Cell density-dependent regulation of matrix metalloproteinase and TIMP expression in differently tumorigenic breast cancer cell lines
Introduction
One of the classic hallmarks of the transformed phenotype in vitro is compromised contact inhibition of cell growth, a phenomenon also linked to acquisition of invasive potential [1]. The matrix metalloproteinases (MMPs) are a large family of metal-dependent matrix-degrading endopeptidases implicated in numerous aspects of tumor progression (reviewed in [2]). In vivo, the activity of these proteases is modulated by their naturally occurring inhibitors, the tissue inhibitors of metalloproteinase (TIMPs) [3]. While TIMPs generally repress MMP activity and activation, TIMP-2 is also involved in the activation process of latent MMP-2 [4].
In addition to regulation through MMP–TIMP interactions, substantial evidence indicates that transcriptional regulation plays an important role in the biology of MMPs. The promoter regions of many MMP genes, including MMP-1, -3, -7, -9, -12 and -13, have in common the presence of a proximal (approximately −70 bp) [5], [6] activating-protein-1 (AP-1) binding site. As previously reported, the MMP-2 promoter contains a distal modified AP-1 binding site that has been shown to preferentially bind Fra-1/JunB complexes [7]. AP-1 is not a single protein, but a menagerie of dimeric proteins that belong to the Jun, Fos, Maf, and ATF sub-families, whereby c-Jun is the most potent transcriptional activator in this group [8]. Although limited, previous data have suggested that cell density may exert effects on expression of major MMPs. However, a clear-cut proof therefore still is missing. Binding of MMP-2 to the cell surface has been found to be reduced [9] in confluent breast cancer cells, and transcription factors involved in MMP regulation, such as Ets, appear to be expressed at higher levels in sparse cultures of endothelial cells [10]. However, the dynamics and the factors regulating MMP expression in vivo are not well understood, as is the role of AP-1 in the regulation of major MMPs in biologically different settings.
In recent studies, we and others provided circumstantial evidence that expressions of major MMPs and in vivo tumorigenicity correlate. This was seen in tumor cell models of low and highly malignant keratinocytes and breast carcinoma cells which had been shown to have different biological behavior in retransplantation studies [11], [12], [13], [14], [15], [16]. Furthermore, in vivo studies on tumor tissue samples have shown that differences in the expression and activity levels of major MMPs occur in those areas with different tumor cell densities [17], [18].
In our present study, we show that, in addition to the correlation with tumorigenicity, tumor cell density regulates the expression of the major MMPs and their naturally occurring inhibitors TIMP-1 and -2. We provide evidence that this MMP regulation correlates with invasive capacity and inversely correlates with c-Jun and c-fos mRNA expression. Finally, we demonstrate that the synthesis of MMPs, as well as invasion, is enhanced after overexpression of c-Jun by transfection of the breast cancer cell lines, while TIMP-1 expression is downregulated. These data demonstrate that cell density can regulate the invasive potential of tumor cells and that AP-1 is a key transcription factor controlling invasiveness in this system.
Section snippets
Cell types and culture conditions
The breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-468 (ATCC), and MDA-MB-435 (kindly donated by Dr. Janet E. Price, M.D. Anderson Cancer Center, Houston, USA) used in this study reflect a stepwise increase in malignant biological behavior on the basis of methodical retransplantation studies (MDA-MB-435 > MDA-MB-231 > MDA-MB-468 > MCF-7 [19], [20]). These cell lines are commonly used for breast cancer studies and therefore are well-defined in their growth, invasive, and metastatic
Reference gene determination
Prior to in vitro cell density experiments, we had to identify the most appropriate reference gene for quantitative RT-PCR. Therefore, the mRNA expression levels of cyclophilin B (CPB), HPRT, PBGD, glucose-6-phosphatate dehydrogenase (G6P), and GAPDH were tested in four cell lines (MCF-7, MDA-MB-468, MDA-MB-231, MDA-MB-435) by quantitative RT-PCR. Assuming equal efficiencies of cDNA synthesis in parallel preparations for all cell densities (SC1 to CON), the cDNA stages were diluted according to
Discussion
The interaction of cells with the extracellular matrix (ECM) is critical for the normal development and function of organisms. The turnover and remodeling of ECM must be highly regulated; uncontrolled proteolysis contributes to the generation of many pathological conditions characterized by excessive degradation of ECM components including tumor growth and metastasis. Previous studies have provided evidence that an enhanced production of MMPs in vivo is associated with a more aggressive growth
Acknowledgments
The technical assistance by Mrs. K. Duchateau is gratefully acknowledged. We thank Dr. Janet Price for her generous gift of the breast cancer cell line MDA-MB-435. Supported by DAAD/CRUI grant program Vigoni to B.B., and Associazione Italiana per la Ricerca sul Cancro (AIRC) and Ministero della Sanità to A.A.
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