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Identification of breast cancer intravasation genes
Tumor Cell Biology VI
Background: Little is known about how metastatic breast cancer cells intravasate the circulatory system of the mammary gland, but approaches to study this process are suggested from gene expression signatures of primary breast cancers. A 70-gene-expression signature is strongly predictive of metastasis and hence poor survival in young patients with lymph node-negative breast cancer. This poor prognosis signature consists of genes regulating cell cycle, invasion, metastasis, and angiogenesis. Objective/Hypothesis: A 95-gene lung metastasis signature that mediates breast cancer metastasis to lung led us to propose that highly lung metastatic human breast cancer sublines differentially express genes encoding intravasation proteins. A powerful way to study intravasation is to use an in vitro model examining the transmigration of cancer cell lines across a monolayer of cells from an endothelial cell line of the primary organ. This assay mimics the correct movement of cancer cells across the endothelium (abluminal to luminal). Our goal is to identify differentially expressed genes encoding intravasation proteins from highly lung metastatic human breast cancer sublines. Specific Aims: 1. Identify genes from a highly lung metastatic human breast cancer subline responsible for differential transmigration across human mammary microvascular endothelial cells (HMMEC). 2. Determine the contribution of each isolated gene to intravasation across HMMEC. 3. Validate the contribution of the most important genes encoding intravasation proteins using an in vivo lung metastasis assay. Study Design: A gain-of-function genetic screen will be employed to identify transfectants that exhibit enhanced transmigration across HMMEC following transfection of a recipient cell line with a retroviral cDNA library from a highly lung metastatic human breast cancer subline. These transfectants harbor cDNAs encoding putative intravasation proteins that may be involved in cell adhesion and/or intravasation itself. RNA interference will be used to silence expression of these genes in the subline to determine their contribution to intravasation across HMMEC and to spontaneous lung metastasis in SCID mice. Lung metastasis in SCID mice will be quantified by in vivo bioluminescent imaging following orthotopic injection of our sublines carrying the luciferase gene and small interfering RNAs (siRNA) knock downs of intravasation genes into the mammary fat pad. Potential Outcome and Benefits of Research: Our research plan will help stimulate development of monoclonal antibodies and small molecule drugs directed against breast cancer intravasation proteins identified here. New potential therapeutics will block the intravasation of the mammary circulatory system by breast cancer cells and prevent their metastasis to distant organs, including lung and bone, a key factor in breast cancer morbidity and mortality.
Metastasis of breast cancer cells requires their successful navigation of several critical steps including escape from the primary breast tumor, invasion of the tumor border, entry (intravasation) of the circulatory system, adhesion to the microvasculature of the target organ, escape (extravasation) of the circulatory system, and infiltration and proliferation into target tissue, including bone and lung. Little is known about how breast cancer cells intravasate the circulatory system of the mammary gland, but approaches to study this process have been derived from gene expression signatures of primary breast tumors. A gene expression signature is defined by the expression pattern of a specific set of genes that may characterize a tumor. A 70-gene-expression signature [52 overexpressed (active) genes and 18 underexpressed (silent) genes] is strongly predictive of metastasis and poor survival in young patients with early breast cancer. This poor prognosis signature consists of genes regulating cell cycle, invasion, metastasis, and angiogenesis (formation of new blood vessels). Gene expression signatures mediating breast cancer metastasis to bone or lung imply that specific genes are expressed in certain primary breast tumors that allow cancer cells to form bone or lung metastases. We postulate that human breast cancer cells with high capacity to form lung metastases specifically express a subset of genes that allow them to readily enter the circulatory system of the mammary gland. We will employ a novel recombinant DNA method (termed gain-of-function) to clone intravasation genes from such cells. These encoded proteins may be involved in cell adherence to the mammary microvasculature and/or intravasation itself. We shall validate the contribution of these intravasation genes using an animal model of lung metastasis. First we will use a recently described genetics tool called RNA interference to silence the expression of these genes in human breast cancer cells with high capacity to form lung metastases. We shall then test if these genetically modified breast cancer cells are impaired in their ability to form lung metastases in nude mice when compared to unmodified breast cancer cells following injection into the mammary fat pad. Our research plan will help stimulate development of monoclonal antibodies and small molecule drugs directed against breast cancer intravasation proteins identified here. New potential therapeutics will block the intravasation of the mammary circulatory system by breast cancer cells and prevent their metastasis to key organs, including lung and bone, a key factor in breast cancer morbidity and mortality.