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The Role of Prox-1 in Lymphangiogenesis and Breast Cancer Metastasis.
Background: The evaluation of patients with breast cancer has relied heavily on the functional analysis of the lymphatic system to direct diagnosis and disease management. In spite of this, little is known about the molecular mechanisms governing lymphangiogenesis. Recent molecular advances have identified a number of key regulators critical for the proper development of the lymphatic system. One such gene, the prox-1 transcription factor, is one of the earliest markers involved in lymphatic differentiation. Genetic ablation of prox-1 generates mice with no functional lymphatics. Furthermore, the loss of prox-1 results in the inability to regulate a number of molecular targets resulting in early lymphatic endothelial cells (LECs) displaying a blood endothelial cell (BEC) phenotype. Overexpression of prox-1 in vitro initiates a differentiation program that upregulates lymphatic specific genes while downregulating blood specific genes. More interestingly, this same study also suggests that terminally differentiated BECs can be reprogrammed to become more lymphatic-like. While the evidence suggests that prox-1 initiates reprogramming and differentiation, whether this occurs in a biologically relevant fashion is unclear. Objective/Hypothesis: Our goal is to generate a novel animal model for lymphangiogenesis based on the gene prox-1 and test its ability to influence rates of breast cancer metastasis. Our hypothesis reasons that the development of a conditional mouse model would address prox-1 mediated endothelial cell reprogramming and plasticity in vivo. This inducible model can be utilized to analyze prox-1 transgenic mice bearing mammary tumors to ask whether rates of metastasis could be influenced by the inducible regulation of lymphatic development. Specific aims: 1) Address the importance of the transcription factor prox-1 in the development, molecular reprogramming and plasticity of the lymphatic vasculature. 2) Investigate the potential to modulate lymphangiogenesis and thereby influence breast cancer cells to metastasize. Study design: The first aim will involve the analysis of transgenic embryos that overexpress prox-1 within the vasculature. Molecular markers for lymphatic and blood specific genes will be used to detect BEC reprogramming to a LEC-like profile. Blood and lymphatic structures from post-natal mice rescued from lethality via the conditional system will also be analyzed for BEC and LEC markers. The second aim will involve the transplant of tumor cells into the mammary fat pad of rescued transgenic mice as well as the interbreeding of MMTV-ErbB2 or polyomavirus mT transgenic mice with the Prox-1 mice to assess the effect of genetically modulating lymphatic development on rates of breast cancer metastasis. The presence of micrometastasis, rates of metastasis into sentinal lymph nodes and distant organs will be assessed. Potential outcomes/Impact: The generation of a conditional system for lymphangiogenesis addresses a need for relevant animal models in this rapidly evolving field. Specific questions related to lymphatic function in breast cancer research can now be addressed utilizing this mouse model to develop innovative ways to stem metastasis and lymphedema. Future results may translate into clinically applicable therapies thereby generating a new front in controlling metastatic breast disease.
In 2005, approximately 232,840 new cases of breast cancer will be diagnosed in the United States and Canada; of those 45,710 will succumb to this disease. While many surgical and adjuvant treatments have been developed that have increased patient survival and decreased morbidity, this disease is still the second leading cause of death in women in North America. The mechanism of metastasis is believed to occur via two vascular systems that feed tumor growth, the well-characterized blood vasculature, and the ill characterized lymphatic vasculature. With the breadth of knowledge in angiogenesis, many chemotherapeutic agents have been developed that molecularly target this process. In contrast, the lymphatic system has not been scrutinized as a direct target for chemotherapeutic intervention due to the gap of knowledge in its molecular function. In spite of this, clinical oncologists rely heavily on the evaluation of the lymphatic system in order to assess patient prognosis and plan disease management. A number of genes have been found to play key roles in its development and function, for example the vascular endothelial growth factor receptor-3 (VEGFR-3), the ligand Angiopoietin-2 and the nuclear protein Prox-1. This latter gene has been dubbed a ‘master regulatory’ switch as suggested by mouse studies that ablate this genes function; Prox-1 knockout mice lack functional lymphatics early in development resulting in lethality. Given the importance of Prox-1 in regulating lymphatic development as well as the clear role of lymphatics in metastasis, our hypothesis reasons that if Prox-1 is indeed a switch in lymphatic development, one could molecularly control lymphangiogenesis in an inducible fashion and potentially control breast cancer metastasis. To this end, our specific aims revolves around two key goals; 1) to generate a novel inducible mouse model based on the gene Prox-1 to manipulate lymphangiogenesis at any time in development and; 2) test the ability to control breast cancer metastasis by regulating lymphangiogenesis via Prox-1. The general methodology for the first aim entails turning on or off Prox-1 expression i.e. regulating lymphatic growth and observing, a) gross changes in physiology; b) general vascular changes and, c) changes in molecular markers associated with either blood or lymphatic vasculature. In our second aim we will transplant tumor cells derived from the c-ErbB2 and the polyomavirus middle-T breast cancer models into the mammary gland of inducible Prox-1 mice in addition to interbreeding c-ErbB2 transgenic mice with Prox-1 mice. Prox-1 expression will be induced or silenced and tumor-bearing mice will be assessed for perivascular micrometastasis and dissemination rates into sentinel lymph nodes and distant organ sites. The potential in lymphangiogenic research in breast cancer is clear; one can control lymphangiogenesis and address issues such as lymphedema and metastasis. From these studies, breast cancer management can see a new partner with antiangiogenic therapies, one that exploits the molecular mechanisms that govern lymphangiogenesis thereby attacking another conduit for metatastic spread related to breast cancer.