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Abstract. MT1-MMP (membrane type-1 matrix metalloproteinase), otherwise known as MMP14 is a proteolytic enzyme known to be involved in degradating extracellular matrix and assist progression of cancer invasion and progression. We investigated the impact of targeting the expression of MT1-MMP in breast cancer and its clinical relevance. Human breast cancer cell line MDA-MB-231 was used. Expression of MT1-MMP in the breast cancer cell line was manipulated by way of retroviral ribozyme transgene. The in vitro invasion, growth and cell migration were determined on cell lines transfected with either the transgene or control plasmid. Protein and message levels of MMP14 was also assessed using immunohistochemistry and real-time quantitative analysis, and correlated with clinical and pathological information of the patients. Retroviral ribozyme transgene to human MT1-MMP successfully knocked down the levels of MT1-MMP mRNA from MDA-MB-231 cells. Reduction of MT1-MMP from the breast cancer cells resulted in significant reduction of in vitro invasiveness and loss of response to an invasion stimulus, HGF, compared with control and wild-type cells. The invasion index for MT1-MMP knockdown cells were 13±3

Introduction: Matrilysin (MMP-7) is a metalloproteinase that is involved in the degradation of extracellular matrix, invasion, and tumor progression. The current study examined if targeting matrilysin using retroviral ribozyme transgenes may have an impact on breast cancer cells and may have clinical implications. Experimental Design: Retroviral hammerhead ribozyme transgenes were designed to specifically target human matrilysin mRNA. The breast cancer cell MDA-MB-231was transfected with either a retroviral matrilysin transgene or a control retroviral transgene. Stably transfected cells were tested for their invasiveness and migratory properties in vitro. The cells were also used in creating a tumor model in athymic nude mice in which the growth of tumors and levels of matrilysin were assessed. In addition, levels of both protein and mRNA of matrilysin were investigated in a cohort of human breast tumors. In human breast tumors, breast cancer cells stained matrilysin at a significantly higher density, compared with normal mammary epithelium. The highest level of matrilysin was seen in high-grade tumors and that from patients with moderate and poor prognosis. Finally, high levels of matrilysin were significantly linked with a poor long-term survival (P = 0.0143). Conclusion: Matrilysin, which is aberrantly expressed in human breast tumors, can be effectively eliminated from breast cancer cells by way of hammerhead ribozyme transgene. Elimination of matrilysin is associated with low invasiveness and slow tumor growth. Taken together, the study suggests that targeting matrilysin may have important therapeutic implications.Matrilysin (MMP-7, putative metalloproteinase I, PUMP1) gene was identified through studies of collagenase-related connective tissue -degrading metalloproteinases produced by human tumors (1). MMP-7, by acting as a proteolytic enzyme, has been indicated in the invasiveness and progression of cancer cells. Matrilysin cleaves extracellular matrix and basement membrane proteins, such as fibronectin, collagen type IV, laminin, and, particularly, elastin, entactin, osteopontin, and cartilage proteoglycan aggregates. Furthermore, matrilysin seems to mediate the proteolytic processing of other molecules (e.g., tumor necrosis factor a precursor, urokinase plasminogen activator; refs. 2, 3).The promoter region of the matrilysin gene contains typical MMP promoter elements, such as AP-1 and PEA3, which mediate responsiveness to growth factors, oncogenes, and phorbol esters. The transcription relation of matrilysin also requires the LEF-1/ h-catenin pathways (4, 5). Matrilysin protein localizes to secretory and ductal epithelium in the endometrium and in various exocrine glands. In the mouse, high constitutive levels of matrilysin mRNA are found in epithelial cells in the uterus, small intestine, and extratesticular ducts, suggesting that matrilysin may have a specific role in normal gland and organ function, a

Tumour endothelial markers (TEMs) are a newly discovered family of endothelial markers associated with tumour specific angiogenesis. This study sought to examine the levels of expression for TEMs in human breast cancer. Breast cancer tissues (n = 120) together with normal background tissues (n = 33) were obtained after surgery. RNA was extracted from frozen sections for gene amplification. The expression of TEMs was assessed using RT-PCR and the quantity of their transcripts was determined using real-time-quantitative PCR (Q-RT-PCR). TEM-7R (P = 0.05) and TEM-8 (P < 0.01) were significantly raised in breast cancer tissues compared with the levels detected in normal background tissues. After a median follow-up of 72.2 months it was found that patients who had recurrent disease and/or who had died from breast cancer had a significantly (P < 0.05) elevated level of TEM-1 compared to those patients who were disease free. In addition, elevated levels of TEM-4, TEM-5, TEM-6, TEM-7 and TEM-7R were also raised in breast cancer tissues. Patients who had developed nodal involvement exhibited significantly (P < 0.05) high levels of TEM-1 and TEM-7R compared to patients who were node negative. Furthermore, the levels of TEMs did not correlate with tumour or histological grade. We conclude that elevated levels of TEM-1, TEM-7R and TEM-8 (but not TEM-2, 4, 5, 6 and 7) are associated with either nodal involvement, and/or disease progression, and may therefore, have a prognostic value in breast cancer.