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First published online 26 April 2005
doi: 10.1242/jcs.02334

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Breast cancer cells induce stromal fibroblasts to express MMP-9 via secretion of TNF- and TGF-ß

¹ Laboratory of Cell Regulation and Carcinogenesis, CCR, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
² Laboratory of Receptor Biology and Gene Expression, CCR, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
³ Cancer and Cell Biology Branch, CCR, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (54K): [in a new window]   Fig. 1. Tumor cells induce MMP-9 expression in fibroblasts. (A) Morphology of a coculture of DFs and CA1a cells. Spindle-shaped fibroblasts (S) surround tumor-cell islets (T). Bar, 200 µm (B) (a) An additional MMP which runs slightly higher on SDS-PAGE (as shown by gelatin zymography) than human MMP-9 was induced in cocultures. (b) Activity of the induced gelatinolytic enzyme was blocked by the addition of 10 mM EDTA to the developing buffer during gelatin zymography. Tissue-culture supernatants used in (a) were taken from cocultures, for (b) they were taken from cultures grown in a Millipore system that separates CA1a cells from DFs by a membrane, which allows exchange of signals but prevents cell-cell contact. (C) In DFs, the gelatinolytic activity was induced with medium conditioned by CA1A cells. Media, conditioned for 4 days by DFs or CA1A cells, were used to stimulate homotypic cultures of DFs or CA1A cells (lanes labeled CA1a and DF). As a negative control, cells were stimulated with media that had been incubated for 4 days in an empty dish to distinguish between the influence of CA1A cells and DFs on the media and the age of the medium (4-day incubation at 37°C) (lanes labeled medium). (D) Gelatin zymography of the tissue-culture supernatant and the pellet obtained by immunoprecipitation. The induced enzyme was removed from the supernatant by immunoprecipitation with an anti-MMP9 antibody but not an anti-MMP2 antibody. (E) The gelatinolytic enzyme was not induced in the medium of CA1A and MMP9KO cells cocultured for 4 days, but was induced in cocultures of CA1A and MMP2KO cells and also in cocultures of CA1A cells and the corresponding wild-type fibroblasts MMP9WT or MMP2WT. Homotypic cultures and also medium kept in empty dishes served as controls.

View larger version (99K): [in a new window]   Fig. 2. The main gelatinolytic enzyme activity is localized to the stromal partner in cocultures of fibroblasts and CA1a cells and also in xenograft tumors, and contains significant levels of MMP-9 and MMP-2 activity. (A-C) Fibroblasts were labeled with DiI (red) before plating, fluorescein-labeled gelatin was used as a substrate (green). Nuclei were stained with DAPI (blue). The instrument settings of the microscope were kept constant for each experiment to allow comparison of the collected fluorescence signals of the different cultures. (A) In situ zymography of homotypic cultures and cocultures of DFs and CA1a cells. (B) Cocultures of MMP2KO or MMP9KO with CA1a cells showed significantly less enzyme activity than cocultures employing the corresponding wild-type fibroblasts. (C) Cocultures of human embryonic-lung fibroblasts (WI-38) and CA1a cells showed a growth pattern similar to the one in the heterologous cultures; however, presumably because of the slow growth of WI-38 cells, there were fewer fibroblasts. Gelatin zymography of culture supernatants of 4-day homotypic and heterotypic cultures showed an induction of MMP-9 in the coculture supernatant. (D) In situ zymography with DQ gelatin on serial sections of a xenograft tumor, formed by CA1A cells in nude mice, shows the main gelatinolyic activity (green) of the specimen localized in tissue that is also -smooth muscle actin-positive, thus exhibiting characteristics of stromal tissue. Tumor cells are cytokeratin-positive and show only a low gelatinolytic activity. Cytokeratin and -smooth muscle actin are detected with a secondary FITC-labeled antibody (green) by indirect immunofluorescence; nuclei are stained with DAPI (blue). Bars, 80 µm.

View larger version (44K): [in a new window]   Fig. 3. In fibroblasts, MMP-9 induction by tumor cells depends on the malignancy of the tumor cell line and on the integrity of TGF-ß-, EGFR- and MAPK-signaling. (A) MMP-9 was induced in cocultures of DFs and 10A, At.1k, CA1h or CA1a cells as early as at day 1. The level of its activity depends on the cell-type and also on the age of the culture. (B) Compared with S3WT and CA1a cells cultured together, the expression of MMP-9 is reduced in cocultures of S3KO and CA1a cells. Similar results were obtained for mammary fibroblasts (mS3KO, mS3WT). Notice, MMP-9-forms of a lower molecular weight, suggesting an activation by proteolytic processing in the cocultures. (C) DFs grown to 80% confluence in 24-well plates with DMEM compl. stimulated with CA1a cells (10,000/well) for 24 hours. The culture-supernatant was analyzed by gelatin zymography. Expression of MMP-9 was induced by CA1a cells within 12 hours. (D) Thirty minutes before addition of CA1a cells, DFs were treated with actinomycin D (10 ng/ml) or cycloheximide (10 ng/ml). Both substances blocked the induction of MMP-9 expression, indicating that it depends on an intact RNA- and protein synthesis. Experimental design as in C. (E) Induction of MMP-9 in serum-starved (0% serum) DFs by CA1A cells or CA1a lysate is modulated by several small-molecule inhibitors. DFs were incubated with inhibitors (LY294002, 20 µM; SB431542, 5 µM; manumycin, 5 µM; AG1478, 10 µM; or SB203580, 10 µM) or DMSO (negative control) for 30 minutes, and stimulated for 24 hours with CA1A cells (10,000/well) or lysates of the same number of cells. The culture-supernatant was analyzed by gelatin zymography. Zymograms shown here were optimized to show the effect of inhibitors on MMP-9 induction and show representative results of five to ten experiments. Notice, whereas MMP-9 levels are influenced by stimulation with inhibitors, MMP-2 levels remain constant. This indicates equal growth of fibroblasts, which was confirmed by fibroblast-monolayer staining as described in Materials and Methods. (F) SB431542 inhibits gelatinolysis in cocultures as shown by in-situ zymography. Cultures were treated with SB431542 (5 µM) or SB203580 (10 µM) for a 24-hour period and then again 1 hour before in-situ zymography. Media used for in-situ zymography were supplemented with SB431542 (5 µm) or SB203580 (10 µM). Bar, 80 µm.

View larger version (30K): [in a new window]   Fig. 4. In fibroblasts, TGF-ß, TNF-, EGF and HGF are involved in the induction of MMP-9 by tumor cells. (A) TGF-ß1, TNF- and, to a lesser extent, EGF induce the expression of MMP-9 in serum-starved (0.5% serum) DFs as shown by gelatin zymography (experimental set up as described in Fig. 3). (B) Indirect immunofluorescence and immunohistochemistry of cocultures of DFs and CA1a shows expression of TGF-ß mainly in tumor cells. The highest immunoreactivity for TNF- is observed in CA1a cells, migrating on the fibroblast monolayer (arrow heads). Bars, 32 µm (TGF-ß) and 100 mm (TNF-). (C,D) Neutralizing TNF- or HGF antibody significantly decreases MMP-9 levels induced by CA1a lysate. CA1a lysate and serum-starved (0.5% FCS), 80% confluent DFs were separately preincubated for 30 minutes with the antibodies, before combining and incubating them for an additional 24 hours. Cell-free culture-supernatants were analyzed by gelatin zymography. (E) Neutralizing HGF antibody slightly reduces levels of MMP-9 induced by TGF-ß1 (experimental set-up as in C). (F) MMP-9 induction by TNF- is decreased by neutralizing HGF antibody (experimental set-up as in C). (H) The EGF-receptor inhibitor AG1478 interfers with TGF-ß- but not TNF–induced MMP-9 induction (experimental set up as in Fig. 3E).

View larger version (39K): [in a new window]   Fig. 5. Model of MMP-9 induction in fibroblasts by breast-cancer cells. Tumor-cell-derived TGF-ß and TNF- directly induce expression of MMP-9, which is augmented indirectly by the effects of these cytokines on HGF- and EGF-signaling.