Extracellular Matrix Remodeling by Bone Marrow ... - ACS Publications

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Extracellular Matrix Remodeling by Bone Marrow Fibroblast-like Cells Correlates with Disease Progression in Multiple Myeloma Astrid Slany,† Verena Haudek-Prinz,† Anastasia Meshcheryakova,‡ Andrea Bileck,† Wolfgang Lamm,§ Christoph Zielinski,§ Christopher Gerner,†,* and Johannes Drach§ †

Faculty of Chemistry, Institute of Analytical Chemistry, University of Vienna, Währingerstraße 38, A-1090 Vienna, Austria Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Austria § Department of Medicine I, Division of Oncology, Medical University of Vienna, Austria ‡

S Supporting Information *

ABSTRACT: The pathogenesis of multiple myeloma (MM) is regarded as a multistep process, in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. Molecular events characteristic for the transition from MGUS to MM are still poorly defined. We hypothesized that fibroblast-like cells in the tumor microenvironment are critically involved in the pathogenesis of MM. Therefore, we performed a comparative proteome profiling study, analyzing primary human fibroblastlike cells isolated from the bone marrow of MM, of MGUS, as well as of non-neoplastic control patients. Thereby, a group of extracellular matrix (ECM) proteins, ECM receptors, and ECM-modulating enzymes turned out to be progressively upregulated in MGUS and MM. These proteins include laminin α4, lysyl-hydroxylase 2, prolyl 4-hydroxylase 1, nidogen-2, integrin α5β5, c-type mannose receptor 2, PAI-1, basigin, and MMP-2, in addition to PDGF-receptor β and the growth factor periostin, which are likewise involved in ECM activities. Our results indicate that ECM remodeling by fibroblast-like cells may take place already at the level of MGUS and may become even more pronounced in MM. The identified proteins which indicate the stepwise progression from MGUS to MM may offer new tools for therapeutic strategies. KEYWORDS: multiple myeloma, tumor−stroma interactions, primary human cells, comparative proteome profiling

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transition from MGUS to MM.5 Until now no adequate molecular or immunohistochemical markers exist that would allow a reliable differentiation between MGUS and MM or prediction of the transition from MGUS to MM.6 We hypothesize that MM progression isat least to a certain degreedependent on the tumor microenvironmentbased on several recent observations.7−13 Indeed, complex mutual interactions in the BM between myeloma cells and surrounding cells, such as stromal cells, endothelial cells, as well as osteoblasts and osteoclasts, have recently been recognized. The cellular communication is most probably required for tumor cell survival, growth, and differentiation.14 The fact that MM cells cannot live in culture by themselves but need a feeder-layer of stromal cells to survive in vitro reinforces the importance of the tumor microenvironment.15 Especially fibroblast-like cells have been described to be involved in the support of myeloma cells.9,16 These stromal cells are furthermore the main contributors of the BM

INTRODUCTION Multiple myeloma (MM), the second most frequent hematological cancer, is characterized by a clonal proliferation of malignant plasma cells within the bone marrow (BM). Clinical manifestations of MM include characteristic features, such as occurrence of nonfunctional monoclonal immunoglobulins, which appear as monoclonal “spikes” in the serum and/or urine, as well as osteolytic bone lesions. Despite recent advances in understanding the disease pathogenesis and in developing novel therapies, MM remains incurable with a median overall survival time of 5 to 6 years.1 MM develops from a premalignant disorder called monoclonal gammopathy of undetermined significance (MGUS), which is characterized by low concentrations of clonal immunoglobulin and may progress to MM with a probability of 1% per year.2 Cytogenetic and molecular studies indicate that chromosomal abnormalities which lead to karyotypic instability already occur in MGUS plasma cells and comprise deletions of 13q and primary immunoglobulin translocations.3 It is now evident that essentially all cases of MM are consistently preceded by MGUS,4 but little is known about the © 2013 American Chemical Society

Received: August 28, 2013 Published: November 20, 2013 844

dx.doi.org/10.1021/pr400881p | J. Proteome Res. 2014, 13, 844−854

Journal of Proteome Research

Article

Medium for 5 min at room temperature, and again washed twice with PBS/BSA/Azid. The following murine monoclonal antibodies (mABs) were used: FITC-conjugated mAbs specific for CD90 (Acris, SM1170F, 1:5), CD45 (Acris, SM3025F, 1:5), CD31 (Acris, BM4047F, 1:5), and CD34 (BD Pharmingen, # 555821, 1:5); phycoerythrin (PE)-conjugated mAbs specific for CD90 (Eubio, SM1170R, 1:3), Fibroblasts/Epithelial cells (FE, Acris, SM1214R, 1:20), and CD54 (BD Pharmingen, # 555511, 1:20). Flow cytometric analysis was performed using a LSRII instrument (BD Biosciences) and the FlowJo software (TreeStar).

extracellular matrix (ECM), which is part of the BM microenvironment and includes fibronectin, laminins, collagens, and proteoglycans. Several reports describe the involvement of fibronectin, as well as of laminins and their receptors (intergrins), in the expansion of diverse tumors,16−19 suggesting that tumor development must go along with ECM remodeling.10 Degradation of collagens in the BM ECM by matrix metalloproteinases (MMPs) has been shown to contribute to MM progression.11 Moreover, the ECM can provide a protective environment against drug effects and thereby allow resistant myeloma cells to emerge.20−22 Fibronectin, for example, has been described to be directly involved in the mechanism of drug resistance of MM cells.22 We intended to investigate in more detail the role of stromal cells in the progression from MGUS to MM. For this purpose, we isolated primary human fibroblast-like cells from BM aspirates of MM and MGUS patients, as well as of control patients without evidence for neoplastic disease, and we performed comparative proteome profiling, as described previously.23−26 Gene expression and antibody array analyses have been conducted for this purpose;10,12,27 however, proteomic analyses may give more comprehensive information about the expression and regulation of relevant proteins and may reveal diagnostic, prognostic, and predictive biomarkers.28,29 The present proteomic study aims to investigate the role of BM fibroblast-like cells in MM progression, focusing on their role in expressing ECM- and ECM-related proteins.

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Cell Fractionation

To obtain the fraction of secreted proteins, cells were washed with EBM-2 (Lonza) without FCS and then cultivated for 24 h in this serum-free medium. After cultivation, the supernatant was collected, sterile filtered (0.2 μm, FP POINT 2-S, Schleicher & Schuell, Whatman) to remove cellular debris, and precipitated by addition of ice-cold ethanol overnight. For the isolation of cytoplasmic proteins, cells were lysed in lysis buffer (10 mM HEPES/NaOH, pH 7.4, 0.25 M sucrose, 10 mM NaCl, 3.5 mM MgCl2, 0.5% Triton X-100, 1 mM EGTA) including protease inhibitors, and fibroblasts were pressed 12 times through a 23g syringe to induce cell lysis.23 The cytoplasmic fraction was separated from nuclei by centrifugation at 2300g for 5 min and precipitated by the addition of icecold ethanol overnight. To obtain the nuclear extract, the remaining pellet was lysed for 10 min on ice with 100 mM Tris/HCl pH 7.4, 1 mM EDTA, pH 7.5, 500 mM NaCl (including protease inhibitors), and afterward diluted in 10 mM Tris/HCl, pH 7.4, 1 mM EDTA pH 7.5, 0.5% NP-40 (including protease inhibitors), and kept on ice for a further 15 min. After centrifugation at 2300g for 5 min the proteins in the supernatant were precipitated by addition of ice-cold ethanol overnight. Afterward, proteins of all fractions were pelletized by centrifugation for 20 min at 4750g at 4 °C and dissolved in sample buffer (7.5 M urea, 1.5 M thiourea, 4% CHAPS, 0.05% SDS, 100 mM DDT) at concentrations of 1− 12 μg/μL.

MATERIALS AND METHODS

Cell Isolation

Fibroblast-like cells were isolated from BM aspirates of six MM, as well as three MGUS patients. Control cells were obtained from BM of three patients tested for MM, but without evidence for neoplastic disease. BM samples were isolated with written consent of each donor and the approval of the Austrian Ethics committee. BM aspirates were filtered through a 40 μm mesh (40 μm Nylon Cell Stainer, BD Falcon), previously saturated with fetal calf serum (FCS, ATCC, # 30-2020) for 24 h. The residue in the filter, including fibroblast-like cells enclosed in fat micelles, was washed out into a culture flask using fibroblast basal medium (FBM, Lonza Clonetics, # CC-3131) supplemented with one FGM BulletKit (Lonza Clonetics, # CC3130) and 10% FCS. The culture flask was placed in an incubator at 37 °C in a humidified atmosphere containing 5% CO2. After 24 h, the medium was discarded, adherent cells were washed with PBS, and fresh medium was added into the culture flask. Fibroblast-like cells were isolated by outgrowth from the remaining cell culture and were further characterized by FACS analysis.

SDS-PAGE for Subsequent Shotgun Analysis

The different protein fractions were loaded separately on 12% polyacrylamid gels. Electrophoresis was performed until complete separation of a prestained molecular marker (Dual Color, Biorad, Hercules, CA). Proteins in the gels were fixed with 50% methanol/10% acetic acid and subsequently silver stained as described below. Bands were then cut into slices of different molecular weights (for secretome 6 slices, for cytoplasm, and for nuclear extract 8 slices), and proteins were digested with trypsin as described below.

FACS Analysis

MS-Compatible Silver Staining Procedure

After reaching at least 75% confluence, cells were detached by trypsin treatment. Collected cells were washed twice with PBS, centrifuged at 300g for 4 min at 4 °C, resuspended in the appropriate volume of PBS to reach a concentration of 5 × 104 to 5 × 105 cells per 50 μL. To block unspecific binding, 1/10 volume of Beriglobin (CSL Behring; diluted 1:8 in PBS/BSA/ Azid) was added and incubated for 10−15 min on ice. 50 μL cell suspensions were transferred into micronic tubes (Thermo Scientific) followed by the addition of 10 μL of primary antibody (directly fluorochrome conjugated) for 30 min at 4 °C. Subsequently, cells were washed twice with PBS/BSA/Azid (1×PBS; 20% BSA; 0.4% Na3N), fixed with 100 μL of Fixation

1D-gels were washed with 50% methanol followed by bidistilled water. Proteins inside the gels were sensitized with 0.02% Na2S2O3 and stained for 20 min with ice-cold 0.1% AgNO3. Gels were then rinsed with bidistilled water, and silver staining was subsequently developed with 3% Na2CO3/0.05% formaldehyde as previously described.30 Digest with Trypsin

The digest with trypsin was performed as described before.31 In brief, gel slices were further cut into smaller pieces; proteins therein were destained, reduced with DTT, and alkylated with iodacetamide before they were digested with trypsin (sequenc845



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PDGFR-β (rabbit, monoclonal, Cell Signaling #3169, 1:500), laminin α-4 (rabbit, polyclonal, Acris AP20549PU-N, 1:500), PAI-1 (mouse, monoclonal, Abnova H00005054-M01, 1:500), P4HA1 (rabbit, polyclonal, Proteintech 12658-1-AP, 1:500), Chitinase 3-like protein 1 (mouse, monoclonal, Acris AM09070PU-N, 1:500), fibulin-2 (rabbit, polyclonal, Gene Tex GTX105108, 1:1000), as well as a HRP-coupled rabbit antimouse antibody (Bio-Rad #1706516, 1:10000) and a HRPcoupled goat antirabbit antibody (CALBIOCHEM #401353, 1:10000) were used for immunodetection. Bioluminescence signals were developed with SuperSignal West Pico Chemoluminescent Substrate (Thermo fibulinScientific). Ponceau S staining intensities were used for normalization.

ing grade, Roche) overnight at 37 °C. After elution, the peptides were forwarded to LC-MS/MS analysis. Mass Spectrometry

Mass spectrometry was performed as described previously.23 In brief, peptides were separated by nanoflow LC using the HPLC-Chip technology from Agilent, equipped with a 40 nL Zorbax 300SB-C18 trapping column and a 75 μm × 150 mm Zorbax 300SB-C18 separation column. For peptide elution we applied a gradient from 0.2% formic acid and 2% ACN to 0.2% formic acid and 40% ACN over 80 min and a flow rate of 400 nL/min. Peptide identification was accomplished by MS/MS fragmentation analysis with an ion trap mass spectrometer (XCT-Ultra, Agilent) equipped with an orthogonal nanospray ion source. The MS/MS data were interpreted by the Spectrum Mill MS Proteomics Workbench software (Version A.03.03, Agilent) searching against the SwissProt/UniProtKB protein database for human proteins (Version 12/2010 containing 20328 entries). Capillary voltage was set to 1.75 kV, peptides were searched within a m/z range from 400 to 1400, and fragmentation was triggered for the four highest peptide candidates, allowing for three independent fragmentations and using a dynamic exclusion list lasting for 1 min. Precursor mass deviation was limited to a maximum of 1.5 Da, the product mass tolerance to maximal 0.7 Da, and the minimum matched peak intensity (%SPI) to 70%. A peptide was included in the result files when its SpectrumMill score was above 13. Peptides scoring between 9 and 13 were only included if precursor m/z value, retention time, and MS2 pattern matched to a reference spectrum scoring above 13. Concerning protein inference, we chose the smallest number of proteins necessary to explain all observed peptides as described for ProteinProphet.32 Furthermore, only proteins identified with at least two distinct peptides were included. Selection of protein identification was also based on robustness. Only peptide identifications reproduced in at least two different donors were included (Tables S1−S3). The false discovery rate was determined by searching against the corresponding reversed database. Our filtering steps led to peptide identifications with consistently less than 1% apparent identifications when searching against the reversed database compared to the search against the true database, demonstrating high data accuracy. For statistical analysis, we used G-statistics referring to the number of distinct peptide identifications per protein (SPSS Statistics software version 17.0). Box plots were generated using SPSS (Figure 4). Data interpretation was further supported by the Griss proteomics database engine (GPDE).33,34 The GPDE software can be downloaded freely from http://sourceforge.net/projects/gpde/. A semiquantitative assessment of the identified proteins (Figure 3, Table 2) was performed by determination of their average “emPAI” (exponentially modified protein abundance index) values according to Ishihama et al.35 Here, the limits for peptide identification were set from 500 to 4500 Da.

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RESULTS

Cell Characterization

Primary human fibroblast-like cells were isolated from BM aspirates of six patients suffering from MM. To get enough cells for subsequent analyses, cells were cultivated for 3 to 5 passages, which corresponded to cultivation periods of 3 to 5 weeks. In the same way, fibroblast-like cells from BM of three patients with MGUS were isolated and investigated. In order to get control data, the corresponding cells from BM of three patients which showed no evidence for a neoplastic disease were gained and analyzed similarly. All cells were then harvested, documented by photography (Figure 1a), and characterized by FACS analysis (Supporting Information Figure S1). Cells from all samples were highly positive for fibroblastspecific markers CD90 or FE, but negative for leukocyte, endothelial cell, and hematopoietic stem cell markers CD45, CD31, and CD34. Furthermore, all samples contained cells positive for CD54 (ICAM-1), indicating inflammatory activation.37 Comparative Proteome Profiling Analyses

Cytoplasmic, nuclear, and secreted protein fractions of all cells were extracted, and each fraction was analyzed independently using shotgun analysis in order to increase the experimental reproducibility of the resulting proteome profiles of primary cells, as described by Gundacker et al.38 Data of all fractions were subsequently combined and resulted in a total of 1811 proteins identified in all fibroblast-like cells taken together, proteins identified with at least two distinct high confident peptides and in at least two different donors (Supporting Information Table S1−S3). 1546 proteins were identified in the cells of MM patients (Supporting Information Table S1), 1546 proteins in cells from MGUS patients (Supporting Information Table S2), and 1779 proteins in cells from non-neoplastic control (Supporting Information Table S3). Interpretation, as well as comparative analysis of the proteome profiles, was supported by our self-designed database, the GPDE (see Material and Methods). Thereby, classification of proteins according to their occurrence in cellular components using gene ontology terms as provided by the UniProt database39 showed very similar distribution patterns for the proteins expressed by MM-derived, MGUS-derived, and control fibroblast-like cells (Figure 1b). Furthermore, data analysis using the GPDE allowed documentation of all protein identifications. Details for the identification of c-type mannose receptor 2 are given as an example in Figure 2. The amino acid sequence of the protein is specified, whereby those sequences which were positively identified can be distinguished (Figure 2a). Sequences of

Western Blot Analyses

Twenty-five micrograms of proteins obtained out of BM fibroblast-like cells were subjected to Western blot analyses. Depending on the protein to be analyzed, one subcellular fraction in which differences between MM- and MGUSderived, as well as control cells, had been robustly determined by shotgun analysis was used. Proteins were separated by SDSPAGE and immunoblotted as previously described.36 Antibodies to periostin (rabbit, polyclonal, Abcam ab14041, 1:500), 846



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Figure 2. GPDE documentation of a selected protein identification. GPDE readouts are shown and suggest safe identification of c-type mannose receptor 2. The amino acid sequence of the protein is specified; underlined sequences were actually identified (a). Sequences of identified peptides are listed including their SpectrumMill identification scores. Peptide sequences which are unique in the human proteome are marked with a tick. The frequency with which each peptide was identified regarding the experiments with positive protein identification is represented by a black-underlayed bar and entitled as “Id.count” (b). An example of a sequence fragmentation of the most frequent peptide which led to the identification of c-type mannose receptor 2 is presented as well (c).

Figure 1. Comparison of fibroblast-like cells isolated from BM of MM, MGUS, and control patients. Fibroblast-like cells were isolated from BM aspirates of MM, MGUS, and control patients. Cells were cultivated for 3 to 5 passages, which approximately corresponds to 3 to 5 weeks. Previous to further analysis, cells were documented by photography (a). Cytoplasmic, nuclear, and secreted protein fractions of the cells were prepared separately and analyzed using a shotgun approach. Resulting data, including all subcellular fractions obtained from MM- and MGUS-derived, as well as control cells, were combined and proteins classified according to their occurrence in cellular components (b).

factors were likewise induced in these cells, which may be indicative for an increased replication rate under cell culture conditions; however, even though in culture MM-related cells tended to grow faster than control cells, no significant differences between these cells regarding their growth behavior could be determined. Applying G-statistics on the number of distinct peptides identified per protein comparing cells from MM patients to non-neoplastic controls, 7 proteins displayed p-values below 0.05 (Figure 3). Considering these proteins in more detail suggested a stepwise alteration in protein expression correlating with disease progression (Figure 4). In order to further investigate this assumption, we also analyzed the data in a semiquantitative way. Therefore, we calculated the “emPAI” (exponentially modified protein abundance index) values for each protein identification according to Ishihama et al.,35 and as described in the Material and Methods. This allowed us to distinguish proteins whose expression rate seemed to increase in a stepwise fashion in fibroblast-like cells of controls and in MGUS-related and MM-related fibroblast-like cells. Thereby a group of ECM-related proteins showed the most consistent progressive pattern. So, several ECM components, ECM

identified peptides are listed, and indications about their unique or repeated occurrence in the human proteome can be retrieved, as well as the frequency with which each peptide was identified regarding the experiments with positive protein identification (Figure 2b). Furthermore, a fragmentation spectrum of one peptide which led to the identification of ctype mannose receptor 2 is exemplified (Figure 2c). Only 14 of the identified 1811 proteins were specifically and reproducibly identified in cells from non-neoplastic donors (Table 1 and Supporting Information Table S3). Only one protein was apparently specific for MGUS patients, namely protein FAM3C (Table 1 and Supporting Information Table S2), described to be involved in liver cancer progression.40 44 proteins were apparently specific for MM patients (Table 1 and Supporting Information Table S1). Those included C−X−C motif chemokine 5, which might be a potential maker protein for activated fibroblast-like cells in MM, as it was found to be consistently secreted by these cells. Some DNA replication 847



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Table 1. Proteins Which Were Selectively Identified in Fibroblast-like Cells Isolated from BM of MM, MGUS, or Control Patientsa accession Control Only: P41208 P27487 P30042 P98095 Q15477 P62807 P17936 Q9UMX5 Q16647 P28300 P78346 P42285 Q9NPA0 Q9H269 MGUS Only: Q92520 MM Only: Q9C0C2 O95861 Q9NZ32 P49753 P30520 Q96BM9 P47895 Q9Y6E2 P51572 P27708 P13861 Q9H0A8 Q7L5N1 P42830 Q9NY33 P49736 P33991 P33992 Q14566 P33993 O60234 P48507 O76003 P63096 P08754 P54652 P52294 Q01628 Q7Z4H8 Q9Y5P6 Q9BV20 Q16539 Q8WVJ2 Q9NRN5 O75915 A6ZKI3 Q9BZK3 Q6P996 O60749 Q9UBE0 Q9Y2Z0 P23919

name

c_expcount

s_expcount

n_expcount

peptides

centrin-2 dipeptidyl peptidase 4 ES1 protein homologue, mitochondrial fibulin-2 helicase SKI2W histone H2B type 1-C/E/F/G/I insulin-like growth factor-binding protein 3 neudesin prostacyclin synthase protein-lysine 6-oxidase ribonuclease P protein subunit p30 superkiller viralicidic activity 2-like 2 UPF0480 protein C15orf24 vacuolar protein sorting-associated protein 16 homologue

0|3 2|3 2|3 1|3 0|3 3|3 0|3 3|3 2|3 0|3 0|3 0|3 0|3 0|3

0|3 0|3 0|3 0|3 0|3 1|3 2|3 0|3 0|3 2|3 0|3 0|3 0|3 0|3

2|3 0|3 0|3 3|3 2|3 0|3 0|3 0|3 0|3 0|3 2|3 2|3 2|3 3|3

2 5 3 9 4 4 2 5 2 3 3 2 4 2

protein FAM3C

0|3

2|3

0|3

5

182 kDa tankyrase-1-binding protein 3′(2′),5′-bisphosphate nucleotidase 1 actin-related protein 10 acyl-coenzyme A thioesterase 2, mitochondrial adenylosuccinate synthetase isozyme 2 ADP-ribosylation factor-like protein 8A aldehyde dehydrogenase family 1 member A3 basic leucine zipper and W2 domain-containing protein 2 B-cell receptor-associated protein 31 CAD protein cAMP-dependent protein kinase type II-α regulatory subunit COMM domain-containing protein 4 COP9 signalosome complex subunit 6 C−X−C motif chemokine 5 dipeptidyl-peptidase 3 DNA replication licensing factor MCM2 DNA replication licensing factor MCM4 DNA replication licensing factor MCM5 DNA replication licensing factor MCM6 DNA replication licensing factor MCM7 Gl ia maturation factor γ glutamate–cysteine ligase regulatory subunit Glutaredoxin-3 guanine nucleotide-binding protein G(i), α-1 subunit guanine nucleotide-binding protein G(k) subunit α heat shock-related 70 kDa protein 2 importin subunit α-1 Interferon-induced transmembrane protein 3 KDEL motif-containing protein 2 mannose-1-phosphate guanyltransferase β methylthioribose-1-phosphate isomerase mitogen-activated protein kinase 14 NudC domain-containing protein 2 olfactomedin-like protein 3 PRA1 family protein 3 protein FAM127A putative nascent polypeptide-associated complex subunit α-like protein pyridoxal-dependent decarboxylase domain-containing protein 1 sorting nexin-2 SUMO-activating enzyme subunit 1 suppressor of G2 allele of SKP1 homologue thymidylate kinase

4|6 2|6 3|6 3|6 4|6 4|6 3|6 3|6 3|6 3|6 2|6 3|6 3|6 2|6 3|6 2|6 2|6 2|6 2|6 3|6 3|6 3|6 4|6 3|6 3|6 3|6 3|6 3|6 4|6 3|6 3|6 3|6 3|6 0|6 4|6 3|6 4|6 3|6 3|6 3|6 3|6 3|6

0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 2|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 3|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6

0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6 0|6

5 4 2 2 6 4 3 3 2 5 2 2 6 2 3 3 6 2 5 6 2 2 4 5 5 9 3 3 7 4 3 2 3 8 3 3 2 3 5 5 5 8

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Table 1. continued accession MM Only: O43294 Q99426

name transforming growth factor β-1-induced transcript 1 protein tubulin-folding cofactor B

c_expcount

s_expcount

n_expcount

peptides

2|6 4|6

0|6 0|6

0|6 0|6

2 3

Proteins which were specifically identified in only one of the three patient groups (control, MGUS, or MM) are listed. “accession”: Swiss-Prot accession numbers; “names”: protein names; “c_expcount”, “s_expcount”, and “n_expcount”: the number of experiments in which the protein has been identified (“c” refers to cytoplasm, “s” to secreted, and “n” to nuclear extract); “peptides”: the number of distinct peptides identified for each protein. All results obtained for any given patient are considered as one experiment here.

a

Figure 3. Comparative analysis of selected ECM- and ECM-related proteins in fibroblast-like cells isolated from BM of MM, MGUS, and control patients. ECM- and ECM-related proteins, including their Swiss-Prot accession numbers (referred to as “Acc.Nr.”), are listed, arranging them into three groups: up-regulated proteins (a), nonregulated proteins (b), and down-regulated proteins (c). “ExpCounts” indicate the rate of patients (for controls, MGUS and MM, respectively) in which the protein has been identified. In the last column, empAI values for all subcellular fractions are visualized by colored cell symbols, with increased color intensities corresponding to increased emPAI values. Expression differences between MM patients and controls were tested using G-statistics (*, p < 0.05).

receptor (PDGFR-β) and the growth factor periostin, which are likewise involved in ECM activities, showed a similar progression-related pattern, in the cytoplasmic and secreted protein fraction, respectively, as well as in the nuclear fractions. This apparent increase in protein expression did not include all ECM proteins. Fibrillin-1 was apparently not regulated with respect to disease progression (Figure 3b), whereas fibulin-2, protein-lysine 6-oxidase, and Chitinase-3-like-protein 1 were found to be down-regulated (Figure 3c). Protein abundances

receptors, and ECM-modulating enzymes had emPAI values that progressively increased from controls to MGUS and further to MM (Figure 3a). Those included PAI-1, nidogen-2, laminin subunit α4, MMP-2, and biglycan, which mainly concerned the secreted protein fractions, as well as basigin, lysyl-hydroxylase 2, prolyl 4-hydroxylase 1, different integrin subunits (α5, αV, and β5), c-type mannose receptor 2, and basigin, which mainly concerned the cytoplasmic protein fractions. Additionally, β-type platelet-derived growth factor 849



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Figure 4. Protein expression correlated with disease progression. Proteins expressed by tumor-associated bone marrow fibroblast-like cells may be correlated with the disease state as demonstrated by the comparison of cells from non-neoplastic donors (con) to the premalignancy MGUS and multiple myeloma (MM). Box plots displaying the number of distinct peptides identified per protein of selected candidate biomarkers were generated using SPSS.

detection by shotgun analysis. Obviously this is due to the higher detection sensitivity of Western blot analysis. Furthermore, down-regulation of Chitinase 3-like protein 1 and fibulin-2 was observed in the secreted protein fraction and cytoplasm of the same cells, respectively.

are visualized in Figure 3 by colored cell symbols representing cytoplasmic, nuclear, and secreted protein fractions, with increased color intensities corresponding to increased emPAI values.34 Furthermore, more information on the proteomics data concerning these proteins is provided in Table 2.

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Confirmation of Data by Western Blot Analyses

DISCUSSION In order to learn more about the contribution of the tumor microenvironment to the progression from MGUS to MM, we have investigated fibroblast-like cells isolated from the BM of MM patients and compared them to those of MGUS patients, as well as of non-neoplastic control persons. All cells were cultivated for a short time period to get enough cells for further analyses. It is worth mentioning that Zdzisinska et al. brought evidence that the phenotype of isolated BM stromal cells from MM patients is hardly changing during cell culture conditions,

The differences in abundances of selected proteins were confirmed independently by Western blot analyses (Figure 5). In each case, one subcellular fraction in which differences had been robustly determined by shotgun experiments was used. By this means, up-regulation of periostin, laminin α4, and PAI-1 in the secreted protein fraction, as well as PDGFR-β and prolyl 4-hydroxylase 1 in the cytoplasm of MGUS- and MMassociated fibroblast-like cells, was confirmed, although a slight expression of PDGFR-β and prolyl 4-hydroxylase 1 was detected in the control samples where there had been no 850



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Table 2. Comparative analysis of selected ECM and ECM-related proteins in fibroblast-like cells isolated from BM of MM, MGUS and control patients. ECM and ECM-related proteins, including their Swiss-Prot accession numbers (referred to as “Acc.Nr.”), are listed, arranging them into three groups: up-regulated proteins (a), non-regulated proteins (b) and downregulated proteins (c). “Peptides” indicate the number of distinct peptides which have been identified for each protein (for controls, MGUS and MM, respectively). Similarly, “Coverage” indicates the respective sequence coverage for each protein (in %), and “Score” the sum of SpectrumMill scores of all identified peptides per protein control acc. nr.

protein name

(a) Up-Regulated Proteins P35613 basigin P21810 biglycan Q9UBG0 C-type mannose receptor 2 P08648 integrin α-5 P06756 integrin α-V P18084 integrin β-5 Q16363 Laminin α4 O00469 lysyl-hydroxylase 2 P08253 MMP-2 Q14112 nidogen-2 P05121 PAI-1 P09619 PDGFR-β Q15063 periostin P13674 prolyl 4-hydroxylase 1 (b) Nonregulated Proteins P35555 fibrillin-1 (c) Down-Regulated Proteins P36222 chitinase-3-like protein 1 P98095 fibulin-2 P28300 protein-lysine 6-oxidase

MGUS

MM

peptides

coverage

score

peptides

coverage

score

peptides

coverage

score

0 0 2 2 0 2 0 1 4 0 9 0 1 0

0.00 0.00 2.03 3.91 0.00 4.26 0.00 1.90 14.85 0.00 21.14 0.00 1.67 0.00

0.00 0.00 47.42 29.31 0.00 47.06 0.00 11.79 92.56 0.00 192.69 0.00 15.01 0.00

3 2 2 4 6 2 6 12 3 8 13 4 1 2

8.83 7.07 1.83 7.82 7.63 4.26 5.43 25.64 9.39 7.85 47.76 4.70 1.67 6.37

46.80 42.12 76.64 76.45 88.67 57.92 215.10 404.91 220.29 136.10 498.47 64.90 29.62 87.21

4 8 13 16 8 4 13 25 22 15 23 10 8 11

13.77 23.91 14.27 20.31 9.92 8.64 12.07 44.37 42.58 18.55 52.99 13.02 17.34 27.72

149.46 162.77 391.03 558.61 245.24 217.02 285.72 1709.89 560.59 263.21 1313.32 305.69 381.63 492.910

4

2.02

117.36

4

2.16

164.74

3

2.02

191.59

16 9 3

45.95 9.80 8.87

675.60 235.93 51.08

9 0 0

29.77 0.00 0.00

198.47 0.00 0.00

0 0 0

0.00 0.00 0.00

0.00 0.00 0.00

Thereafter, FACS analysis demonstrated very similar characteristics of all cells. Importantly, isolated BM fibroblastlike cells from control persons showed the same inflammatory activation state as cells derived from MGUS and MM patients. This was demonstrated by FACS analysis using CD54 antibody. Furthermore, we performed proteome profiling of the cells using an LC-MS/MS based shotgun approach. A first rather rough classification of all identified proteins according to gene ontology cellular component terms showed very similar distribution patterns for the three cell types. A semiquantitative analysis of the proteome profiles revealed subtle but important differences therein. So, ECM- and ECMrelated proteins appeared to be differently regulated in fibroblast-like cells isolated from the BM of MM and MGUS patients or control persons. These proteins are very important, as tumor progression is evidently accompanied by ECM remodeling41 and fibroblasts are the main contributors of ECM- and ECM-related proteins. In the present work, we focused on these proteins and found out that many of them appeared to be up-regulated in BM fibroblast-like cells from MGUS patients when compared to control cells, and even more in cells from MM patients. However, we were able to rule out general up-regulation of proteins, as fibrillin-1 showed no regulation, and fibulin-2, protein-lysine 6-oxidase, and Chitinase 3-like protein 1 were found to be down-regulated in MGUSand MM-derived cells when compared to controls. Interestingly, loss of fibulin-2 has been described in the context of breast cancer, where it appears to correlate with cancer progression,42 and protein-lysine 6-oxidase is supposed to play a role in tumor suppression.43 Secreted proteins which were found by us to be up-regulated in the above-mentioned progressive pattern included periostin

Figure 5. Confirmation of up- and down-regulation of certain ECMand ECM-related proteins by Western blot analysis. Proteins obtained out of BM fibroblast-like cells, taking one subcellular fraction in which differences between MM- and MGUS-derived, as well as control cells, had been robustly determined by shotgun analysis, were analyzed by Western blot experiments. So, in the secretome of the cells, periostin, laminin α4, and PAI-1 proved to be progressively up-regulated from controls to MGUS and further to MM. In the same way, PDGFR-β and prolyl 4-hydroxylase showed to be up-regulated in the cytoplasmic fraction of the cells. In contrast, down-regulation of Chitinase 3-like protein 1 (in the secretome) and fibulin-2 (in the cytoplasmic fraction) was observed in MGUS- and MM-associated cells.

as they observed an unaffected malignant phenotype of these cells over a period of several weeks of cell culture.11 851



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Another interesting observation in this context was the upregulation of basigin in MM- as well as in MGUS-related cells. This protein, also called “extracellular matrix metalloproteinaseinducer”, is highly expressed on the one hand by tumor cells, stimulating adjacent fibroblasts to produce matrix metalloproteinases.50 On the other hand this protein seems to be induced also in fibroblasts themselves, as a consequence of a positive feedback mechanism.51 We therefore hypothesize that in a first step basigin could be up-regulated in the stage of MGUS and that in a second step basigin could induce upregulation of MMPs such as MMP-2, when MGUS progresses to MM. Indications about overexpression of basigin in MM plasma cells and its involvement in regulating the myeloma cell proliferation have been published recently.52 MMPs are involved in one of two possible pathways for collagen degradation, which takes place in the ECM. An alternative pathway occurs via internalization of collagens followed by intracellular lysosomal degradation.53 Therefor, binding to c-type mannose receptor 2 is a preliminary and fundamental step. This receptor is mainly expressed on the surface of stromal fibroblasts54 and has been described to play an important role in ECM-remodeling and tissue invasion caused by certain tumors.54,55 We found increased amounts of c-type mannose receptor 2 in the cytoplasm of MM-derived BM fibroblast-like cells, whereas the amount of this receptor in MGUS-derived cells was the same as in controls. Up-regulation of this protein could therefore indicate progression from MGUS to MM. Furthermore, Western blot analyses were performed in order to confirm our results. These experiments independently showed that periostin, PDGFR-β, laminin α4, PAI-1, and prolyl 4-hydroxylase 1 were up-regulated in MGUS- and MMassociated cells. Chitinase 3-like protein 1 and fibulin-2 were mainly detectable in control cells, whereas in MGUS- and MMrelated cells the protein seemed partly or completely downregulated, again confirming our previous observations.

and PAI-1, proteins which are known to be involved in ECM activities and which have been described in the context of MM.44,45 Biglycan as well as nidogen-2 were also found by us to be progressively up-regulated in the secretome of fibroblast-like cells of MGUS and MM patients, but they were not present in control cells. Interestingly, biglycan has been mentioned in a very recent paper about targeting adhesion molecules as a strategy in MM therapy.46 As concerns nidogen-2, until now rather little is known about this protein in relation to MM, even though we observed that up-regulation of this protein strongly correlates with MM progression. We observed furthermore up-regulation of laminin α4 in the secreted protein fraction of MGUS- as well as MM-derived cells. This protein has been described to be highly secreted by bone marrow stromal cells in the proximity of certain tumors.17,19 Laminins are known to receive and transmit signals from tumor, as well as from tumor-associated, cells. These signals are transmitted via integrins, transmembrane receptors on cell surfaces consisting of one α and one β subunit, which are reorganized during tumor progression.17 We found up-regulation of certain integrin isoforms, namely α5, αV, and β5, in the cytoplasmic protein fractions of MGUS- and MM-derived cells. Interestingly, integrin α5β5 has been described by Gregoretti et al. as being highly expressed by MM-supporting stromal cells.47 Another remarkable protein in this context, which was also found to be up-regulated, was PDGFR-β. Veevers-Lowe et al.48 demonstrated that interactions between integrins and PDGFRβ are essential for cell migration, and they observed coimmunoprecipitation of PDGFR-β together with α5-integrin. Bissell also described PDGFR-β in a recent review about the role of the microenvironment in cancer progression as being involved in the progression of certain tumors, such as breast, colon, and prostate cancer.7 The increased expression of this protein was described to correlate with negative prognosis, as well as decreased survival of patients suffering from these diseases.49 Remarkably, we found PDGFR-β already to be induced in MGUS-related BM fibroblast-like cells, and it was even more up-regulated in the corresponding cells from MMsamples, whereas expression of the protein was not or only in extremely low quantities detectable in the controls. We therefore propose PDGFR-β, as a marker protein for the gradual progression in the MM pathogenesis. Other targets of our investigations were ECM-degrading enzymes, as ECM remodeling needs ECM degradation as a preliminary step for reorganization. One important candidate therefor is matrix metalloproteinase-2 (MMP-2), a type IV collagenase which has been described to be highly expressed in peritumoral fibroblasts of MM patients.11 We were able to confirm this observation. Indeed, MMP-2 was found by us to be highly secreted by BM fibroblast-like cells from MM patients. Increased amounts were also measured in the nuclear extract of these cells. It should be mentioned that secreted proteins can be found in the nuclear protein fraction as well, as during the secretion process they may accumulate in the endoplasmic reticulum, which remains attached to the nuclear membrane during the isolation procedure. In addition, increased amounts of MMP-2 were also measured in the cytoplasm of both MGUS- and MM-associated cells. This could be indicative of a higher turnover and synthesis rate of this protein in these cells, besides the increased secretion in MMrelated cells.

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CONCLUSION AND OUTLOOK Proteome profiling of BM fibroblast-like cells allowed us to reveal that stromal cell-derived ECM remodeling and potentially associated tumor promotion in the bone marrow take place already at the level of MGUS. We were able to identify marker proteins which may indicate a stepwise transition from MGUS to MM. Monitoring of such marker proteins could provide important prognostic and predictive information about tumor progression, independent of the tumor cell state. Consequently, this study may be an essential first step in the development of an individualized assessment strategy focusing on the state of the tumor microenvironment.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. FACS analysis of fibroblast-like cells isolated from BM of MM, MGUS, and control patients. Cells were characterized by FACS analysis, which showed that cells were positive for fibroblast-specific markers CD90 and FE, but negative for leukocyte, endothelial cell, and hematopoietic stem cell markers CD45, CD31, and CD34, respectively. All samples contained cells which were inflammatory activated, as demonstrated by positive CD54-staining. Tables S1−S3: Proteins identified by shotgun analysis in fibroblast-like cells isolated from BM of MM (Table S1), MGUS (Table 852



Article

(9) Mitsiades, C. S.; Mitsiades, N. S.; Munshi, N. C.; Richardson, P. G.; Anderson, K. C. The role of the bone microenvironment in the pathophysiology and therapeutic management of multiple myeloma: interplay of growth factors, their receptors and stromal interactions. Eur. J. Cancer 2006, 42 (11), 1564−73. (10) Corre, J.; Mahtouk, K.; Attal, M.; Gadelorge, M.; Huynh, A.; Fleury-Cappellesso, S.; Danho, C.; Laharrague, P.; Klein, B.; Reme, T.; Bourin, P. Bone marrow mesenchymal stem cells are abnormal in multiple myeloma. Leukemia 2007, 21 (5), 1079−88. (11) Zdzisinska, B.; Walter-Croneck, A.; Kandefer-Szerszen, M. Matrix metalloproteinases-1 and -2, and tissue inhibitor of metalloproteinase-2 production is abnormal in bone marrow stromal cells of multiple myeloma patients. Leuk. Res. 2008, 32 (11), 1763−9. (12) Garayoa, M.; Garcia, J. L.; Santamaria, C.; Garcia-Gomez, A.; Blanco, J. F.; Pandiella, A.; Hernandez, J. M.; Sanchez-Guijo, F. M.; del Canizo, M. C.; Gutierrez, N. C.; San Miguel, J. F. Mesenchymal stem cells from multiple myeloma patients display distinct genomic profile as compared with those from normal donors. Leukemia 2009, 23 (8), 1515−27. (13) Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144 (5), 646−74. (14) Hideshima, T.; Mitsiades, C.; Tonon, G.; Richardson, P. G.; Anderson, K. C. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat. Rev. Cancer 2007, 7 (8), 585−98. (15) Degrassi, A.; Hilbert, D. M.; Rudikoff, S.; Anderson, A. O.; Potter, M.; Coon, H. G. In vitro culture of primary plasmacytomas requires stromal cell feeder layers. Proc. Natl. Acad. Sci. U. S. A. 1993, 90 (5), 2060−4. (16) Pagnucco, G.; Cardinale, G.; Gervasi, F. Targeting multiple myeloma cells and their bone marrow microenvironment. Ann. N.Y. Acad. Sci. 2004, 1028, 390−9. (17) Givant-Horwitz, V.; Davidson, B.; Reich, R. Laminin-induced signaling in tumor cells. Cancer Lett. 2005, 223 (1), 1−10. (18) Pierce, R. A.; Griffin, G. L.; Mudd, M. S.; Moxley, M. A.; Longmore, W. J.; Sanes, J. R.; Miner, J. H.; Senior, R. M. Expression of laminin alpha3, alpha4, and alpha5 chains by alveolar epithelial cells and fibroblasts. Am. J. Respir. Cell Mol. Biol. 1998, 19 (2), 237−44. (19) Patarroyo, M.; Tryggvason, K.; Virtanen, I. Laminin isoforms in tumor invasion, angiogenesis and metastasis. Semin. Cancer Biol. 2002, 12 (3), 197−207. (20) Dalton, W. S.; Hazlehurst, L.; Shain, K.; Landowski, T.; Alsina, M. Targeting the bone marrow microenvironment in hematologic malignancies. Semin. Hematol. 2004, 41 (2 Suppl 4), 1−5. (21) Hazlehurst, L. A.; Argilagos, R. F.; Emmons, M.; Boulware, D.; Beam, C. A.; Sullivan, D. M.; Dalton, W. S. Cell adhesion to fibronectin (CAM-DR) influences acquired mitoxantrone resistance in U937 cells. Cancer Res. 2006, 66 (4), 2338−45. (22) Vincent, T.; Mechti, N. Extracellular matrix in bone marrow can mediate drug resistance in myeloma. Leuk. Lymphoma 2005, 46 (6), 803−11. (23) Gundacker, N. C.; Haudek, V. J.; Wimmer, H.; Slany, A.; Griss, J.; Bochkov, V.; Zielinski, C.; Wagner, O.; Stockl, J.; Gerner, C. Cytoplasmic proteome and secretome profiles of differently stimulated human dendritic cells. J. Proteome Res. 2009, 8 (6), 2799−811. (24) Haudek-Prinz, V. J.; Klepeisz, P.; Slany, A.; Griss, J.; Meshcheryakova, A.; Paulitschke, V.; Mitulovic, G.; Stockl, J.; Gerner, C. Proteome signatures of inflammatory activated primary human peripheral blood mononuclear cells. J. Proteomics 2012, DOI: 10.1016/j.jprot.2012.07.012. (25) Slany, A.; Haudek, V. J.; Zwickl, H.; Gundacker, N. C.; Grusch, M.; Weiss, T. S.; Seir, K.; Rodgarkia-Dara, C.; Hellerbrand, C.; Gerner, C. Cell characterization by proteome profiling applied to primary hepatocytes and hepatocyte cell lines Hep-G2 and Hep-3B. J. Proteome Res. 2010, 9 (1), 6−21. (26) Wimmer, H.; Gundacker, N. C.; Griss, J.; Haudek, V. J.; Stattner, S.; Mohr, T.; Zwickl, H.; Paulitschke, V.; Baron, D. M.; Trittner, W.; Kubicek, M.; Bayer, E.; Slany, A.; Gerner, C. Introducing the CPL/MUW proteome database: interpretation of human liver and

S2), and control patients (Table S3), respectively. Shotgun analysis results from cytoplasmic, nuclear, and secreted protein fractions of fibroblast-like cells obtained from BM of MM, MGUS, and control patients, respectively, are listed. Proteins which were specifically and reproducibly identified in cells from control persons, as well as from MGUS patients or MM patients are highlighted in bold in the corresponding table. “accession”: Swiss-Prot accession numbers; “names”: protein names; “c_expcount”, “s_expcount”, and “n_expcount”: the number of experiments in which the protein has been identified (“c” refers to cytoplasm, “s” to seceted, and “n” to nuclear extract); “peptides”: the number of distinct peptides identified for each protein; “c_empai”, “s_ empai”, and “n_empai”: the emPAI values for each protein per fraction; “coverage”: sequence coverage for each protein obtained by our analysis; “score”: sum of SpectrumMill scores of all identified peptides per protein. All results obtained for any given patient are considered as one experiment here. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +43 1 4277 52302. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS This work was funded by the Austrian National Bank “Jubiläumsfondsprojekt Nr. 13674” and supported by the “Initiative Krebsforschung”. We would like to thank Editha Bayer, Thomas Mohr, and Rosa-Maria Weiss for excellent technical assistance.

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