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Regulating of MMP Secretion During the Invasion of Breast Cancer Cells
Tumor Cell Biology II
The common cause of death from breast cancer is due to metastasis. Importantly, the metastasis mechanisms of breast carcinomas are not well defined. Extracellular matrix (ECM) is crucial for regulation of breast cancer cell migration and metastasis. The breach of the biological barriers, such as basement membranes is an important step in cancer cell invasion and metastasis, and usually involves a localized degradation of ECM via the secretion of matrix metalloproteinases (MMPs). Furthermore, ECM degradation can result in the release of ECM-associated growth factors required for tumor growth and metastasis. Thus, identification of the mechanisms that control the synthesis, secretion and activation of MMPs is crucial for understanding the processes governing breast tumor development. MMP-9 is a member of the MMP endopeptidases. Elevated levels of active MMP-9 have been correlated with poor prognosis in women with breast cancer. MMP-9 is secreted by tumor, immune, and stroma cells, and is known for its role in tumor metastasis. However, while the transcriptional regulation and activation of MMP-9 is well understood, very little is known about the mechanisms of MMP-9 secretion. We propose that blocking MMP secretion may provide an undescribed and efficacious mechanism by which to suppress metastasis in breast cancer cells. Multiple proteins are known to regulate protein targeting and secretion. Interestingly, recent studies have identified several proteins families that specifically regulate secretion of various hormones neurotransmitters. Unfortunately, similar studies have not been undertaken for the MMPs and the identities of the proteins that regulate MMP secretion are essentially unknown. Thus, the main goal of this project is to identify the molecular machinery that regulates MMP-9 secretion during the metastasis of breast cancer cells. We propose three different aims designed to identify the proteins regulating MMP-9 secretion using a combination of biochemical , cell biological and whole animal techniques that are well established in either Dr. Prekeris or Dr. Schedin laboratories. The RNAi screen will be done in collaboration with Dr. Jagath Junutula (Genentech Inc). In aim #1 we will identify the proteins regulating MMP-9 transport and secretion using an existing siRNA library specific for membrane transport proteins. In aim #2 we will test the effect of putative regulators of MMP-9 secretion on breast cancer cell motility and invasion using in vitro assays. Finally, in Aim #3 we will test the effect of putative MMP-9 secretion regulators on growth and metastasis of breast tumors using mammary fat pad xenograft model. The completion of this project will identify and characterize the molecules that regulate MMP-9 secretion. Since very little information about MMP secretion is currently available, the data from this project will lay the foundation for the new direction in future studies on regulation of breast cancer cell metastasis.
Epithelial carcinomas are the most common cancers types that include breast tumors. In 2006, an estimated 211,240 American women will be diagnosed with breast cancer, and an estimated 40,410 women will die from this disease. Prognosis of the disease is correlated to the tumor metastasis, that is the most common cause of death among breast cancer patients. The base of the metastasis is the ability of cancer cells to enter blood stream or lymphatics and form secondary tumors in other tissues. Thus, the development of new anti-cancer therapies relies on our understanding of the mechanisms regulating the cell ability to migrate and invade other tissues. Extracellular matrix (ECM) is crucial for regulation of breast cancer cell migration. Besides providing the substrate for the adhesion of the migrating cells, ECM also serves as a barrier for the cell motility. The breach of this biological barrier is an important step in cancer cell invasion and metastasis, and usually involves a localized degradation of ECM via the secretion of proteins known as matrix melalloproteinases (MMPs). MMPs are the large family of endopeptidases that are capable of cleaving multiple ECM molecules. MMP-2 and MMP-9 are the members of this family and are well known for their role in tumor growth and metastasis. In recent years several MMP inhibitors have been developed as a potential anti-cancer drugs. Unfortunately, the results of clinical trials were disappointing because of the numerous side effects of the drugs. Most of the MMP-based drugs were designed to inhibit the activity of extracellular activated MMPs. Thus, the inhibition of MMP release may be used as an alternative and more specific approach. Unfortunately we know very little about the molecules that regulate the release of MMPs. Identification and characterization of these molecules is the focus of this proposal. To achieve that we will use a several novel technique that allows the selective elimination of various regulatory molecules from the breast cancer cells, known as RNAi, followed by the test of the invasive ability of these cells in vitro and in vivo. The main strength of this proposal is a collaboration between three laboratories, each of them with extensive experience in the different aspects of this project. Dr. Rytis Prekeris (project PI) has an extensive expertise in the field of molecular and cell biology membrane traffic. Dr. Pepper Schedin has an extensive expertise in breast cancer biology and xenograft assays (the measurement of metastasis in animals). Finally, Dr. Jagath Junutula (will not be supported by this grant) has been instrumental in creating siRNA library that will allow the rapid identification of candidate proteins, to be tested in this project. The completion of this project not only will identify and characterize the molecules that regulate MMP-9 secretion, but also will lay the foundation for the new direction in future studies on regulation of breast cancer cell metastasis.