Pleiotropic functions of the tumor- and metastasis-suppressing matrix metalloproteinase-8 in mammary cancer in MMTV-PyMT transgenic mice (original) (raw)
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Cancer Research, 2004
Previous work in our laboratory led to the cloning, from the same parent tumor cell line (MDA-MB-435), of two human breast cancer cell lines (M-4A4 and NM-2C5) with opposite metastatic phenotypes. Additional investigations revealed that the nonmetastatic cell line NM-2C5 overexpressed the neutrophil collagenase, matrix metalloproteinase (MMP)-8, relative to its partner. Because other studies have implicated the MMP family in promoting tumor metastasis, we investigated the apparently paradoxical expression of MMP-8 in these cell lines. By genetic engineering, we inverted its relative levels of expression in the two partners and studied the effects on the behavior of the tumors that they generated in athymic mice. Knock-down of expression in NM-2C5 cells by transduction with a sequence encoding a specific ribozyme and overexpression of MMP-8 in M-4A4 cells by retroviral transduction both strikingly changed metastatic performance in opposite directions, indicating that this gene plays a role in the regulation of tumor metastasis.
Cancer Research, 2008
Collagenase-2 (matrix metalloproteinase-8, MMP-8) is an MMP mainly produced by neutrophils and associated with many inflammatory conditions. We have previously described that MMP-8 plays a protective role in cancer through its ability to regulate the inflammatory response induced by carcinogens. Moreover, it has been reported that experimental manipulation of the expression levels of this enzyme alters the metastatic behavior of human breast cancer cells. In this work, we have used mutant mice deficient in MMP-8 and syngenic melanoma and lung carcinoma tumor cells lines overexpressing this enzyme to further explore the putative antimetastatic potential of MMP-8. We report herein that MMP-8 prevents metastasis formation through the modulation of tumor cell adhesion and invasion. Thus, tumor cells overexpressing MMP-8 have an increased adhesion to extracellular matrix proteins, whereas their invasive ability through Matrigel is substantially reduced when compared with control cells. Analysis of MMP-8 in breast cancer patients revealed that the expression of this metalloproteinase by breast tumors correlates with a lower incidence of lymph node metastasis and confers good prognosis to these patients. On this basis, we propose that MMP-8 is a tumor protective factor, which also has the ability to reduce the metastatic potential of malignant cells in both mice and human. [Cancer Res 2008;68(8):2755-63]
International Journal of Molecular Medicine, 2008
Matrix metalloproteinases (MMPs) are a family of extracellular proteinases whose contributions to cancer progression have been studied because of their matrixdegrading abilities and elevated expression in advanced stage tumors. Recent findings suggest a role for MMPs during the multiple stages of tumor progression including establishment and growth, migration, invasion, metastasis, and angiogenesis. MMP-9 regulation at the molecular level can be studied by measuring the effect(s) of a variety of physiological and pharmacological agents on cells. Multiple signaling molecules such as protein kinase C, pertussis toxin-sensitive guanine nucleotide-binding protein G, and protein tyrosine kinases are known to mediate the secretion of MMPs in cell lines. We previously reported an upregulation of MMP-9 in T cells of mammary tumor-bearing mice. In this study, pharmacologic inhibitors were used to dissect the signaling pathways involved in the upregulation of MMP-9 in the splenic T cells of normal and mammary tumor-bearing mice. Staurosporine, a protein kinase inhibitor, stimulated MMP-9 secretion by normal T lymphocytes, while the constitutively high levels of MMP-9 produced by tumor bearers' T cells were decreased by Genistein, a specific tyrosine kinase inhibitor, and Rottlerin, a PKC inhibitor. Using a NF-κB specific probe to the murine MMP-9 promoter, electromobility shift assays of nuclear proteins from normal and tumor bearers' splenic T cells revealed a pattern of higher intensity bands from the tumor bearers' nuclear extracts, indicating a greater amount of these transcription factors bound to the recognition motif. When mammary tumor bearers' T cells were cultured with the NF-κB inhibitors, N-p-Tosyl-L-lysine chloromethyl ketone hydrochloride and Bay 11-7082, there was a subsequent decreased production of MMP-9. These results suggest that the tumor burden may be activating various signaling pathways within splenic T lymphocytes to upregulate MMP-9 expression.
Breast Cancer Research, 2013
Introduction: Despite continued improvements in diagnosis, surgical techniques, and chemotherapy, breast cancer patients are still overcome by cancer metastasis. Tumor cell proliferation, invasion and metastasis are mediated, at least in part, through degradation of basement membrane by neutral matrix metalloproteinases (MMP) produced by tumor and stromal cells. Evidence suggests that MMP-9 plays a significant role in breast tumor cell invasion and metastasis. DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression. Methods: The application of anti-MMP-9 DNAzyme (AM9D) for the treatment of primary and metastatic breast cancer was evaluated in vitro and in vivo using MDA-MB-231 cells and the MMTV-PyMT transgenic breast cancer mouse model. Spontaneously developed mammary tumors in MMTV-PyMT transgenic mice were treated intratumorally with naked AM9D, once a week for 4 weeks. The stability of DNAzyme was determined in vitro and in vivo using fluorescently labeled DNAzyme. Results: AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%. Weekly intratumoral treatment of spontaneously developed mammary tumors in MMTV-PyMT transgenic mice was sufficient to significantly reduce the rate of tumor growth and final tumor load in a dose dependent and statistically significant manner (P < 0.05). This decrease in tumor growth was correlated with decreased MMP-9 protein production within the treated tumor tissues. Tumors treated with AM9D were also less vascularized and contained more apoptotic cells compared to control and untreated tumors. Conclusions: These results show that targeting and down regulation of MMP-9 by AM9D could prove useful as a therapy against breast carcinoma tumor growth and invasion.
Differentiation, 2003
The multi-step nature of metastasis poses difficulties in both design and interpretation of experiments to unveil the mechanisms causing the process. In order to facilitate such studies, we have previously derived a pair of breast tumor cell lines that originate from the same breast tumor but which have diametrically opposite metastatic capabilities. In this system, the monoclonal cell line M-4A4 is metastatic to the lungs of athymic mice, whereas clone NM-2C5 is equally tumorigenic but non-metastatic. Here, we report that representational difference analysis (RDA) of cDNA obtained from the two clonal populations revealed an increased expression of tyrosinase-related protein-1 (TYRP-1) and the matrix metalloproteinase-8 (MMP-8) genes in the non-metastatic cell line. RNA and protein analyses in cultured cells and in primary xenograft tissues confirmed that the non-metastatic cell line expresses TYRP-1 and MMP-8 at levels that are at least 20-fold higher than the metastatic counterpart. Other members of the MMP family (MMP-9 and MMP-2) and the tissue inhibitor of metalloproteinase-2 (TIMP-2) were found to be expressed at similar levels in both populations. The effects of MMP-8 and TYRP-1 on in vitro invasion and migration were assessed in cells whose expression of these genes was altered by stable transduction with sense and antisense constructs. Specific downregulation of MMP-8 in non-metastatic NM-2C5 cells resulted in a 2.5-fold increased capacity to invade through Matrigel.
Loss of collagenase-2 confers increased skin tumor susceptibility to male mice
Nature Genetics, 2003
We generated mice deficient in collagenase-2 (Mmp8), an MMP mainly produced by neutrophils in inflammatory reactions and detected in some malignant tumors 1,4 . Loss of Mmp8 did not cause abnormalities during embryonic development or in adult mice. Contrary to previous studies with MMP-deficient mice, however, the absence of Mmp8 strongly increased the incidence of skin tumors in male Mmp8 -/mice. Female Mmp8 -/mice whose ovaries were removed or were treated with tamoxifen were also more susceptible to tumors compared with wild-type mice. Bone marrow transplantation experiments confirmed that Mmp8 supplied by neutrophils was sufficient to restore the natural protection against tumor development mediated by this protease in male mice. Histopathological analysis showed that mutant mice had abnormalities in the inflammatory response induced by carcinogens. Our study identifies a paradoxical protective role for Mmp8 in cancer and provides a genetic model to evaluate the molecular basis of gender differences in cancer susceptibility.
British Journal of Cancer, 2007
An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinase (MMP)-1, -2, -7, -9, -11, -13 and -14, tissular inhibitors of metalloproteinase (TIMP)-1, -2 and -3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast were performed. To identify specific groups of tumours with distinct expression profiles the data were analysed by unsupervised hierarchical cluster analysis by each cellular type. We did not find well-defined cluster of cases for tumour cells or fibroblastic cells. However, for mononuclear inflammatory cells the dendogram shows a first-order division of the tumours into two distinct MMP/TIMP molecular profiles, designated group 1 (n ¼ 89) and group 2 (n ¼ 42). Matrix metalloproteinase-7, -9, -11, -13 and -14, and TIMP-1 and -2, were identified as showing significant high expression in group 2 compared with group 1. Multivariate analysis demonstrated that clustering for mononuclear inflammatory cells was the most potent independent factor associated with distant relapse-free survival (group 2: 5.6 (3.5 -9.6), Po0.001). We identify a phenotype of mononuclear inflammatory cells infiltrating tumours, which is associated with the development of distant metastasis. Therefore, this finding suggests that these host inflammatory cells could be a possible target for inhibition of metastasis.
Proteolytic activity and remodeling of the extracellular matrix are important players in tumor progression. However, to date the role of the extracellular matrix in tumor regression remains unresolved. To address this, we used a progesterone-dependent in vivo mouse mammary tumor line, C4-HD, which regresses in response to hormone therapy. Within the first 72 hours of treatment, massive apoptosis was accompanied by changes in the staining patterns of laminin and collagens I, III, and IV. We thus hypothesized that an increase in matrix metalloproteinase (MMP) activity could be involved in this process. This indeed was the case as the activities of MMP-2, -9, and -3 increased in regressing tumors, coinciding with the peak of apoptosis. Moreover, cell-cell interactions were disrupted during early hours of regression with E-cadherin levels reduced and fragmentation products detected during regression. Analysis of -catenin revealed that although total levels within the tissue did not change, this molecule switched from being involved in cell-cell adhesion in the growing tumor to being expressed in the reactive stroma during regression. Our data provide a novel role for proteolytic activity in tumor regression and question the underlying principle for using MMP inhibitors in cancer treatment. (Am J Pathol 2006, 168:270 -279; DOI: 10.2353/ ajpath.2006.050012)
Clinical & experimental metastasis, 2000
We have investigated the gelatinase profiles and invasiveness of clonal tumour sublines derived from a spontaneously arising mammary tumour in a Balb/cfC3H mouse. The 67NR. 66c14 and 4T1.2 sublines have low, intermediate and high metastatic potential respectively. In Boyden chamber studies, Matrigel invasion was seen to be progressively higher in the more metastatic lines 4T1.2>66c14>67NR, consistent with MMP-2 activation potential, MMP-9 secretion, and migration over either type I or IV collagen, which were low in both 67NR and 66c14 cells compared to 4T1.2 cells. These attributes are consistent with those seen in human breast cancer cell lines which appear to have undergone an epithelial-mesenchymal transition (EMT) as indicated by vimentin expression. We were, however, surprised to find vimentin expression, MT1-MMP expression and stellate Matrigel outgrowth in the non-invasive, non-metastatic 67NR cells. indicating that they had undergone an EMT despite not being invasive. ...