André Lochter

The Stromal Proteinase MMP3/Stromelysin-1 Promotes Mammary Carcinogenesis

Matrix metalloproteinases (MMPs) are invariably upregulated in the stromal compartment of epithelial cancers and appear to promote invasion and metastasis. Here we report that phenotypically normal mammary epithelial cells with tetracycline-regulated expression of MMP3/stromelysin-1 (Str1) form epithelial glandular structures in vivo without Str1 but form invasive mesenchymal-like tumors with Str1. Once initiated, the tumors become independent of continued Str1 expression. Str1 also promotes spontaneous premalignant changes and malignant conversion in mammary glands of transgenic mice. These changes are blocked by coexpression of a TIMP1 transgene. The premalignant and malignant lesions have stereotyped genomic changes unlike those seen in other murine mammary cancer models. These data indicate that Str1 influences tumor initiation and alters neoplastic risk.

Proteinases in bone resorption: obvious and less obvious roles

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.

Expression of Autoactivated Stromelysin-1 in Mammary Glands of Transgenic Mice Leads to a Reactive Stroma During Early Development

Extracellular matrix and extracellular matrix-degrading matrix metalloproteinases play a key role in interactions between the epithelium and the mesenchyme during mammary gland development and disease. In patients with breast cancer, the mammary mesenchyme undergoes a stromal reaction, the etiology of which is unknown. We previously showed that targeting of an autoactivating mutant of the matrix metalloproteinase stromelysin-1 to mammary epithelia of transgenic mice resulted in reduced mammary function during pregnancy and development of preneoplastic and neoplastic lesions. Here we examine the cascade of alterations before breast tumor formation in the mammary gland stroma once the expression of the stromelysin-1 transgene commences. Beginning in postpubertal virgin animals, low levels of transgene expression in mammary epithelia led to increased expression of endogenous stromelysin-1 in stromal fibroblasts and up-regulation of other matrix metalloproteinases, without basement membrane disruption. These changes were accompanied by the progressive development of a compensatory reactive stroma, characterized by increased collagen content and vascularization in glands from virgin mice. This remodeling of the gland affected epithelial-mesenchymal communication as indicated by inappropriate expression of tenascin-C starting by day 6 of pregnancy. This, together with increased transgene expression, led to basement membrane disruption starting by day 15 of pregnancy. We propose that the highly reactive stroma provides a prelude to breast epithelial tumors observed in these animals.

Misregulation of Stromelysin-1 Expression in Mouse Mammary Tumor Cells Accompanies Acquisition of Stromelysin-1-dependent Invasive Properties

Stromelysin-1 is a member of the metalloproteinase family of extracellular matrix-degrading enzymes that regulates tissue remodeling. We previously established a transgenic mouse model in which rat stromelysin-1 targeted to the mammary gland augmented expression of endogenous stromelysin-1, disrupted functional differentiation, and induced mammary tumors. A cell line generated from an adenocarcinoma in one of these animals and a previously described mammary tumor cell line generated in culture readily invaded both a reconstituted basement membrane and type I collagen gels, whereas a nonmalignant, functionally normal epithelial cell line did not. Invasion of Matrigel by tumor cells was largely abolished by metalloproteinase inhibitors, but not by inhibitors of other proteinase families. Inhibition experiments with antisense oligodeoxynucleotides revealed that Matrigel invasion of both cell lines was critically dependent on stromelysin-1 expression. Invasion of collagen, on the other hand, was reduced by only 40-50%. Stromelysin-1 was expressed in both malignant and nonmalignant cells grown on plastic substrata. Its expression was completely inhibited in nonmalignant cells, but up-regulated in tumor cells, in response to Matrigel. Thus misregulation of stromelysin-1 expression appears to be an important aspect of mammary tumor cell progression to an invasive phenotype.

The Significance of Matrix Metalloproteinases during Early Stages of Tumor Progressiona

ABSTRACT: Matrix metalloproteinases (MMPs) orchestrate tissue remodeling and play diverse roles during organ development. They are produced excessively during the course of various pathological conditions, including solid tumors. An important function of MMPs during tumor progression is to provide the proteolytic activity that is necessary both for tumor cells to invade extracellular matrix (ECM) and for neovascularization of tumor tissue by endothelial cells. Recently, independent studies in transgenic animals suggest that MMPs may, in addition, promote very early stages of tumor progression. To investigate this possibility further, we have analyzed the consequences of MMP overexpression in functionally normal and nontumorigenic mouse mammary epithelial cells in culture. Our observations demonstrate that the MMP stromelysin‐1 (SL‐1) triggers an epigenetic molecular program in mammary epithelial cells that results in a number of phenotypic alterations that eventually culminate in the generation of a malignant tumor‐cell phenotype.

Transforming Growth Factor-β-induced Osteoblast Elongation Regulates Osteoclastic Bone Resorption through a p38 Mitogen-activated Protein Kinase- and Matrix Metalloproteinase-dependent Pathway

Transforming growth factor-β (TGF-β) is a powerful modulator of bone metabolism, and both its anabolic and catabolic effects on bone have been described. Here we have tested the hypothesis that TGF-β-induced changes in osteoblast shape promote bone resorption by increasing the surface area of bone that is accessible to osteoclasts. The addition of TGF-β1 to MC3T3-E1 cells resulted in cytoskeletal reorganization, augmented expression of focal adhesion kinase, and cell elongation, accompanied by an increase in the area of cell-free substratum. TGF-β1 also triggered activation of Erk1/2 and p38 mitogen-activated protein (MAP) kinase. The p38 MAP kinase inhibitor PD169316, but not an inhibitor of the Erk1/2 pathway, abrogated the effect of TGF-β1 on cell shape. The matrix metalloproteinase inhibitor GM6001 also interfered with osteoblast elongation. Treatment of MC3T3-E1 cells seeded at confluence onto bone slices to mimic a bone lining cell layer with TGF-β1 also induced cell elongation and increased pit formation by subsequently added osteoclasts. These effects were again blocked by PD169316 and GM6001. We propose that this novel pathway regulating osteoblast morphology plays an important role in the catabolic effects of TGF-β on bone metabolism.

An odyssey from breast to bone: multi-step control of mammary metastases and osteolysis by matrix metalloproteinases.

Development of metastases distant to the primary site of solid tumors marks late stages of tumor progression. Almost all malignant mammary tumors are carcinomas arising from the breast epithelium, but the morphological and molecular alterations in the mammary stroma surrounding the premalignant and the growing tumor contribute to its conversion into neoplastic tissue. Two parameters are critical for initiation of the metastatic process and access of tumor cells to the circulation. These are the ability of tumor cells to invade the basement membrane and the stroma, and the neovascularization of breast tumor tissue. A major site for development of distant metastases is the skeleton. After colonizing the bone, tumor cells promote a cascade of events leading to recruitment of osteoclasts and subsequent osteolytic bone destruction. A ubiquitous theme of neoplastic progression of breast tumors is the overproduction of matrix metalloproteinases. In this review, we summarize the recent insights into the functional consequences of matrix metalloproteinase expression and activation during malignant conversion in the breast, and after bone colonization. The current literature supports the hypothesis that matrix metalloproteinases play a key role in the metastatic expansion of most, if not all, mammary tumors and in the ensuing bone loss.

Transcriptional regulation of stromelysin-1 gene expression is altered during progression of mouse mammary epithelial cells from functionally normal to malignant

The matrix metalloproteinase stromelysin-1 plays a central role during mammary gland development and tumor progression. To gain insight into the regulation of stromelysin-1 gene expression, the murine stromelysin-1 promoter was cloned and transfected into mouse mammary epithelial cells displaying various degrees of malignancy. A reconstituted basement membrane inhibited stromelysin-1 promoter activity in functionally normal cells, had little effect on moderately malignant cells and up-regulated the promoter in highly malignant cells. Spreading of normal and malignant cells was reduced by a reconstituted basement membrane, compared to a plastic substratum. Preventing spreading by maintenance of cells in suspension culture, regulated stromelysin-1 promoter activity in a manner similar to that on a reconstituted basement membrane. Conversely, increasing spreading by augmenting substratum adhesivity up-regulated stromelysin-1 promoter activity in tumor cells. In cells with reduced spreading in the presence of reconstituted basement membrane and in suspension culture, actin stress fibers were replaced by cortical actin bundles. In tumor cells, but not in functionally normal cells, treatment with phorbol diesters also resulted in accumulation of cortical actin and increased stromelysin-1 promoter activity. Consistent with an epithelial-to-mesenchymal conversion, regulation of stromelysin-1 gene expression in highly malignant cells was similar to its regulation in mammary fibroblasts. We conclude that the switch in transcriptional regulation of stromelysin-1 expression that occurs during epithelial-to-mesenchymal transition and conversion to tumorigenicity is related to altered regulation of signals from the cytoarchitecture.