1612 Rita Polati1 Jessica Brandi1 Irene Dalai2 Alberto Zamo` 2 ∗ Daniela Cecconi1
Electrophoresis 2015, 36, 1612–1621
Research Article
Tissue proteomics of splenic marginal zone lymphoma
1 Proteomics
and Mass Spectrometry Laboratory, Department of Biotechnology, University of Verona, Verona, Italy 2 Department of Pathology and Diagnostics, Pathological Anatomy, University of Verona, Verona, Italy
Received June 4, 2014 Revised March 27, 2015 Accepted April 2, 2015
Splenic marginal zone lymphoma (SMZL) is a rare chronic B lymphoproliferative disease, whose molecular pathogenesis has still not been well established. For the first time, a proteomic approach was undertaken to analyse the protein profiles of SMZL tissue. 1D and 2D Western blot, immunohistochemical analysis, and functional data mining were also performed in order to validate results, investigate protein species specific regulation, classify proteins, and explore their potential relationships. We demonstrated that SMZL is characterized by modulation of protein species related to energetic metabolism and apoptosis pathways. We also reported specific protein species (such as biliverdin reductase A, manganese superoxide dismutase, beta-2 microglobulin, growth factor receptor-bound protein 2, acidic leucine-rich nuclear phosphoprotein 32 family member A, and Set nuclear oncogene) directly involved in NF-kB and BCR pathways, as well as in chromatin remodelling and cytoskeleton. Our findings shed new light on SMZL pathogenesis and provide a basis for the future development of novel biomarkers. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD001124. Keywords: Lymphocytes / Splenic marginal zone lymphoma / Tissue proteomics DOI 10.1002/elps.201400329
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Splenic marginal zone lymphoma (SMZL) is a rare chronic B lymphoproliferative disease, constituting less than 2% of non-Hodgkin lymphomas. It affects mainly elderly patients with a median age of 65 years without gender predilection. This lymphoma primarily manifests itself with an increase in peripheral blood lymphocyte counts and splenomegaly, while the peripheral superficial lymph nodes are often not swollen [1]. The diagnosis of SMZL relies on a combination of histopathological analysis and immunophenotyping of the spleen samples, although bone marrow biopsies are frequently the first - and often the only - sample analysed by pathologists, since splenectomy is not clinically required in Correspondence: Dr. Daniela Cecconi, Mass Spectrometry & Proteomics Lab, Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy FAX: +39 045 8027929 E-mail: [email protected]
a large percentage of patients [2]. Several clinical and biological factors have been proposed for identifying prognostic subgroups in this lymphoma entity, but the results have not been reproducible [3–6]. Recently, the SMZL Working Group proposed a prognostic index based on the combination of four factors: hemoglobin level, platelet count, elevated lactate dehydrogenase, and the presence of extrahilar lymphadenopathy (outside the splenic and hepatichila) [7]. There are also no standard criteria for SMZL treatment [8]. At the genetic and molecular level, SMZL is a heterogeneous disease. Deletion of the long arm of chromosome 7 (7q), in particular of the region at 7q31-32, and gains of the long arm of chromosome 3 (3q), which are the most common cytogenetic alterations, are recurrent in only 20–30% and 10–20% of cases, respectively, and genes targeted by these lesions are mostly unknown. Whole-genome sequencing analysis identified recurrent somatic NOTCH2 mutations in about a quarter of those cases of SMZL [9], in addition other gene mutations in various
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Abbreviations: GO, gene ontology; PCA, principal component analysis; SMZL, splenic marginal zone lymphoma C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Additional corresponding author: Alberto Zamo, ` MD. Department of Pathology and Diagnostics, Anatomia Patologica, University of Verona, Largo L. Scuro 10, 37134 Verona, Italy, E-mail: [email protected] `
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signalling pathways have been identified [10, 11]. However, knowledge of SMZL molecular pathogenesis is still limited. To date, no proteomic study has been conducted to identify modulated protein markers (the true effectors of the molecular pathways). In this study, we deal with SMZL for what we believe is the first time from a proteomic point of view. We analysed the proteome that characterizes the spleen tissue of SMZL patients by investigating the specific regulation of protein species, thus exploiting the full potential of 2DE/MS analysis [12,13]. The use of solid tissues represents the best possible option for the proteomic analysis of hematologic tumors [14]. Although tissue proteomics is very challenging (due to the limited sample availability, and possible interference from plasma or non-cancer cells), it nevertheless provides more specific information on the deregulated molecular pathways in a solid tumor (which may also depend on the tissue microenvironment in which the tumor is located). By using a classical proteomic approach we were able, for the first time, to obtain insights into the deregulated proteome of SMZL.
2 Materials and methods
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were collected separately from each sample and protein concentrations were determined using the Bradford Protein Assay. The samples were then incubated with 5 mM tributyl phosphine (Sigma) and 20 mM acrylamide (Sigma) for 60 min at room temperature to reduce disulphide bonds and alkylate the cysteine thiolic groups. The reaction was blocked by the addition of 10 mM DTT (Sigma).
2.3 Two-dimensional polyacrylamide gel electrophoresis The spleen protein sample from each individual was analysed separately. Each 2D-PAGE gel was prepared with 800 g of proteins mixed with 450 l of 2DE buffer. The first dimension separation was performed in 17 cm, pH 3–10 non-linear IPG (Bio-Rad) and the total product time ×voltage applied was 70 000 Vh for each strip. The second dimensional separation was performed using 10–20% T 2.6% C gradient SDS-PAGE (18 cm × 20 cm × 1.5 mm), applying 40 mA for each gel for 3 min, then 2 mA/gel for 1 h, and 20 mA/gel until the track dye, bromophenol-blue, reached the anodic end of the gels. After 2DE, the proteins were detected by the pure ruthenium chelate RuBPS (Cyanagen).
2.1 Patients and tumour biopsies For proteomics analysis, a total of four SMZL tissue samples, diagnosed according to WHO criteria [15] were obtained from frozen spleen biopsies of various patients retrieved from the archives of the Department of Pathology and Diagnostics, University of Verona, Verona, Italy. For comparative purposes, four non-neoplastic spleens (removed for trauma or pancreas surgery) were also included. Western blot validation was performed on an independent sample set composed of four SMZL tissue samples, and four non-neoplastic spleen samples. Immunohistochemical analysis was performed on 15 SMZL samples and 5 non-neoplastic spleens. All the tissue samples were collected from patients who had signed informed consent forms.
2.2 Tissue preparation and protein extraction for proteomics analysis 10-m-thick tissue sections were cut from samples (20 slices for each biopsy) using a cryostat, and then placed in 400 L of 2DE buffer containing 7 M urea (Bio-Rad), 2 M thiourea (Sigma), 3% w/v CHAPS (Sigma), 20 mM Tris (Sigma), and 1x Complete, Mini, EDTA-free protease inhibitors (Roche) and stored at –80°C until analysis. Each frozen spleen tissue was homogenized and solubilized by sonication for 30 seconds × 5 times on ice and protein, precipitated by overnight incubation with acetone:methanol (8:1) at - 20°C. Pellets were subsequently collected from each sample by centrifugation at 11 000 × g for 15 min, resuspended in 2DE buffer, and centrifuged again at 21 000 × g for 40 min to remove insoluble tissue debris and nucleic acids. Supernatants C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.4 Image analysis and statistical treatment Digitized gel images were used to estimate the amount of different protein species in each analytical gel using PDQuest software (Bio-Rad, version 7.3). Each gel was analysed for spot detection, background subtraction, and protein spot OD intensity quantification. After manual editing, a match set was created in which the protein spots from different gels are matched to each other and are included in a synthetic image, called a match set standard (master 2DE gels). This artificial composite image contains all the information the software needs to analyse the gels quantitatively, qualitatively, or statistically. Spot quantity values were normalised in each gel dividing the raw quantity of each spot by the total quantity of all the spots included in the standard gel. Univariate and multivariate statistical analyses were carried out. Principal component analysis (PCA) was performed with Simca-P+ software (Umetrics, version 12) using logtransformed, normalized spot volumes of individual gels. The transformed spot volumes were thus mean-centered and scaled to unit variance to minimize the bias for spots of large volume on the PCA results. The resulting spot quantity values for the principal components were used to identify the protein species responsible for the variability between the two groups (control and SMZL). Student’s t-test was also performed to assess the statistical significance of modulated protein species. Based on the average spot volume ratio, spots whose relative amount is changed at least 1.5 fold (increase or decrease) between controls and SMZL samples at a 95% confidence level (t test; www.electrophoresis-journal.com
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p ⬍ 0.05) were considered to be significant. Spots were selected for identification based on significant differences in t-test and/or absolute spot loading values on principal components (PCs).
2.5 Protein identification by nanoHPLC Chip Ion Trap mass spectrometry Spots selected for identification were carefully cut out from 2D stained gels and subjected to in-gel trypsin digestion as previously described [16]. Peptides from 5 L of each sample were then separated by reversed phase nano-HPLCChip technology (Agilent Technologies, Palo Alto, CA, USA) online-coupled with a 3D ion trap mass spectrometer (model Esquire 6000, Bruker Daltonics, Bremen, Germany). The chip was composed of a Zorbax 300SB-C18 (43 mm × 75 m, with a 5 m particle size) analytical column and a Zorbax 300SBC18 (40 nL, 5m) enrichment column. The complete system was fully controlled by ChemStation (Agilent Technologies) and EsquireControl (Bruker Daltonics) software. The scan range used was from 300 to 1800 m/z. For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The three most abundant peptides of each m/z were selected for further isolation and fragmentation. The MS/MS scanning was performed in the normal resolution mode at a scan rate of 13.000 m/z per second. A total of five scans were averaged to obtain an MS/MS spectrum. The peak lists were searched using MASCOT (version 2.4.01, http:/www.matrixsciences.com) against all entries of the NCBInr database (38 032 689 sequences; 13 525 028 931 residues). The following parameters were set: trypsin digestion, up to one missed cleavage; fixed and variable modifications: propionamide (Cys) and oxidation (Met), respectively; peptide and fragment tolerances: ± 0.9 Da and ± 0.9 Da, respectively, and peptide charges: +1, +2 and +3. For protein identification, at least two peptides were considered. Only significant hits, as identified by the MASCOT probability analysis (p ⬍ 0.05) were accepted.
2.6 Mono and two-dimensional immunoblotting analyses Confirmation of the proteomic data by immunoblot analysis was performed on an independent sample set of splenic biopsies. Samples were analysed separately in 1D-WB and as a pool in 2D-WB. We investigated the modulation of Anp32a, Acadm, Grb2, Sod2, Nutf2, and Set. The splenic tissue extracts were separated by SDS-PAGE under denaturing and reducing conditions on 12%T polyacrylamide gels; and by 2DE on a 7 cm long IPG strip (pH 3–10 non-linear) and 10–20%T mini gels. The separated proteins (20 g of protein/lane for 1D-WB, and 100 g of protein/gel for 2D-WB) were transferred to a PVDF membrane through electroblotting. The membranes were treated with primary and secondary C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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antibodies at the appropriate dilutions (see Supporting Information Table 1) and bound antibodies were detected by the ECL Western blotting detection system (Millipore, USA). Equal sample loading was confirmed by anti-tubulin antibody. Western blots were scanned by a ChemiDoc instrument (Bio-Rad), and quantitative analysis of specific signals was performed using Quantity One software (Bio-Rad). In order to investigate the 2D-WB patterns of immuno-detected protein species, the ProMoST tool (Protein Modification Screening Tool) [17] was used. Since ‘trains of spots’ are in most cases attributable to phosphorylation or deamidation [18] (although other less likely PTMs, such as acetylation or methylation, could also be involved), modification of the protein pI by Ser.pSer, Thr-.pThr, Tyr-.pTyr, Gln-.Glu, or Asn-.Asp conversion was analysed. To create the theoretical 2DE gel, the pH range was set from 3 to 10 with steps of 0.5, and a molecular weight range was set from 10 to 200 kDa with steps of 20 kDa. 2.7 Immunohistochemical analysis Tissue sections were incubated for 15 min at 95°C in a pH 6 buffer (Bond Epitope Retrieval Solution 1), then labeled with the antibody for the protein SET (dilution 1:100) or ACADM (dilution 1:300) and processed with a detection system, Bond Polymer Refine kit using an automated stainer (Leica Bond Max). 2.8 Functional data mining Functional annotation of identified proteins was performed according to Gene Ontology (GO) using the FatiGO online tool from Babelomics (Babelomics 4.2; http://babelomics. bioinfo.cipf.es/fatigo.html). GOcellular component, biological process and molecular function were analysed by comparing the GO terms of identified proteins against the rest of the genome. Fisher’s exact test detected the significant over-representation of GO terms in the submitted dataset. Protein interactions were assessed using the STRING 9.0 database (http://string-db.org). We retrieved at least medium confidence (score 0.4) interactions including all the prediction methods implemented in STRING (excepted text mining). No additional white nodes were inserted in order to reduce noise, and network depth was kept to the minimum value (1) to exclude as many false positive interactions as possible.
3 Results 3.1 Proteomic investigation of splenic marginal zone lymphoma tissue Protein profiling of spleen tissues was undertaken to determine proteome changes in SMZL by comparing matched normal and disease biopsies. An average of 464 (± 2.5%) protein spots with an apparent molecular mass between 10 and 200 kDa and a pI between 3 and 10 were detected by 2DE www.electrophoresis-journal.com
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Figure 1. Representative 2DE images obtained using spleen proteins of SMZL patients (on the left) and controls (on the right). Identified protein spots are indicated by white arrows and numbered as specified in Supporting Information Table 2.
(Fig. 1). Protein patterns from SMZL and control splenic tissues were compared by multivariate statistical analysis to identify protein species responsible for correlated variations, and investigating intra- and inter-group relationships. The PCA indicated that there were two distinct groups of maps from the two experimental conditions, SMZL and control (Supporting Information Fig. 1A). This indicates that there is sufficient natural variation in the proteomes to distinguish between the two groups, increasing the confidence that potential disease biomarkers can be found in this dataset. The strongest separation was observed in the first and second PCs, which accounted for most of the variation in the datasets, i.e. 23.5 and 18.6%, respectively. In addition, the loading plot obtained from PCA led to the identification of variables (spots) that were more suited for explaining relationships in the dataset which contribute the most to the separation of the two experimental groups (Supporting Information Fig. 1B). Individual proteins were also analysed by t-test analysis (p ⬍ 0.05) to significantly identify different intensities between the two experimental groups. Supported by multivariate and univariate analyses, a total of 32 spots (17 up-regulated and 15 down-regulated) from the 2DE analysis were identified as significantly differentially regulated in the spleens of SMZL patients (Fig. 1). All these differentially regulated spots were successfully picked up and proteins were identified by nanoHPLC-Chip/MS (Supporting Information Table 2). The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [19] with the dataset identifier PXD001124 and DOI 10.6019/PXD001124.
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3.2 Western blot validation of various deregulated protein species To validate our proteomic results, we measured the protein levels by 1D-WB of six selected targets of interest in the spleens of four SMZL samples and four controls (Fig. 2). We confirmed increased amounts of proteins of the Acadm, Sod2, Nutf2, and Grb2 in SMZL tissue samples. The upregulation of Anp32a, limited to the 14.8 kDa isoform [20], was also confirmed (however one control sample also showed marked immunodetection of Anp32a). Unfortunately, we did not confirm Set modulation, which could be ascribed to the overlap of several protein forms in the 1D separation which may have an opposite modulation (but not detected as significant by 2DE). To deepen the investigation of protein species-specific modulation, the selected proteins listed above were also analysed by 2D-WB (Fig. 3) with the support of ProMoST in silico analysis to interpret any pI shift. 2D-WB results revealed four signals for Sod2 at pI from 6.5 to 7.8, and the absence of the unmodified Sod2 form (pI of 8.25) in both, SMZL as well as in normal spleens. As indicated by ProMoST in silico analysis, the four immunodetected protein species suggested a pattern of phosphorylation. The most acid protein spot (containing at least one protein species probably with four phosphorylations) appears to be induced in SMZL. Obviously, such PTM should be confirmed by MS, or by Western blot with specific anti-phospho-tyr antibodies. In addition, 2D-WB analysis with anti-Grb2 antibody revealed seven immunodetected signals in SMZL and only five in normal spleens, which confirmed that this protein is more abundant in the SMZL spleen tissue.
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and deamidated (pI 5.77) Grb2 forms, and the two other spots could contain at least one protein species, probably one-fold phosphorylated (pI 5.75), and at least one protein species, probably di-phosphorylated (pI 5.57). The 2D-WB analysis obtained with the anti-Acadm antibody indicated eight immunodetected spots (pI ranging from 5.5 to 7.5) in SMZL and, although much less evident, also in the control samples. This confirmed the increased amount of Acadm in the SMZL tissue, suggesting, as indicated by ProMoST in silico analysis, a pattern of phosphorylation and the absence of the unmodified form (pI of 8.12) for this protein. In addition, by 2D-WB analysis with anti-Nutf2 antibody, we found that, in SMZL and normal spleens, there were two spots, one containing the unmodified protein species (pI 5.1) and the other containing at least one protein species, probably one-fold phosphorylated (pI 4.9) as suggested by ProMoST analysis. The amount of the putative one-fold phosphorylated Nutf2 form was increased in the SMZL tissue at the expense of the unmodified protein species. Finally, the Set molecules were depicted as a collection of more than 15 different protein spots in SMZL, and more than 10 in normal spleen tissue. These immunodetected spots could be classified into three groups with different ranges of Mw: ⬎30 kDa (protein species with PTM) which were less abundant in SMZL; 30 kDa (intact or differentially spliced forms) and ⬍30 kDa (cleaved, truncated forms) which were more abundant in SMZL. A similar pattern with multiple spots was also found in the 2D-WB of Anp32a carried out on several electroblotted membranes. Also in this case the complicated 2D-WB pattern could be ascribed to PTM, splice variants, and cleaved/truncated forms of Anp32a. 3.3 Immunohistochemical analysis
Figure 2. (A) Monodimensional Western blot analysis in normal (C) and tumour spleen tissues (SMZL). Tubulin was used as a loading control. (B) Densitometric analysis of the Western blots was carried out using Quantity One software. The intensity of the bands was normalized to the intensity of the respective tubulin band. The values obtained were plotted as mean optical density (OD) ± sd from three independent experiments. White bars represent SMZL and the grey bars, the control samples.
2D-WB suggested that the SMZL spleen tissue is characterized by the appearance of two Grb2 species (pI 6.2 and 6.5) with a shift to more basic pI values (compared to the unmodified Grb2 form), and by the increasing four protein spots (with pI ranging from 5.5 to 6). The analysis carried out with ProMoST suggested that two of these four protein spots could contain the unmodified (pI 5.95)
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Of the modulated proteins, the nuclear oncogene Set and the enzyme Acadm were selected for further evaluation by immunohistochemical analysis. We found that Set (Fig. 4A) was expressed in almost all cells of the tumour SMZL spleen tissue in all the tested cases (15/15). In the normal spleen, on the other hand, it was expressed only by centroblasts of germinal centers, to a lower extent in the mantle, and then again expressed in the marginal zone. Acadm (Fig. 4B) was also expressed in almost all cells of the tumour SMZL spleen tissue in the majority of tested cases (14/15), while in the normal spleen it was only revealed in germinal centers. All the 15 tested cases for Set and Acadm are shown in Supporting Information Figs. 3 and 4, respectively. 3.4 Functional characterization of the proteins identified and protein–protein interaction analyses By gene-annotation enrichment analysis, we found that many of the SMZL identified proteins were located in the following
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Figure 3. Bidimensional Western blot analysis in normal (C) and tumour spleen tissues (SMZL).
Figure 4. (A) Immunohistochemical analysis of Set and (B) Acadm in spleen tissues. Panels on the upper line represent controls, while those of the line below indicate SMZL samples. On the left there are 200x magnifications and on the right 400x. Arrows indicate centroblasts, while the letters GC, the germinal center, m mantle and MZ the marginal zone. Specific staining was dark, and counterstaining was light grey.
“cellular components”: vesicles (membrane-bounded, and cytoplasmic), perinuclear region, and phagocytic cup. “Biological processes” enriched among the deregulated proteins of SMZL spleens were: response to organic substance, response to nutrient levels, monosaccharide metabolic process, protein oligomerization, generation of precursor metabolites and energy, regulation of apoptosis, regulation of programmed cell death, cellular amino acid derivative metabolic process, and glucose catabolic process. Concerning statistically enriched GO term in the category “molecular function”, we found the term oxygen binding to be significant (Supporting Information Table 3). In order to better understand whether the protein species identified in this study had any previously described interactions, we analysed the protein interaction networks using the String 9.0 database. This revealed that there were significant interactions between some of the proteins identified (Supporting Information Fig. 2). There was a core interaction network involving Anp32a, Set, Gapdh, Pkm2, Eno1, Actb, Coro1a. In addition, of the identified proteins, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
also ApoA1, Alb, P4hb, and Capns1 formed a separate interaction network.
4 Discussion The present study is, we believe, the first to directly show the proteomic alterations of spleen occurring with SMZL. An analysis carried out at the splenic tissue level is very important, since the biopsies (unlike body fluids that reflect modifications occurring far from the tumour) enable molecular information directly linked to the tumour biology to be detected. Using a 2DE/MS based approach, we were able to find, for the first time, the differences in the splenic proteome of patients with SMZL and to identify disease-specific changes in the protein species pattern. Knowledge of the genetic lesions associated with SMZL is heterogeneous, thus it is quite difficult to fit our proteomic results with the genetic data available [9]. It is not possible to speculate whether there is a www.electrophoresis-journal.com
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connection between the deregulated protein species and the gene copy number aberrations, therefore the protein species dysregulations reported here should be attributed to other phenomena (such as epigenetic or splicing events, PTMs, etc.). Network analysis revealed connectivity between some of the identified proteins, and two main clusters emerging from the list (Supporting Information Fig. 2). The functional relevance of some of these proteins, as well as their impact on SMZL deregulated pathways is discussed below. Notably, as indicated by GO enrichment analysis (Supporting Information Table 3) the first cluster of proteins revealed by network analysis has a role in metabolism, while the second cluster represents proteins involved in cytoplasmic vesicles. Collectively, gene-annotation enriched analysis (Supporting Information Table 3) suggests that the modulation in spleen proteome associated with SMZL predominantly involves changes in energy metabolism (GO:0031667, GO:0005996, GO:0006091, GO:0006575, GO:0006007) and apoptosis pathways (GO:0042981, GO:0043067). The changes in energy metabolism may involve an increase in the glycolytic pathway (as suggested by the up-regulation of Eno1 and Gpdh) and a stimulation of the -oxidation pathway (as indicated by the up-regulation of Acadm). Acadm was found up-regulated in a subgroup of diffuse large B cell lymphoma [21], and it has been suggested that the -oxidation pathway is required to ensure the turnover of the tumour cells [22]. The role of Acadm in lymphoid cells is poorly understood. However the presence of high levels of the protein in germinal centre cells (Fig. 4B) also suggests a potential role in B-cell receptor (BCR) signalling.
4.1 Deregulated SMZL proteins involved in the NF-B pathway Interestingly, many of the identified protein species belong to SMZL deregulated processes already highlighted by genetic studies (such as NF-kB and BCR pathways, chromatin remodelling, and cytoskeleton) [9,11]. It has been demonstrated that genetic lesions of the NF-B pathway provide a molecular basis for the pathogenesis of more than 30% of SMZLs [23], thus implying that NF-kB activation is a major contributor to the pathogenesis of this disease [9, 11]. NF-kB transcription factors are involved not only in the apoptotic pathway but also in the regulation of energy metabolism [24], which we found to be deregulated (Supporting Information Table 3). Concerning the proteins involved in this pathway, we found that SMZL patients have a higher level of one protein species of biliverdin reductase A (BLVRA) (SSP5501, +3.54). Blvra acts as a reductase and as a ser/thr/tyr kinase, and its overexpression enhances the activation of NF-kB and also the NF-kB-activated iNOS gene [25]. Blvra removes biliverdin, which is an inhibitor of NF-kB, and also acts in the MAPK and PI3K/Akt pathways (implicated in the p65 phosphorylation and activation of NF-kB) [26]. The formation of the ternary complex of biliverdin reductase/protein kinase C␦/ERK2 protein is also C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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essential for the activation of NF-B [27]. Interestingly, Blvra is up-regulated in different types of cancer cells: its overexpression enhances resistance to chemotherapeutics [28], and targeting Blvra overcomes multidrug resistance in leukemia cells [29]. This study also led to the identification of one protein species of manganese superoxide dismutase (SOD2), which was up-regulated in SMZL patients (SSP7302, +2.94). This finding was confirmed by 1D-WB immunoblot analysis (Fig. 2). The induction of SOD2 transcription in response to the activation of NF-kB is well documented [30]. The transcriptional activity of the SOD2 gene is enhanced by the p65 subunit [31]. Notably, Sod2 overexpression enhances the migratory potential of tumour cells, contributing to their increased metastatic behaviour [32]. Sod2 was also detected as one of the most up-regulated proteins in OxPhos cell lines of diffuse large B cell lymphoma [21]. In addition, although Sod2 is known to be regulated at the post-translational level, our 2D-WB results demonstrated that in SMZL, the changes in Sod2 modifications do not predominate over changes in the total amounts of Sod2 (Fig. 3). Our results also indicate that one protein species of beta2 microglobulin (B2M) is up-regulated in SMZL patients (SSP4104, +1.70). B2m is a chaperone of major histocompatibility complex (MHC) class I (-like) molecules, which play a central role in antigen presentation, immunoglobulin transport, and iron metabolism. It is connected to the NF-kB pathway since p50 and p65 subunits bind to the B2M promoter and transactivate it in lymphoid and myeloid cells [33]. Interestingly, the plasma levels of B2m correlate negatively with outcome in non-Hodgkin’s lymphoma [34]. In addition, a marked increase in plasma B2m levels was found in 78% of SMZL patients [35]. it is worth noting that antibodies against B2m induce apoptosis in solid tumours and in hematological malignant cells (such as multiple myeloma, Burkitt lymphoma, mantle cell lymphoma, T-cell and myelogenous leukemia cell lines) [36]. We also identified two protein species (ApoA1 and Phb) whose down-regulation was in agreement with the presence of the NF-kB signalling pathway activated in SMZL. The level of two protein species of apolipoprotein A-I (APOA1) was lower in SMZL patients (SSP2305, −2.22; SSP1305, 1.75). Interestingly, ApoA1 also has an anti-inflammatory activity, reducing the expression of chemokines and chemokine receptors via modulation of NF-kB pathway. Mimetic peptides of ApoA1 suppress phosphorylation of IkBalpha (an upstream mediator of the NF-kB pathway), nuclear p65 expression, and p65 DNA binding activity [37]. Accordingly, in vivo studies have demonstrated that ApoA1–infused mice had lower levels of p65 and p50 subunits [38]. The other SMZL down-regulated protein species we reported was prohibitin (PHB, SSP2408, −1.51). Interestingly, Phb overexpression correlates with the reduction in NF-kB nuclear translocation, NF-kB DNA binding, and NF-kB–mediated gene transcription. This protein inhibits the activation of NF-kB via a mechanism involving a reduction in importin 3, a protein implicated in p50/p65 nuclear translocation [39]. www.electrophoresis-journal.com
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4.2 Deregulated SMZL proteins involved in BCR signalling Of the genes deregulated in SMZLs, there has been a special focus on those involved in BCR signalling [11,40]. Of particular interest is our observation that the amount of one protein species of growth factor receptor-bound protein 2 (GRB2) was higher in SMZL patients (SSP4307, +2.70). Grb2 upregulation was also revealed by 1D-WB analysis (Fig. 2B). Grb2 is an adapter protein that mediates BCR signalling pathways. It is associated with LIME, a transmembrane adaptor required for BCR-signalling [41]. Interestingly, strong cytoplasmic Grb2 expression has already been found by immunohistochemical analysis in the neoplastic cells of SMZL, as well as in other B-cell non-Hodgkin lymphomas [42]. Our results coincide with these observations and provide a validation with a completely independent approach. 2D-WB analysis (Fig. 3) indicated that, by using same experimental conditions (i.e., amount of total protein loaded, antibodies dilution, exposure time to the x-rays, etc . . . ), seven and five different spots could be immunodetected in SMZL and control samples, respectively. All the seven Grb2 species are induced in SMZL, in particular two species with more basic pI values seem to be particularly interesting since (by using the experimental conditions indicated in Section 2) they are not detected in control samples, suggesting a very low level of expression in normal spleen tissue. Future research could lead to the characterization of these two more basic Grb2 protein species, and clarify their role in SMZL tissue, and possibly their involvement in BCR signalling pathways.
4.3 Deregulated SMZL proteins involved in chromatin remodelling Consistent with the hypothesis that the SMZL genetic signature is characterized by deregulated chromatin remodelling [9, 11], here we showed the up-regulation of both one protein species of acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A, SSP0401, +4.05), and one species of Set nuclear oncogene (SET, SSP0601, +4.13). Anp32a and Set were the most SMZL overexpressed protein species identified by our proteomic approach. Notably, we showed that Set is differently localized in the spleen tissue of SMZL compared to the control spleen (Fig. 4A). It is expressed in almost all of the neoplastic cells, while in the control sample it is localized only in the centroblasts of germinal centers, and in sparse lymphoid cells of the mantle and marginal zones. Anp32a and Set act as inhibitors 1 and 2 of the tumour suppressor protein phosphatase 2A (I1PP2A and I2PP2A). They are also components of the inhibitor of the histone acetyl transferase complex that binds to histones and masks them from being acetylated [43] thus leading to transcriptional repression. The tumorigenic role of Anp32a [44] and Set [45] has been already demonstrated in several types of cancers. It is worth mentioning that Set oncogene has been recently suggested as a new treatment C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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molecular target in B-cell malignancies [46]. Interestingly, Set fragments (which were found to be increased in SMZL) prevent the inhibition of the NME1 [47] (an oncogene typical of B-cell lymphoma [48]), and are involved in the inhibition of the tumour suppressor PP2A [49].
4.4 Deregulated SMZL proteins involved in cytoskeleton organization The cytoskeleton organization is the fourth cell process that was found to be notably altered in SMZL [11] by wholeexome sequencing analysis. Accordingly, the data obtained here indicate the down-regulation of specific protein species involved in cytoskeleton organization. Of these, one protein species of beta actin (ACTB) was found to be less expressed in SMZL patients (SSP 1603, −1.70). Actb is a component of the cytoskeleton, and a mediator of internal cell motility. Its reorganization has been shown to be important for BCR activation [50], which confirms that these processes are critical to SMZL development., One protein species of coronin 1A (CORO1A) was also down-regulated in SMZL patients (SSP4603, −2.44). Coro1a, is an actin-binding protein selectively expressed in hematopoietic cells, which plays a crucial role in the immune response through reorganization of the actin cytoskeleton [51]. We also found a lower level of one protein species of calpain small subunit 1 (CAPNS1, SSP1304, −1.64) in SMZL patients. Calpains are endopeptidases, which have numerous functions including, but not limited to, remodelling of the cytoskeleton. One protein species of L-plastin (LCP1, SSP1809, −1.85) was also found to be down-regulated in our study. It is a leukocyte-specific protein that cross-links actin filaments into tight bundles, increasing the stability of actin-based structures. Interestingly, LCP1 −/− mice have been found to be deficient in marginal zone B cells [52]. Finally, we also identified a protein species of nuclear transport factor 2 (NUTF2), which is instead up-regulated in SMZL tissue (SSP1001, +2.42). Nutf2 is indirectly involved in the cytoskeleton organization. It facilitates transport into the nucleus of the filamentous actin-capping protein (CapG), a mediator of cross-talk between the actin cytoskeleton and microtubulebased organelles [53]. An increased amount of Nutf2 in SMZL was confirmed by 1D-WB and by 2D-WB (Figs. 2 and 3).
4.5 Conclusions This is the first study to shed light on the proteome modulations of SMZL, thereby opening up new ground for the development of possible diagnostic as well as therapeutic markers of SMZL. Our findings indicate that SMZL is characterized by changes in energy metabolism and apoptosis pathways. We identified for the first time the deregulated protein species involved in NF-kB, and BCR pathways, as well as in chromatin remodelling and cytoskeleton. Of particular interest www.electrophoresis-journal.com
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are biliverdin reductase A, manganese superoxide dismutase, beta-2 microglobulin, growth factor receptor-bound protein 2, acidic leucine-rich nuclear phosphoprotein 32 family member A, Set nuclear oncogene, and nuclear transport factor 2. Although it is difficult to expand the number of biopsy samples due to the rare incidence of these histological lymphoma entities, the results warrant further validation in larger cohorts. Validation should include the use of independent techniques, also in view of the fact that the 2DE modulations observed may be slightly incorrect due to spot overlapping. This work was supported by AIRC, Fondazione Cariverona and from intramural funds of the University of Verona. All authors declare no financial and no commercial conflicts of interest.
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Research Article
Tissue proteomics of splenic marginal zone lymphoma
1 Proteomics
and Mass Spectrometry Laboratory, Department of Biotechnology, University of Verona, Verona, Italy 2 Department of Pathology and Diagnostics, Pathological Anatomy, University of Verona, Verona, Italy
Received June 4, 2014 Revised March 27, 2015 Accepted April 2, 2015
Splenic marginal zone lymphoma (SMZL) is a rare chronic B lymphoproliferative disease, whose molecular pathogenesis has still not been well established. For the first time, a proteomic approach was undertaken to analyse the protein profiles of SMZL tissue. 1D and 2D Western blot, immunohistochemical analysis, and functional data mining were also performed in order to validate results, investigate protein species specific regulation, classify proteins, and explore their potential relationships. We demonstrated that SMZL is characterized by modulation of protein species related to energetic metabolism and apoptosis pathways. We also reported specific protein species (such as biliverdin reductase A, manganese superoxide dismutase, beta-2 microglobulin, growth factor receptor-bound protein 2, acidic leucine-rich nuclear phosphoprotein 32 family member A, and Set nuclear oncogene) directly involved in NF-kB and BCR pathways, as well as in chromatin remodelling and cytoskeleton. Our findings shed new light on SMZL pathogenesis and provide a basis for the future development of novel biomarkers. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD001124. Keywords: Lymphocytes / Splenic marginal zone lymphoma / Tissue proteomics DOI 10.1002/elps.201400329
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Splenic marginal zone lymphoma (SMZL) is a rare chronic B lymphoproliferative disease, constituting less than 2% of non-Hodgkin lymphomas. It affects mainly elderly patients with a median age of 65 years without gender predilection. This lymphoma primarily manifests itself with an increase in peripheral blood lymphocyte counts and splenomegaly, while the peripheral superficial lymph nodes are often not swollen [1]. The diagnosis of SMZL relies on a combination of histopathological analysis and immunophenotyping of the spleen samples, although bone marrow biopsies are frequently the first - and often the only - sample analysed by pathologists, since splenectomy is not clinically required in Correspondence: Dr. Daniela Cecconi, Mass Spectrometry & Proteomics Lab, Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy FAX: +39 045 8027929 E-mail: [email protected]
a large percentage of patients [2]. Several clinical and biological factors have been proposed for identifying prognostic subgroups in this lymphoma entity, but the results have not been reproducible [3–6]. Recently, the SMZL Working Group proposed a prognostic index based on the combination of four factors: hemoglobin level, platelet count, elevated lactate dehydrogenase, and the presence of extrahilar lymphadenopathy (outside the splenic and hepatichila) [7]. There are also no standard criteria for SMZL treatment [8]. At the genetic and molecular level, SMZL is a heterogeneous disease. Deletion of the long arm of chromosome 7 (7q), in particular of the region at 7q31-32, and gains of the long arm of chromosome 3 (3q), which are the most common cytogenetic alterations, are recurrent in only 20–30% and 10–20% of cases, respectively, and genes targeted by these lesions are mostly unknown. Whole-genome sequencing analysis identified recurrent somatic NOTCH2 mutations in about a quarter of those cases of SMZL [9], in addition other gene mutations in various
∗
Abbreviations: GO, gene ontology; PCA, principal component analysis; SMZL, splenic marginal zone lymphoma C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Additional corresponding author: Alberto Zamo, ` MD. Department of Pathology and Diagnostics, Anatomia Patologica, University of Verona, Largo L. Scuro 10, 37134 Verona, Italy, E-mail: [email protected] `
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signalling pathways have been identified [10, 11]. However, knowledge of SMZL molecular pathogenesis is still limited. To date, no proteomic study has been conducted to identify modulated protein markers (the true effectors of the molecular pathways). In this study, we deal with SMZL for what we believe is the first time from a proteomic point of view. We analysed the proteome that characterizes the spleen tissue of SMZL patients by investigating the specific regulation of protein species, thus exploiting the full potential of 2DE/MS analysis [12,13]. The use of solid tissues represents the best possible option for the proteomic analysis of hematologic tumors [14]. Although tissue proteomics is very challenging (due to the limited sample availability, and possible interference from plasma or non-cancer cells), it nevertheless provides more specific information on the deregulated molecular pathways in a solid tumor (which may also depend on the tissue microenvironment in which the tumor is located). By using a classical proteomic approach we were able, for the first time, to obtain insights into the deregulated proteome of SMZL.
2 Materials and methods
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were collected separately from each sample and protein concentrations were determined using the Bradford Protein Assay. The samples were then incubated with 5 mM tributyl phosphine (Sigma) and 20 mM acrylamide (Sigma) for 60 min at room temperature to reduce disulphide bonds and alkylate the cysteine thiolic groups. The reaction was blocked by the addition of 10 mM DTT (Sigma).
2.3 Two-dimensional polyacrylamide gel electrophoresis The spleen protein sample from each individual was analysed separately. Each 2D-PAGE gel was prepared with 800 g of proteins mixed with 450 l of 2DE buffer. The first dimension separation was performed in 17 cm, pH 3–10 non-linear IPG (Bio-Rad) and the total product time ×voltage applied was 70 000 Vh for each strip. The second dimensional separation was performed using 10–20% T 2.6% C gradient SDS-PAGE (18 cm × 20 cm × 1.5 mm), applying 40 mA for each gel for 3 min, then 2 mA/gel for 1 h, and 20 mA/gel until the track dye, bromophenol-blue, reached the anodic end of the gels. After 2DE, the proteins were detected by the pure ruthenium chelate RuBPS (Cyanagen).
2.1 Patients and tumour biopsies For proteomics analysis, a total of four SMZL tissue samples, diagnosed according to WHO criteria [15] were obtained from frozen spleen biopsies of various patients retrieved from the archives of the Department of Pathology and Diagnostics, University of Verona, Verona, Italy. For comparative purposes, four non-neoplastic spleens (removed for trauma or pancreas surgery) were also included. Western blot validation was performed on an independent sample set composed of four SMZL tissue samples, and four non-neoplastic spleen samples. Immunohistochemical analysis was performed on 15 SMZL samples and 5 non-neoplastic spleens. All the tissue samples were collected from patients who had signed informed consent forms.
2.2 Tissue preparation and protein extraction for proteomics analysis 10-m-thick tissue sections were cut from samples (20 slices for each biopsy) using a cryostat, and then placed in 400 L of 2DE buffer containing 7 M urea (Bio-Rad), 2 M thiourea (Sigma), 3% w/v CHAPS (Sigma), 20 mM Tris (Sigma), and 1x Complete, Mini, EDTA-free protease inhibitors (Roche) and stored at –80°C until analysis. Each frozen spleen tissue was homogenized and solubilized by sonication for 30 seconds × 5 times on ice and protein, precipitated by overnight incubation with acetone:methanol (8:1) at - 20°C. Pellets were subsequently collected from each sample by centrifugation at 11 000 × g for 15 min, resuspended in 2DE buffer, and centrifuged again at 21 000 × g for 40 min to remove insoluble tissue debris and nucleic acids. Supernatants C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.4 Image analysis and statistical treatment Digitized gel images were used to estimate the amount of different protein species in each analytical gel using PDQuest software (Bio-Rad, version 7.3). Each gel was analysed for spot detection, background subtraction, and protein spot OD intensity quantification. After manual editing, a match set was created in which the protein spots from different gels are matched to each other and are included in a synthetic image, called a match set standard (master 2DE gels). This artificial composite image contains all the information the software needs to analyse the gels quantitatively, qualitatively, or statistically. Spot quantity values were normalised in each gel dividing the raw quantity of each spot by the total quantity of all the spots included in the standard gel. Univariate and multivariate statistical analyses were carried out. Principal component analysis (PCA) was performed with Simca-P+ software (Umetrics, version 12) using logtransformed, normalized spot volumes of individual gels. The transformed spot volumes were thus mean-centered and scaled to unit variance to minimize the bias for spots of large volume on the PCA results. The resulting spot quantity values for the principal components were used to identify the protein species responsible for the variability between the two groups (control and SMZL). Student’s t-test was also performed to assess the statistical significance of modulated protein species. Based on the average spot volume ratio, spots whose relative amount is changed at least 1.5 fold (increase or decrease) between controls and SMZL samples at a 95% confidence level (t test; www.electrophoresis-journal.com
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p ⬍ 0.05) were considered to be significant. Spots were selected for identification based on significant differences in t-test and/or absolute spot loading values on principal components (PCs).
2.5 Protein identification by nanoHPLC Chip Ion Trap mass spectrometry Spots selected for identification were carefully cut out from 2D stained gels and subjected to in-gel trypsin digestion as previously described [16]. Peptides from 5 L of each sample were then separated by reversed phase nano-HPLCChip technology (Agilent Technologies, Palo Alto, CA, USA) online-coupled with a 3D ion trap mass spectrometer (model Esquire 6000, Bruker Daltonics, Bremen, Germany). The chip was composed of a Zorbax 300SB-C18 (43 mm × 75 m, with a 5 m particle size) analytical column and a Zorbax 300SBC18 (40 nL, 5m) enrichment column. The complete system was fully controlled by ChemStation (Agilent Technologies) and EsquireControl (Bruker Daltonics) software. The scan range used was from 300 to 1800 m/z. For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The three most abundant peptides of each m/z were selected for further isolation and fragmentation. The MS/MS scanning was performed in the normal resolution mode at a scan rate of 13.000 m/z per second. A total of five scans were averaged to obtain an MS/MS spectrum. The peak lists were searched using MASCOT (version 2.4.01, http:/www.matrixsciences.com) against all entries of the NCBInr database (38 032 689 sequences; 13 525 028 931 residues). The following parameters were set: trypsin digestion, up to one missed cleavage; fixed and variable modifications: propionamide (Cys) and oxidation (Met), respectively; peptide and fragment tolerances: ± 0.9 Da and ± 0.9 Da, respectively, and peptide charges: +1, +2 and +3. For protein identification, at least two peptides were considered. Only significant hits, as identified by the MASCOT probability analysis (p ⬍ 0.05) were accepted.
2.6 Mono and two-dimensional immunoblotting analyses Confirmation of the proteomic data by immunoblot analysis was performed on an independent sample set of splenic biopsies. Samples were analysed separately in 1D-WB and as a pool in 2D-WB. We investigated the modulation of Anp32a, Acadm, Grb2, Sod2, Nutf2, and Set. The splenic tissue extracts were separated by SDS-PAGE under denaturing and reducing conditions on 12%T polyacrylamide gels; and by 2DE on a 7 cm long IPG strip (pH 3–10 non-linear) and 10–20%T mini gels. The separated proteins (20 g of protein/lane for 1D-WB, and 100 g of protein/gel for 2D-WB) were transferred to a PVDF membrane through electroblotting. The membranes were treated with primary and secondary C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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antibodies at the appropriate dilutions (see Supporting Information Table 1) and bound antibodies were detected by the ECL Western blotting detection system (Millipore, USA). Equal sample loading was confirmed by anti-tubulin antibody. Western blots were scanned by a ChemiDoc instrument (Bio-Rad), and quantitative analysis of specific signals was performed using Quantity One software (Bio-Rad). In order to investigate the 2D-WB patterns of immuno-detected protein species, the ProMoST tool (Protein Modification Screening Tool) [17] was used. Since ‘trains of spots’ are in most cases attributable to phosphorylation or deamidation [18] (although other less likely PTMs, such as acetylation or methylation, could also be involved), modification of the protein pI by Ser.pSer, Thr-.pThr, Tyr-.pTyr, Gln-.Glu, or Asn-.Asp conversion was analysed. To create the theoretical 2DE gel, the pH range was set from 3 to 10 with steps of 0.5, and a molecular weight range was set from 10 to 200 kDa with steps of 20 kDa. 2.7 Immunohistochemical analysis Tissue sections were incubated for 15 min at 95°C in a pH 6 buffer (Bond Epitope Retrieval Solution 1), then labeled with the antibody for the protein SET (dilution 1:100) or ACADM (dilution 1:300) and processed with a detection system, Bond Polymer Refine kit using an automated stainer (Leica Bond Max). 2.8 Functional data mining Functional annotation of identified proteins was performed according to Gene Ontology (GO) using the FatiGO online tool from Babelomics (Babelomics 4.2; http://babelomics. bioinfo.cipf.es/fatigo.html). GOcellular component, biological process and molecular function were analysed by comparing the GO terms of identified proteins against the rest of the genome. Fisher’s exact test detected the significant over-representation of GO terms in the submitted dataset. Protein interactions were assessed using the STRING 9.0 database (http://string-db.org). We retrieved at least medium confidence (score 0.4) interactions including all the prediction methods implemented in STRING (excepted text mining). No additional white nodes were inserted in order to reduce noise, and network depth was kept to the minimum value (1) to exclude as many false positive interactions as possible.
3 Results 3.1 Proteomic investigation of splenic marginal zone lymphoma tissue Protein profiling of spleen tissues was undertaken to determine proteome changes in SMZL by comparing matched normal and disease biopsies. An average of 464 (± 2.5%) protein spots with an apparent molecular mass between 10 and 200 kDa and a pI between 3 and 10 were detected by 2DE www.electrophoresis-journal.com
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Figure 1. Representative 2DE images obtained using spleen proteins of SMZL patients (on the left) and controls (on the right). Identified protein spots are indicated by white arrows and numbered as specified in Supporting Information Table 2.
(Fig. 1). Protein patterns from SMZL and control splenic tissues were compared by multivariate statistical analysis to identify protein species responsible for correlated variations, and investigating intra- and inter-group relationships. The PCA indicated that there were two distinct groups of maps from the two experimental conditions, SMZL and control (Supporting Information Fig. 1A). This indicates that there is sufficient natural variation in the proteomes to distinguish between the two groups, increasing the confidence that potential disease biomarkers can be found in this dataset. The strongest separation was observed in the first and second PCs, which accounted for most of the variation in the datasets, i.e. 23.5 and 18.6%, respectively. In addition, the loading plot obtained from PCA led to the identification of variables (spots) that were more suited for explaining relationships in the dataset which contribute the most to the separation of the two experimental groups (Supporting Information Fig. 1B). Individual proteins were also analysed by t-test analysis (p ⬍ 0.05) to significantly identify different intensities between the two experimental groups. Supported by multivariate and univariate analyses, a total of 32 spots (17 up-regulated and 15 down-regulated) from the 2DE analysis were identified as significantly differentially regulated in the spleens of SMZL patients (Fig. 1). All these differentially regulated spots were successfully picked up and proteins were identified by nanoHPLC-Chip/MS (Supporting Information Table 2). The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [19] with the dataset identifier PXD001124 and DOI 10.6019/PXD001124.
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3.2 Western blot validation of various deregulated protein species To validate our proteomic results, we measured the protein levels by 1D-WB of six selected targets of interest in the spleens of four SMZL samples and four controls (Fig. 2). We confirmed increased amounts of proteins of the Acadm, Sod2, Nutf2, and Grb2 in SMZL tissue samples. The upregulation of Anp32a, limited to the 14.8 kDa isoform [20], was also confirmed (however one control sample also showed marked immunodetection of Anp32a). Unfortunately, we did not confirm Set modulation, which could be ascribed to the overlap of several protein forms in the 1D separation which may have an opposite modulation (but not detected as significant by 2DE). To deepen the investigation of protein species-specific modulation, the selected proteins listed above were also analysed by 2D-WB (Fig. 3) with the support of ProMoST in silico analysis to interpret any pI shift. 2D-WB results revealed four signals for Sod2 at pI from 6.5 to 7.8, and the absence of the unmodified Sod2 form (pI of 8.25) in both, SMZL as well as in normal spleens. As indicated by ProMoST in silico analysis, the four immunodetected protein species suggested a pattern of phosphorylation. The most acid protein spot (containing at least one protein species probably with four phosphorylations) appears to be induced in SMZL. Obviously, such PTM should be confirmed by MS, or by Western blot with specific anti-phospho-tyr antibodies. In addition, 2D-WB analysis with anti-Grb2 antibody revealed seven immunodetected signals in SMZL and only five in normal spleens, which confirmed that this protein is more abundant in the SMZL spleen tissue.
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and deamidated (pI 5.77) Grb2 forms, and the two other spots could contain at least one protein species, probably one-fold phosphorylated (pI 5.75), and at least one protein species, probably di-phosphorylated (pI 5.57). The 2D-WB analysis obtained with the anti-Acadm antibody indicated eight immunodetected spots (pI ranging from 5.5 to 7.5) in SMZL and, although much less evident, also in the control samples. This confirmed the increased amount of Acadm in the SMZL tissue, suggesting, as indicated by ProMoST in silico analysis, a pattern of phosphorylation and the absence of the unmodified form (pI of 8.12) for this protein. In addition, by 2D-WB analysis with anti-Nutf2 antibody, we found that, in SMZL and normal spleens, there were two spots, one containing the unmodified protein species (pI 5.1) and the other containing at least one protein species, probably one-fold phosphorylated (pI 4.9) as suggested by ProMoST analysis. The amount of the putative one-fold phosphorylated Nutf2 form was increased in the SMZL tissue at the expense of the unmodified protein species. Finally, the Set molecules were depicted as a collection of more than 15 different protein spots in SMZL, and more than 10 in normal spleen tissue. These immunodetected spots could be classified into three groups with different ranges of Mw: ⬎30 kDa (protein species with PTM) which were less abundant in SMZL; 30 kDa (intact or differentially spliced forms) and ⬍30 kDa (cleaved, truncated forms) which were more abundant in SMZL. A similar pattern with multiple spots was also found in the 2D-WB of Anp32a carried out on several electroblotted membranes. Also in this case the complicated 2D-WB pattern could be ascribed to PTM, splice variants, and cleaved/truncated forms of Anp32a. 3.3 Immunohistochemical analysis
Figure 2. (A) Monodimensional Western blot analysis in normal (C) and tumour spleen tissues (SMZL). Tubulin was used as a loading control. (B) Densitometric analysis of the Western blots was carried out using Quantity One software. The intensity of the bands was normalized to the intensity of the respective tubulin band. The values obtained were plotted as mean optical density (OD) ± sd from three independent experiments. White bars represent SMZL and the grey bars, the control samples.
2D-WB suggested that the SMZL spleen tissue is characterized by the appearance of two Grb2 species (pI 6.2 and 6.5) with a shift to more basic pI values (compared to the unmodified Grb2 form), and by the increasing four protein spots (with pI ranging from 5.5 to 6). The analysis carried out with ProMoST suggested that two of these four protein spots could contain the unmodified (pI 5.95)
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Of the modulated proteins, the nuclear oncogene Set and the enzyme Acadm were selected for further evaluation by immunohistochemical analysis. We found that Set (Fig. 4A) was expressed in almost all cells of the tumour SMZL spleen tissue in all the tested cases (15/15). In the normal spleen, on the other hand, it was expressed only by centroblasts of germinal centers, to a lower extent in the mantle, and then again expressed in the marginal zone. Acadm (Fig. 4B) was also expressed in almost all cells of the tumour SMZL spleen tissue in the majority of tested cases (14/15), while in the normal spleen it was only revealed in germinal centers. All the 15 tested cases for Set and Acadm are shown in Supporting Information Figs. 3 and 4, respectively. 3.4 Functional characterization of the proteins identified and protein–protein interaction analyses By gene-annotation enrichment analysis, we found that many of the SMZL identified proteins were located in the following
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Figure 3. Bidimensional Western blot analysis in normal (C) and tumour spleen tissues (SMZL).
Figure 4. (A) Immunohistochemical analysis of Set and (B) Acadm in spleen tissues. Panels on the upper line represent controls, while those of the line below indicate SMZL samples. On the left there are 200x magnifications and on the right 400x. Arrows indicate centroblasts, while the letters GC, the germinal center, m mantle and MZ the marginal zone. Specific staining was dark, and counterstaining was light grey.
“cellular components”: vesicles (membrane-bounded, and cytoplasmic), perinuclear region, and phagocytic cup. “Biological processes” enriched among the deregulated proteins of SMZL spleens were: response to organic substance, response to nutrient levels, monosaccharide metabolic process, protein oligomerization, generation of precursor metabolites and energy, regulation of apoptosis, regulation of programmed cell death, cellular amino acid derivative metabolic process, and glucose catabolic process. Concerning statistically enriched GO term in the category “molecular function”, we found the term oxygen binding to be significant (Supporting Information Table 3). In order to better understand whether the protein species identified in this study had any previously described interactions, we analysed the protein interaction networks using the String 9.0 database. This revealed that there were significant interactions between some of the proteins identified (Supporting Information Fig. 2). There was a core interaction network involving Anp32a, Set, Gapdh, Pkm2, Eno1, Actb, Coro1a. In addition, of the identified proteins, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
also ApoA1, Alb, P4hb, and Capns1 formed a separate interaction network.
4 Discussion The present study is, we believe, the first to directly show the proteomic alterations of spleen occurring with SMZL. An analysis carried out at the splenic tissue level is very important, since the biopsies (unlike body fluids that reflect modifications occurring far from the tumour) enable molecular information directly linked to the tumour biology to be detected. Using a 2DE/MS based approach, we were able to find, for the first time, the differences in the splenic proteome of patients with SMZL and to identify disease-specific changes in the protein species pattern. Knowledge of the genetic lesions associated with SMZL is heterogeneous, thus it is quite difficult to fit our proteomic results with the genetic data available [9]. It is not possible to speculate whether there is a www.electrophoresis-journal.com
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connection between the deregulated protein species and the gene copy number aberrations, therefore the protein species dysregulations reported here should be attributed to other phenomena (such as epigenetic or splicing events, PTMs, etc.). Network analysis revealed connectivity between some of the identified proteins, and two main clusters emerging from the list (Supporting Information Fig. 2). The functional relevance of some of these proteins, as well as their impact on SMZL deregulated pathways is discussed below. Notably, as indicated by GO enrichment analysis (Supporting Information Table 3) the first cluster of proteins revealed by network analysis has a role in metabolism, while the second cluster represents proteins involved in cytoplasmic vesicles. Collectively, gene-annotation enriched analysis (Supporting Information Table 3) suggests that the modulation in spleen proteome associated with SMZL predominantly involves changes in energy metabolism (GO:0031667, GO:0005996, GO:0006091, GO:0006575, GO:0006007) and apoptosis pathways (GO:0042981, GO:0043067). The changes in energy metabolism may involve an increase in the glycolytic pathway (as suggested by the up-regulation of Eno1 and Gpdh) and a stimulation of the -oxidation pathway (as indicated by the up-regulation of Acadm). Acadm was found up-regulated in a subgroup of diffuse large B cell lymphoma [21], and it has been suggested that the -oxidation pathway is required to ensure the turnover of the tumour cells [22]. The role of Acadm in lymphoid cells is poorly understood. However the presence of high levels of the protein in germinal centre cells (Fig. 4B) also suggests a potential role in B-cell receptor (BCR) signalling.
4.1 Deregulated SMZL proteins involved in the NF-B pathway Interestingly, many of the identified protein species belong to SMZL deregulated processes already highlighted by genetic studies (such as NF-kB and BCR pathways, chromatin remodelling, and cytoskeleton) [9,11]. It has been demonstrated that genetic lesions of the NF-B pathway provide a molecular basis for the pathogenesis of more than 30% of SMZLs [23], thus implying that NF-kB activation is a major contributor to the pathogenesis of this disease [9, 11]. NF-kB transcription factors are involved not only in the apoptotic pathway but also in the regulation of energy metabolism [24], which we found to be deregulated (Supporting Information Table 3). Concerning the proteins involved in this pathway, we found that SMZL patients have a higher level of one protein species of biliverdin reductase A (BLVRA) (SSP5501, +3.54). Blvra acts as a reductase and as a ser/thr/tyr kinase, and its overexpression enhances the activation of NF-kB and also the NF-kB-activated iNOS gene [25]. Blvra removes biliverdin, which is an inhibitor of NF-kB, and also acts in the MAPK and PI3K/Akt pathways (implicated in the p65 phosphorylation and activation of NF-kB) [26]. The formation of the ternary complex of biliverdin reductase/protein kinase C␦/ERK2 protein is also C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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essential for the activation of NF-B [27]. Interestingly, Blvra is up-regulated in different types of cancer cells: its overexpression enhances resistance to chemotherapeutics [28], and targeting Blvra overcomes multidrug resistance in leukemia cells [29]. This study also led to the identification of one protein species of manganese superoxide dismutase (SOD2), which was up-regulated in SMZL patients (SSP7302, +2.94). This finding was confirmed by 1D-WB immunoblot analysis (Fig. 2). The induction of SOD2 transcription in response to the activation of NF-kB is well documented [30]. The transcriptional activity of the SOD2 gene is enhanced by the p65 subunit [31]. Notably, Sod2 overexpression enhances the migratory potential of tumour cells, contributing to their increased metastatic behaviour [32]. Sod2 was also detected as one of the most up-regulated proteins in OxPhos cell lines of diffuse large B cell lymphoma [21]. In addition, although Sod2 is known to be regulated at the post-translational level, our 2D-WB results demonstrated that in SMZL, the changes in Sod2 modifications do not predominate over changes in the total amounts of Sod2 (Fig. 3). Our results also indicate that one protein species of beta2 microglobulin (B2M) is up-regulated in SMZL patients (SSP4104, +1.70). B2m is a chaperone of major histocompatibility complex (MHC) class I (-like) molecules, which play a central role in antigen presentation, immunoglobulin transport, and iron metabolism. It is connected to the NF-kB pathway since p50 and p65 subunits bind to the B2M promoter and transactivate it in lymphoid and myeloid cells [33]. Interestingly, the plasma levels of B2m correlate negatively with outcome in non-Hodgkin’s lymphoma [34]. In addition, a marked increase in plasma B2m levels was found in 78% of SMZL patients [35]. it is worth noting that antibodies against B2m induce apoptosis in solid tumours and in hematological malignant cells (such as multiple myeloma, Burkitt lymphoma, mantle cell lymphoma, T-cell and myelogenous leukemia cell lines) [36]. We also identified two protein species (ApoA1 and Phb) whose down-regulation was in agreement with the presence of the NF-kB signalling pathway activated in SMZL. The level of two protein species of apolipoprotein A-I (APOA1) was lower in SMZL patients (SSP2305, −2.22; SSP1305, 1.75). Interestingly, ApoA1 also has an anti-inflammatory activity, reducing the expression of chemokines and chemokine receptors via modulation of NF-kB pathway. Mimetic peptides of ApoA1 suppress phosphorylation of IkBalpha (an upstream mediator of the NF-kB pathway), nuclear p65 expression, and p65 DNA binding activity [37]. Accordingly, in vivo studies have demonstrated that ApoA1–infused mice had lower levels of p65 and p50 subunits [38]. The other SMZL down-regulated protein species we reported was prohibitin (PHB, SSP2408, −1.51). Interestingly, Phb overexpression correlates with the reduction in NF-kB nuclear translocation, NF-kB DNA binding, and NF-kB–mediated gene transcription. This protein inhibits the activation of NF-kB via a mechanism involving a reduction in importin 3, a protein implicated in p50/p65 nuclear translocation [39]. www.electrophoresis-journal.com
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4.2 Deregulated SMZL proteins involved in BCR signalling Of the genes deregulated in SMZLs, there has been a special focus on those involved in BCR signalling [11,40]. Of particular interest is our observation that the amount of one protein species of growth factor receptor-bound protein 2 (GRB2) was higher in SMZL patients (SSP4307, +2.70). Grb2 upregulation was also revealed by 1D-WB analysis (Fig. 2B). Grb2 is an adapter protein that mediates BCR signalling pathways. It is associated with LIME, a transmembrane adaptor required for BCR-signalling [41]. Interestingly, strong cytoplasmic Grb2 expression has already been found by immunohistochemical analysis in the neoplastic cells of SMZL, as well as in other B-cell non-Hodgkin lymphomas [42]. Our results coincide with these observations and provide a validation with a completely independent approach. 2D-WB analysis (Fig. 3) indicated that, by using same experimental conditions (i.e., amount of total protein loaded, antibodies dilution, exposure time to the x-rays, etc . . . ), seven and five different spots could be immunodetected in SMZL and control samples, respectively. All the seven Grb2 species are induced in SMZL, in particular two species with more basic pI values seem to be particularly interesting since (by using the experimental conditions indicated in Section 2) they are not detected in control samples, suggesting a very low level of expression in normal spleen tissue. Future research could lead to the characterization of these two more basic Grb2 protein species, and clarify their role in SMZL tissue, and possibly their involvement in BCR signalling pathways.
4.3 Deregulated SMZL proteins involved in chromatin remodelling Consistent with the hypothesis that the SMZL genetic signature is characterized by deregulated chromatin remodelling [9, 11], here we showed the up-regulation of both one protein species of acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A, SSP0401, +4.05), and one species of Set nuclear oncogene (SET, SSP0601, +4.13). Anp32a and Set were the most SMZL overexpressed protein species identified by our proteomic approach. Notably, we showed that Set is differently localized in the spleen tissue of SMZL compared to the control spleen (Fig. 4A). It is expressed in almost all of the neoplastic cells, while in the control sample it is localized only in the centroblasts of germinal centers, and in sparse lymphoid cells of the mantle and marginal zones. Anp32a and Set act as inhibitors 1 and 2 of the tumour suppressor protein phosphatase 2A (I1PP2A and I2PP2A). They are also components of the inhibitor of the histone acetyl transferase complex that binds to histones and masks them from being acetylated [43] thus leading to transcriptional repression. The tumorigenic role of Anp32a [44] and Set [45] has been already demonstrated in several types of cancers. It is worth mentioning that Set oncogene has been recently suggested as a new treatment C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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molecular target in B-cell malignancies [46]. Interestingly, Set fragments (which were found to be increased in SMZL) prevent the inhibition of the NME1 [47] (an oncogene typical of B-cell lymphoma [48]), and are involved in the inhibition of the tumour suppressor PP2A [49].
4.4 Deregulated SMZL proteins involved in cytoskeleton organization The cytoskeleton organization is the fourth cell process that was found to be notably altered in SMZL [11] by wholeexome sequencing analysis. Accordingly, the data obtained here indicate the down-regulation of specific protein species involved in cytoskeleton organization. Of these, one protein species of beta actin (ACTB) was found to be less expressed in SMZL patients (SSP 1603, −1.70). Actb is a component of the cytoskeleton, and a mediator of internal cell motility. Its reorganization has been shown to be important for BCR activation [50], which confirms that these processes are critical to SMZL development., One protein species of coronin 1A (CORO1A) was also down-regulated in SMZL patients (SSP4603, −2.44). Coro1a, is an actin-binding protein selectively expressed in hematopoietic cells, which plays a crucial role in the immune response through reorganization of the actin cytoskeleton [51]. We also found a lower level of one protein species of calpain small subunit 1 (CAPNS1, SSP1304, −1.64) in SMZL patients. Calpains are endopeptidases, which have numerous functions including, but not limited to, remodelling of the cytoskeleton. One protein species of L-plastin (LCP1, SSP1809, −1.85) was also found to be down-regulated in our study. It is a leukocyte-specific protein that cross-links actin filaments into tight bundles, increasing the stability of actin-based structures. Interestingly, LCP1 −/− mice have been found to be deficient in marginal zone B cells [52]. Finally, we also identified a protein species of nuclear transport factor 2 (NUTF2), which is instead up-regulated in SMZL tissue (SSP1001, +2.42). Nutf2 is indirectly involved in the cytoskeleton organization. It facilitates transport into the nucleus of the filamentous actin-capping protein (CapG), a mediator of cross-talk between the actin cytoskeleton and microtubulebased organelles [53]. An increased amount of Nutf2 in SMZL was confirmed by 1D-WB and by 2D-WB (Figs. 2 and 3).
4.5 Conclusions This is the first study to shed light on the proteome modulations of SMZL, thereby opening up new ground for the development of possible diagnostic as well as therapeutic markers of SMZL. Our findings indicate that SMZL is characterized by changes in energy metabolism and apoptosis pathways. We identified for the first time the deregulated protein species involved in NF-kB, and BCR pathways, as well as in chromatin remodelling and cytoskeleton. Of particular interest www.electrophoresis-journal.com
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are biliverdin reductase A, manganese superoxide dismutase, beta-2 microglobulin, growth factor receptor-bound protein 2, acidic leucine-rich nuclear phosphoprotein 32 family member A, Set nuclear oncogene, and nuclear transport factor 2. Although it is difficult to expand the number of biopsy samples due to the rare incidence of these histological lymphoma entities, the results warrant further validation in larger cohorts. Validation should include the use of independent techniques, also in view of the fact that the 2DE modulations observed may be slightly incorrect due to spot overlapping. This work was supported by AIRC, Fondazione Cariverona and from intramural funds of the University of Verona. All authors declare no financial and no commercial conflicts of interest.
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