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Departments of Pathology [H. W., W. S., L. H., L. R., G. N. F., W. Z.], Biochemistry and Molecular Biology [W. L., H. H.], and Neuro-Oncology [Y. Z.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and Department of Pathology, The University of Texas-Houston Medical School, Houston, Texas 77030 [H. W.]
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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The acquisition of invasive potential through proteinase expression is an essential event in tumor progression. Among proteinases, MMP-2 and MMP-9 have been shown to be increased in GBMs and are thought to play a key role in facilitating the invasion of GBM cells through brain parenchyma (2 , 3) . Other molecules, such as secreted protein acidic and rich in cysteine and extracellular matrix protein fibronectin have also been implicated in GBM invasion (4 , 5) .
In previous studies, we searched for the gene activities associated with GBM using cDNA microarray profiling followed by tissue microarray validation and identified IGFBP2 overexpression as one of the most frequent molecular events characteristic of GBM (6, 7, 8) . This finding has been subsequently confirmed independently by two different laboratories, and high levels of IGFBP2 expression have been shown to be associated with poor survival in patients with diffuse gliomas (9 , 10) . IGFBP2 is normally predominantly expressed in fetal tissues and is involved in brain development (11, 12, 13, 14) . After birth, IGFBP2 expression decreases significantly in glial cells (15 , 16) . The subsequent differential induction of IGFBP2 expression in the brain has been associated with a variety of pathological conditions, including hypoxia, regeneration, trauma, and tumors of the central nervous system (17) . IGFBP2 is associated with other malignancies as well. In ovarian cancer, the increase in IGFBP2 have been found to correlate positively with the serum tumor marker CA125 (18) . Similarly, increased serum levels of IGFBP2 have been proposed as a prognostic marker for prostate cancer (19) . In addition, the overexpression of IGFBP2 increases the tumorigenicity of Y-1 adrenocortical tumor cells and epidermoid carcinoma cells (20 , 21) . However, the molecular mechanisms by which IGFBP2 enhances tumor cell growth and increases tumorigenicity remain undefined.
To elucidate the cellular pathways affected by elevated levels of IGFBP2 in high-grade gliomas, we established stable SNB19(BP2) clones that overexpress IGFBP2. Although IGFBP2 overexpression in these clones did not affect their growth rates, it enhanced cell invasion in an in vitro invasion assay. Comparison of gene expression profiles of the stable IGFBP2 expressing clones with parental cells or vector-transfected cells revealed increased expression of MMP-2, as well as several other invasion-related genes. Furthermore, IGFBP2 overexpression also correlated with MMP-2 overexpression in 68 glioblastoma tissues. Our results demonstrate for the first time that overexpression of IGFBP2 in glioma cells leads to increased MMP-2 expression and an increased invasion.
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Materials and Methods |
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Determination of Cell Growth.
A total of 5 x 104 cells in 5 ml of DMEM/F12 medium supplemented with 10% FBS were seeded into T25 tissue culture flasks. Every 24 h for 7 days, a set of three flasks were removed, rinsed with PBS, trypsinized, and resuspended. Three independent cell counts were obtained and the mean cell counts were calculated. The number of cells were plotted against time to obtain a growth curve for the cell population.
In Vitro Chemoinvasion Assay.
Chemoinvasion was measured using 24-well BioCoat Matrigel invasion chambers (Becton Dickinson Labware, Bedford, MA) with an 8-µm pore polycarbonate filter coated with matrigel. The lower compartment contained 0.6 ml of DMEM/F12 medium with 0.5% FBS as chemoattractants or serum-free DMEM/F12 medium as a control. In the upper compartment, 5 x 104 cells/well were placed in triplicate wells and incubated for 24 h at 37°C in humidified incubator with 5% CO2. After incubation, the cells that had passed through the filter into the lower wells were stained with Giemsa (Fisher Scientific, Orangeburg, NY) and counted under a microscope in five predetermined fields. All assays were repeated at least three times. The differences in the invasion rates between control cell lines and stable SNB19(BP2) lines were analyzed using a two-tailed Student’s t test.
RNA Isolation and Microarray Analysis.
Total RNA was isolated from SNB19 glioma cells and stable SNB19(BP2) cells using TRI reagent (MRC, Cincinnati, OH). Microarray was carried out as described previously (22
, 23)
using the Pathway array containing 1500 functionally well-characterized genes in duplicate.4
Hybridized arrays were scanned with a GeneTAC LSIV scanner (Genomic Solutions, Ann Arbor, MI). The images were quantified using the imaging software ArrayVision from Imaging Research Inc. (Ontario, Canada), and the significantly differentially expressed genes were identified as described (24)
. For differentially expressed genes, a smoothed T value cutoff of 2.0 was used.
LightCycler Real-time Quantitative RT-PCR.
Total RNA was extracted using TRI reagent. One microgram of total RNA was reverse transcribed under standard condition in a reaction mixture containing all four deoxynucleoside triphosphates, 100 unit of Superscript II Plus RNase H- Reverse Transcriptase (Life Technologies, Inc., Grand Island, NY) and 100 µM random hexamer primers (Pharmacia Biotech, Buckinghamshire, United Kingdom). PCR reactions were performed with 4 µl of reverse transcriptase product serving as template DNA (synthesized cDNA) and 6 µl of master-mix. The master-mix of the following reaction components was prepared: 4 µl of water, 1 µl of primer mix (4 mM MgCl2 and 0.4 µM forward and reverse primers), and 1 µl of LightCycler Fast DNA Master SYBR Green I (Roche Diagnostic, Mannheim, Germany). Capillaries were closed, centrifuged, and placed into the rotor. Real-time PCR procedures and the primer sequences of the internal control gene GAPDH were described in detail in Nakanishi et al. (25)
. The primer sequences for MMP-2 were as follows: 5'-CAAAAACAAGAAGACATACATCTT-3' and 5'-GCTTCCAAACTTCACGCTC-3'. The specific PCR condition for MMP-2 was 95°C for 1 min, followed by 50 cycles at 95°C for 0 s, 60°C for 5 s, and 72°C for 10 s. Fluorescence was measured at 80°C for 2 s. Each quantification reaction was performed in triplicate, and the mean values were used to calculate the ratios of MMP-2 to GAPDH, with a value of 1 used as the control. All assays were repeated at least three times.
Immunoblotting.
Production of IGFBP-2 and MMP-2 by stable SNB19(BP2) cells was analyzed by Western blotting. Briefly, cells were incubated in serum-free medium for 24 h, after which time the medium was clarified by centrifugation and concentrated using spin columns (Amicon, Beverly, MA). Equal amounts of proteins in the concentrated samples were resolved by 10% SDS-PAGE, electroblotted onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech, Chicago, IL), blocked in 5% skim milk in 1x TBS, and probed with the following primary antibodies: anti-IGFBP-2 polyclonal antibody C-18 (Santa Cruz Biotechnology, Inc.) and anti-MMP-2 monoclonal antibody Ab-3 (Oncogene Research Products, Cambridge, MA). Proteins were detected with an enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech, Piscataway, NJ).
Gelatin Zymography.
Gelatin zymography was performed as described previously with minor modifications (26)
. Equal numbers of cells (1 x 106) were seeded onto 100-mm plates in DMEM/F12 containing 10% FBS and cultured overnight. Cells were then washed twice with PBS and incubated with serum-free DMEM/F12 medium for 24 h. The medium was collected after the removal of cell debris and concentrated using Amicon spin columns. Concentrated samples with equal amounts of proteins were mixed with SDS sample buffer without reducing agent and subjected to 7.5% SDS-PAGE containing 0.1% gelatin A. After electrophoresis, the gels were washed four times in 2.5% Triton X-100 for 1 h at room temperature to remove SDS and then incubated for 24–48 h at 37°C in buffer containing 5 mM CaCl2 and 1 µM ZnCl2. The proteins in the gel were stained with Coomassie Brilliant Blue. Proteolytic activity in the gel was visualized as clear bands (zones of gelatin degradation) against the blue background of stained gelatin.
Luciferase Reporter Assays.
A luciferase reporter plasmid driven by 1716 bp of the human MMP-2 promoter (27)
and an internal Renilla luciferase control vector (pRL-TK) were used in this study. Stable SNB19(BP2) cells were seeded into 6-well tissue culture plates and the next day transfected with 0.5 µg of the MMP-2 luciferase reporter construct and 0.5 µg of the pRL-TK control using the FuGENE 6 reagent. After 24 h, cells were lysed and cell extracts assayed in triplicate with an enhanced luciferase assay kit (PharMingen, San Diego, CA). The luciferase activity from the MMP-2 vector was normalized to the Renilla luciferase activity.
Brain Tumor Tissue Microarray.
A human glioma tissue microarray was constructed using formalin-fixed, paraffin-embedded archival tissue blocks as described previously (7)
. Duplicate samples were collected from different sites of the most representative tumor regions. The tissue microarray included tissues from 68 GBM patients. Expression levels of IGFBP2, and MMP-2 were evaluated by a standard indirect immunoperoxidase procedure (ABC-Elite; Vector Laboratories, Burlingame, CA). In brief, antigen retrieval was performed by treatment in a steamer for 25 min. Anti-IGFBP2 (C-18, Santa Cruz Biotechnology, Inc.) was used at a 1:1000 dilution at 4°C overnight. Anti-MMP-2 monoclonal antibody Ab-6 (Oncogene Research Products) was used at a 1:50 dilution at 4°C overnight. Secondary antibody incubation was performed at room temperature for 60 min. Mayer’s hematoxylin nuclear staining was used as a counterstain. Tumors with none or few (<5% of the neoplastic cells) IGFBP2 or MMP-2 cytoplasmic staining were regarded as negative. Tumors with strong cytoplasmic staining in 
5% of the neoplastic cells were regarded as positive. The statistical analysis was performed using Fisher’s exact test.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. The SNB19(BP2) c1 clone showed a level of IGFBP2 protein similar to that of the parental or vector control cells. Very low IGFBP2 expression was detected in c4 clone, whereas high levels of IGFBP2 expression were detected in c5 and c8 clones. Despite the differences in the levels of IGFBP2, these clones did not show marked differences in their growth rates (Fig. 1B)
. We then tested whether IGFBP2 expression affected glioma invasion using a matrigel in vitro invasion assay, with 0.5% FBS used as chemoattractant to stimulate cells to penetrate through the matrigel and migrate through the filters. The matrigel contains laminin, collagen type IV, heparin sulfate proteoglycan, entactin, which are extracellular matrix compositions of normal brain and gliomas. The invasion assay revealed prominent differences between IGFBP2 overexpressing c5 and c8 clones and the vector-transfected cells both with and without chemoattractant (P < 0.01; Fig. 1D
). Significantly higher rates of invasion were observed in IGFBP2 overexpressing cells (c5 and c8 clones) when 0.5% FBS used as chemoattractant, they had a 15–24-fold higher invasion rate than that of the vector-transfected cells (P < 0.01). In contrast, there was no difference between the c1 and c4 clones and the vector- transfected cells (P > 0.05; Fig. 1, C and D
). Because clone c4 had essentially undetectable IGFBP2 expression, it provided an additional independent system to verify the observed effects of IGFBP2 on cell invasion. SNB19(BP2) c4 cells were, therefore, transfected with IGFBP2 expressing vector and selected with puromycin for subclones that express IGFBP2. Three subclones were derived from this selection, named SNB19(BP2) c4.1, SNB19(BP2) c4.6, and SNB19(BP2) c4.10. The levels of IGFBP2 expression in these clones were evaluated by Western blotting using cell medium with anti-IGFBP2 antibody. As shown in Fig. 2
, SNB19(BP2) c4.1 and SNB19(BP2) c4.10 subclones expressed high levels of IGFBP2, whereas SNB19(BP2) c4.6 subclone expressed only moderate levels of IGFBP2. Invasion assays were carried out to compare these subclones with the parental SNB19(BP2) c4 clone. Again, significantly higher rates of invasion were observed between IGFBP2 overexpressing c4.1 and c4.10 subclones and the parental cells both with and without chemoattractant (P < 0.01; Fig. 2
). When 0.5% FBS was used as chemoattractant, 4–7-fold higher rates were observed in IGFBP2 overexpressing cells (c4.1 and c4.10) that invaded through the membrane than in parental cells (P < 0.01); in contrast, there was no difference between the c4.6 subclone and the parental cells (P > 0.05; Fig. 2
). Thus, the results consistently, in two independently derived cell lines, demonstrated that increased IGFBP2 expression in human SNB19 cells could facilitate tumor cell invasion in vitro.
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. IGFBP2, as expected, is one of the genes induced in the high expressing clones (see growth factor section in Table 1
). The altered genes, which are consistently associated with the changes in cell invasion (Figs. 1
and 2
), included invasion related genes MMP-2, fibronectin 1, thrombospondin 2, integrin 
5 and 6. Cell cycle inhibitor p21 gene was also induced, although we did not see major growth alterations in the IGFBP2 overexpressing clones.
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. In contrast, there was no difference in MMP-2 RNA levels between the clones expressing low levels of IGFBP2 (c1 and c4) and vector-transfected control cells. We also observed a consistent 3–5-fold increase in the MMP-2 mRNA levels in the IGFBP2 overexpressing SNB19(BP2) c4.1 and SNB19(BP2) c4.10 clones (Fig. 3A)
. Only a slightly increased MMP-2 mRNA level was observed in the SNB19(BP2) c4.6 clone, which had low IGFBP2 expression (Fig. 3A)
. To ascertain that the increase of MMP-2 mRNA levels in IGFBP2 high expressing clones was not attributable to a general increase mRNA expression by IGFBP2 overexpression and was a specific phenomenon, we examined the expression levels of platelet-derived growth factor receptor (PDGFR) in those clones by real time RT-PCR. We did not observe significant differences in platelet-derived growth factor receptor expression between clones expressing high (c5 and c8) and low (vector-transfected control, c1, and c4) levels of IGFBP2 (data not shown), indicating that the effect of IGFBP2 on MMP-2 expression was specific.
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, MMP-2 protein was significantly higher in IGFBP2 overexpressing clones (c5 and c8) than in vector-transfected control. In contrast, there was no difference in MMP-2 protein level between IGFBP2 low expressing clones (c1 and c4) and vector-transfected control. Consistently, gelatin zymography assay showed a stronger lytic zone at the molecular mass of 72 kDa, demonstrating the elevated MMP-2 enzyme activity in IGFBP2 overexpressing clones (Fig. 3C)
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To investigate whether the increase in MMP-2 expression in SNB19(BP2) cells might be attributable to increased MMP-2 gene transcription, we examined the activities of an MMP-2 promoter-luciferase reporter construct after transient transfection into SNB19(BP2) cells and compared the activities with those in parental cells. MMP-2/luciferase activities were 3–6-fold higher in SNB19(BP2) c5 and c8 than in parental control cells, but there was no such difference between clones expressing low level of IGFBP2 (c1 and c4) with vector-transfected control cells (Fig. 3D)
. Taken together, these results support a role for IGFBP2 in transcriptionally up-regulating MMP-2 gene expression.
IGFBP-2 Overexpression Correlated with MMP-2 Expression in GBM.
To further confirm the significance of the in vitro observations showing that IGFBP2 regulates MMP-2 gene expression, a human GBM tissue microarray containing 68 GBMs was constructed using formalin-fixed, paraffin-embedded archival tissue blocks. The expression of IGFBP2 and MMP-2 was evaluated by immunohistochemistry under identical standardized conditions for all patient samples on consecutive tissue sections cut from the array block. The immunohistochemistry staining for IGFBP2 and MMP-2 was successful in all 68 GBMs and the duplicate cores from the same case had identical expression pattern. Examples of both positive and negative staining are shown in Fig. 4, A–D
. Quantification of the staining showed that among GBMs that were positive for IGFBP2, 87.5% (49 of 56) also showed an increase in MMP-2 expression and only 12.5% (7 of 56) did not show MMP-2 expression. In contrast, GBMs that were negative for IGFBP2 staining, 42% (5 of 12) did not have MMP-2 expression (Fig. 4E)
. Strong IGFBP2 immunopositivity was associated with MMP-2 immunopositivity (P < 0.05; Fisher’s exact test). These data further support the notion that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and glioma cell invasion.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Using stable SNB19 cell lines that express different levels of IGFBP2, we found that increased IGFBP2 had no effect on cellular proliferation, but did result in significantly increased cellular invasion in the in vitro cell invasion assay. Our microarray studies showed that IGFBP2 overexpressing clones overexpressed MMP-2 as well. MMP-2 is thought to play a key role in tumor progression by degrading the of extracellular matrix, thereby facilitating tumor cell invasion into surrounding normal tissues (28) . We then further validated this finding by real time RT-PCR, Western blotting, and enzymatic assays, which confirmed that the MMP-2 protein level and the protease activity were both elevated in IGFBP2 overexpressing clones. Furthermore, tissue array and immunohistochemistry analysis showed a significant correlation between the overexpression of IGFBP2 and elevated MMP-2 expression in 68 GBMs. Therefore, our studies for the first time provided a molecular mechanism through which IGFBP2 functions as an important invasion-promoting gene.
Our data also showed a positive correlation between IGFBP2 expression and the MMP-2 promoter activity. This transcriptional regulation is not likely attributable to a direct action of IGFBP2, because we did not detect IGFBP2 staining in the nucleus by immunohistochemistry (data not shown). This is contrast to the behavior of IGFBP5, which can translocate to the nucleus by a nuclear localization signal and may directly affect transcription (29)
. IGFBP2 may instead serve as an upstream signal regulator that activates downstream transcription factors important for the up-regulation of MMP-2. Underscoring this possibility is the fact that among the genes altered in IGFBP2 overexpressing cells are a number of transcription factors (Table 1)
. Potentially, those transcription factors may be responsible for IGFBP2 mediated MMP-2 up-regulation.
Because IGFs are involved in tumor cell migration and invasion through their regulatory effects on MMP-2 expression (30 , 31) , the effect of IGFBP2 on MMP-2 may be achieved through enhancement of IGF-dependent pathway, although we cannot exclude the possibility of IGF-independent effect of IGFBP2. It is noteworthy that both negative and positive effects of IGFBP2 on IGF-dependent cell proliferation have been reported (32 , 33) , and IGFBP2 has been shown to augment both IGF-dependent and IGF-independent anti-apoptotic functions in the developing mouse limb and brain (34 , 35) . In this regard, it would be important to understand whether the regulation of MMP-2 by IGFBP2 is mediated through an IGF-dependent or independent pathway.
Tumor cell invasion involves a series of complicated steps, including the acquisition of cell motility and cell adhesion properties and the production of extracellular protease. Tumor cell adhesion to the basement membrane is mediated by integrin, which is a member of a family of transmembrane receptor heterodimers with and ß subunits. Extracellular matrix proteins, such as fibronectin and thrombospondin, stimulate motility through integrin receptors (36)
. Therefore, it is interesting that cell adhesion molecule integrins and motility factors fibronectin 1 and thrombospondin 2 were also induced in IGFBP2 overexpressing clone (Table 1)
. This suggests that IGFBP2 affects multiple genes in the cell invasion pathways. This potentially robust action of IGFBP2 may explain the frequent induction of IGFBP2 in multiple types of advanced cancer.
Taken together, using cellular, genomics, and molecular approaches, we have documented a strong association between high levels of IGFBP2 expression, increased expression of invasion gene MMP-2, and an increased invasion potential. These results shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The work was supported partially by a grant from the Texas Higher Education Coordinating Board, the Tobacco Settlement Fund as appropriated to The University of Texas M. D. Anderson Cancer Center by the Texas Legislature, and a donation from Kadoorie Foundation. 
2 To whom requests for reprints should be addressed, at Cancer Genomics Core Lab, Department of Pathology, Box 85, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 745-1103; Fax: (713) 792-5549; E-mail: wzhang{at}mdanderson.org
3 The abbreviations used are: GBM, glioblastoma multiforme; IGF, insulin-like growth factor; IGFBP2, insulin-like growth factor binding protein 2; MMP-2, matrix metalloproteinase-2; FBS, fetal bovine serum; RT-PCR, reverse-transcription PCR; GAPDH, glyceraldehydes-3-phosphate dehydrogenase.
4 Internet address: www.mdanderson.org/
genomics.
Received 2/24/03. Accepted 6/ 5/03.
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