Identification of Gliotropic Factors That Induce Human Stem Cell

Apr 7, 2009 - malignant gliomas remains poorly understood. ... clinically relevant information regarding tumor malignancy, and should also prove appro...
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Identification of Gliotropic Factors That Induce Human Stem Cell Migration to Malignant Tumor Jeung Hee An,† Soo Youn Lee,‡ Jeong Yong Jeon,‡ Kyung Gi Cho,§ Seong U. Kim,|,# and Myung Ae Lee*,‡ BK 21 Center for Intelligent Nanostructured Core Material Technology, Department of Chemical & Biomolecular Engineering, Sogang University, Seoul, 123-742, Korea, Brain Disease Research Center, School of Medicine, Ajou University, Suwon, 443-721 Korea, Department of Neurosurgery, Ajou University, Suwon, 443-721 Korea, Division of Neurology, Department of Medicine, UBC Hospital, University of British Columbia, Vancouver, Canada, and Medical Research Institute, Chungang University College of Medicine, Seoul, Korea Received January 9, 2009

Neural stem cells are mobile, are attracted to regions of brain damage, and can migrate a considerable distance to reach a glioma site. However, the molecular basis of the progression of gliotropism to malignant gliomas remains poorly understood. With the use of clinically and histologically assessed glioma cells, we have assessed their protein and gene profiles via proteomics and microarray approaches, and have identified candidate genes from human glioma tissues. This research is expected to provide clues to the molecular mechanisms underlying the migration of neural stem cells (F3 cell) to glioma sites. The expression of 16 proteins was shown to have increased commonly in human glioma tissues. Among them, the expression of annexin A2, TIMP-1, COL11A1, bax, CD74, TNFSF8, and SPTLC2 were all increased in human glioma cells, as confirmed by Western blotting and immunohistochemical staining. In particular, annexin A2 effects an increase in migration toward F3 and glioblastoma cells (U87 cell) in a Boyden chamber migration assay. An ERK inhibitor (PD98057) and a CDK5 inhibitor (rescovitine) inhibited 50% and 90% of annexin A2-induced migration in F3 cells, respectively. A similar chemotactic migration was noted in F3 and U87 cells. These results demonstrated that 7 candidate proteins may harbor a potential glioma tropism factor relevant to the pathology of malignant glioma. These results reveal that this novel molecular approach to the monitoring of glioma may provide clinically relevant information regarding tumor malignancy, and should also prove appropriate for highthroughput clinical screening applications. Keywords: gliotropism factor • proteomics • microarray • migration • Neural stem cell

Introduction Metastasis, the well-known phenomenon of the organspecific spread of tumor cells, is largely a mechanical process, which is either directed passively as the result of size constraints, or occurs as the consequence of a fertile environment provided by the organ in which tumor cells can proliferate.1 Additionally, brain metastasis is known to be dependent on multiple cell-cell and cell-matrix interactions, which allow tumor cells to detach from the primary tumor, arrest at distant sites, transmigrate across the endothelial lining into the parenchymal tissue, and ultimately form secondary tumors.2,3 Generally, adhesion molecules such as vascular endothelial * To whom correspondence should be addressed. Brain Disease Research Center, School of Medicine, Ajou University, Suwon, 443-721 Korea. Phone: 82-02-3273-0357. Fax: 82-02-3273-0331. E-mail: [email protected]. † Sogang University. ‡ Brain Disease Research Center, School of Medicine, Ajou University. § Department of Neurosurgery, Ajou University. | University of British Columbia. # Chungang University College of Medicine. 10.1021/pr900020q CCC: $40.75

 2009 American Chemical Society

growth factor (VEGF), the ligands of lymphocyte functionassociated antigen-1, intercellular adhesion molecule-1, and integrin expression have been identified as crucial for metastasis-related molecular genes and chemoattraction factors.4,5 Neural stem cells are mobile, are attracted to regions of brain damage, and can migrate a considerable distance to reach a glioma site.6 Interestingly, glioma and neural stem cells (NSCs) evidence similar migration properties.7 In particular, stromal cell-derived factor-1 (SDF-1), chemokine monocyte chemoattractive protein, vascular endothelial growth factor (VEGF), and others have been demonstrated to regulate the migration of NSCs.8-11 Recently, a number of in vivo and in vitro studies have also demonstrated that NSCs have a unique capacity to migrate throughout the brain and to aggressively target invading tumor cells, such as glioma cells.7,10 Stem cells of various lineages have been recognized as attractive vehicles to improve the delivery of therapeutic genes to glioma cells.12 Neural stem/ progenitor cells can be engineered to generate therapeutic molecules, and also harbor the potential to overcome these limitations and engraft stably into the brain.13 Journal of Proteome Research 2009, 8, 2873–2881 2873 Published on Web 04/07/2009

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Table 1. Clinical Characteristics of Analyzed Patients sample code

T1 T2

Glioblastoma Glioblastoman

T3 T4 T5 T6 T7 T8 T9 T10 N1 N2 N3

Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoman Anaplastic oligodendroglioma Glioblastoma Mengioma Mengioma Mengioma

N5 N6 N7 N8 a

group

region

age

sex

Frontal Rt. Parietal and splenium of corpus callosum Frontal Lt. frontal Frontal Lt. temporal Lt. insular Rt. Frontal Lt. Frontal Frontal margin tumor area margin tumor area margin tumor area

69 65

F M

69 33 68 28 53 73 56 52 69 65 46

F M M F F F F M F M M

margin margin margin margin

68 36 55 47

M M M F

tumor tumor tumor tumor

area area area area

T, tumor tissues; N, normal tissues; F, female; M, male.

We hypothesized that genes secreted from human glioma tissues may be putative chemoattractants, and may provide clues to the molecular pathway involved in the migration of NSCs to glioma cells. In this study, we demonstrate that highthroughput screening using proteomic and microarray techniques should provide a more comprehensive overview of the interaction of tumor-related proteins, the interplay among processes, and the context in which a tumor-specific molecule or pathway may operate in the mechanisms inherent to glioblastoma. Indeed, these approaches have been applied to the tropism molecular pathway in the migration of stem cells to tumors. In this study, we have evaluated the tumortropic factor profiles of NSCs via proteomics and microarray techniques, and have identified genes associated with the gliotropic properties of NSCs. Our results showed that seven candidate gliomatropism genes may function as useful molecular indicators; this constitutes a significant step forward in our current understanding of these gliomas. Our findings help to elucidate the complex process of glioma-specific metastasis formation, which depends heavily on the interplay between adhesion molecules and transmigration-associated proteins.

Materials and Methods Glioma Tissues and Cells. All patients undergoing surgical treatment at Ajou University Hospital for primary brain cancer between 2005 and 2007 were invited to participate in an ethical and legal guidelines program. The clinical and pathologic data of the samples are provided in Table 1. Tumor tissues and their nontumoral tissue counterparts were resected. Among the patients participating in the broad protocol, 15 were analyzed on the basis of the presence of a tumor diagnosed as a grade IV (n ) 10) glioblastoma of any histologic type, and the availability of a fresh-frozen material sample. All tissue samples were evaluated by a neuropathologist. Patients’ ages at diagnosis varied from 27 to 73 years. The protocol for this clinical study was reviewed and approved by the Ajou University Medical Center. Each patient signed written informed consent and institutional policies and regulations forms, as a condition of registering for participation in the study. 2874

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The U87 and HEK293T cell lines were purchased from the American type Culture Collection. Human neural stem cells (NSCs) were established as previously described7,14 and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, and 10 µg/mL of penicillin-streptomycin (Gibco, NY), then incubated at 37 °C in an incubator with an atmosphere of 5% CO2/95% air. Microarray Analysis. Total RNA was isolated as previously described from 100 mg of glioma tissue using TRI reagent (Molecular Research Center, Cincinnati, OH) in accordance with the manufacturer’s instructions, then cleaned using an RNeasy kit (Qiagen, Valencia, CA).15 Contaminating DNA was degraded enzymatically with RNase free DNase (Qiagen, Valencia, CA). RNA was quantified and analyzed for integrity using an RNA 6000 Nano-Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). First- and second- strand cDNA synthesis, biotin-labeled cRNA synthesis, and cRNA fragmentation and hybridization reactions were conducted via one cycle with a cDNA synthesis kit (Affymetrix, Santa Clara, CA). Eight micrograms of purified RNA was used for cDNA synthesis. We studied gene expressions using Affymetrix Human Genome HG-U133A and HG-U133B Gene Chip expression arrays (45 000 array features covering >28 000 UniGene clusters). Initial gene expression data analysis was done using Microarray Suite 5.0 software (Affymetrix 2001). GenPlex v1.8 software (ISTECH, Inc., Goyang City, South Korea) was utilized for data analysis. The MAS5 algorithm was employed for expression summary and signal calculation. Global saline normalization using a GCOS algorithm was conducted, after which the normalized data were log2-tranformed. Fold change and Welch t test were applied for the selection of differentially expressed genes. The difference was more than 2-fold, and the significance level was 0.05. For better visualization and comparison, two methods were utilized for the detection of differentially expressed genes: the 2-fold differentially expressed genes were clustered via hierarchical clustering with Pearson correlation; the top 200 differentially expressed genes with the greatest fold changes were classified into functional subgroups via Gene Ontology analysis. Proteomics. Human tumor tissues were suspended in sample buffer containing 40 mM Tris-HCl, 7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 0.2% (v/v) Bio-Lytes, and endonuclease. The suspensions were sonicated for approximately 30 s and centrifuged for 1 h at 100 000g to remove DNA, RNA, and any particulate materials. The supernatants contained the total glioma proteins solubilized in sample buffer. 2-DE was conducted with a Bio-Rad Electrophoresis system.16 Total protein in liver tissue (1 mg) was utilized for each electrophoresis. Aliquots of glioma tissue proteins in sample buffer were applied to immobilized pH 3-10 nonlinear immobilized pH gradient (IPG) strips. Isoelectric focusing electrophoresis (IEF) was conducted at 100 000 Vh. Following IEF, the strips were equilibrated for 10 min in 6 M urea, 2.5% (w/v) SDS, 2% (w/v) DTT, 5 mM TBP, 50 mM Tris-HCl (pH 6.8), and 20% (v/v) glycerol. The second dimensions were analyzed on 9-18% linear gradient polyacrylamide gels using a Protean XL system (Bio-Rad) at 15 mA per gel and 20 °C. Immediately after electrophoresis, the gels were fixed in 40% methanol and 5% phosphoric acid, and then stained for 24 h with Coomassie blue G 250. The stained gels were scanned with a GS-800 calibrated densitometer (Bio-Rad). The digitized gel images were normalized and comparatively analyzed using the PDQUEST program (V. 6.2, Bio-Rad). For mass spectrometry fingerprinting, protein

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Figure 1. Microphotographs showing hematoxylin-eosin staining of human glioma and normal tissues. ×100: Bars represent 100 µm.

spots were directly excised from the gels, destained with 50% acetonitrile in 25 mM NH4HCO3, and dried in a speed vacuum concentrator (Savant). The dried gel pieces were reswelled with 50 mM NH4HCO3 (pH 8.0) containing 100 ng/µL trypsin, and then incubated for 17 h at 37 °C. The supernatant peptide mixtures were extracted with 50% acetonitrile in 5% TFA and dried in a speed vacuum concentrator. The peptide mixtures were then dissolved in 4 µL of 50% acetonitrile in 0.1% TFA. Aliquots (0.5 µL) were then applied to large disks and permitted to air-dry. The matrix utilized was R-cyano-4-hydroxycinnamic acid. Spectra were acquired using a Voyager-DETM STR Biospectrometry Workstation System (Applied Biosystems, Foster City, CA) in positive reflector mode. Peptide masses were obtained for the range 850-3000 Da. Mass spectra were recorded using 50-150 shots depending on the single-to-noise ratio acquired from each sample. For on-target digested samples, tryptic autolysis peptides were utilized as an internal standard. The resulting data were evaluated using an Applied Biosystem GPS Explorer v3.6 system (Applied Biosystems), which utilizes the MASCOT 2.1 (www.matrixscience.com) database search engine for PMF and PMF identification for the MS data. A search was conducted against the Swiss-Prot database. Protein identifications were established using the following parameters: taxonomy was Homo sapiens, the enzyme was trypsin, the number of missed cleavage sites was allowed up to 1, the fixed modification was carbamidomethylation of cysteine, the variable modification was oxidation of methionine, monoisotopic peptide mass tolerance was 50 ppm, and the fragment ion tolerance was 0.3 Da. Protein functions were obtained from the Gene Ontology consortium (http://www. Geneontolry.org) and Swiss-Prot database. Western Blot Analysis. Protein extracts from normal and tumoral tissues from nine patients were separated in parallel via 12% SDS-PAGE. The proteins were transferred to PVDF membranes. After blocking, the membranes were incubated with anti-annexin A2 (BD Transduction), TIMP-1, BAX (Cell Signaling), CD74 (Stressgen, MI), TNFRSF8 (RD Systems), serine palmitoyltransferase, and COL11A1 (Abcam, Cambridge Science Park, U.K.) with 5% BSA in TBST buffer overnight at 4 °C. After washing, the membranes were incubated for 1 h at room temperature in either an anti-rabbit or anti-mouse IG antibody conjugate (1:2000, Santa Cruz Biotechnology). Immunoblots were detected with an ECL Western blotting detection system (Amersham International, Little Chalfont, U.K.) and visualized after exposure to film. Detectable proteins were quantified via densitometry (Raytest, Straubenhardt, Germany). Immunohistochemical Analysis and Immunocytochemistry Stain. Immunohistochemical analysis was analyzed in accordance with the previously described method.10 Four micrometer-thick sections of paraffin-embedded tumor tissues were utilized for immunohistochemical staining with the

Figure 2. Graphical representation of the distribution (percentage) of genes by functional category. (A) Identification of proteins in human normal and glioma tissues using proteomics; (B) identification of genes in human normal and glioma tissues by microarray analysis.

streptavidin-biotin peroxidase technique (Universal LSAB-HRP kit, DAKO, Carpinteria, CA). The frozen sections were then rehydrated by a sequential series of concentrated alcohols. At the final stage, the peroxidase reaction was visualized using 3-3-diaminobenzeidinetetrahydrochloride. The sections were then counterstained with hematoxylin, dehyhydrated, and mounted. Adherent cultures were grown on fibronectin-coated glass slides. Cells were fixed in 4% paraformadehyde (15 min), rinsed in PBS, and labeled with annnexin A2 primary antibody using standard immunocytochemical techniques. Images were acquired on a Zess LSM510 Axioplan-2 upright confocal microscope using LSM image software (Carl Zeiss, Jena, Germany). Boyden Chamber Migration Assay. In vitro migration assays were conducted as previously described.7 A migration kit from BD Falcon with 24-well culture plates was used. Each well of the plates was separated into two chambers by an insert membrane of 8-µm pores. One day before the assay, the cells (4 × 104 cells) were seeded into each upper chamber. The next day, recombinant Annexin A2 (R & D Systems, Inc., Minneapolis, MN) was added to DMEM media in the lower chamber at the indicated concentrations. After 12 h of incubation at 37 °C, migrating cells on the bottom of the insert membrane and nonmigrating cells on the upper side of the Journal of Proteome Research • Vol. 8, No. 6, 2009 2875

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membrane were fixed with 4% PFA at 10 min, and then processed for immunocytochemistry. The migration was quantified via the analysis of at least 10 random fields per filter for each independent experiment using Olympus microscopy image software. Statistical analyses were conducted using Student’s t test.

Results Gliomas Sampled in This Study Evidenced Typical Patterns. The human glioblastoma tissue samples were routinely hematoxylin-eosin (H&E) stained (Figure 1). The staining was consistent with a grade IV malignant glioma, evidencing palisades and extensive tumor neovascularization (Figure 1). Additionally, our results revealed features indicative of glioblastoma microvascular proliferation, nuclear atypia, the presence of giant cells, and pseudopalisading necrosis. The available clinical information is included in Table 1. The tumor patients had, in total, 10 grade IV neuroblastomas. Normal tissues were acquired at the margin tumor area from 3 neuroblastoma, 1 oligodendroglioma, and 3 meningioma patients. The median age of the patients was 53, and all patients had previously undergone surgical resection. Similar Pattern of Gene Expression Profiles between Proteomics and Microarray Analysis. In the present study, we compared the gene expression profiles of neuroblastoma and normal tissues in order to identify genes exhibiting differential expression levels. A microarray approach was utilized to evaluate the level of gene expression of the human glioma tissues, wherein we obtained gene profiles for 3 tumor patients with metastasis and 3 normal samples. Affymetrix microarray was utilized as a single-label system. The acquired data were initially preprocessed via image analysis using GenPlex software. The Affymetrix microarray was excluded from analysis in cases in which >50% of all genes were accompanied by absent or bad flags. The selected genes exhibited differential expression in tumor samples as compared to normal tissues. We prepared tissue lysates from 8 glioma and 5 normal tissues, and separated the proteins using 2DE. In our 2-DE image analysis, averages of 1000 protein spots per gel were detected. With the use of the PDQUEST program, the change in expression was considered significant if the intensity of the corresponding spot differed reproducibly by more than 3-fold in relative volume (% V) between the glioma and normal tissues. In our computer analysis, we detected 99 differential 2-DE spots, 70 up-regulated and 23 down-regulated, in the glioma tissues. Ninety-nine gel spots were analyzed via MALDITOF/MS and identified by PMF. Our success rate was approximately 70%. In our analysis, 200 genes and 99 proteins were selected via microarray and proteomic techniques, respectively. We categorized the identified proteins according to the functional based on universal GO annotation terms (Figure 2A). They yielded similar results, showing that modules and gene ontology terms overlapped for the 10 categories obtained on significant genes from proteomics and microarray analyses. Their similar molecular functions were categorized as protein binding activity, catalytic activity, transcription regulator activity, structural molecule activity, enzyme regulator activity, or transporter activity. Among them, transporter genes were more abundant in tumors than in normal tissues, and this is associated with the migration capacity of stem cells against glioma. 2876

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Figure 3. 2D-Gel electrophoresis of human glioma proteins (A) and quantitation of the levels of identified proteins in human glioma tissues as compared to normal tissues (B). (A) The proteins (1 mg) from the whole glioma cells were extracted and separated on pI 3-10 nonlinear immobilized pI-gradient strips, followed by 9-18% polyacrylamide gel. The gel was stained with Coomassie’s brilliant blue G250. The identified proteins are listed in Table 2. (B) Relative level of identified proteins with increasing expression from normal tissues to tumor tissues.

Figure 4. Western blotting analysis of annexin A2, TIMP-1, COL11A1, Bax, CD74, TNFSF8, SPTLC2, and actin in human glioma tissues.

The expression of 14 proteins increased commonly in human glioma tissues, as was shown by proteomic and microarray analyses. These proteins were H-2 class II histocompatibility antigen (CD74), caspase 7, TIMP1 protein, spindle and kinetochore-associated protein 2, A1(XI) collagen, tumor necrosis factor ligand superfamily member 8, claudin-1, blood vessel epicardial substance, Sec1 family domain containing protein, Annexin A2, C13orf18 protein, EH-domain containing protein

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Specific Adhesion Genes in Human Malignant Glioma Table 2. List of Identified Proteins of Human Glioma Tissues gene name

coverage (%)

MW/pI

accession no.

no.

protein name

1

CD74

22

30205/6.5

P04441

protein binding, transmenbrane

2

H-2 class II histocompatibility antigen TIMP1 protein

TIMP1

18.3

18847/8.3

P01033

3

MOUSE A1(XI) collagen

Col11a1

23.4

17863/3.8

Q8VIA5

4 5 6

Col11a1 Col11a1 TNFSF8

16.1 23.4 9

12821/3.8 17863/3.8 26017/7.6

Q8VIA4 Q80WR4 P32971

7

A1(XI) collagen A1(XI) collagen Tumor necrosis factor ligand superfamily member 8 Annexin A2

Anxa2

25

38677/7.6

p07356

8

Apoptosis regulator Bax

BAX

30

24220/7.7

Q07814

9

Serine palmitoyltransferase 2

SPTLC2

26

62925/7.9

O15270

enzyme inhibitor activity, metalloendopeptidase inhibitor activity, protein binding extracellular matrix structral constuent, tensil strenth,structure molecule activity extracellular matrix structral constuent extracellular matrix structral constuent cytokine activity, tumor necrosis factor receptor binding calcium-dependent phospholipid binding, cytoskeletal protein binding, phospholipase inhibitor activity BH3 domain binding, lipid binding, protein binding protein heterodimerization activity acyltransferase activity, pyridoxal phosphate binding, serine C-palmitoyltransferase activity

4, bax, and serine palmitoyltransferase 2. Correlations between the microarray and proteomics techniques in commonly expressed genes were evaluated via Pearson correlation analysis. The statistical correlation was r ) 0.7647, p < 6.148 × 10-6 (Figure 2B). The data revealed a high correlation between microarray and proteomics data in the glioma tissue data. Identification of Gliotropism-Related Genes in Human Glioma Tissues. The secreted genes may be associated with gliotropism and/or brain metastasis. Quantifications of the

function

levels of annexin A2, TIMP-1, CD74, Col11a1, bax, TNSF8 and SPTLC2 were conducted in the glioma, via genomic and proteomic analyses (Figure 3A, Table 2). In particular, partial 2-DE images for annexin A2, TIMP-1, CD74, Col11a1, TNSF8 and SPTLC2 are shown in Figure 3B. Among them, the levels of annexin A2, TIMP-1, CD74, and Col11a1 expression were higher in primary glioma than in adjacent normal tissues. By way of contrast, levels of bax, TNSF8, and SPTLC2 expression were increased slightly in the glioma tissues as compared to the normal tissues (Figure 3). First, the expression level of annexin a2 observed in the proteomics and genomics assays were increased by 9.9-fold and 32.4-fold in the glioma tissue as compared to the normal tissues, respectively. Second, the expression of Col11a1 protein was also upregulated by an average of 5.7-fold and 21.2-fold in the proteomics and genomics assays, respectively (Figure 3B). Seven candidate proteins were validated via Western analysis and immunohistochemical staining, evidencing a significant increase in annexin A2, TIMP-1, CD74, Col11a1, TNSF8, and SPTLC2 in glioma tissues (Figures 4 and 5). Interestingly, annexin a2 was identified by proteomic and cDNA microarrays, and was located at 38.6 kDa/pI 7.6 in the 2-DE gels obtained from human neuroblastoma tissues. The expression level of annexin A2 exhibited the highest level of alteration in the glioma. Clearly, the expression level of annexin A2 significantly altered patterns in glioma tissue upon Western blotting analysis (Figure 4). In the immunohistochemical stain assay, Annexin A2 was prominently located in the cytoplasm

Figure 5. Microphotographs showing immunohistochemical results for annexin A2, TIMP-1, Bax, TNFSF8, and SPTLC2 in human glioma and normal tissues. ×100: Bars represent 100 µm.

Figure 6. Immunodetection of Annexin A2 protein on the surfaces of F3 and U87 cells. Journal of Proteome Research • Vol. 8, No. 6, 2009 2877

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Figure 7. Human recombinant annexin A2 increased the migration activity of neural stem cell F3 (A) and U87 neuroblastoma cells (B), not but of mouse fibroblast NI3T3 cells (C) in a dose-dependent manner in Boden chamber assays. Column percentages of control; bar, SD. Statistical comparisons are calculated between annexin treatment cells and DMEM controls (*, p < 0.001).

and the cell membrane on the tissue sections of neuroblastoma tissue, but not the nearly normal tissue, as is shown in Figure 5. In this 2DE study, we detected the presence of multiple Col11a1 protein spots with slightly varying pI that ran along horizontal lines in the glioma tissue (Figure 3A), and their quantitation is presented in Figure 3B. These proteins are not detected in normal brain tissues, but are exclusively present in human neuroblastoma cells. These spots with the same pI but different MW (17.8 kDa/pI 3.8; 12.8 kDa/pI 3.8, respectively) were identified via MS. However, Col11a1 isoforms would not have been detected on Affymetrix microarray analysis. The shift in pI of the proteins suggests the presence of a variety of phosphorylation states of the proteins. Western blotting demonstrated that the expression of Col11a1 was upregulated more profoundly in human glioma tissues than in normal tissues, and may be specific marker proteins in malignant glioma tissues. In particular, the levels of TIMP-1 and CD74 expression were higher in glioma tissues, as indicated by proteomic analysis. Immunohistochemical analysis for cytoplasmic TIMP-1 protein 2878

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expression exhibited a statistically significant difference in mean expression between the tumor and normal controls. Frozen sections of the normal and metastatic human tumor specimens from 10 individual patients were evaluated for bax, TNSF8, and SPLP2 immunoreactivity. We noted high levels of TNSF8 and SPLP2 in the cytoplasm and cell membranes of human neuroblastoma tissues. Additionally, the expression level of SPLP2 observed in the proteomics and genomics assays were increased by 5.72-fold and 7.56-fold in the glioma tissue as compared to normal tissues, respectively. However, the Western blotting analysis results did not evidence any distinctive tumor markers because SPLP2 expression was noted in the normal tissues. Annexin A2 Stimulates the Migration of NSCs but Not of HEK293 Cells. Annexin A2 is located on the surfaces of tumor cells, and may perform a pivotal function in extracellular proteolysis, as well as in tumor cell invasion and metastasis. The expression of annexin A2 was principally located in the cell membranes of the F3 and U87 cell (Figure 6). We selected U87 human glioma cells and F3 human neural stem cells as putative soluble chemotactic factors. Additionally, we selected

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Figure 8. Neural stem cells (A) and tumors (B) involve similar ERK and CDK signal pathway mechanisms. Human recombinant annexin A2 was assessed via transwell migration assays, using F3 and U87 cells. The ERK inhibitor, PD98057, and the CDK inhibitor, rescovitin, inhibited annexin A2-induced migration in neural stem cells (F3) and neuroblastoma cells (U87 cells).

the nontumor cell lineshuman embryonic kidney (HEK293) cellssas a low migration control. The number of cells that migrated through the membrane to annexin A2 did so in an apparently dose-dependent manner (Figure 6A). At concentrations of 20 ng/mL or higher, their migration rate was significantly higher than that observed for unconditional medium. In the U87 glioma cells, recombinant annexin A2 also induced a significant increase in migration capacity in a dose-dependent fashion (Figure 6B, from 20 to 200 ng/mL, respectively). However, recombinant human annexin A2 exhibited no migration capacity in HEK293 cells (Figure 6C). Interestingly, the glioma and neural stem cells (NSCs) also evidenced similar migration capacity patterns. These data reveal that annexin A2 may function as the primary chemotactic factor generated by glioma and NSCs. As MAPK and CD5K are involved in annnexin A2-induced chemotactic signaling, we evaluated the migration effects of annexin A2 on MAPK and CD5K inhibitor treatment cells. The ERK inhibitor, PD98057, inhibited 59% and 63% of annexin A2induced migration in F3 and U87 cells, respectively (Figure 7A,B). Additionally, the CDK5 inhibitor (rescovitin) repressed 90% of annexin A2-induced chemotaxis in F3 and U87 cells, respectively (Figure 8). These results showed that tumor and stem cells have a common signaling pathway. However, the HEK293 cell line does not appear to influence annexin A2stimulated migration capacity.

Discussion Heterogeneous metastatic formation of glioma consists of a complex series of events involving multiple cell-cell and

cell-matrix interaction, which are mediated by the interplay of a variety of cell adhesion molecules, chemokines and chemokine receptors, growth factors and growth factor receptors, hormones, neurotransmitters, and proteases.17,18 Thus, tumor cells utilize some metastasis-associated molecular genes and chemoattractive molecules, which perform functions during physiological processes including neurogenetic diseases and malignant tumors.19 In glioma tissues, our knowledge of the specific expression and molecular characterization of gliotropism-related genes, as well as their molecular mechanisms, remains limited. In our study, the proteomics and microarray results concerning the sites of genetic alterations should provide insight into the pathogenesis of glioma and should facilitate the establishment of new molecular targets for diagnosis and therapy. Our results demonstrate that the genes secreted from glioma cells stimulated the migration capacity of NSCs. The results of a previous report indicated that NSCs seek out tumor foci that have infiltrated far from the main tumor mass, a common occurrence with glioma.20 We also noted that the NSCs migrate freely through the tumor, but slowed down without direction in normal adult brain parenchyma, the environment of which appears to be less conducive to their migration. Our previous results show that NSCs armed with oncolytic molecules are capable of effectively and specifically delivering these molecules to regions of glioma cell invasion, thus, increasing survival duration.20 Recently, the migration of NSCs has been shown to be induced by a variety of signals derived from multiple sources, including attractants, adhesion and substrate molecules, chemokines, and so forth.8-10 Journal of Proteome Research • Vol. 8, No. 6, 2009 2879

research articles In this study, we focused principally on the functions of annexin A2 in the regulation of NSC migration. Most interestingly, human recombinant annexin A2 was shown to elicit the selective migration of NSCs in an in vitro transwell assay. Annexin A2 was of particular interest because of the increasing number of reports suggesting that annexin A2 may be a metastatis tumor marker for malignant tumors. Several studies have previously demonstrated that increased levels of annexin A2 were correlated with advanced glioblastoma.21,22 Our results showed that the level of annexin A2 was more profoundly increased in human glioma tissues than in normal tissues, and this was confirmed via Western blotting and immunohistochemical staining. Many factors are involved in the brain tumor tropisms of NSCs and their interactions within the tumor environment.7-11 As gliomas progress and invade, an extensive modulation of the extracellular matrix ensues.23 We attempted to determine which factors were upregulated in glioma cells and might function as soluble chemotactic factors for the induction of glioma tropisms in NSCs. Interestingly, our previous findings show that NIH3T cells did not migrate to tumor regions in animal models.20 In the current study, recombinant annexin A2 did not induce the migration of HEK293T cells. These results show that cell migration to brain tumors occurs in a cell-specific fashion. Annexin A2 belongs to a family of Ca2+ dependent phosphate lipid binding proteins that have been shown to be associated with endosomes, secretory granule, and the plasma membrane in different cell types.24-27 In general, annexin has been shown to exist as a monomer, a heterodimer with S100A10, or a heterotetramer. Annexin A2 exists in the cell cytoplasm as a monomer (heavy chain of 36 kDa or p36) or in a complex with a light chain of 11 kDa (p11), a member of the S100 protein family.28,29 The formation of Annexin a2 heterotetramer enables its association with lipid rafts on the plasma membrane, where it has been proposedtobeinvolvedinregulationandmembranetrafficking.30,31 Thus, annexin a2 performs a pivotal role in cell surface fibrinolysis, angiogenesis, cancer metastasis, and neurite outgrowth.32-35 In this study, annexin A2 was more abundant in the cell cytoplasm and membranes of the glioma tissues than was the case in normal tissues. Additionally, the immunocytochemical staining results demonstrated that annexin expression is detected on the membrane region in the NSCs and U87 glioblastoma cells. Our results suggest that the presence of annexin A2 at the plasma membrane is required for the process of exocytosis in stem cells. Even though the physiological function of annexin A2 has yet to be well-characterized, it may participate in calcium-dependent exocytosis, endocytosis, and cell-cell adhesion.36,37 In this study, treatment with chemical inhibitors of ERK inhibitors (PD98057) and CDK5 inhibitors (rescovitine) induced a reduction of 59% and 90% in annexin A2-induced migration in NSC cells, respectively. Recently, CDK5 regulates neuronal migration and modulates the duration of ERK activation.38,39 Further study will be required to definitively assess the association between annexin A2 and CDK5 and ERK signaling in cell migration. Generally, annnexin A2 on the cell surface has been demonstrated to function as a receptor or binding protein for proteases (cathepsin B, the plasminogen tissue activator) and proteins in the extracellular matrix (collagen and tenascin C).29,40 In U87 neuroblastoma cells, human recombinant annexin A2 increased the migration capacity, and treatment with chemical inhibitors of PD98057 and rescovitine reduced annexin A2-induced migration by 63% and 90%, respectively. 2880

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An et al. These results show that stem cells and tumor stem cells exhibit identical migration signal pathway patterns. Our results also show that annexin A2 underpins a novel mechanism of stem cell tropism to neuroblastoma cells, and also that the other proteinssTIMP-1, CD74, Col11a1, bax, TNSF8, and SPTLC2smay constitute new potential tropism factors in the pathology of malignant glioma. Our findings demonstrate that this new molecular approach to the monitoring of glioma may provide clinically relevant information regarding tumor malignancy, and may prove suitable for high-throughput clinical screening applications. The results of this study suggest that NSCs can utilize annexin A2 as an informative migration cue. The identification and characterization of gliotropism-associated proteins for various phenotypes using proteomics and genomics is expected to prove pivotal in the development of effective stem cell-based targeted cancer therapy. In addition, annexin A2 may provide critical clues to our understanding of the migration of neural stem cells under physiological and pathophysiological conditions.

Acknowledgment. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD)′′(KRF-2006- 532-C00010). References (1) Dittar, T.; Heyder, C.; Gloria-Maercker, E.; Hatzmann, W.; Za¨nker, K. S. Adhesion molecules and chemokines: the naviagation system for circulating tumor (stem) cell to metastasize in an organ-specific manner. Clin. Exp. Metastasis 2008, 25, 11–32. (2) Juliano, R. L.; Varner, J. A. Adhesion molecules in cancer: the role of integrins. Curr. Opin. Cell Biol. 1993, 5, 812–818. (3) Nathoo, N.; Chahlavi, A.; Barnett, G. H.; Toms, L. A. Pathobiology of brain metastases. J. Clin. Pathol. 2005, 58, 237–242. (4) Salmaggi, A.; Bolardi, A.; Gelati, M.; Russo, A.; Calatozzolo, C.; Ciusani, E.; Sciacca, F. L.; Ottolina, A.; Parati, E. A.; La Porta, C.; Alessandri, G.; Marras, C.; Croci, D.; De Rossi, M. Glioblastomaderived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype. Glia 2006, 54, 850–860. (5) Gengras, M. C.; Roussel, E.; Bruner, J. M.; Branch, C. D; Moser, R. P. Comparison of cell adhesion molecule expression between glioblastoma multiforme and autologous normal brain tissue. J. Neuroimmunol. 1995, 57, 143–153. (6) Benedetti, S.; Pirola, B.; Pollo, B.; Magrassi, L.; Bruzzone, M. G.; Rigamonti, D.; Galli, R.; Selleri, S.; Di Meco, F.; De Fraja, C.; Vescovi, A.; Cattaneo, E.; Finocchiaro, G. Gene therapy of experimental brain tumors using neural progenitor cells. Nat. Med. 2000, 6, 447–450. (7) Kendall, S. E.; Najbauer, J.; Johnston, H. F.; Merz, M. Z.; Li, S.; Bowers, M.; Garcia, E.; Kim, S. U.; Barish, M. E.; Aboody, K. S.; Glackin, C. A. Neural stem cell targeting of glioma is dependent on phosphoinositide 3-kinase signaling. Stem Cells 2008, 26, 1575– 1586. (8) Imitola, J.; Raddassi, K.; Park, K. I.; Mueller, F. J.; Nieto, M.; Tenq, Y. D.; Frenkel, D.; Li, J.; Sidman, R. L.; Walsh, C. A.; Synder, E Y.; Khoury, S. J. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1R/CXC chemokine receptor 4 pathway. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 18117– 18122. (9) Widera, D.; Holtkamp, W.; Entschladen, F.; Niggemann, B.; Za¨nker, K.; Kaltschmidt, B.; Kaltschmidt, C. MCP-1 induces migration of adult neural stem cells. Eur. J. Cell Biol. 2004, 83, 381–387. (10) Schmidt, N. O.; Przylecki, W.; Yang, W.; Ziu, M.; Teng, Y.; Kim, S. U.; Black, P. M.; Aboody, K. S.; Carroll, R. S. Brain tumor tropism of transplanted human neural stem cells is induced by vascular endothelial growth factor. Neoplasia 2005, 7, 623–629. (11) Jurvansuu, J.; Zhao, Y.; Leung, D. S.; Boulaire, J.; Yu, Y. H.; Ahmed, S.; Wang, S. Transmenbrane protein 18 enhances the tropism of neural stem cells for glioma cells. Cancer Res. 2008, 68, 4614–4622. (12) Aboody, K. S.; Brown, A.; Rainov, N. G.; Bower, K. A.; Liu, S.; Yang, W.; Small, J. E.; Herrlinger, U.; Ourednik, V.; Black, P. M.; Breakefield, X. O.; Snyder, E. Y. Neural stem cells display extensive tropism for pathology in adult brain: Evidence from intracranial gliomas. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12846–12851.

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