Calcium/calmodulin-dependent protein kinase type II subunit alpha Histone deacetylase 6 Mitotic checkpoint protein BUB3 Carboxypeptidase E Insulin-2 Secretogranin-3 Secretogranin-2 Proenkephalin-B Islet amyloid polypeptide Secretogranin-1 Chromogranin-A

P09535 P21661 P57774 Q9QXV0 P12961 P23591 P63239 Q9WVJ2 P11798 Q9Z2V5 Q9WVA3 Q00493 P01326 P47867 Q03517 O35417 P12968 P16014 P26339

8.98 8.47 8.03 7.27 7.17 7.03 6.95 6.74 6.73 6.28 6.20 2.72 2.51 2.24 2.16 1.90 1.79 1.66 1.50

0.008 o0.001 0.013 0.036 0.001 0.016 0.017 0.002 0.004 0.042 0.019 0.007 0.036 0.001 o0.001 o0.001 0.002 o0.001 0.040

SPþ SPþ SPþ SPþ SPþ SP  SPþ SP  SP  SP  SP  SPþ SPþ SPþ SPþ SPþ SPþ SPþ SPþ

a

Comparison of the proteins present in the extracellular buffer after exposure to either 0.1 or 25 mM glucose for 30 min (n ¼ 3). Quantitative changes expressed by log2 ratio. Cutoff threshold for significant changes: Log2 RZ 1.0. c Only peptides with a significant (po0.05, Student's t-test) increase are included. d Classical secretory proteins containing a signal peptide in the protein sequence were predicted with SignalP 4.1 (SPþ). Non-classical secretory proteins were predicted by SecretomeP 2.0 or by the term “extracellular region” in the Gene Ontology database (SP  ). b

only five of the secretory proteins were increased upon stimulation (Table 2). The short isoform of RNA-binding protein FUS was shared with the thapsigargin stimulated group, while Septin-2 and Tropomyosin alpha-3 chain were shared with the imatinib and thapsigargin stimulated group. In the secretome analysis using thapsigargin as stimulation 432 proteins were identified in all replicate experiments (Supplementary Table 4S). Of these 217 proteins were predicted as secretory by either classical (43) or non-classical (174) mechanisms. Subsets of significantly changed proteins are presented in Table 2 and Supplementary Table 5S. Unconditioned culture media was searched for any background signals to exclude contaminations originating from the sample preparations.

Secretome of imatinib-treated GIST882 cells There is a growing understanding that manipulation of the c-KIT receptor can alter secretion activity [37]. We therefore sought to investigate if pre-incubation with therapeutic levels of imatinib, 1100 ng/ml for two hours, before thapsigargin stimulation could change the secretory pattern. In this group, 526 proteins were detected in total, whereof 258 were considered secretory by classical (50) and non-classical (208) pathways (Supplementary Table 6S). Compared to thapsigargin stimulation this led to a radical quantitative change in released proteins (Table 2, Supplementary Table 5S). Insulin-like growth factor-binding protein 7, neutral alpha-glucosidase AB and adenylyl cyclaseassociated protein 1 were significantly changed (Log2 RZ1.0) in both groups (Table 2). Furthermore, DAVID functional annotation showed that proteins exclusive for imatinib treatment were

enriched for translational and transcriptional properties (Table 3), in contrast to the functions of the proteins secreted in un-stimulated samples (Table 4).

Validation of proteomic data Results obtained from LC-MS/MS were compared to some of the highest expressed proteins in the conditioned media by using Western blot as an orthogonal method. First, the antibody dilutions was optimized, with HSP60 and PEA15 being positive in both cell lysates and all stimulatory conditions, while HSP27 was positive in cell lysate (n¼1, data not shown). Next, the experiments were repeated in triplicates, and as expected, HSP60, HSP27, and PEA15 were detected in all cell lysates. Moreover, in agreement with the mass spectrometry data, HSP60 and HSP27 were detected in the GIST882 secretome from the thapsigargin and the combined imatinib/thapsigargin stimulated groups. In samples containing culture medium (CM) or potassium, only LC-MS/MS was sensitive enough to detect HSP60 and HSP27 in the secretome. PEA15 was found by both methods in potassium and thapsigargin stimulated groups, but only by Western blot in samples containing CM or stimulated by both thapsigargin and imatinib (Fig. 3).

Discussion The first secretome study was published about a decade ago, and technological advances since then have resulted in the identification of secreted biomarker proteins from several different cancer

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

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Fig. 2 – Visualization of all proteins identified in conditioned media from the GIST882 cell line. In total, 764 proteins were identified from all conditions. Protein interaction networks based on respective gene names were generated by the open source software platform Cytoscape. CM; culture medium, Tg; thapsigargin, IM; imatinib.

types. Mass spectrometry-based proteomic analyses of conditioned media, under well-defined experimental conditions, have aided the biomarker discovery significantly. Several of the candidates have also been validated in clinical samples [38]. GIST882 is the first established immortalized GIST cell line, representing an imatinib-sensitive cell line with a KIT mutation, and is commonly used for in vitro studies of GIST [24]. Although no single cell line can mimic the heterogeneity seen among tumors, we chose GIST882 knowing that 80% of GISTs are imatinib-sensitive and contain KIT mutations, much like GIST882. Our aim was to determine if GIST cells actively secrete proteins, which could serve as guidance for targeted analysis of patient serum or plasma. By applying a cell culture model system the secretome from GIST882 was determined using LC–MS, and the findings were compared to Western blot findings. To validate the methodology of peptide secretion by proteomics we used a pancreatic β-cell line, MIN6m9 cells, exposed to different glucose concentrations as a model system. In accordance with the literature, we detected not only an increase in insulin in the extracellular buffer, but also higher concentrations of proteins that have been previously reported to be present in the insulin granules of the β-cell, such as insulin-like growth factor II, proenkephalin-B and islet amyloid polypeptide, as well as

members of the extended granin family (chromogranin A, neuroendocrine protein 7B2/secretogranin 5, proSAAS, segretogranin  1, 2, and  3) [34]. Several of the released granule components are involved in (pro)insulin processing (neuroendocrine convertase 1/proprotein convertase 1, neuroendocrine convertase 2/proprotein convertase 2, carboxypeptidase E, neuroendocrine protein 7B2/secretogranin 5 and proSAAS ) [26,39] and modulation of β-cell function (islet amyloid polypeptide/ amylin, insulin-like growth factor II, proenkephalin-B and proneuropeptide Y) [36,40–44]. Most of these factors are also present in the granule proteomes of the insulin-producing INS-1E cells [35]. In summary, the majority of the proteins that were elevated by glucose stimulation in our experiments are known to be part of the secretory process and/or associated to insulin granules. Based on these findings, we conclude that the proteomic experimental approach is appropriate for exploring possible regulated protein release from GIST cells. In a number of tissues intracellular Ca2þ is the second messenger in control of secretory events. Stimulus-secretion coupling commonly refers to the increase in ([Ca2þ]i), and we have previously found that KCl and thapsigargin effectively triggers a brisk increase in cytoplasmic free Ca2þ concentrations and ATP release from GIST882 cells [9]. By exposing the cells to the same

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700

E X PE R IM EN TA L C ELL R E S EA RC H

701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760

Q4

7

] (]]]]) ]]]–]]]

Table 2 – Stimulation increases the release of subsets of proteins from the GIST882 cells. Protein name

Potassiume Isoform short of RNA-binding protein FUS Isoform 2 of glucosidase 2 subunit beta Septin-2 Tropomyosin alpha-3 chain Isoform 2 of Tryptophan-tRNA ligase, cytoplasmic

Thapsigarginf Isoform 3 of DENN domain-containing protein 5B Trypsin-1 Isoform 2 of inter-alpha-trypsin inhibitor heavy chain H4 Isoform short of RNA-binding protein FUS Astrocytic phosphoprotein PEA-15 Calpain small subunit 1 Isoform 2 of proteasome subunit alpha type-3 Isoform 2 of proteasome subunit beta type-5 Apolipoprotein A-I Isoform cytoplasmicþperoxisomal of peroxiredoxin-5, mitochondrial Glutathione S-transferase Mu 3 NEDD8 Fibulin-1 Glutaredoxin-1 D-dopachrome decarboxylase Dihydropyrimidinase-related protein 2 Isoform 2 of protein CDV3 homolog Adenylyl cyclase-associated protein 1 Adenylate kinase isoenzyme 1 Serine-tRNA ligase, cytoplasmic Barrier-to-autointegration factor Neutral alpha-glucosidase AB Peptidyl-prolyl cis-trans isomerase FKBP4 Insulin-like growth factor-binding protein 7 Acyl-CoA-binding protein Imatinib followed by thapsigarging Nuclease-sensitive element-binding protein 1 Proteasome subunit alpha type-5 Protein disulfide-isomerase Transcription elongation factor B polypeptide 1 40S ribosomal protein S20 Aldose reductase Succinyl-CoA:3-ketoacid coenzyme A transferase 1, mitochondrial Glutamate dehydrogenase 1, mitochondrial Mesencephalic astrocyte-derived neurotrophic factor Isoform 3 of Src substrate cortactin Protein Niban Isoform short of ES1 protein homolog, mitochondrial Alcohol dehydrogenase class-3 Keratin, type I cytoskeletal 9 Chloride intracellular channel protein 4 Histidine triad nucleotide-binding protein 2, mitochondrial Isoform C of Prelamin-A/C Septin-2 Glyoxylate reductase/hydroxypyruvate reductase ATP synthase subunit alpha, mitochondrial Isoform CD34-T of hematopoietic progenitor cell antigen CD34 40S ribosomal protein S19 50 (30 )-deoxyribonucleotidase, cytosolic type GrpE protein homolog 1, mitochondrial Cyclin-dependent kinase inhibitor 2A, isoforms 1/2/3 26S proteasome non-ATPase regulatory subunit 6 Isoform 3 of aldehyde dehydrogenase family 16 member A1

Accession number (Uniprot)

Log2 Ra

pValueb

P35637-2 P14314-2 Q15019 P06753 P23381-2

14.49 13.67 12.19 1.60 1.35

0.012 0.008 0.003 0.042 0.009

Q6ZUT9-3 P07477 Q14624-2 P35637-2 Q15121 P04632 P25788-2 P28074-2 P02647 P30044-2

16.49 14.93 14.68 14.59 14.00 13.53 12.68 10.30 5.88 3.56

0.050 0.040 o0.001 0.005 o0.001 0.008 0.010 0.039 o0.001 0.005

P21266 Q15843 P23142 P35754 P30046 Q16555 Q9UKY7-2 Q01518 P00568 P49591 O75531 Q14697 Q02790 Q16270 P07108

3.54 3.25 3.19 2.32 2.14 2.03 1.78 1.64 1.59 1.57 1.54 1.44 1.36 1.18 1.01

o0.001 0.016 o0.001 0.012 0.011 o0.001 0.029 0.030 0.023 0.009 0.030 0.033 0.020 0.002 0.002

P67809 P28066 P07237 Q15369 P60866 P15121 P55809 P00367 P55145 Q14247-3 Q9BZQ8 P30042-2 P11766 P35527 Q9Y696 Q9BX68 P02545-2 Q15019 Q9UBQ7 P25705 P28906-2 P39019 Q8TCD5 Q9HAV7 P42771 Q15008 Q8IZ83-3

14.88 14.28 13.41 13.28 13.27 13.20 13.19 13.17 13.04 12.94 12.80 12.51 12.50 12.44 12.39 12.33 12.19 12.10 11.92 11.91 11.89 11.85 11.61 11.58 11.44 11.43 11.38

o0.001 0.002 0.004 o0.001 o0.001 o0.001 o0.001 0.016 0.007 o0.001 o0.001 0.001 0.033 0.003 0.031 0.010 0.032 0.003 o0.001 0.045 0.030 o0.001 0.045 0.035 0.042 0.037 0.001

Secretoryc

SPþ SP 

SP  SPþ SPþ SP  SP  SP  SPþ SP 

SPþ

ExoCartad

O O O O O

O O O O O O O O O O O O O

SP  SP  SP  SPþ SPþ SP  SP  SPþ SP  SP  SPþ

O O O O O O O

O O O O O O O

SPþ O O SPþ

SP  SP 

SP  SPþ SP  SP  SP  SP  SP 

O O O O O O O O O

O O

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820

8

821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880

E XP E RI ME N TAL CE L L R ES E ARC H

] (]]]]) ]]]–]]]

Table 2 (continued ) Protein name

Accession number (Uniprot)

Log2 Ra

pValueb

Secretoryc

ExoCartad

Galectin-3 Nebulin ATP synthase subunit beta, mitochondrial Spectrin beta chain, non-erythrocytic 1 Adenylyl cyclase-associated protein 1 Tropomyosin alpha-3 chain Isoform 2 of Tropomyosin alpha-1 chain Neutral alpha-glucosidase AB Malate dehydrogenase, mitochondrial Dynactin subunit 2 UPF0556 protein C19orf10 Isoform 2 of F-actin-capping protein subunit beta Aspartate aminotransferase, mitochondrial Insulin-like growth factor-binding protein 7 30 (20 ),50 -bisphosphate nucleotidase 1 Aldehyde dehydrogenase, mitochondrial T-complex protein 1 subunit theta Vimentin Protein DJ-1

P17931 P20929 P06576 Q01082 Q01518 P06753 P09493-2 Q14697 P40926 Q13561 Q969H8 P47756-2 P00505 Q16270 O95861 P05091 P50990 P08670 Q99497

11.25 10.13 2.61 1.89 1.82 1.74 1.62 1.46 1.38 1.36 1.27 1.22 1.20 1.09 1.08 1.08 1.05 1.05 1.04

o0.001 0.041 0.011 0.049 0.009 0.011 0.040 0.028 0.001 0.022 0.017 0.042 0.027 0.005 0.008 0.003 0.046 0.004 0.003

SP 

O O O O O O O O O O

SP 

SP  SP  SPþ SP  SPþ SP  SP  SPþ SP  SP  SP  SP 

O O O O O O O

a

Significant quantitative changes expressed by log2 ratio. Cutoff threshold for significant changes: Log2 R Z 1.0. Only peptides with a significant (po0.05, ANOVA) increase are included. c Classical secretory proteins containing a signal peptide in the protein sequence were predicted with SignalP 4.1 (SPþ). Non-classical secretory proteins were predicted by SecretomeP 2.0 or by the term “extracellular region” in the Gene Ontology database (SP  ). d Enriched exosomal proteins identified by ExoCarta. e Proteins enriched extracellularly after 60 mM potassium (KCl) exposure for 5 min (n ¼3), compared to control (n ¼3). f Proteins enriched extracellularly after 2 μM thapsigargin exposure for 5 min (n ¼ 4), compared to control (n ¼3). g stimulatory conditions we hypothesized would non-classical proteins canthapsigargin potentially5 occur through(n4), thecompared release of Proteins present extracellularly after two hoursthis of 1100 ng/ml increase imatinib pre-incubation followed by 2 μM min exposure protein secretion. nano-meter sized exosomes or microvesicles (MVs), together to control (n3). In total, the proteomic characterization of the b

Table 3 – Functional clustering of secretory proteins exclusively found in imatinib pre-treated GIST882 samples followed by thapsigargin stimulation. Biological function

p-Value

Molecules

Count 17

Translation

0.001

Regulation of RNA metabolic process Regulation of transcription, DNAdependent RNA biosynthetic process Transcription

0.001

Translational elongation Regulation of transcription

0.011 0.020

Ribosome biogenesis Regulation of transcription from RNA polymerase II promoter Negative regulation of nitrogen compound metabolic process Negative regulation of RNA metabolic process

0.027 0.032

P60866, P49411, Q12904, P82909, P39019, Q9NSE4, P54577, P23588, Q15046, O60739, Q8N5X7, P62241, P42677, P23396, P62854, P26373, P56537 Q15369, P52597, P12956, Q13185, P27797, Q99471, P42771, Q13951, Q9Y2T7, P67809, P62854, P63279, P46108, Q13263 Q15369, Q9Y2T7, P12956, P67809, Q13185, Q99471, P27797, P42771, P63279, P46108, Q13263, Q13951 O00625, Q9Y2T7, P67809, P42771, P10155, Q13951 Q15369, O00625, Q9Y2T7, P67809, Q13185, Q92945, P42771, P10155, Q9Y6K9, Q13263, Q13951 P60866, P49411, P39019, P62241, P42677, P23396, P62854, P26373 Q15369, P12956, Q13185, Q99471, P27797, P42771, Q13951, Q9Y2T7, P67809, Q92945, P23396, Q9Y6K9, P63279, P46108, Q13263 P39019, Q8NI36, P42771, P56537 Q15369, P12956, P27797, P46108, Q13263, Q13951

0.032

Q13185, P27797, P63279, P23396, P62854, Q13263

6

0.033

Q13185, P27797, P63279, P62854, Q13263

5

0.003 0.004 0.005

serum-free CM identified 764 proteins, 51.3% deemed secretory by Gene Ontology annotation, SignalP or SecretomeP terms (Fig. 2). The majority of the secreted species were annotated as released by non-classical mechanisms. The extracellular export of these

14 12 6 11 8 15 4 6

called extracellular vesicles (EVs). MVs are 0.1–1 mm in diameter and form through budding off the plasma membrane into the extracellular milieu [45]. As part of their formation cytosolic components are captured in the lumen and plasma membrane

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940

E X PE R IM EN TA L C ELL R E S EA RC H

941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000

9

] (]]]]) ]]]–]]]

Table 4 – Functional clustering of all GIST882 secretory proteins found in un-stimulated samples. Biological function

p-Value

Response to wounding

0.001

Response to organic substance

0.001

Response to hormone stimulus

0.003

Response to inorganic substance

0.005

Positive regulation of multicellular organismal process Negative regulation of programmed cell death Negative regulation of apoptosis

0.007

Negative regulation of cell death

0.012

Response to endogenous stimulus Regulation of response to external stimulus Immune response

0.017

Response to extracellular stimulus Defense response

0.026

Response to nutrient levels Regulation of cell death

0.029 0.033

Regulation of apoptosis

0.033

Regulation of programmed cell death

0.033

Response to reactive oxygen species Regulation of growth Cellular response to oxidative stress Regulation of cell activation Heart development Regulation of leukocyte activation Regulation of cell growth Liver development Regulation of body fluid levels Regulation of inflammatory response Regulation of MAP kinase activity MAPKKK cascade hemostasis Oxidation reduction

0.012

Molecules

Count

P02751, P02749, P02765, P00441, P14174, P30086, P02461, P09493, P30044, P04179, P01008, P08758, P01024, P07996, P01023, P32119, P0C0L4 Q16270, P00441, P31153, P30153, Q9UBY9, P30086, P00505, P23528, P02461, P05091, P61604, O94788, P30048, P68032, P18065, P09104, P08758, P07996, P01023, P32119, P31146, P02765, P02452, P50454, P10809, O75874, P67775, P02771, P04792, P00918 Q16270, P02765, P31153, P02452, P30086, P05091, O75874, O94788, P18065, P09104, P07996, P01023, P00918 P08294, P00441, P02452, P30086, P62158, P09493, P30044, P04179, P30048, P18065, P07996, P01023, P32119, P00918, Q06830 P09493, P02749, P10809, P00441, P02765, P07996, P00918

17 30

13 15 7

P02749, P02768, P00441, P14174, P23528, P13693, P30044, P10809, P09211, P04179, P30048, P11021, P08758, P07996, P04792, P32119 P02749, P02768, P00441, P14174, P23528, P13693, P30044, P10809, P09211, P04179, P30048, P11021, P08758, P07996, P04792, P32119 P02749, P02768, P00441, P14174, P23528, P13693, P30044, P10809, P09211, P04179, P30048, P11021, P08758, P07996, P04792, P32119 Q16270, P02765, P31153, P02452, P30086, P23528, P05091, O75874, O94788, P18065, P09104, P07996, P01023, P00918 P36955, P02749, P02765, P01008, P01024, P07996, P01023

16

12

0.037

P31146, P10809, P10319, P01893, P14174, P06744, P01024, P07996, P32119, P0C0L4, P68371, Q06830 Q16270, P02768, P04179, P00441, O94788, P01008, P00338, P02452, P18065, P11021, P01023 P02751, P31146, P02765, Q14019, P14174, P68371, P30044, P10319, P01024, P07996, P01023, P32119, P0C0L4, Q06830 Q16270, P02768, P04179, P00441, O94788, P01008, P02452, P18065, P11021, P01023 P00441, P30153, P14174, P23528, P68371, P61604, P51397, P30044, P30048, Q13564, P09382, P11021, O43707, P15559, P08758, P07996, P32119, Q06830, P02768, P02749, P13693, P10809, P09211, P04179, P67775, P04792 P00441, P30153, P14174, P23528, P68371, P61604, P51397, P30044, P30048, Q13564, P09382, P11021, O43707, P15559, P08758, P07996, P32119, Q06830, P02768, P02749, P13693, P10809, P09211, P04179, P67775, P04792 P00441, P30153, P14174, P23528, P68371, P61604, P51397, P30044, P30048, Q13564, P09382, P11021, O43707, P15559, P08758, P07996, P32119, Q06830, P02768, P02749, P13693, P10809, P09211, P04179, P67775, P04792 P09493, P30044, P04179, P00441, P30048, P02452, P32119, Q06830

0.041 0.041

Q16270, P00441, P67775, P06733, P18065, P24593, P30153 P09493, P30044, P04179, P00441, P30048, P32119, Q06830

7 7

0.044 0.044 0.044

P31146, P10809, P00441, P14174, P07996, P32119 P09493, P11310, P04179, O94788, P68032, P02461 P31146, P10809, P00441, P14174, P07996, P32119

6 6 6

0.044 0.045 0.045 0.045

Q16270, P67775, P06733, P18065, P24593, P30153 P11310, P04179, O94788, P02771, P05091 P02749, P01008, P06744, P08758, P02461 P36955, P02765, P01008, P01024, P01023

6 5 5 5

0.045

P00441, P67775, P30153, P07996, P32119

5

0.045 0.045 0.049

P00441, P67775, P30153, P07996, P32119 P02749, P01008, P06744, P08758, P02461 P00441, P09622, Q13162, P16152, P05091, P30043, P11310, P30044, O94788, O43488, P30048, P15559, P32119, Q06830, P08294, P40926, P00390, P00338, O60701, P07195, P31937, O75874, P04179, Q9NVS9, O75828

0.012

0.019 0.022

0.026

proteins in the surrounding membrane. The MV composition and release are dynamic and vary between cell types, activation status, ([Ca2þ]i) concentrations, and the degree of cellular stress

16 16 14 7

11 14 10 26

26

26

8

5 5 25

[46–48]. Furthermore, the lipid bi-layered exosomes (30–100 nm) are smaller than MVs and constitute another potential source of protein markers [49]. Proteins of these origins have been shown

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060

10

+T g IM

G IS T8 82

IS T8 82 G

IS T8 82

+

+

Tg

K+

M C + G

IS T8 82 G

Cell lysate

60 kDa

HSP27

27 kDa

PEA15

15 kDa

HSP60

60 kDa

HSP27

27 kDa

PEA15

15 kDa

HSP60 HSP27 PEA15

Proteomics

Secreted

HSP60

Western blot

1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120

E XP E RI ME N TAL CE L L R ES E ARC H

Fig. 3 – Western blot validation of HSP27, HSP60, and PEA-15 in the secretome of the GIST882 cell line. HSP27; heat shock protein beta-1, HSP60; 60 kDa heat shock protein, PEA-15; astrocytic phosphoprotein, Tg; thapsigargin, IM; imatinib, CM; conditioned media.

to impact tumor physiology, pathogenesis, intercellular communication and disease progression in several cancers, including tumors of the gastrointestinal tract [50] and sarcomas other than GIST [51–53]. By searching Exocarta [33] we found that the majority of all proteins, including the non-classically released proteins, have an exosomal origin (Supplementary Tables 4–7). The possibility of EV existence in GIST cells adds another complexity to the analysis and warrants further investigations. To allow comparisons between the studied conditions the proteins were quantitated as described in the Material and Methods section. By comparing the stimulated groups with the control group we revealed subsets of proteins being significantly released upon stimulation. In fact, the majority of the significantly elevated secretory proteins in the thapsigargin group (Table 2) have already been acknowledged in other tumor types, for example; trypsin and PEA-15 in colon cancers [54,55], RNAbinding protein FUS (fused in sarcoma) in myxoid liposarcoma [56], calpain small subunit 1, insulin-like growth factor-binding protein 7, and Acyl-CoA-binding protein in hepatocellular carcinomas [57–59], proteasome subunit alpha type-3 in esophageal cancer [60], inter-alpha-trypsin inhibitor heavy chain H4, fibulin1, and protein CDV3 homolog in breast cancer [61–63], Apo A-I in ovarian cancer [64], Peroxiredoxin-5 in human lung carcinoma cells [65], glutathione S-transferase Mu 3 in gastric cancer [66], and neutral alpha-glucosidase AB in head and neck cancer [67]. It is also noteworthy that blocking the mutated c-KIT protein with imatinib radically changed the protein release pattern (Tables 2 and 4, Fig. 2). This could have clinical implications when searching for biomarkers in imatinib-treated versus non-treated GIST patients. Interestingly, the most increased protein following imatinib treatment was nuclease-sensitive element-binding protein 1 (Y-box-binding protein 1) which is known for its involvement in the acquisition of global drug resistance through increased MDR1 (ABCB1) expression [68]. KCl treatment on the

] (]]]]) ]]]–]]]

other hand induced only minor quantitative changes (Table 2), where Septin-2 has been implicated in renal cell carcinomas [69], tropomyosin-3 in hepatocellular carcinoma [70], and tryptophantRNA ligase in colorectal cancer [71]. The greater effect observed with thapsigargin treatment can possibly be explained by an irreversible block of the SERCA pump which prolongs Ca2þ signaling and consequently also cell secretion, while depolarization of the plasma membrane by high KCl causes a transient ([Ca2þ]i) increase [72]. Proteins expressed at high levels in each group, as detected by proteomics, were compared to Western blot analysis (Fig. 3). Although the analysis of conditioned media has proven to be a successful strategy the method has some apparent drawbacks. Cell cultures are in vitro discovery systems that differ at the molecular level from in vivo situations. Even though this makes direct correlations between conditioned media and clinical samples difficult, it enables the determination of low abundant proteins, which is a challenge when using proteomics-based methods on complex body fluids. There are studies suggesting that stromal cells in the tumor microenvironment might be important for the tumor cell secretome [73]. Since GIST is a stromal tumor, co-culture of GIST cells with a stromal component could possibly change or enhance the protein secretion even further. In conclusion, we show for the first time that GIST cells indeed contain a secretome signature made up by both classically and non-classically released proteins. In the search for suitable biomarkers in the more complex GIST patient samples, this study aids in the understanding of basic GIST secretome characteristics.

Conflict of interest The authors declare no conflict of interest.

Acknowledgments The study was financially supported by the Swedish Research Council, the Novo Nordisk Foundation, the Swedish Cancer Society, funds from Karolinska Institutet, the Swedish Society of Medicine (Bengt Ihre grant), the Tore Nilsson Foundation, the Thuring Foundation, the Jeansson Foundation, Magn. Bergvall Foundation, and the Cancer Research Foundations of Radiumhemmet. Financial support was also provided through the Regional Agreement on Medical Training and Clinical Research (ALF) Q2 between the Stockholm County Council and Karolinska Institutet. Q3

Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2015.05.004.

references [1] WHO Classification of Tumours of Soft Tissue and Bone. Pathology and Genetics of Tumours of Soft Tissue and Bone, in: B.J. Fletcher CDM, Hogendoorn PCW, F Mertens (Eds.), IARC Press, Lyon, 2013.

Please cite this article as: E. Berglund, et al., Secretome protein signature of human gastrointestinal stromal tumor cells, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.004

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