• tenascin-C;
  • TGF-β;
  • matrix metalloproteinase;
  • breast cancer


  1. Top of page
  2. Abstract
  6. Acknowledgements

Tenascin-C (TN-C) and matrix metalloproteinases (MMPs) are highly expressed in cancer tissues and probably promote cell migration during cancer progression. TN-C and MMPs are often co-localized in areas of active tissue remodeling in pathologic conditions, suggesting reciprocal regulation. To investigate whether TN-C regulates MMPs expression in cancer cells, we first exposed mammary cancer cells derived from TN-C-deficient mice to TN-C and examined MMPs expression. TN-C was then compared with fibronectin (FN), laminin (LN), basic fibroblast growth factor (b-FGF) and transforming growth factor-beta1 (TGF-β1). Results of endpoint RT-PCR, quantitative real-time RT-PCR and gelatin zymography demonstrated that TN-C, strongly and dose dependently, upregulates MMP-9 expression in murine mammary cancer cells. TN-C weakly induced MMP-2, MMP-3 and MMP-13. FN and LN induced MMP-9 to lesser extents compared with TN-C. b-FGF had no effect on MMP-9 expression. TGF-β1 induced MMP-9 expression in a dose-dependent manner, and this induction was significantly enhanced by addition of TN-C. TN-C and TGF-β1 also upregulated MMP-9 expression in the human breast cancer cell line MDA-MB-231. Neutralization with specific anti-TGF-β1 antibody showed decreased expression of MMP-9, indicating that TGF-β controls the baseline MMP-9 expression by a direct autocrine mechanism. Under neutralization of TGF-β, addition of TN-C still upregulated MMP-9. Conversely, neutralization of endogenous TN-C (in a TN-C-positive mammary cancer cell line) downregulated TGF-β-induced MMP-9 expression. Thus, TN-C induces MMP-9 expression directly and by collaboration with TGF-β. These findings reveal a novel role of TN-C in breast cancer progression. © 2003 Wiley-Liss, Inc.

Tumor invasion and subsequent development of metastasis are complex processes that involve multiple and coordinated steps: dissolution of basement membranes, modulation of tumor cell adhesion, migration and growth at both the primary and the secondary sites. Tumor cells that produce and/or induce factors that can assist in these steps have an advantage for successful invasion and development of metastasis. Tenascin-C (TN-C), a large 6-armed glycoprotein of the extracellular matrix (ECM), is suspected of playing a significant role in cancer invasion. In a variety of malignant tumor tissues, TN-C is highly expressed by tumor cells themselves and stromal cells such as fibroblasts and endothelial cells.1, 2, 3, 4 Previous histopathologic studies using antibodies and nucleotide probes have demonstrated that higher TN-C expression is associated with tumor progression and an unfavorable outcome of patients.4, 5, 6, 7 In breast cancer, cases with dense TN-C deposition also show a high risk of distant metastasis, local and distant recurrence and adverse patient outcome.5, 8 The progressive nature of tumors producing TN-C may be attributed to the properties of TN-C that promote cell migration and proliferation, as evaluated from in vitro studies.9, 10

Matrix metalloproteinases (MMPs), a class of zinc-dependent enzymes, have been implicated in a proteolytic cascade for ECM degradation. They are collectively capable of degrading all the constituents of the ECM. Like TN-C, MMPs are actively produced by both tumor and stromal cells in cancer tissues including breast cancers, suggesting an involvement in tumor invasion and metastasis.11, 12, 13, 14 MMPs are secreted as proenzymes and require activation for their proteolytic activity by the MMP cascade and serum-derived serine proteinases.15 Genes of most MMPs are inducible. Growth factors and oncogenic transformation are among the effectors.16, 17, 18, 19 Transforming growth factor-β (TGF-β) may be an effector or a suppressor depending on the target cell types.20, 21, 22 However, in advanced cancers, increased expression of MMPs is considered to be regulated by the cancer tissue microenvironment in which the TGF-β system plays an important role.23

Thus, both TN-C and MMPs are upregulated in breast cancer tissues. It is tempting to consider that TN-C could induce MMPs expression and activity. Supporting this hypothesis, upregulation of MMPs was observed in synovial fibroblasts plated on a mixed substrate of fibronectin (FN) and TN-C but not on FN alone.24 In addition, during development and remodeling of mammary tissues, TN-C deposition coincides with the expression of MMPs.25, 26 To explore the regulatory pathways of MMPs in breast cancer tissues, we examined the effects of TN-C on the induction of MMPs in murine and human mammary cancer cells. In our study, we found that TN-C induces expression of some MMPs, especially MMP-9, in mammary cancer cells and that TN-C could enhance effects of TGF-β on MMP-9 induction, indicating a new function of TN-C in cancer progression.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Animals, cells and cell culture

TN-C-deficient mice originally generated by Saga et al.27 were backcrossed with GRS/A mice for more than 12 generations and were congenic. Mammary cancer cell lines were established from a murine mammary tumor (MMT) spontaneously developed in the TN-C-deficient homozygote, then cloned and named 5E, 7E and 1C. Other mammary cancer cell lines named 4B8 and 6E7 were cloned from MMT of a wild-type GRS/A mouse.28 Human breast cancer cell line MDA-MB-231 was obtained from the American Type Culture Collection. These cell lines were maintained in Iscove's modified Dulbecco's medium (IMDM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, Gaithersburg, MD) at 37°C under a 5% CO2 atmosphere. For induction experiments, cells were plated in Falcon 6-well plates (Becton Dickinson, Franklin Lakes, NJ) at a concentration of 5 × 105 cells/well in 2 ml of 0.1% FBS IMDM and incubated for 48 hr. The medium was then replaced with 2 ml of medium containing the substance under investigation for MMP-9 induction: Basic fibroblast growth factor (b-FGF; 50 ng/ml; Wako Life Science, Osaka, Japan), TGF-β1 (5 ng/ml; Boehringer Mannheim, Mannheim, Germany), FN (20 μg/ml, Gibco-BRL, Gaithersburg, MD), Laminin (LN; 20 μg/ml; Gibco-BRL) and TN-C (10 μg/ml). The incubation was continued for another 24 hr. TN-C was isolated and purified from conditioned medium of the human glioma cell line U-251MG.9

For cultures on ECM substrates, the wells were coated as previously reported.24 Freshly trypsinized cells were washed 3 times in a 0.1% FBS IMDM medium and plated for 24 hr in wells coated with FN (30 μg/ml), TN-C (10 μg/ml) or FN and TN-C substrate mixture.

Conditioned media for gelatin zymography were collected, spun at 2,000g for 5 min at 4°C to remove cellular debris and used immediately or stored at −80°C. Total RNA was isolated from the cells as described below.

Gelatin zymography

The secreted metalloproteinases were detected by zymography as previously reported.29 Briefly, 20 μl of conditioned medium was mixed with an equal volume of sample buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 0.2% bromophenol blue). Fifteen microliters of this mixture was then applied to a 10% SDS-polyacrylamide gel containing 1 mg/ml of gelatin. After electrophoresis the gels were washed twice in 100 mM NaCl, 2.5% Triton X-100, 50 mM Tris-HCl buffer (pH 7.5) and incubated for 24 hr at 37°C in 5 mM CaCl2, 50 mM Tris-HCl buffer (pH 8.0). The gels were then stained in 0.1% Coomassie Brilliant Blue R250 in 40% methanol and 10% acetic acid for 1 hr and destained in the same solution without the dye. Gelatinolytic enzymes were detected as clear bands against the blue background of the gels. Gels were scanned in a digital scanner, and densitometric measurement was performed with NIH Image (version 1.56) software.

Analysis of MMP mRNAs using reverse transcription polymerase chain reaction

Total RNAs were extracted with Isogen (Wako Life Science, Osaka, Japan), a premixed RNA isolation reagent based on the acid guanidium thiocyanate-phenol-chloroform extraction method, following the supplied protocol. Reverse transcription (RT) polymerase chain reaction (PCR) (total RNA 1 μg/reaction) was performed using First Strand cDNA Synthesis Kit and oligo-dT primers (Boehringer Mannheim). Aliquots of cDNA were subjected to PCR using the primers shown in Table I for 27 cycles. An initial denaturation step was employed at 95°C for 10 min, followed by repeat cycles of 94°C for 30 sec (denaturation), 55°C for 30 sec (annealing) and 72°C for 1 min (elongation). The samples were then incubated at 72°C for 7 min and stored at 4°C. The amplified products were analyzed by electrophoresis in 2% agarose gels, stained with ethidium bromide (0.1 μg/ml) and detected by ultraviolet light. The images were captured on a digital camera and stored on a disk using Documentation System (Amersham Pharmacia Biotech, Buckingamshire, UK). The bands were quantitated by NIH Image (version 1.56) software.

Table I. Oligonucleotide Primers for RT-PCR Amplification and Detection
mRNAPrimer sequence Product size (bp)Position on cDNA
  1. MMP-, matrix metalloproteinase-; TGF-β1, transforming growth factor-beta 1.

 Backward5′-CAGTACCAGTGTCAGTATCAGC-3′ 1415–1394
 Backward5′-AAAACCCTCTTGGTCTGCGG-3′ 1793–1774
 Backward5′-ATCTCATTACCAACACCACTCC-3′ 1882–1861
 Backward5′-AGCTTTTCACACATCAGTAAGC-3′ 2526–2505
 Backward5′-GAACGCTGGCAGTAAAGCAGTC-3′ 1976–1955

Quantitative real-time RT-PCR

Quantitative real-time RT-PCR was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems/Perkin-Elmer, Foster City, CA). The TaqMan PCR Universal Master Mix reagent, MicroAmp optical tubes and MicroAmp caps were also from Applied Biosystems. The PCR primers and the TaqMan fluorogenic probes were designed using the Primer Express software program (Applied Biosystems) and ordered from Takara Biomedicals (Shiga, Japan). Their sequences are shown in Table II. The sequences of primers and probes for human MMP-9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been previously reported.30 The standard curve used was based on serial dilutions of one cDNA sample from RT products ranging from 10−1 to 10−4. Amplification mixes (50 μl) contained the sample cDNA (1 μl, around 10 ng), 200 nM each primer (1 μl), 40 nM probe (0.2 μl), TaqMan PCR Universal Master Mix reagent (25 μl) and sterile double distilled water (21.8 μl). The thermal cycling conditions comprised 2 min at 50°C, 10 min at 95°C and 40 cycles at 95°C for 15 sec and 60°C for 1 min. GAPDH was used as the housekeeping gene.

Table II. Oligonucleotide Primers and Probes Used for Real-Time PCR Amplification and Detection
mRNAPrimer and probe sequences Product size (bp)Position on cDNA
  1. MMP-9, matrix metalloproteinase-9; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

MMP-9Forward primer5′-CGTGTCTGGAGATTCGACTTGA-3′871945–1967
 Backward primer5′-TGGAAGATGTCGTGTGAGTTCC-3′ 2051–2030
GAPDHForward primer5′-ATGGCCTTCCGTGTTCCTAC-3′85715–734
 Backward primer5′-TGATGTCATCATACTTGGCAGG-3′ 799–778


The levels of TGF-β1 in conditioned media of TN-C-null cells (treated or not with 10 μg/ml of TN-C) were assayed using a kit obtained from R&D Systems (Duoset®, Minneapolis, MN), according to the supplied protocol.

Neutralization of TGF-β and TN-C bioactivities

For TGF-β neutralization, TN-C-null cells were treated with 50 μg/ml of monoclonal anti-TGF-β antibody (clone 1D11, R&D Systems, Minneapolis, MN) or 1 μg/ml of recombinant human latent associated peptides (LAP) (R&D Systems). The antibody and LAP are capable of neutralizing TGF-β1, β2 and β3. In another set of experiments TN-C (1 μg/ml) was added to the medium at the time of TGF-β neutralization. For TN-C neutralization, wild-type TN-C-positive cells 6E7 were treated with 10 μg/ml of a rabbit polyclonal, affinity purified, human anti-TN-C antibody31 or a control normal rabbit IgG, in the presence or absence of TGF-β1 (5 ng/ml).

Statistical analysis

The results are expressed as the mean ± the standard deviation (SD) of at least 3 experiments and comparisons are based on the unpaired t-test (Student's t-test). A p-value < 0.05 was considered significant.


  1. Top of page
  2. Abstract
  6. Acknowledgements

TN-C induces expression of MMPs, especially MMP-9, in murine mammary cancer cell lines derived from TN-C-deficient mice

Initially we examined the expression of MMP-2, MMP-3, MMP-9, MMP-11, MMP-13 and MMP-14 by RT-PCR in TN-C-null cancer cells grown under serum starvation conditions with or without the addition of 10 μg/ml of TN-C. As shown in Figure 1, the addition of TN-C resulted in significant upregulation of MMP-9 and apparent upregulation of MMP-2, MMP-3 and MMP-13 mRNAs. MMP-11 and MMP-14 were almost not affected by the addition of TN-C. Among MMPs expressed by mammary cancer cells, it was previously reported that MMP-9 is upregulated coincidentally with malignant transformation and tumor progression.32, 33 With this in mind, we decided to further test MMP-9 upregulation in different TN-C-null cancer cell lines using quantitative real-time PCR. As shown in Figure 2a, 2 cell lines, 5E and 1C, exhibited increased expression of MMP-9 mRNA by TNC, whereas 7E did show constitutive overexpression of MMP-9. To confirm this finding at the protein level, gelatin zymography was performed on the culture medium of all 3 cell lines. A prominent gelatinolytic activity consistent with the latent form of murine MMP-9 (105 kDa) was observed in all cell lines after treatment with 10 μg/ml TN-C (Fig. 2b). Cell line 7E secreted a noticeable amount of MMP-9 even without TN-C treatment. The 7E cells might have undergone a phenotypical change possibly to compensate for the TN-C-null condition. This cell line was withdrawn from subsequent experiments in our study.

thumbnail image

Figure 1. Expression of MMPs in TN-C-null cancer cells after TN-C treatment. Serum-starved cells were incubated with or without TN-C (10 μg/ml) for 24 hr. Total RNA was isolated from the cells and analyzed by RT-PCR. Sequences in cDNA were amplified with specific primers for MMP-2, MMP-3, MMP-9, MMP-11, MMP-13, MMP-14 and β-Actin cDNA. (a) The products were separated on 2% agarose gels and stained with ethidium bromide. (b) Densitometric measurements of data shown in (a) are expressed as the ratio of the level of each MMP mRNA to the level of β-Actin mRNA present in that sample.

Download figure to PowerPoint

thumbnail image

Figure 2. Expression of MMP-9 in TN-C-null cancer cells. Serum-starved cells from clone 5E, 1C and 7E were incubated with or without TN-C (10 μg/ml) for 24 hr. (a) Total RNA was isolated from the cells, subjected to reverse transcription and analyzed by real-time PCR. The results are expressed as the ratio of the level of MMP-9 mRNA to the level of GAPDH mRNA present in untreated cells of the respective clones. (b) The results of gelatin zymography of the conditioned media from the same experiment.

Download figure to PowerPoint

ECM proteins induce MMP-9 in murine mammary cancer cells

We compared the effect of TN-C on the induction of MMP-9 to those of other ECM glycoproteins, FN and LN. The addition of soluble FN and LN increased MMP-9 expression in the cancer cells. Whereas TN-C increased MMP-9 mRNA more than 5-fold, FN showed a 4-fold increase and LN showed a 3-fold increase. These differences were statistically significant (Fig. 3a). Induction of MMP-9 mRNA was also translated by a proportional increase in the gelatinolytic activities in the conditioned medium (data not shown). Next, although we examined the synergistic effects in a combination of soluble TN-C and FN or LN, no synergy was observed. It is well known that cells in culture react differently to ECM proteins, depending on whether the proteins are soluble or coated to a surface. When cells were cultured on substrates of TN-C, FN or a mixture of FN and TN-C, the highest expression of MMP-9 was observed on TN-C alone. A mixture of FN and TN had a higher expression than FN alone (Fig. 3b and c) but lower than TN-C alone.

thumbnail image

Figure 3. Induction of MMP-9 by TN-C, FN and LN in TN-C-null cancer cells. (a) Soluble ECM proteins were added to serum-starved cells, and MMP-9 mRNA expression was analyzed by real-time PCR. Lane 1, control; lane 2, TN-C 10 μg/ml; lane 3, FN 20 μg/ml; lane 4, LN 20 μg/ml; lane 5, TN 1 μg/ml; lane 6, FN 2 μg/ml; lane 7, LN 2 μg/ml; lane 8, TN-C 1 μg/ml + FN 2 μg/ml; lane 9, TN 1 μg/ml + LN 2 μg/ml. The results are expressed as x-fold induction of MMP-9 mRNA in each sample compared with the control. (b) Cells grown in a 10% FCS containing medium were trypsinized, washed 3 times in a serum-free medium and plated in wells coated with FN (30 μg/ml), TN-C (10 μg/ml) or a mixed substratum of FN and TN-C for 24 hr. The results of real-time PCR are expressed as the ratio of the level MMP-9 mRNA to the level of GAPDH mRNA present in the untreated well. The error bars indicate the SD of 3 experiments with 3 different RNA samples. Induction by TN-C is significantly stronger than that by FN and LN (unpaired t-test, p < 0.05). Cont., control. (c) A representative zymogram of conditioned media from 1 experiment in (b).

Download figure to PowerPoint

TN-C enhances the effect of TGF-β1 on MMP-9 induction in mammary cancer cells

TN-C was compared with b-FGF and TGF-β1. b-FGF was previously reported to induce MMP-9 in cancer cells.34, 35 However, in mammary cancer cells used in our study, it had no effect on the expression of MMP-9 mRNA. In contrast, 5 ng/ml of TGF-β1 induced a 4-fold increase, not significantly different from TN-C (Fig. 4a). The gelatinolytic activity in the conditioned medium was also increased with no apparent difference between TGF-β1 and TN-C (Fig. 4β). We then studied the effect of TN-C and TGF-β1 concentrations on MMP-9 upregulation and found that both TN-C and TGF-β1 increased MMP-9 expression in a dose-dependent manner (Fig. 5a). In addition, using a human breast cancer cell line, MDA-MB-231, we confirmed the effect of TN-C on MMP-9 induction in human cells. As shown in Figure 5b, treatment of human cells with TN-C and TGF-β1 increased the MMP-9 mRNA expression 7-fold and 5-fold, respectively. Because TGF-β1 is a known inducer of ECM proteins including TN-C,36 we tested TN-C induction by TGF-β1 using real-time PCR in wild-type murine mammary cancer cells (4B8 and 6E7). Treatment of the wild-type cells with TGF-β1 increased the expression of TN-C mRNA and MMP-9 mRNA up to 7-fold and 53-fold, respectively (data not shown). Having found that TN-C induces MMP-9 and that both TN-C and MMP-9 are induced by TGF-β1, we decided to compare the TN-C and TGF-β1 effects on other MMPs expression in TN-C-null and wild-type cancer cells. We found that MMP-9 was the only MMP induced by both TN-C and TGF-β1 in all the cell lines examined (Table III). To further clarify this close association between TN-C, TGF-β1 and MMP-9, we examined whether TN-C could modulate the effects of TGF-β1 on MMP-9 expression. When TN-C-null cells were treated with lower but effective concentrations of TGF-β1 (0.1 ng/ml) and TN-C (1 μg/ml), a synergistic effect on MMP-9 induction by TN-C and TGF-β1 was observed (Fig. 6). Next, the endogenous expression of TGF-β1 was tested by RT-PCR and ELISA. By RT-PCR, all the cell lines showed a constitutive expression of TGF-β1 that was not apparently affected by the presence or absence of TN-C treatment (data not shown). In the ELISA assay after activation by acidic treatment, the conditioned media of TN-C-null cells treated with or without 10 μg/ml of TN-C also showed significant levels of total TGF-β1 (1.27 and 1.67 ng/ml respectively, latent TGF-β1 included). Therefore, using a neutralizing antibody of TGF-β and latent associated polypeptide (LAP), we tried to minimize the endogenous TGF-β1's effect and clarify whether the TN-C effect on MMP-9 expression is direct or indirect by enhancing an autocrine effect of TGF-β1 secreted by the cancer cells. Treatment of TN-C-null cells with the monoclonal antibody significantly decreased the expression of MMP-9 mRNA from the baseline level and after TN-C treatment. The treatment with LAP showed the same tendency, although the difference was not significant. In the condition where TGF-β activity was neutralized, the addition of TN-C still showed a 3-fold increase in MMP-9 mRNA (Fig. 7a). Furthermore, neutralization of endogenously secreted TN-C by wild-type cells with a polyclonal anti-TN-C antibody significantly reduced the TGF-β1-induced upregulation of MMP-9 (Fig. 7b), although the upregulation of TN-C mRNA by TGF-β1 was not affected (data not shown). Thus, MMP-9 expression in breast cancer cells could be directly regulated by TGF-β1 and TN-C, and the TGF-β1 effect on MMP-9 expression is evidently enhanced by the addition of TN-C.

thumbnail image

Figure 4. TN-C and TGF-β1 induce MMP-9 in TN-C-null cancer cells. Serum-starved cells were treated with TN-C (10 -μg/ml), TGF-β1 (5 ng/ml), b-FGF (50 ng/ml) or nothing (control) for 24 hr. (a) Total RNA extracted from the cells was reverse transcribed and analyzed by real-time PCR. Results are expressed as x-fold induction compared with untreated cells. The error bars indicate the SD of 3 experiments with 3 different RNA samples. The difference between TN-C and TGF-β1 was not statistically significant (unpaired t-test). (b) A representative zymogram of conditioned mediums from one experiment in (a). Cont., control.

Download figure to PowerPoint

thumbnail image

Figure 5. MMP-9 induction by TN-C and TGF-β1 is dose-dependent and is also observed in human breast cancer cells. (a) Serum-starved TN-C-null cancer cells were incubated with increasing doses of TN-C or TGF-β1 for 24 hr. The results of real-time PCR are expressed as x-fold induction compared with untreated cells. (b) Serum-starved MDA-MB-231 cancer cells were incubated with TN-C (10 μg/ml), TGF-β1 (5 ng/ml) or nothing (control) for 24 hr. The results are expressed as the ratio of the level MMP-9 mRNA to the level of GAPDH mRNA present in the untreated cells. The error bars indicate the SD of at least 3 experiments with 3 different RNA samples.

Download figure to PowerPoint

Table III. Induction of MMPs by TN-C and TGF-β1 in Murine Mammary Cancer Cells
  • MMP-, matrix metalloproteinase-; TN-C, tenascin-C (10 μg/ml); TGF-β1, transforming growth factor-beta 1 (5 ng/ml).–+, induction present (at least a doubling of MMP-9 level compared with untreated cells); −, induction absent.

  • 1

    TN-C-null cells.

  • 2

    Wild-type cells.

thumbnail image

Figure 6. Combined effects of TN-C and TGF-β1 on MMP-9 expression in TN-C-null cancer cells. Serum-starved cells were incubated with TN-C (1 μg/ml) in the presence or absence of TGF-β1 (0.1 ng/ml) for 24 hr. The results of real-time PCR are expressed as x-fold induction compared with untreated cells. The error bars indicate the SD of 3 experiments with 3 different RNA samples. The difference between TN-C alone and TN-C combined with TGF-β1 is significant (unpaired t-test, p < 0.05).

Download figure to PowerPoint

thumbnail image

Figure 7. Neutralization of endogenous TGF-β and TN-C. (a) TN-C-null cancer cells were treated with 50 μg/ml of monoclonal anti-TGF-β antibody or 1 μg/ml of recombinant human LAP (TGF-β1), with or without the addition of TN-C (1 μg/ml). (b) TN-C-positive cancer cells (6E7) were treated with 10 μg/ml of a polyclonal anti-TN-C antibody or 10 υg/ml of control normal rabbit IgG, with or without the addition of TGF-β1 (5 ng/ml). The results of real-time PCR are expressed as the ratio of the level MMP-9 mRNA to the level of GAPDH mRNA present in the untreated cells. The error bars indicate the SD of 3 experiments with 3 different RNA samples. Inhibitions of MMP-9 expression by anti-TGF-β and anti-TN-C were significant (unpaired t-test, p < 0.05).

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  6. Acknowledgements

Our present study of MMPs expression in mammary cancer cells has demonstrated that TN-C upregulates MMP-9 expression in mammary cancer cells and enhances TGF-β-induced MMP-9 expression. TN-C, TGF-β and MMP-9 are all known to be upregulated in breast cancer tissues and expressed in both the stromal and the epithelial compartments of tumors.1, 2, 5, 11, 31, 37–40 TGF-β is known to play an important role in tumor progression through selection of malignant cells that escape TGF-β-induced cell growth inhibition and through the upregulation of ECM and protease expression by cancer cells.41 In agreement, our results also show that the mammary cancer cells used in our study constitutively express TGF-β1 mRNA and that the addition of TGF-β1 upregulates the TN-C and MMP-9 mRNAs expression in these cells. Breast cancer invasion is accompanied by the loss of basement membrane23 and the accumulation of TN-C at the invasion front.2, 8 MMP-9 is known, on the other hand, to participate in the proteolysis of basement membrane materials.15 Interestingly, TN-C, which is not a permanent component of the basement membrane, is known to resist MMP-9 proteolytic activity.42, 43 TN-C deposition and MMPs upregulation are coincidentally shown during development and in remodeling tissues.25, 26, 44, 45 By showing that TN-C upregulates MMP-9 in mammary cancer cells, our study confirms and extends the findings of previous reports that placed TN-C upstream in the regulation of some MMPs, including MMP-9, in the synovial fibroblasts and interstitial cells of heart valves.24, 45 By comparing TN-C to FN and LN, we have shown that TN-C is the most determinant ECM factor for MMP-9 upregulation resulting from cell-ECM interactions studied so far. The results of our experiments with soluble and substrate-bound TN-C, FN and LN indicate that MMP-9 upregulation is strongly dependent on TN-C concentration when mammary cancer cells are exposed to mixtures of ECM proteins. Thus, in addition to the previously reported role of TN-C in cancer cell growth and migration,9, 10 the accumulation of TN-C at the focal site of breast cancer invasion probably also contributes to the degradation of the basement membrane by focally upregulating MMP-9 expression.

Although we have observed an induction of MMP-2, MMP-3, MMP-9 and MMP-13 after TN-C treatment of the mammary cancer cells used in our study, our results concentrate on MMP-9 because its upregulation was the most evident. Other studies have also shown particular expression patterns of MMP-9 among the various MMPs described in breast cancers.11, 46, 47 For instance, the MMP-9 gene seems to be inducible in breast cancer cells in culture, whereas the MMP-2 gene seems to be constitutively expressed.32 The MMP-9 distribution in breast cancer tissues is more focal and restricted to some breast cancers, whereas MMP-2 expression tends to be diffuse and ubiquitous.12, 48 Furthermore, several lines of evidence implicate MMP-9 in breast cancer progression.17, 33, 49

Our data showing that TGF-β upregulates MMP-9 in mammary cancer cells are in agreement with a previous report that showed induction in human bone-metastasizing breast cancer cells.22 TGF-β has also been reported to induce MMP-2 in fibroblasts, and MMP-9 in colon and prostatic cancer cells.20, 21, 50, 51 Considering that TGF-β and TN-C are expressed together in the tumor microenvironment and that both induce MMP-9, we investigated whether their effects were synergistic on MMP-9 induction. We showed that TN-C enhances the effects of TGF-β on MMP-9 expression. By neutralizing constitutively expressed TGF-β with a monoclonal antibody, we have shown a specific downregulation of MMP-9 mRNA. Other MMPs were not affected on real-time PCR (data not shown). This strongly suggests that TGF-β plays a primary role in the control of baseline MMP-9 mRNA level, via a direct autocrine mechanism. Our data also showed that TN-C was still capable of inducing MMP-9 mRNA, despite the addition to the culture of neutralizing antibodies against TGF-β. In addition, blocking of endogenously secreted TN-C with a polyclonal antibody resulted in the downregulation of TGF-β-induced MMP-9 expression. These findings indicate that TN-C can directly stimulate MMP-9 synthesis and that signaling from separated receptors for TGF-β and TN-C could enhance MMP-9 induction. Because TGF-β also stimulates TN-C expression, secreted TGF-β and TN-C could synergistically activate MMP-9 expression of cancer cells in an autocrine manner, followed by progression of cancer cell invasion and metastasis.

As other inducers of MMP-9 in mammary cells, epidermal growth factor (EGF) and amphiregulin in SKBR-3 cells, insulin-like growth factor I (IGF-I), bcl-2 overexpression in MCF-7 cells and FN fragments (FN120) in normal mammary cells have been reported.52, 53, 54, 55 Recently, interactions between TN-C and other growth factor signaling pathways have been found. Epidermal growth factor-like repeats of TN-C can bind and directly activate epidermal growth factor receptor (EGFR).56 TN-C was also shown to act through the αvβ3 integrin to promote EGFR phosphorylation in vascular smooth muscle cells.44 Thus, further studies are necessary to explore the functional domains and receptors of TN-C and intracellular signaling for the induction of MMP-9 expression in cancer cells. In addition, the TN-C-null cell lines used in our study can be considered useful for the investigation of TN-C functional domains.


  1. Top of page
  2. Abstract
  6. Acknowledgements

We thank Dr. E.C. Gabazza for help with the ELISA assay.


  1. Top of page
  2. Abstract
  6. Acknowledgements
  • 1
    Lightner VA, Marks JR, McCachren SM. Epithelial cells are an important source of tenascin in normal and malignant human breast tissue. Exp Cell Res 1994;210: 17784.
  • 2
    Yoshida T, Matsumoto E, Hanamura N, Kalembeyi I, Katsuta K, Ishihara A, Sakakura T. Co-expression of tenascin and fibronectin in epithelial and stromal cells of benign lesions and ductal carcinomas in the human breast. J Pathol 1997;182: 4218.
  • 3
    Hanamura N, Yoshida T, Matsumoto E, Kawarada Y, Sakakura T. Expression of fibronectin and tenascin-C mRNA by myofibroblasts, vascular cells and epithelial cells in human colon adenomas and carcinomas. Int J Cancer 1997;73: 105.
  • 4
    Jones FS, Jones PL. The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling. Dev Dyn 2000;218: 23559.
  • 5
    Ishihara A, Yoshida T, Tamaki, Sakakura T. Tenascin expression in cancer cells and stroma of human breast cancer and its prognostic significance. Clin Cancer Res 1995;1: 103541.
  • 6
    Jahkola T, Toivonen T, Nordling S, von Smitten K, Virtanen I. Expression of tenascin-C in intraductal carcinoma of human breast: relationship to invasion. Eur J Cancer 1998;34: 168792.
  • 7
    Kressner U, Lindmark G, Tomasini-Johansson B, Bergstrom R, Gerdin B, Pahlman L, Glimelius B. Stromal tenascin distribution as a prognostic marker in colorectal cancer. Br J Cancer 1997;76: 52630.
  • 8
    Jahkola T, Toivonen T, von Smitten K, Nordling S, von Boguslawki K, Haglund C, Nevanlinna H. Tenascin-C expression in the invasion border of early breast cancer: a predictor of local and distant recurrence. Br J Cancer 1998;78: 150713.
  • 9
    Yoshida T, Yoshimura E, Numata H, Sakakura Y, Sakakura T. Involvement of tenscin-c in migration and proliferation of laryngeal cancer cells. Virchows Arch 1999;435: 496500.
  • 10
    Orend G, Chiquet-Ehrismann R. Adhesion modulation by antiadhesive molecules of the extracellular matrix. Exp Cell Res 2000;261: 10410.
  • 11
    Soini Y, Hurskainen T, Hoyhtya M, Oikarinen A, Autio-Harmainen H. 1994. 72 kd and 92 kd type IV collagenase, type IV collagen and laminin mRNAs in breast cancer: a study by in situ hybridization. J Histochem Cytochem 1994;42: 94551.
  • 12
    Heppner KJ, Matrisian LM, Jensen RA, Rodgers WH. Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host response. Am J Pathol 1996;149: 27382.
  • 13
    El-Shabrawi Y, Neda Ardjomand, Radner H, Ardjomand N. MMP-9 is predominantly expressed in epitheliod and not spindle cell uveal melanoma. J Pathol 2001;194: 2016.
  • 14
    Kumaki F, Matsui K, Kawai T, Ozeki Y, Yu Z-X, Ferrans VJ, Travis WD. Expression of matrix metalloproteinases in invasive pulmonary adenocarcinoma with bronchioalveolar component and atypical adenomatous hyperplasia. Am J Pathol 2001;159: 212535.
  • 15
    Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000;18: 113549.
  • 16
    Bond M, Fabunmi RP, Baker AH, Newby AC. Synergetic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B. FEBS Lett 1998;435: 2934.
  • 17
    Lee PH, Hwang J-J, Murphy G, Margot MI. Functional significance of MMP-9 in tumor necrosis factor-induced proliferation and branching morphogenesis of mammary epithelial cells. Endocrinology 2000;141: 376473.
  • 18
    Himelstein BP, Lee EJ, Sato H, Seiki M, Muschel RJ. Transcriptional activation of matrix metalloproteinase-9 gene in H-ras and v-myc transformed rat embryo cell line. Oncogene 1997;14: 19958.
  • 19
    Nagase H, Woessner JF. Matrix metalloproteinases. J Biol Chem 1999;274: 214914.
  • 20
    Overall CM, Wrana JL, Sodek J. Independent regulation of collagenase, 72-kDa progelatinase, and metalloendopeptidase inhibitor expression in human fibroblasts by transforming growth-factor-β. J Biol Chem 1989;264: 18609.
  • 21
    Shimizu S, Nishikawa Y, Kuroda K, Takagi S, Kozaki K, Hyuga S, Saga S, Matsuyama M. Involvement of transforming growth factor β1 in autocrine enhancement of gelatinase B secretion by murine metastatic colon carcinoma cells. Cancer Res 1996;56: 336670.
  • 22
    Duivenvoorden WC, Hirte HW, Singh G. Transforming growth factor β1 acts as an inducer of matrix metalloproteinase expression and activity in human bone-metastasizing cancer cells. Clin Exp Metastasis 1999;17: 2734.
  • 23
    Hagerdon HG, Bachmeier BE, Nerlich AG. Synthesis and degradation of basement membranes and extracellular matrix and their regulation by TGF-β in invasive carcinomas [review]. Intern J Oncol 2001;18: 66981.
  • 24
    Tremble P, Chiquet-Ehrismann R, Werb Z. The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts. Mol Cell Biol 1994;5: 43953.
  • 25
    Talhouk RS, Chin JR, Unemori EN, Werb Z, Bissel MJ. Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture. Development 1991;112: 43949.
  • 26
    Jones PL, Boudreau N, Myers CA, Erickson HP, Bissell MJ. Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells: localization of active regions using recombinant tenascin fragments. J Cell Sci 1995;108: 51927.
  • 27
    Saga Y, Yagi T, Ikawa Y, Sakakura T, Aizawa S. Mice develop normally without tenascin. Genes Devel 1992;6: 182131.
  • 28
    Saga Y, Tsukamoto T, Jing N, Kusakabe M, Sakakura T. Murine tenascin: cDNA cloning, structure and temporal expression of isoforms. Gene 1991;104: 177.
  • 29
    Mori M, Minori K, Shiraishi T, Fujie T, Baba K, Kusumoto H, Haraguchi M, Ueo H, Akiyoshi T. Analysis of MT1-MMP and MMP-2 expression in human gastric cancers. Int J Cancer 1997;74: 31621.
  • 30
    Van Trappen PO, Ryan A, Carroll M, Lecoeur C, Goff L, Gyselman VG, Young BD, Lowe DG, Pepper MS, Shepherd JH, Jacobs IJ. A model for co-expression pattern of genes implicated in angiogenesis and tumour cell invasion in cervical cancer. Br J Cancer 2002;87: 53744.
  • 31
    Kalembeyi I, Yoshida T, Iriyama K, Sakakura T. Analysis of tenascin mRNA expression in the murine mammary gland from embryogenesis to carcinogenesis: an in situ hybridization study. Int J Dev Biol 1997;41: 56973.
  • 32
    Giambernardi TA, Grant GM, Taylor GP, Hay RJ, Maher VM, McCormick JJ, Klebe RJ. Overview of matrix metalloproteinase expression in cultured human cells. Matrix Biol 1998;16: 48396.
  • 33
    Kupferman ME, Fini ME, Muller WJ, Weber R, Cheng Y, Muschel RJ. Matrix metalloproteinase 9 promoter activity is induced coincident with invasion during tumor progression. Am J Pathol 2000;157: 177783.
  • 34
    Andersen K, Maelandsmo GM, Hovig E, Fodstad O, Loennechen T, Windberg JO. Interleukin-1 alpha and basic fibroblast growth factor induction of matrix metalloproteinases and their inhibitors in osteosarcoma cells is modulated by the metastasis associated protein CAPL. Anticancer Res 1998;18: 3299303.
  • 35
    Hazan RB, Philips GR, Qiao RF, Norton L, Aaronson SA. Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion and metastasis. J Cell Biol 2000;148: 77990.
  • 36
    Chiquet-Ehrismann R, Kalla P, Pearson CA. Participation of tenascin and transforming growth factor-beta in reciprocal epithelial-mesenchymal interactions of MCF7 cells and fibroblasts. Cancer Res 1989;49: 43225.
  • 37
    Mackie EJ, Chiquet-Ehrismann R, Pearson CA, Inaguma Y, Taya K, Kawarada Y, Sakakura T. Tenascin is a stromal marker of epithelial malignancy in the mammary gland. Proc Natl Acad Sci USA 1987;84: 46215.
  • 38
    Howeedy AA, Virtanen I, Laitinen L, Gould MS, Koukoulis GK, Gould VE. Differential distribution of tenascin in the normal, hyperplastic and neoplastic breast. Lab Invest 1990;63: 798806.
  • 39
    Gorsch SM, Memoli VA, Stukel TA, Gold Li, Arrick BA. Immunohistochemical staining for transforming growth factor beta1 associates with disease progression in human breast cancer. Cancer Res 1992;52: 694952.
  • 40
    Lebeau A, Nerlich AG, Sauer U, Lichtinghagen R, Lohrs U. Tissue distribution of major matrix metalloproteinases and their transcripts in human breast carcinomas. Anticancer Res 1999;19: 425764.
  • 41
    Derynck R, Akhurst RJ, Balmain A. TGF-β signaling in tumor suppression and cancer progression. Nat Genet 2001;29: 11729.
  • 42
    Imai K, Kusakabe M, Sakakura T, Nakanishi I, Okada Y. Susceptibility of tenascin to degradation by matrix metalloproteinases and serine proteinases. FEBS Lett 1994;352: 2168.
  • 43
    Siri A, Knauper V, Veirana, N, Caocci F, Murphy G, Zardi L. Different susceptibility of small and large human tenascin-C isoforms to degradation by matrix metalloproteinases. J Biol Chem 1995;270: 86504.
  • 44
    Jones PL, Crack J, Rabinovitch M. 1997. Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alfaVbeta3 integrin to promote epidermal growth factor receptor phosphorylation and growth. J Cell Biol 1997;139: 27993.
  • 45
    Jian B, Jones PL, Li Q, Mohler III ER, Schoen FJ, Levy RJ. Matrix metalloproteinase-2 is associated with tenascin-C in calcific aortic stenosis. Am J Pathol 2001;159: 3217.
  • 46
    Remacle AG, Noel A, Duggan C, McDermott E, O'Higgins N, Foidart JM, Duffy MJ. Assay of matrix metalloproteinases types 1, 2, 3 and 9 in breast cancer. Br J Cancer 1998;77: 92631.
  • 47
    Brummer O, Athar S, Riethdorf L, Loning T, Herbst H. Matrix-metalloproteinases 1, 2 and 3 and their tissue inhibitors 1 and 2 in benign and malignant breast lesions: an in situ hybridization study. Virchows Arch 1999;435: 56673.
  • 48
    Jones JL, Glynn P, Walker RA. Expression of MMP-2 and MMP-9, their inhibitors, and the activator MT1-MMP in primary breast carcinomas. J Pathol 1999;189: 1618.
  • 49
    van't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AAM, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002;415: 5305.
  • 50
    Sehgal I, Thompson TC. 1999. Novel regulation of type IV collagenase (matrix metalloproteinase-9 and -2) activities by transforming growth factor-β1 in human prostate cancer cell lines. Mol Biol Cell 1999;10: 40716.
  • 51
    Festuccia C, Angelucci A, Gravina GL, Villanova I, Teti A. Osteoblast-derived TGF-β1 modulates matrix degrading protease expression and activity in prostate cancer cells. Int J Cancer 2000;85: 40715.
  • 52
    Kondapaka SB, Fridman R, Reddy KB. Epidermal growth factor and amphiregulin upregulate matrix metalloproteinase-9 (MMP-9) in human breast cancer cells. Int J Cancer 1997;70: 7226.
  • 53
    Mira E, Manes S, Lacalle RA, Marquez G, Martinez-A C. Insulin-like growth factor I-triggered cell migration and invasion are mediated by matrix metalloproteinase-9. Endocrinology 1999;140: 165764.
  • 54
    Ricca A, Biroccio A, Del Bufalo D, Mackay AR, Santoni A, Cippitelli M. bcl-2 over-expression enhances NF-kB activity and induces mmp-9 transcription in human MCF7ADR breast cancer cells. Int J Cancer 2000;86: 18896.
  • 55
    Schedin P, Strange R, Mitrenga T, Wolfe, Kaeck M. Fibronectin fragments induce MMP activity in mouse mammary epithelial cells: evidence for a role in mammary tissue remodeling. J Cell Sci 2000;113: 795806.
  • 56
    Swindle CS, Tran KT, Johnson TD, Banerjee P, Mayes AN, Griffith L, Wells A. Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor. J Cell Biol 2001;154: 454968.