Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway
Stromelysin-3 (ST-3) is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized.
The present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed.
Using both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts.
These data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour fibroblasts leads to the stimulation of the IGF-1R pathway in carcinoma cells, thus enhancing their proliferative capacity. In addition, further different cellular processes seem to be activated by ST-3, possibly accounting for the dual role of ST-3 in tumour progression and metastasis.
KeywordsBreast Cancer Cell Line Soluble Growth Factor Breast Cancer Associate Gene Estradiol Pellet Mock Transfected Control Cell
Stromelysin-3, MMP: matrix metalloprotease, IGFBP: insulin like growth factor binding protein, ECM: extracellular matrix, EMT: epithelial mesenchymal transition, FISH: Fluorescence in situ hybridisation
The human protein family of matrix metalloproteinases (MMPs) includes at least 25 members that are believed to play a role in the various stages of tumourigenesis [1, 2]. Most of these zinc-dependent endopeptidases cleave more than one extracellular matrix (ECM) component and each ECM component is generally cleaved by more than one MMP. Aside from matrix degradation, MMPs play an important role in releasing growth factors, such as TGF-β or VEGF, or other biologically active factors [3, 4]. The effect of over-expression or inhibition of MMPs in tumours might be complex, as these proteases are able to promote and impede tumour relevant processes such as proliferation, apoptosis, angiogenesis and metastasis .
Stromelysin-3 (ST-3, MMP-11) was originally identified as a breast cancer associated gene, which is over-expressed in more than 90% of invasive breast carcinomas . Recent studies have shown that ST-3 over-expression is found in most human carcinomas . Expression of the gene has also been detected in fibroblasts at the invasion front of tumours in close proximity to carcinoma cells . Furthermore, it has been shown that ST-3 expression is induced in fibroblasts by co-culturing with breast carcinoma cells [8, 9]. Although ST-3 was discovered more than a decade ago and assigned to the family of MMPs due to sequence homologies, no ECM component has yet been identified that is cleaved by full length human ST-3 . However, ST-3 has been found to cleave IGFBP-1, thereby releasing IGF-1 . IGF-1 is known to stimulate proliferation, enhancing survival and migration of cancer cells . Despite a limited knowledge of ST-3 substrates and mode of action, an in vitro study has demonstrated that ST-3 over-expression in MCF-7 cells is capable of increasing cell survival under serum starvation conditions . However, the mechanism of ST-3 activation during tumourigenesis remains unclear. Various in vivo studies reveal a ST-3 specific function in tumourigenesis e.g. ST-3 enhanced tumour take of MCF-7 cells in nude mice when injected subcutaneously . Other studies, performed in an ST-3 knock out mouse model and in human xenograft models of ST-3 over-expressing MCF-7 cells demonstrate that the activation of ST-3 leads to a lower percentage of apoptotic cells in the induced tumours [15, 16]. According to these results, the generation of MMTV-ras transgenic mice in an ST-3 deficient genotype results in the development of less and smaller primary tumours . Interestingly, these mice developed more lung metastases. The question has therefore arisen, whether ST-3 over-expression may cause different functional effects in different stages of tumourigenesis and thereby play a dual role [17, 18], or whether compensation by other MMP family members could account for the observed effect. Indeed, it has been shown that during involution, ST-1 and ST-2 is up-regulated in matrilysin knock out mice, and that matrilysin and ST-2 expression is elevated in ST-1 deficient mice .
In the present study, the influence of ST-3 on cell lines of different stages of breast cancer progression is investigated. To this end, the induction of endogenous ST-3 expression and the effect of ST-3 over-expression on the non-invasive cell line MCF-7 as well as on the highly invasive and dedifferentiated cell line MDA-MB-231 is analysed both in vitro and in vivo. Furthermore, the impact of ST-3 over-expression on the gene expression pattern of breast cancer cells was determined.
Primary fibroblasts were isolated from human breast tumours (kindly provided by Dr. Têtu, Centre Hospitalier Universitaire de Quebec, Canada). MDA-MB-231 cells and primary breast tumour fibroblasts were maintained in DMEM/F12. MCF-7 cells were maintained in RPMI w/o phenol red (Gibco, Germany) supplemented with 0.002 U/ml insulin and 0.1 nM Estradiol (E2). For MDA-MB-468, RPMI 1640 (Gibco, Germany) was used. These media contained 10% FCS. MCF-10A cells were maintained in DMEM/F12 (Gibco, Germany) with 5% heat inactivated horse serum, 20 ng/ml EGF, 100 ng/ml cholera toxin, 0.002 U/ml insulin and 500 ng/ml hydrocortisone. For 3D matrigel cultures, 96 well plates were pre-coated with matrigel (BD Bioscience, 10 mg/ml). 75 μl of a cell/matrigel suspension containing 1 × 103 cells/μl were added to each well. For co-cultures, 1 × 104 fibroblasts were seeded on cover slips. When the cells reached confluence, epithelial cells were seeded on top. At day three, co-cultures were incubated with 10 μg/ml Brefeldin A for 5 hours and investigated by immunocytochemistry.
Immunocytochemistry was carried out as described previously . Briefly, cells grown on cover slips were washed with PBS, fixed with 4% PFA and permeabilized in 0.2% Triton-X100/PBS. Cover slips were incubated with the primary and, after washing, with the secondary antibodies for 20 min at RT. For immunohistochemical staining, paraffin sections were dewaxed and dehydrated by graded ethanol. After blocking, sections were incubated with the primary and secondary antibodies. As primary antibodies goat α-(human vimentin) (1:30, Santa Cruz, Germany), rabbit α-(human cytokeratin) (1:100, Novocastra, Newcastle) and mouse α-(human ST-3) (1:50, Clone SL3.05, Neomarkers, CA) were used. Secondary antibodies were donkey-α-(mouse IgG) Rhodamin Red (1:100, Dianova, Germany) and donkey α-(goat IgG)Cy5 (1:400, Dianova, Germany). Stainings were performed as triple stainings on the same section. Samples were examined using a Leica TCS SL confocal microscope (Leica, Germany).
Generation of ST-3 over-expressing clones
ST-3 cDNA was amplified by performing a PCR and cloning into the mammalian expression vector pcDNA3.1V5HisTOPO (Invitrogen, Karlsruhe, Germany). Expression of ST-3 protein was confirmed by in vitro transcription/translation in reticulocyte lysate (Promega). The ST-3 or empty expression plasmid was transfected into MCF-7 and MDA-MB-231 cells. 6 μl Fugene (Boehringer, Mannheim, Germany) was mixed with 196 μl Optimem (Gibco, Karlsruhe, Germany) and incubated for 5 min at room temperature. After supplementation with 2 μg DNA and a further incubation period of 15 min, this mixture was applied to a 60% confluent 6-well plate. On the following day, transfected cells were seeded at a density of 4000 cells/cm2 and selected by supplementation of media with G418 (750 μg/ml for MDA-MB-231 and 600 μg/ml for MCF-7). Transfection efficiencies were determined using a β-Gal plasmid and were in the range of 10–20%. Three independently derived and randomly chosen cell clones were isolated.
Cell culture supernatant containing 0.1% FCS was collected after 48 hours of culture and concentrated 20 times by Biomax 5 k filter (Fa. Millipore, MA). Western blotting was carried out according to manufacturer's instructions utilizing the Novex gel system (Invitrogen, Germany) and 4–12% gradient SDS-PAGE. 12 μl of the concentrated supernatants were applied and equal protein loading was confirmed by Ponceau staining after transfer onto nitrocellulose membranes. Immunoreactive proteins were detected by secondary antibody binding (donkey α-(mouse IgG)) conjugated to horseradish peroxidase, (1:50.000, Amersham Bioscience, Germany) followed by incubation with ECL-Plus (0.1 ml/cm2, Amersham Biosciences, Germany). The primary antibody, mouse anti-ST-3 (Clone SL3.05, Neomarkers, CA) was used at a dilution of 1:300.
Cells of either three ST-3 or three mock clones were detached by trypsin, counted and mixed in equal parts. For each well, a total number of 4.5 × 104 cells (i.e. 1.5 × 104 cells per clone) were mixed with 75 μl matrigel and applied to matrigel coated 96-well plates. Gels were allowed to solidify for 30 min at 37°C before supplementation with 100 μl of cell culture media. After 7 days of 3D culture, cell numbers were determined by means of a MTS assay (Fa. Promega, Germany). The applicability of the MTS test was determined beforehand by confirming the linearity of measurement of known cell numbers in 3D cultures (data not shown). All assays were conducted at least 3 times with triplicates.
In vivotumourigenesis assay
8–12 week-old female immunocompromised NMRI nude/nude mice were obtained from Harlan Winkelmann (Borchem, Germany). 2 × 106 cells consisting of equal parts of either three ST-3 or mock clones were mixed with 50 μl matrigel and injected into the mammary fat pad. Growth factor depleted matrigel was prepared as described by Taub et al . Tumour take and tumour growth was determined by the measurement of tumour burdens estimated from calliper measurements twice a week. The minimum tumour size was defined as 3 × 3 mm. Animals were sacrificed when tumours were larger than 1000 mm3. Animals receiving MCF-7 tumour cells were supplemented with 17-beta Estradiol pellets (IRA, FI) that were implanted subcutaneously one week in advance. Animals were sacrificed according to the guidelines of the "Deutsches Tierschutzgesetz".
Microarray gene expression analysis
Cells were grown for 3 days in 3D matrigel cultures as described above. RNA-isolation was carried out using TriFast (peqLab, Erlangen, Germany). The integrity of resuspended total RNA was measured on a Bioanalyzer gel (Agilent Technologies). For the microarray experiment, 500 ng of total RNA was reverse transcribed with an oligo-(dT)-T7 promoter primer and Moloney murine leukemia virus-reverse transcriptase (MMLV-RT) to synthesize first and second strands of cDNA. Fluorescent antisense cRNA was synthesized with simultaneous incorporation of either cyanine 3-cytidine 5'-triphosphate (3-CTP) or cyanine 5-CTP. The labelling efficiency was verified with a Nanodrop photometer (Kisker). Before hybridisation, 2 μg of each labelled cRNA product were fragmented and mixed with control targets and hybridisation buffer according to the supplier's protocol (Agilent Technologies). Hybridisations were done overnight for ~19 h at 60°C and arrays were subsequently washed. The scanning resolution was 5-μm using a DNA microarray laser scanner (Agilent). Experiments were then repeated, performing a colour swap dye reversal. Features were extracted with an image analysis tool (Agilent Technologies, version A 6.1.1) using default settings. Data analysis was conducted on the Rosetta Inpharmatics Platform Resolver Build version 4.0. Expression patterns were identified by stringent data analysis using anticorrelation of the dye reversal ratio profiles and a 2-fold expression cut-off. Combining the first and the second criteria of analysis, data points were filtered out with a low p value (p < 0.01). To conduct functional categorizing, all genes with a minimum of two-fold absolute changes were submitted to the Database for Annotation, Visualization and Integrated Discovery (DAVID). Pathway mapping analysis for these genes was done by BioRag (Bioresource for array genes ).
For all statistical analyses, a significance level of p ≤ 0.05 was employed. To test differences in means of cell numbers and tumour volumes, a two sided T-test was used. Differences of tumour take curves were evaluated by means of a log rank test. To conduct these statistical analyses, the software package SPSS 12.0 was employed.
Induction of ST-3 expression in co-cultures of breast epithelial cells and fibroblasts
Characterisation of ST-3 over-expressing clones
Functional analysis in vivo
Analysis of cell proliferation in in vitro3D matrigel cultures
Analysis of ST-3 influence on gene expression pattern
Pathways affected by ST-3 over-expression in MDA-MB-231*
MAPK signalling pathway
Wnt signalling pathway
thousand and one amino acid protein kinase
Jak-STAT signalling pathway
Ubiquitin mediated proteolysis
Integrin-mediated cell adhesion
* analysed by BioRag
It has been shown in the present study that ST-3 induction by an immortalized, non-tumourigenic cell line is similar to the induction by tumourigenic breast cancer cell lines. The stimulation of ST-3 expression in breast cancer fibroblasts by different breast tumour cell lines was recently described on mRNA and protein level and is in line with our results [8, 9]. Cell-cell contacts seem to be beneficial, but not essential to induce ST-3 expression, since ST-3 was induced in direct co-cultures and to a lower extent in indirect co-cultures . PKC seems to be involved in the induction of ST-3 expression in normal pulmonary fibroblasts by non-small cell lung cancer cells . The observation that non-tumourigenic cells are also able to induce ST-3 expression might indicate that the association of ST-3 expression and invasive properties of carcinomas is a consequence of invasive epithelial cells coming into contact with fibroblasts rather than being the cause of invasion. The observation, both by our group as well as others, that ST-3 over-expression does not lead to an enhanced invasiveness of cells (data not shown and ) provides further strength to this hypothesis. Normal as well as tumor derived breast epithelial cells expressed low amounts of ST-3. An epithelial expression of ST-3 was already described in gastric and pancreatic carcinoma [29, 30] and seems to also occur in breast tissue.
ST-3 significantly enhanced tumour take as well as tumour growth rates. Thus, the results indicate that ST-3 is not only involved in tumour formation, as it was postulated by previous studies , but additionally in tumour progression. This was true for MCF-7, the breast cancer cell line also used in former in vivo experiments [14, 31], and for MDA-MB-231 cells. MCF-7 is a rather well differentiated tumour cell line, reflected by its oestrogen-dependent growth. This cell line is non-invasive with characteristics of epithelial cells (e.g. E-cadherin expression and a lack of ST-3 expression). In contrast, MDA-MB-231 is a tumour cell line that has undergone epithelial-mesenchymal transition and thus acquired fibroblastoid characteristics (e.g. morphology, invasiveness, lack of E-Cadherin and expression of low amounts of ST-3). MDA-MB-231 cell growth is independent of the presence of oestrogen. Thus, ST-3 seems to have similar effects on breast cancer cells derived from more differentiated tumours and on cells from more aggressive and dedifferentiated lesions.
The described in vitro results indicate that ST-3 over-expression leads to an accelerated cell proliferation. Indeed, ST-3 protein expression has been found to be up-regulated in human invasive breast carcinomas and in co-expression to clinically-used proliferation markers . Since ST-3 has been shown to be capable of releasing IGF-1 bound to IGFBP-1, thereby stimulating proliferation of BAF/3 cells , this signalling pathway was investigated in the present study. The IGF-1 signalling cascade seemed to be the only or predominant pathway responsible for the ST-3 effect in the MCF-7 cell line, possibly via the ERK/MAPK pathway. It was described that ST-3 over-expression in MCF-7 cells results in the activation of p42/p44 MAP kinase . In contrast, additional pathways seem to be involved in MDA-MB-231 cells. In this respect, it is notable that there seems to be cross-talk between IGF-1 and the estrogen receptor .
Transcriptional profiling revealed that cancer related processes such as cell proliferation, cell motility and cell metabolism are affected by ST-3 over-expression. Most notably, cell proliferation and cell cycle progression seem to be influenced, an observation that has been confirmed by our in vitro model. ST-3 leads to an up-regulation of the small GTPases of the Rho family, RhoC and cdc42, which have an important role in cancer progression, including proliferation, invasion, and metastasis of breast tumours . Cdc42 and RhoC are induced in response to integrin activation suggesting a role for ST-3 in mediating cell-matrix adhesion, a prerequisite for cell migration. Rho GTPases also play a role in the induction of MMP expression [35, 36, 37] and, conversely, blocking Rho function can inhibit transcription of MMPs . Whether this is also true for ST-3 activated Rho GTPases still needs to be proven. Interestingly, MMP-28 expression was induced in ST-3 overexpressing cells. This MMP has been described to be involved in EMT in lung carcinoma cells  and could additionally contribute to the observed effect.
However, no induction of gene expression of growth factors could be detected in the presence of ST-3, suggesting that ST-3 might not be involved in de novo growth factor synthesis, which is crucial for autocrine activation of signalling pathways as a characteristic feature for tumour cells . Moreover, ST-3 seems to be involved in the release and activation of soluble growth factors bound by ECM components. This hypothesis is also supported by our in vivo data and the finding that ST-3 can activate extracellular IGF bound to IGFBP-1, which strengthened its definition as a MMP . Thus, our results suggest that ST-3 enhances rather than initiates tumourigenesis by either activating ECM-bound signaling components or at least by synergistic effects with ECM containing growth factors such as IGF-1. Our gene expression data also support a role in ST-3 activation of Wnt-signalling components, such as FZD7 and DVL3, which is in line with the activation of the Wnt pathway by ST-1/MMP-3 via E-cadherin cleavage, resulting in EMT and thus enhanced invasive properties of mammary tumour cells .
This study shows that ST-3 expression in fibroblasts is induced not only by epithelial cells of tumour origin, but also by non-transformed cells. Results from an orthotopic in vivo model as well as from in vitro 3D assays have demonstrated that ST-3 leads to an enhanced tumourigenic potency in cell lines of different dedifferentiation status. The mechanisms by which ST-3 exerts its function seem to involve activation of IGF-1 signalling and potentially MAPK and PI3-kinase pathways. Clearly, further studies are needed to confirm and investigate the impact of ST-3 on these pathways, but our data provide some novel clues for the mechanisms of ST-3 activation in breast cancer cells.
Agilent scanner, Agilent and Rosetta software were kindly supported by Hans-Joachim Mollenkopf, Microarray Core Facility, Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany. We further thank Dr. Têtu (Centre Hospitalier Universitaire de Quebec, Canada) for providing the fibroblasts. Parts of this study were conducted at metaGen Pharmaceuticals GmbH, Berlin, Germany.
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