ARTICLE

Coexpression of Glucose Transporter 1 and Matrix Metalloproteinase-2 in Human Cancers

Satoshi Ito, Toshio Fukusato, Takahiro Nemoto, Hisahiko Sekihara, Yousuke Seyama, Shunichiro Kubota

Affiliations of authors: S. Ito, Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo, Japan, and Third Department of Internal Medicine, Yokohama City University School of Medicine, Japan; T. Fukusato, Department of Pathology, Faculty of Medicine, Gunma University, Maebashi, Japan; T. Nemoto, Y. Seyama, S. Kubota, Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo; H. Sekihara, Third Department of Internal Medicine, Yokohama City University School of Medicine.

Correspondence to: Shunichiro Kubota, M.D., Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan (e-mail: kubota{at}bio.m.u-tokyo.ac.jp).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background: Cancer cells express higher levels of glucose transporter proteins (Gluts) than do normal cells. Glut-1 overexpression is associated with invasiveness. Because matrix metalloproteinase-2 (MMP-2) is also overexpressed in cancer cells and is associated with invasiveness, we tested the hypothesis that Glut-1 may regulate MMP-2 expression. Methods: We transiently transfected Glut-1 complementary DNA (cDNA) or Glut-1 antisense oligonucleotides in the human rhabdomyosarcoma cell line RD and analyzed MMP-2 mRNA expression and cell invasiveness. Empty vector and sense oligonucleotides were used for controls. We analyzed MMP-2 promoter activity in transfectants with a luciferase reporter assay and with p53 and Ets-1 gel mobility shift assays. Eight human cancer cell lines and 80 human cancer specimens were analyzed for coexpression of Glut-1 and MMP-2 by western blot and immunohistochemical analyses, respectively. Results: Overexpression of Glut-1 in RD cells increased MMP-2 expression 4.3-fold (95% confidence interval [CI] = 3.7-fold to 4.9-fold) and invasiveness 3.2-fold (95% CI = 2.6-fold to 3.8-fold) relative to control transfected cells. Conversely, suppression of Glut-1 expression by antisense oligonucleotides decreased MMP-2 expression by 71.5% (95% CI = 71.1% to 71.9%) and invasiveness by 53.0% (95% CI = 47.5% to 58.5%). Glut-1-mediated MMP-2 expression involved the binding of the transcription factor p53 but not Ets-1. All eight human cancer cell lines coexpressed Glut-1 and MMP-2 by western blotting, and 45 of 80 human tumor tissues coexpressed Glut-1 and MMP-2 by immunohistochemistry. Conclusions: MMP-2 expression and cell invasiveness are tightly associated with Glut-1 expression in human cancer cell lines. Because suppression of Glut-1 decreased MMP-2 expression and cancer cell invasion, Glut-1 could be a target for therapy of various cancers that overexpress Glut-1.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cancer cells often take up more glucose than do normal cells (1). Glucose uptake is mediated by glucose transporters (Gluts) (2). These proteins belong to a family of homologous proteins (Glut-1–7) that differ in their tissue distribution and affinity for glucose (35). Although Glut-1 is not expressed in normal epithelial tissues or benign epithelial cell tumors, it is widely expressed in various human cancers (6). High Glut-1 expression is associated with increased invasiveness of cell lines in vitro and in vivo (7,8) and with poor prognosis of patients with colorectal carcinoma (9,10), non-small-cell lung carcinoma (11), breast cancer (12), lung cancer (13), and thyroid cancer (14). Although overexpression of Glut-1 in various cancer cells increases glucose uptake, which is associated with increased cellular proliferation and increased malignant potential (6,9,10,1214), the relationship between Glut-1 expression and cancer cell invasion has not been assessed. The increased invasiveness and metastasis associated with Glut-1 overexpression are unlikely to be explained by Glut-1 overexpression alone because, to invade and metastasize, tumor cells must degrade basement membrane and extracellular matrix. These activities are carried out by matrix metalloproteinases (MMPs). Increased glucose uptake mediated by Glut-1 is not directly associated with degradation of basement membrane or extracellular matrix.

The process of metastasis consists of many steps, of which invasion through the basement membrane of blood vessels is one of the most important (15). MMPs are thought to be important in degrading basement membrane because their activity is associated with invasive and metastatic potential (16). These structurally related enzymes consist of at least 24 members (17,18) that are classified on the basis of their substrate specificities: collagenases, gelatinases, stromelysins, membrane-type MMPs, and unclassified MMPs (17,18). MMP activity is controlled at multiple levels. MMP genes are transcriptionally regulated in response to growth factors, cytokines, and oncogenes (18). In addition, MMPs are secreted as inactive precursors or zymogens that require proteolytic processing to release the active enzyme. This processing is achieved by proteinases such as plasmin or membrane-type MMP (18,19).

One MMP, MMP-2 (a 72-kd metalloproteinase) specifically cleaves type IV collagen, the major component of basement membranes (18). In human tumors, MMP-2 expression has been directly associated with cancer invasiveness and metastasis (2025). Because Glut-1 is overexpressed in cancer cells, and because this overexpression is associated with metastasis, we hypothesized that Glut-1 regulates the expression of proteins involved in basement membrane and extracellular matrix degradation, such as MMP-2. In this article, we report on our testing of this hypothesis by modulating the expression of Glut-1 and examining the effects on MMP-2 expression and invasiveness.


    METHODS
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell Culture

All cell lines used in this study were obtained from the Human Science Research Resources Bank (Osaka, Japan) and grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). We used the following cancer cell lines: RD (rhabdomyosarcoma), YKG1 (glioblastoma), T98G (glioblastoma), A172 (glioblastoma), A549 (lung carcinoma), MKN45 (gastric carcinoma), HT-1080 (fibrosarcoma), MCF7 (breast carcinoma), and MDA-MB-231 (breast carcinoma).

Generation of Glut-1 Complementary DNA, Glut-1 Expression Vectors, and Antisense Oligonucleotides

Total RNA was isolated from human MDA-MB-231 breast cancer cells as described (26). Reverse transcription–polymerase chain reaction (RT–PCR) was used to generate Glut-1 complementary DNA (cDNA) from 800 ng of total RNA, according to the manufacturer's instructions (RNA PCR Kit; TaKaRa, Otsu, Japan). The sequences for the Glut-1 sense and antisense primers were 5`-TCAGAGTCGCAGTGGGAGTC-3` and 5`-ACTCACACTTGGGAATCAGC-3`. A 1599-bp product was amplified by PCR and cloned into the TA cloning site of the pTargeT expression vector (Promega, Tokyo, Japan). The sequence of the entire coding region of Glut-1 was confirmed by using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan) to make sure that Glut-1 was correctly amplified by PCR. The control vector consisted of a pTargeT expression vector without Glut-1 cDNA. Glut-1 phosphorothioate oligonucleotides (sense, 5`-CGGGCCAAGAGTGTGC TAAA-3` and antisense, 5`-TGACGATACCGGAGCCAATG 3`) were synthesized in an automated DNA synthesizer (model 380B; Applied Biosystems).

Transfection With Glut-1 Antisense Oligonucleotides or Glut-1 Expression Vector

RD cells (1 x 106) were plated into 100-mm dishes and allowed to adhere overnight. The next day, the cells were transiently transfected by exposure to a mixture containing oligonucleotides (20 µM) or containing Glut-1 cDNA (2 µg) or control DNA expression vectors and LipofectAMINE PLUS Reagent (Life Technologies, Inc., Tokyo, Japan) in 6.5 mL of DMEM for 3 hours at 37 °C in a 5% CO2 incubator, according to the manufacturer's instructions. After the transfection, the mixture was removed and replaced with DMEM containing 10% FCS.

Analysis of the Expression of Glut-1, MMP-2, {beta}-actin, and Membrane Type-MMP by RT–PCR and Northern Blotting

Thirty-six hours after transfection with Glut-1 sense or antisense oligonucleotides or Glut-1 expression vector, total RNA from RD cells was isolated using the acid guanidinium–phenol–chloroform method. mRNA expression of Glut-1, MMP-2, {beta}-actin, and membrane type (MT)-MMP was measured by RT–PCR starting with 800 ng of total RNA and specific primers, according to the manufacturer's instructions (RNA PCR kit, version 2.1; TaKaRa). We used the sense and antisense primers MMP-2, 5`-TTCAAGGACCGGTTCATTTGGCGGACTGTG-3` and 5`-TTCCAAACTTCACGCTCTTCAGACTTTGGTT-3`; MT-MMP, 5`-AACAACCAGAAGCTGAAGGTA-3` and 5`-ACCTTGTC CAGCAGGGAACG-3`; {beta}-actin, 5`-CGTGGGGCGCCCCAG GCACCA-3` and 5`-TTGGCCTTGGGGTTCAGGGGG-3`; and Glut-1, 5`-CGGGCCAAGAGTGTGCTAAA-3` and 5`-TGACGAT ACCGGAGCCAATG-3`. PCR was carried out in a Gene Amp PCR Thermal Cycler 9600 (PerkinElmer, Fremont, CA) for 23 cycles, with each cycle consisting of denaturation at 94 °C for 30 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 30 seconds. The PCR products were subjected to electrophoresis through a 2% agarose gel, which was then stained with ethidium bromide to visualize the bands. The sequence of the amplified PCR products was verified to make sure that PCR was correctly performed. NIH Image software (version 1.61; National Institutes of Health, Bethesda, MD) was used to quantify the intensities of the bands. To compare differences between samples, the relative intensity of each band was normalized to the intensity of the {beta}-actin band amplified from the same sample.

Northern blotting was performed using 10 µg of total RNA isolated from RD cells, RD cells transfected with Glut-1 sense or antisense oligonucleotides, and RD cells transfected with Glut-1 or control DNA expression vectors. RNAs were separated by electrophoresis through a 1% formaldehyde/agarose/MOPS gel and transferred to a nylon membrane. The northern blot was hybridized with riboprobes for Glut-1 and MMP-2, as described (26). To generate the probes, cDNAs for Glut-1, MMP-2, or {beta}-actin were cloned into the SK multiple cloning site of the pBluescript II vector (TOYOBO, Tokyo, Japan). Glut-1 cDNA contained the sequence from 62 to 1660 base positions of the gene (GenBank accession number K03195), MMP-2 cDNA contained the sequence from 1415 to 1907 base positions of the gene (GenBank accession number J03210), and {beta}-actin cDNA contained the sequence from 112 to 354 base positions of the gene (GenBank accession number AB004047). Glut-1, MMP-2, and {beta}-actin riboprobes were generated by in vitro transcription using the T7 polymerase binding site of each cloning vector and an in vitro transcription kit (Roche Japan, Tokyo). Specific riboprobes were labeled with digoxigenin–deoxyuridine triphosphate (dUTP) by random priming (Roche Japan). The mRNA bands on the northern blot were quantified using NIH Image software, and the intensities of the MMP-2 and Glut-1 mRNA bands were normalized to the intensities of the control {beta}-actin bands from the same samples.

Gelatin Zymography

Sixty hours after transfection with Glut-1 or control DNA expression vectors, RD cells were detached with trypsin and plated on 12-well cluster dishes at a concentration of 5 x 105 cells/well. The cells were adhered in the presence of serum, which was then replaced with serum-free medium. After 24 hours, the serum-free conditioned media were collected, and aliquots (45-µL) were assayed for MMP-2 and MMP-9 activities by gelatin zymography, as described (27), and the data were quantified using NIH Image software. An MMP-1 enzyme assay was performed using an enzyme-linked immunosorbent assay (ELISA) kit (Amersham Biosciences, Tokyo, Japan), according to the manufacturer's recommended protocol.

Western Blotting

Cells were cultured in complete growth medium until they were approximately 80%–90% confluent. The cells were washed twice with phosphate-buffered saline (PBS), collected with cell scrapers, homogenized, and extracted with 50 mM Tris–HCl buffer (pH 7.5) containing proteinase inhibitors (5.0 µg/mL phenylmethylsulfonyl fluoride, 2.0 µg/mL leupeptin, 2.0 µg/mL aprotinin). The protein concentration of the extracts was measured using a Bradford assay kit (Bio-Rad, Hercules, CA). Extracted proteins (100 µg) were separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with specific rabbit polyclonal antibodies against human Glut-1 and MMP-2 (Santa Cruz Biotechnology, Santa Cruz, CA), as described (28). Protein bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences). Band intensities were quantified using NIH Image software.

Invasion Assay

The invasiveness of RD, YKG1, and T98G cells (2 x 105) transfected with Glut-1 sense or antisense oligonucleotides was assayed 60 hours after transfection by using a Boyden chamber as described (27). Invasiveness was measured by counting the number of cells that penetrated the polycarbonate filters (Neuro Probe, Inc., Gaithersburg, MD). The invasiveness of each cell line was expressed as a percentage of the control (cells transfected with sense oligonucleotides). Transfected cells were assayed in triplicate for invasiveness, and the experiments were repeated twice.

In some experiments, antibodies were added to neutralize MMP-2 activity. In these experiments, 60 hours after transfection with the Glut-1 cDNA or control DNA expression vectors, RD cells were incubated with anti-MMP-2 antibody (456 µg/mL) and then tested in the invasion assay, as described (27). This antibody concentration effectively neutralizes MMP-2 activity (27). In control experiments, we substituted immunoglobulin G (IgG; 456 µg/mL) of the same species and isotype as the anti-MMP-2 antibody.

Cloning of the Human MMP-2 Promoter

We identified and cloned the human MMP-2 promoter from human placental DNA by using RT–PCR with specific primers for human MMP-2 promoter, as described (29). The PCR product was cloned into the pGL3 vector (Promega), which was then sequenced. The constructs used for the experiments were pGL3a, containing the full-length MMP-2 promoter (from positions –1659 to +57); pGL3b, containing a deletion mutant starting at position –1079 upstream of the transcription start site (from positions –1079 to +57); and pGL3e, containing no MMP-2 promoter.

Luciferase Assay

RD cells were detached with trypsin, plated on 12-well cluster dishes at a concentration of 5 x 105 cells/well, and cultured for 12 hours in complete medium. One microgram of pGL3a, pGL3b, or pGL3e was cotransfected with 0.1 µg of pRL-CMV vector, which has a cytomegalovirus promoter in front of a renilla luciferase gene. After 48 hours, luciferase activity was measured in lysates from the transfected cells, according to the manufacturer's recommended protocol (Dual-Luciferase Reporter Assay System; Promega). The activity of renilla luciferase was used to normalize any variation in transfection efficiency. After the firefly and luciferase activities in cell lysates were sequentially quantified, the promoter activity of each plasmid construct was calculated as the firefly–renilla luciferase activity ratio. The transfection efficiency of RD cells using the pCMV{beta} vector (Clontech, Tokyo, Japan), which contains the {beta}-galactosidase gene, was 75.0% ± 5.8%.

Preparation of Nuclear Extracts and Gel Mobility Shift Assays

Forty-eight hours after transfection, nuclear extracts were prepared, as described (28), from untreated control RD cells, from transfection control RD cells (those treated with LipofectAMINE PLUS Reagent or with control vector), or from RD cells transfected with Glut-1 cDNA or Glut-1 sense or antisense oligonucleotides. The protein concentration in the nuclear extracts was measured using the Bradford assay kit (Bio-Rad). For each gel–mobility shift assay, 2 µg of each nuclear extract was used.

Consensus or mutant p53 oligonucleotides and consensus or mutant Ets-1 family oligonucleotides (Santa Cruz Biotechnology) were used as probes and labeled with DIG-11–2`,3`-dideoxyribonucleoside uridine 5`-triphosphate (DIG-11-ddUTP) by using the terminal deoxynucleotidyl transferase method, according to the manufacturer's recommended protocol (Roche Japan). Binding reaction mixtures (20 µL containing 2 µg of nuclear extract, 0.4 ng of labeled probe, 20 mM HEPES [pH 7.6], 5 mM EDTA, 10 mM (NH4)2SO4, 1 mM dithiothreitol, 0.2% [w/v] Tween 20, 30 mM KCl, and 1 µg of poly [dI-dC]) were mixed on ice and incubated for 15 minutes at room temperature. For the competition experiments, 125- or 250-fold molar excess of the unlabeled probe was added to the mixture before incubation.

The protein–DNA complexes were resolved by electrophoresis through a 6% polyacrylamide gel in 0.25x Tris–borate–EDTA buffer (22.25 mM Tris, 22.25 mM borate, 0.5 mM EDTA [pH 8.0]) for 2 hours at 4 °C. The protein–DNA complexes were transferred to a nylon membrane (Roche Japan) by electroblotting, and the protein–DNA complexes were detected with a chemiluminescence detection kit (Roche Japan).

To test the specificity of the protein–DNA binding reaction, nuclear extracts were preincubated with 25 µg of mouse monoclonal anti-p53 antibody (BD Biosciences, Tokyo, Japan), 25 µg of mouse monoclonal anti-Ets-1 antibody (BD Biosciences), or 25 µg of control mouse IgG (Cosmo Bio, Tokyo, Japan) for 1 hour before the addition of the DNA probe.

Immunohistochemical and Fluorescence Analyses

Tumor tissues of various organs from 80 Japanese patients were examined. The tumors had been surgically resected at the Second Department of Surgery, Gunma University Hospital, and its affiliated hospitals from 1992 through 2000. The mean age of the patients was 58.1 years (range = 5–89 years). Thirty-four patients were male and 46 were female. Tumors were histologically classified according to the World Health Organization International Histological Classification of Tumors (30). The study protocol conformed to the ethical guidelines of the Gunma University School of Medicine.

Archival formalin-fixed, paraffin-embedded tissue sections (3-µm thick) were deparaffinized and immunostained by following a standard protocol. Briefly, after inhibiting endogenous peroxidase activity with a 3% aqueous hydrogen peroxide–methanol solution for 20 minutes and blocking the section with 10% normal rabbit serum, we incubated the tissue sections overnight with primary antibodies (polyclonal goat anti-MMP-2 and anti-Glut-1 antibodies, 1 : 500 dilution, Santa Cruz Biotechnology) at 4 °C. The sections were processed by the streptavidin–biotin–peroxidase method according to the manufacturer's recommended protocol (Histofine SAB kit; Nichirei, Japan) that used diaminobenzidine as the chromogen. Nuclei were lightly counterstained with hematoxylin. To confirm the specificity of the MMP-2 or Glut-1 immunostaining, nonimmune serum was substituted for the primary antibodies and used to stain additional tissue sections.

For the purpose of this study, tissue sections were considered negative for MMP-2 or Glut-1 expression if 100% of the cells were Glut-1 or MMP-2 negative by immunohistochemistry. If any expression was detected, tissue sections were considered Glut-1 or MMP-2 positive.

Immunofluorescence staining was performed on breast cancer tissue sections by using a combination of rabbit polyclonal anti-Glut-1 (1 : 500 dilution; Santa Cruz Biotechnology) and mouse monoclonal anti-MMP-2 (1 : 500 dilution; Fuji, Toyama, Japan) antibodies. After the tissue sections were incubated with the combination of primary antibodies for 1 hour at room temperature, they were rinsed extensively in PBS and then incubated with rhodamine-conjugated goat anti-rabbit IgG (1 : 1000 dilution; Santa Cruz Biotechnology) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1 : 1000; Santa Cruz Biotechnology) for 1 hour at room temperature. After extensive rinsing in PBS, the sections were examined under a fluorescence microscope (IX70; Olympus, Tokyo, Japan).

Effect of Glucose on Glut-1 and MMP-2 mRNA Expression

To determine the time-dependent and concentration-dependent effects on Glut-1 and MMP-2 mRNA expression, RD cells (1 x 106 cells/dish) were plated in 100-mm dishes and allowed to adhere for 2 hours. Next, the medium was changed to DMEM containing 2% FCS in which the glucose concentrations were adjusted to very low (2.7 mM), low (5.6 mM), or high (25 mM). RD cells were incubated for various periods of time before RNA was extracted and examined by northern blot analysis for expression of Glut-1, MMP-2, and {beta}-actin mRNAs. The mRNA bands on the northern blot were quantified by using NIH Image software, and the fold of intensities of the MMP-2 and Glut-1 mRNA bands were normalized to the intensities of the control {beta}-actin bands from the same samples. After image analysis, the data from three samples per group were expressed as fold induction relative to {beta}-actin.

Statistical Analysis

The data were provided as means of three experiments with 95% confidence intervals (CIs). All experiments were performed three times, unless stated otherwise. All statistical analyses were performed by using Statview 4.5 software (Abacus Concepts, Inc., Berkeley, CA). The statistical significance of differences between treatment groups was determined by the use of analysis of variance (ANOVA) followed by Fisher's partial least-squares difference test. All P values were two-sided, and P values of less than .05 were considered statistically significant.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of MMP-2 and Glut-1 in Various Cell Lines

We first examined the expression of MMP-2 and Glut-1 in eight cancer cell lines by western blotting. All of the cell lines coexpressed MMP-2 and Glut-1 proteins (Fig. 1, AGo). The relative band intensities of four cell lines (A549, T98G, YKG1, and RD) were comparable for MMP-2 and Glut-1 expression levels (lower panel). The relative band intensities in three cell lines (A172, MCF7, and MDA-MB-231) were higher for MMP-2 expression than for Glut-1 expression. The relative band intensities for MKN45 cells were higher for Glut-1 expression than for MMP-2 expression. The results suggest that MMP-2 and Glut-1 were coexpressed at the protein level in various cancer cell lines.



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Fig. 1. Expression of glucose transporter 1 (Glut-1) and matrix metalloproteinase-2 (MMP-2) in eight cancer cell lines and effects of inhibition or overexpression of Glut-1 on MMP-2 and membrane type (MT)-MMP expression in the rhabdomyosarcoma cell line RD. A) Whole-cell lysates from lung (A549), glioblastoma (A172, T98G, and YKG1), breast (MCF7 and MDA-MB-231), gastric (MKN45), and rhabdomyosarcoma (RD) cancer cell lines were examined for MMP-2 and Glut-1 protein expression by western blot analysis by using specific goat polyclonal antibodies against Glut-1 and MMP-2 (upper panel). Levels of expression were quantified using NIH Image software (lower panel). Data are expressed as mean and 95% confidence intervals of three independent experiments. Open bars represent Glu-1 expression. Closed bars represent MMP-2 expression. BD) mRNA was isolated from RD cells (control), from RD cells transfected with Glut-1 sense or antisense oligonucleotides (B), from RD cells transfected with control (mock) or Glut-1 expression vectors (C and D), or from RD cells treated with transfection reagents (lipo; BD). mRNA was amplified by reverse transcription–polymerase chain reaction (RT–PCR) with specific primers for Glut-1, MMP-2, MT-MMP, and {beta}-actin. Representative ethidium bromide stained gels are shown in the upper panels. mRNA levels were quantified by densitometry and expressed relative to the level of {beta}-actin (lower panels). The size of the PCR products was determined by comparison with the 100-bp DNA ladder marker (M). In all cases data are expressed as mean and 95% confidence intervals of three experiments.

 
Effect of Transfection of Glut-1 Antisense Oligonucleotides and Glut-1 cDNA on MMP-2 mRNA Expression

We next used RT–PCR analysis to evaluate the effect of altering Glut-1 on mRNA expression levels of MMP-2. To inhibit Glut-1 expression, we transfected RD cells, which constitutively express Glut-1, with Glut-1 antisense oligonucleotides. Transfection with specific Glut-1 antisense oligonucleotides decreased mRNA levels of Glut-1 and MMP-2 but not of {beta}-actin (Fig 1, BGo). Compared with control cells transfected with Glut-1 sense oligonucleotides, the level of Glut-1 mRNA decreased by 68.8% (95% CI = 68.0% to 69.6%; P<.001), and the level of MMP-2 mRNA decreased by 71.5% (95% CI = 71.1% to 71.9%; P<.001) in cells transfected with Glut-1 antisense oligonucleotides.

Next, we examined the effect of overexpressing Glut-1 mRNA in RD cells by transfecting the cells with a Glut-1 expression vector. Compared with cells transfected with control vector, the level of Glut-1 mRNA expression increased by 5.4-fold (95% CI = 4.6-fold to 6.2-fold; P<.001) and the level of MMP-2 mRNA increased by 4.3-fold (95% CI = 3.7-fold to 4.9-fold; P<.001) (Fig. 1, CGo). Overexpression of Glut-1 mRNA did not have a statistically significant effect on the level of MT-MMP mRNA expression (Fig. 1, DGo). These results suggest that MMP-2 but not MT-MMP expression is associated with Glut-1 expression in RD cells.

To determine whether the association between Glut-1 and MMP-2 was unique to RD cells, we transfected other cancer cell lines with Glut-1 antisense oligonucleotides and examined the effect on Glut-1 and MMP-2 mRNAs. Decreased expression of Glut-1 and MMP-2 mRNAs was observed in the human glioblastoma cell lines YKG1 and T98G after transfection with Glut-1 antisense oligonucleotides (Fig. 2Go). Compared with control cells transfected with Glut-1 sense oligonucleotides, the level of Glut-1 mRNA decreased by 68.8% (95% CI = 67.4% to 70.2%; P<.001), and the level of MMP-2 mRNA decreased by 71.5% (95% CI = 67.1% to 75.9%; P<.001) in YKG1 cells transfected with Glut-1 antisense oligonucleotides (Fig. 2, AGo). Similar results were obtained with T98G cells. Compared with T98G cells transfected with Glut-1 sense oligonucleotides, the level of Glut-1 mRNA decreased by 74.1% (95% CI = 73.8% to 74.4%; P<.001), and the level of MMP-2 mRNA decreased by 65.9% (95% CI = 65.7% to 66.1%; P<.001) in T98G cells transfected with Glut-1 antisense oligonucleotides (Fig. 2, BGo). The results suggest that the association between the expression of Glut-1 and MMP-2 is not unique to RD cells.



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Fig. 2. Effect of inhibiting expression of glucose transporter 1 (Glut-1) on matrix metalloproteinase-2 (MMP-2) expression in glioblastoma cell lines YKG1 (A) and T98G (B). mRNA was isolated from untreated cells (control), from cells transfected with Glut-1 sense or antisense oligonucleotides, or from cells treated with transfection reagents (lipo). mRNA was amplified by reverse transcription–polymerase chain reaction (RT–PCR) with specific primers for Glut-1, MMP-2, and {beta}-actin. Representative ethidium bromide-stained gels are shown in the upper panels. mRNA levels were quantified by densitometry and expressed relative to the level of {beta}-actin (lower panels). The size of the PCR products was determined by comparison with the 100-bp DNA ladder marker (M). In all cases, data are expressed as mean and 95% confidence intervals of three experiments.

 
To confirm the results of RT–PCR analysis, we transfected RD cells with expression vectors carrying Glut-1 cDNA or Glut-1 antisense oligonucleotides and assessed Glut-1 and MMP-2 expression by northern blot analysis (Supplementary Fig. 1Go, available on line at the Journal's Web site, http://jncicancerspectrum.oupjournals.org/jnci/content/vol94/issue14/index.shtml). The molecular sizes of Glut-1 and MMP-2 transcripts were 2.8 and 3.1 kb, respectively (31,32). The levels of Glut-1 and MMP-2 mRNAs expressed in Glut-1 antisense oligonucleotide transfectants were 37.3% (95% CI = 34.4% to 40.2%; P<.001) and 47.3% (95% CI = 44.1% to 50.5%; P<.001), respectively, lower than those in control cells. The levels of Glut-1 and MMP-2 mRNAs expressed in Glut-1 expression vector transfectants were 2.1-fold (95% CI = 1.9-fold to 2.3-fold; P<.001) and 2.7-fold (95% CI = 2.6-fold to 2.8-fold; P<.001), respectively, higher than those in untreated cells. Thus, we confirmed the results of RT–PCR analysis.

Effect of Glut-1 Antisense Oligonucleotides and Glut-1 cDNA on MMP Protein Expression

Next, to assess the effect of Glut-1 on MMP-2 protein activity, which is a direct reflection of MMP-2 protein expression, we measured the gelatinolytic activity in the supernatants of Glut-1-overexpressing RD cells by zymography. RD cells secrete the latent, inactive form of MMP-2 (27). Neither activated MMP-2 nor the related molecule MMP-9 was detected in culture supernatants from control RD cells (Fig. 3Go, lane 1). RD cells that overexpressed Glut-1 secreted 4.0-fold (95% CI = 3.96-fold to 4.06-fold) more active MMP-2 than did untransfected cells (P<.001). By contrast, RD cells transfected with Glut-1 antisense oligonucleotides secreted only 29.4% (95% CI = 27.5% to 31.3%) of the level of active MMP-2 secreted by untreated cells (P<.001). Thus, MMP-2 expression was regulated at the mRNA level, and this corresponded to changes at the protein level.



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Fig. 3. Effects of inhibition or overexpression of glucose transporter-1 (Glut-1) on matrix metalloproteinase-2 (MMP-2) protein activity in the rhabdomyosarcoma cell line RD. MMP-2 and MMP-9 protein expression from culture supernatants was assessed by gelatin zymography (25). Equivalent volumes of supernatants from untreated RD cells (lane 1), RD cells treated with transfection reagents (lane 2), RD cells transfected with a Glut-1 expression vector (lane 3), RD cells transfected with a control vector (lane 4), RD cells transfected with Glut-1 sense oligonucleotides (lane 5), RD cells transfected with Glut-1 antisense oligonucleotides (lane 6), and the human fibrosarcoma cell line HT-1080 (lane 7) were tested. The supernatant from HT-1080 cells, which express both MMP-2 and MMP-9, was used as a positive control. A representative experiment of three independent experiments is shown.

 
To confirm the specificity of the association between Glut-1 expression and MMP-2 activity, we also measured MMP-1 activity. There were no differences in MMP-1 activity, as measured in a specific ELISA, among RD cells transfected with the Glut-1 expression vector or Glut-1 antisense oligonucleotides relative to control cells (data not shown).

Effect of Glut-1 on Transcriptional Activity of MMP-2

To elucidate the mechanism by which Glut-1 may regulate MMP-2 expression, we next evaluated whether Glut-1 could influence the MMP-2 gene promoter. We subcloned the full-length MMP-2 promoter region into pGL3e (pGL3a), transiently cotransfected the vector along with pRL-CMV into RD cells, and measured the resulting luciferase activity after 48 hours. We compared this luciferase activity with that from cells transfected with the control vector pGL3e (containing no MMP-2 promoter) and pRL-CMV. Fig. 4, AGo, shows the relative luciferase activity expressed as the ratio of firefly luciferase (from the pGL3 vector) to renilla luciferase (from the pRL-CMV vector) detected in cell lysates. Cells transfected with pGL3e had very low levels of the firefly luciferase activity. However, cells transfected with pGL3a containing the full-length MMP-2 promoter (from positions –1659 to +57) expressed statistically significantly more luciferase activity (2.1-fold; 95% CI = 1.6-fold to 2.6-fold; P = .002) than did control cells.



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Fig. 4. Effect of overexpressing glucose transporter 1 (Glut-1) on matrix metalloproteinase-2 (MMP-2) promoter activity. A) Rhabdomyosarcoma cell line RD was transfected with control (mock) or Glut-1 expression vectors (Glut1) or treated with the transfection reagents (lipo) and then cotransfected with pRL containing the renilla luciferase gene and with the pGL3e firefly luciferase vector, pGL3a containing the entire MMP-2 promoter region (from positions –1659 to +57 ), or pGL3b containing a partial MMP-2 promoter region (from positions –1079 to +57). The fold induction of firefly luciferase activity to renilla luciferase activity was measured after 48 hours. Data from three independent experiments are expressed as means ± 95% confidence intervals. B and C) Effect of Glut-1 expression on MMP-2 promoter activity was studied by a gel mobility shift assay with consensus oligonucleotide probes for p53 (B) and Ets-1 (C) DNA binding factors. A 25-bp double-stranded oligonucleotide probe containing the consensus p53 binding site (B) or the Ets-1 binding site (C) were incubated with nuclear extracts from untreated RD cells (lane 1), untransfected RD cells treated with transfection reagents (lane 2), RD cells transfected with the control vector (lane 3), RD cells transfected with a Glut-1 expression vector (lane 4), RD cells transfected with Glut-1 sense oligonucleotides (lane 5), or RD cells transfected with Glut-1 antisense oligonucleotides (lane 6). The protein–DNA complexes (B, upper two bands, and C, upper band) were separated from the free probe (bottom bands) on a 5% polyacrylamide gel. Competition assays were performed by adding unlabeled oligonucleotide probes or mouse monoclonal antibodies to p53 or Ets-1 to the nuclear extracts before adding the labeled probes. The labeled probes were incubated with nuclear extracts from untreated RD cells (lane 7), untreated RD cells and a 125-fold molar excess of unlabeled oligonucleotide probe (lane 8), untreated RD cells and a 250-fold molar excess of unlabeled oligonucleotide probe (lane 9), untreated RD cells and a 125-fold molar excess of unlabeled mutant oligonucleotide probe (lane 10), untreated RD cells and a 250-fold molar excess of unlabeled mutant oligonucleotide probe (lane 11), untreated RD cells and 2 µg of anti-p53 or anti-Ets family antibody (lane 12), or untreated RD cells and 2 µg of immunoglobulin G (IgG) (lane 13). Untreated RD cells were incubated with labeled mutant oligonucleotide probe (lane 14), and untreated RD cells were incubated with labeled mutant oligonucleotide probe and a 125-fold molar excess of unlabeled oligonucleotide (lane 15). Representative gels from three independent experiments are shown.

 
To identify the responsive element in the MMP-2 promoter sequence, we constructed pGL3b, which contains a deletion starting at position –1079 upstream of the transcription start site (deleted region included positions –1079 to +57). After cotransfecting this construct with pRL-CMV into RD cells, we measured the luciferase activity. Similar levels of luciferase activity were detected in lysates from Glut-1 transfectants, control (i.e., pGL3e) transfectants, and control transfection reagent-treated cells (Fig. 4, AGo). The result suggested that the –1659 to –1079 region of the MMP-2 promoter is involved in Glut-1-mediated MMP-2 transcription.

Identification of Factors Associated With Glut-1-Mediated MMP-2 Transcriptional Activation

The region spanning from –1659 to –1079, which appeared to have a crucial role in Glut-1-mediated MMP-2 transcriptional activity, consists of one p53 binding site and one Ets-1 binding site (29). To determine whether either transcription factor is important for Glut-1-mediated MMP-2 promoter activity, we performed gel mobility shift assays by using oligonucleotide probes containing Ets-1 or p53 binding consensus sequences. Nuclear extracts from untreated RD cells contain p53 binding factors that interact with specific p53 oligonucleotide consensus sites, generating two specific bands (Fig. 4, BGo). The intensity of the bands increased with increasing endogenous MMP-2 transcriptional activity (Fig. 4, BGo). There was no substantial difference in the intensity of the bands among untreated RD cells, control transfection reagent-treated RD cells, control-transfected RD cells, and Glut-1 sense oligonucleotide-transfected RD cells. By contrast, there was an increase in the intensity of the bands derived from Glut-1-transfected RD cells and a decrease in the intensity of the bands derived from Glut-1 antisense oligonucleotide-transfected RD cells.

Nuclear extracts from untreated RD cells contain an Ets-1 binding factor that interacts with a specific Ets-1 oligonucleotide consensus site, generating a single specific band (Fig. 4, CGo). There was no difference in the intensity of the Ets-1 bands among untreated RD cells, control reagent-treated RD cells, control transfected RD cells, Glut-1-transfected RD cells, or among Glut-1 sense or antisense oligonucleotide-transfected RD cells.

The p53 and Ets-1 protein–DNA complexes were specific because their formation was completely inhibited by 125-fold or 250-fold molar excesses of unlabeled oligonucleotide probe or 250-fold molar excess of unlabeled mutated oligonucleotide probe and partially inhibited by a 125-fold molar excess of unlabeled mutated oligonucleotide probe (Fig. 4, B and CGo). When this mutated oligonucleotide was used as a probe, fewer low-mobility complexes were formed relative to those formed in the presence of the nonmutated oligonucleotide, and these were completely inhibited by a 125-fold molar excess of unlabeled oligonucleotide probe (Fig. 4, B and CGo, lanes 10–15). Furthermore, we confirmed the identity of the bands by preincubating the nuclear extracts with antibodies to p53 or Ets-1 for 1 hour at room temperature. This incubation prevented the interaction between the protein and the DNA probe, as evidenced by a lack of slower mobility bands. Preincubation with nonimmune IgG had no effect on the formation of p53 and Ets-1 protein–DNA complexes (Fig. 4, B and CGo, lane 13).

Effect of Glut-1 Expression on Cell Invasion

Because MMP-2 is involved in cell invasion, we next examined the functional consequences of suppressing Glut-1 expression in RD, T98G, and YKG1 cells in a Boyden chamber invasion assay (27). The invasion of RD, T98G, and YKG1 cells transfected with Glut-1 antisense oligonucleotides was suppressed by 53.0% (95% CI = 47.5% to 58.5%; P = .007), 55.1% (95% CI = 47.5% to 62.7%; P = .01), and 52.4% (95% CI = 47.6% to 57.2%; P = .001), respectively, compared with cells transfected with Glut-1 sense oligonucleotides (Fig. 5, AGo). The magnitude of the suppression of invasion was similar to MMP-2 mRNA suppression seen in RD, T98G, and YKG1 cells—68.8%, 71.5%, and 65.9%, respectively (Fig. 2Go).



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Fig. 5. Effects of inhibiting or overexpressing glucose transporter 1 (Glut-1) on tumor cell invasiveness. A) Rhabdomyosarcoma cell line RD and glioblastoma cell lines YKG1 and T98G were transfected with Glut-1 sense or antisense oligonucleotides, and invasiveness was assayed using a Boyden chamber as described in the "Materials and Methods" section (25). The experiment was performed in triplicate twice. B) RD cells, transfected with a control (mock) or Glut-1 expression vector, were tested for invasiveness by using a Boyden chamber in the presence of anti-matrix metalloproteinase-2 (MMP-2) antibody (456 µg/mL) or control immunoglobulin G (IgG) (456 µg/mL). Invasiveness was expressed as a percentage of the control (cells transfected with sense oligonucleotides in A or with control vector in B). Bars in both A and B represent the means and 95% confidence intervals of three independent experiments.

 
We next studied whether Glut-1 overexpression stimulated RD cell invasion and, if so, whether MMP-2 was involved in this behavior. As shown in Fig. 5, BGo, RD cells transfected with Glut-1 cDNA were 3.2-fold (95% CI = 2.6-fold to 3.8-fold) more invasive than control transfected cells (P = .002). This invasiveness was mediated, at least in part, by MMP-2 because the addition of anti-MMP-2 antibodies, but not control IgG antibodies, statistically significantly suppressed the increased invasion in Glut-1-transfected cells (P<.001).

Expression of Glut-1 and MMP-2 in Tissue Sections

Because all the cancer cell lines we tested coexpressed Glut-1 and MMP-2 in vitro, we next examined whether human cancers expressed MMP-2 and Glut-1 in the same cells by immunohistochemistry and immunofluorescence. In Fig. 6, AGo, the upper and lower panels show the typical staining patterns for MMP-2 and Glut-1, respectively, in a human lung squamous cell carcinoma. By immunofluorescence, we detected expression of MMP-2 and Glut-1 in the same cells (Fig. 6, BGo). The results of MMP-2 and Glut-1 expression analyses in 80 human cancer specimens analyzed by immunoperoxidase staining by using anti-MMP-2 and Glut-1 antibodies are summarized in Table 1Go. Of the 80 cancer specimens, 45 coexpressed Glut-1 and MMP-2, 19 expressed neither Glut-1 nor MMP-2, 14 expressed Glut-1 but not MMP-2, and two expressed MMP-2 but not Glut-1. The relationship between the expression of Glut-1 and MMP-2 was statistically significant (P<.001).



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Fig. 6. Expression of glucose transporter 1 (Glut-1) and matrix metalloproteinase-2 (MMP-2) in human cancers. Expression of Glut-1 and MMP-2 in tissue sections from 80 human cancers was determined by immunohistochemistry (A) and by immunofluorescence (B). A) Representative tissue sections from a lung cancer specimen were immunostained by using 0.2% (vol/vol) anti-MMP-2 (A, upper panel) or Glut-1 (A, lower panel) antibody and developed by the diaminobenzidine (DAB)-based colorimetric method. Nuclei were lightly counterstained with hematoxylin. Magnification is x200. B) Representative tissue sections from a breast cancer specimen were immunostained by using a combination of primary rabbit polyclonal anti-Glut-1 antibodies and mouse monoclonal anti-MMP-2 antibodies. After incubation with the primary antibodies, the sections were extensively washed with phosphate-buffered saline and then incubated with rhodamine-conjugated goat anti-rabbit immunoglobulin G (IgG) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The sections were examined with a fluorescence microscope (model IX70; Olympus, Tokyo, Japan). Upper panel shows expression of MMP-2; middle panel shows expression of Glut-1; lower panel shows the digitally merged combination image of the other two panels. Bar = 20 µm.

 

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Table 1. Coexpression of glucose transporter 1 (Glut-1) and matrix metalloproteinase-2 (MMP-2) in 80 human cancer specimens analyzed by immunohistochemistry*
 
Effect of Glucose on Glut-1 and MMP-2 mRNA Expression

To assess the effect of glucose on Glut-1 and MMP-2 mRNA expression, RD cells were incubated with varying concentrations of glucose (2.7–25 mM) for 0, 36, and 60 hours, and total RNA was extracted. Glut-1 and MMP-2 mRNA expression was analyzed by northern blotting. As shown in Fig. 7Go, glucose did not affect Glut-1 and MMP-2 mRNA expression at any of the tested concentrations or incubation times.



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Fig. 7. Effect of glucose on glucose transporter 1 (Glut-1) and matrix metalloproteinase-2 (MMP-2) mRNA expression. A) Rhabdomyosarcoma cell line RD was incubated in medium containing very low (2.7 mM), low (5.6 mM), and high (25 mM) glucose for 0, 36, and 60 hours. At each time point, total RNA was isolated and analyzed by northern blot for Glut-1, MMP-2, and {beta}-actin mRNA expression. Ethidium bromide-stained ribosomal RNA is shown for a loading comparison (A, upper panel). B) mRNA levels were quantified using NIH Image software and expressed relative to {beta}-actin levels. Bars represent the means and 95% confidence intervals of three independent experiments.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we investigated the hypothesis that Glut-1 regulates MMP-2 expression. In RD rhabdomyosarcoma cells, the mechanism by which Glut-1 regulates MMP-2 appears to involve binding of p53 to the MMP-2 promoter. Although p53 is the most common gene for abnormalities in human cancer (33) and RD cells contain a p53 mutation (codon 248 C->T) (34), Glut-1 overexpression increased MMP-2 promoter activity. The data are consistent with a previous report (29) that both mutant and wild-type p53 enhanced MMP-2 promoter activity, although the magnitude of activation was smaller for mutant p53 than for wild-type p53.

Glut-1 gene expression can be induced in cancer cells by oncogenes such as activated Ras and Src, autocrine growth factor loops, and local hypoxia (9,35,36). Glut-1 expression appears to reflect the biologic behavior of cancer cells because the expression of Glut-1 is regulated by conditions that are related to cell proliferation, differentiation, and nutritional starvation. The results of overexpressing Glut-1 are consistent with increased glucose uptake, increased adaptive changes to glycolytic metabolism, and increased cellular proliferation of cancer cells. For example, overexpression of Glut-1 was associated with increased uptake of 2-fluorodeoxyglucose as measured by positron emission tomography of pancreatic cancer cells (37). Younes et al. (38) reported that Glut-1 expression is increased in breast carcinomas with higher histologic grades and higher proliferative activity. However, the direct mechanism by which Glut-1 expression is associated with poor prognosis has not been clearly understood.

In this study, we analyzed expression of Glut-1 and MMP-2 in eight cancer cell lines and 80 cancer specimens. All eight cancer cell lines coexpressed Glut-1 and MMP-2, albeit to various levels. Although the reason for the variability in expression levels is not clear, it may be a result of the difference in the sensitivity of the MMP-2 regulatory machinery induced by Glut-1 in various cancer cells. However, we cannot exclude the possibility that other mechanisms in addition to Glut-1 regulation are also involved in the regulation of MMP-2 expression in A172, MCF7, MDA-MB-231, and MKN45 cells. As summarized in Table 1Go, there was a statistically significantly frequent coexpression of Glut-1 and MMP-2 in 80 human tumor tissues.

To our knowledge, this is the first report to show a statistically significant association between Glut-1 and MMP-2 expression. It is important to note that the relationship between Glut-1 and MMP-2 is independent of available glucose because three different concentrations of glucose did not alter Glut-1 or MMP-2 mRNA expression (Fig. 7Go). The overexpression of MMP-2 that results from overexpression of Glut-1 may contribute to neoplastic invasiveness. This finding is consistent with the fact that Glut-1 overexpression is strongly associated with increased tumor cell invasion, increased metastasis, and poor clinical prognosis (914). Thus, the data further support our idea that Glut-1 modulates MMP-2 expression, which could promote tumor cell invasion. Because MMP-2 is closely associated with neoplastic invasiveness, regulation of MMP-2 expression or activity may be a possible target for therapeutic intervention. In this study, loss of Glut-1 expression suppressed MMP-2 expression in invasive human rhabdomyosarcoma and glioblastoma cell lines, with a concomitant suppression of cell invasion. Because a high frequency of human cancers coexpressed Glut-1 and MMP-2 (Table 1Go), suppression of Glut-1 expression may be a rational approach to suppressing the metastasis of various human cancers that overexpress Glut-1.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Supported by a grant from the Ministry of Education, Science, Sports, and Culture of Japan.

We are grateful to Satoko Yamashita for her excellent technical assistance.


    REFERENCES
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 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Manuscript received December 4, 2001; revised May 3, 2002; accepted May 22, 2002.


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