Research Grants Awarded
Cell-Cell Interactions And Eph/Eprhin Signaling Axis In Mammary Gland Branching Morphogenesis And Tumorigenesis
Scientific rationale: The structure of a tissue or organ is critical for its function. Loss of tissue architecture is a prerequisite for, and one of the defining characteristics of, most cancers. Thus, defining the mechanisms involved in generating and maintaining cell and tissue specificity is crucial to understanding how homeostasis is lost during tumorigenesis. Two tyrosine receptor kinases, EphA2 and EphB4, are normally expressed in mammary glands, but are frequently over-expressed in breast cancers and appear to play a role in tumor progression and transformation of breast cells. These receptors are known to play a crucial role during branching morphogenesis in a number of tissues such as kidney and the lung; however, their roles in mammary gland are not well understood. EphA2 and EphB4 are normally expressed in the two compartments of this organ, i.e. the luminal and the myoepithelial cells, respectively. Their expression, which is estrogen dependant, is induced at puberty and undergoes cyclical changes during the estrous cycle. I have preliminary analysis to show that Eph expression is significantly higher in what is referred to as ?basal? in human breast cancer cell lines. Our laboratory recently provided a further subdivision on the basis of morphological characterizations in three dimensional (3D) cultures that track with RNA array signatures. EphA2 receptor was especially highest in the group referred to as ?stellate?, which belongs to the four distinct morphological groups (Grape-like, Mass, Round and Stellate) and which all displays metastatic and invasive properties. There is ample evidence that signaling pathways involved in tumorigenesis are a usurpation of the similar pathways in normal mammary gland invasion during development. Because of the potential role of Eph receptors in invasion and the metastasis process, I will initially characterize their function in mammary branching morphogenesis.
I expect that by understanding this receptor family, which regulates tissue morphogenesis, we will be able to elucidate additional mechanisms for how loss of tissue architecture and tumorigenesis are linked.
Objective/Hypothesis: We hypothesize that EphA2 and EphB4 are important signaling entities during mammary branching morphogenesis and that their dysregulation contributes to the malignant state.
Research aims and design: (1) Characterize the expression levels, localization and function of EphA2 and EphB4 in mammary branching morphogenesis. Using physiologically relevant 3D culture assays of mammary cell lines as well as mouse primary organoids, I propose to determine the effect of Eph activation on branching using recombinant ephrin ligands. Combinations of over-expression and knockdown of Eph-related proteins will also be used to characterize these processes. (2) Determine the signaling pathways involved in Eph-related functions during morphogenesis. These will be identified utilizing specific inhibitors of key signaling pathways known to be involved in epithelial branching. Other potential downstream effectors such as metalloproteinases (MMPs) will also be investigated. (3) Determine levels of Eph signaling in our models of human breast cancer progression and metastatic breast cancer cell lines, and using state of the art technologies, determine what role they may play in invasion.
Potential outcomes and relevance of research: This study will shed light on the mechanisms by which Eph/ephrin signaling regulates mammary branching morphogenesis, a normal, regulated process of invasion. This study will provide insights on the consequences of deregulation of this pathway in breast cancer, and will suggest appropriate intracellular targets for effective inhibition of invasive breast cancers.
The National Cancer Institute estimates that more than 1,500 people die each day from cancer making it a second-leading cause of death in the United States exceeded only by heart disease. In the US, breast cancer is the third most common cause of cancer death (after lung cancer and colon cancer) and it is estimated that 40,910 women will die from this disease in 2007. In 2004, an article published in Fortune Magazine, entitled ?Why we?re losing the war on cancer?, argued that the number of heart disease-related deaths per 100,000 US Citizens has steadily declined since 1950, whereas the number of cancer related deaths has remained essentially constant. While the cause for this unfortunate disparity is undoubtedly multifactorial, one might speculate that part of the explanation lies in the models used to understand and cure cancer.
Most experiments using human cancer cells are performed on flat 2-dimensional (2D) substrata, assuming that cell monolayers reflect the essential physiology of organ. Relative to normal cells, human cancer cells proliferate faster in most 2D culture experiments and will form tumors when injected into mice. In these types of experiments the cancer cells tend to respond usually to the toxic drugs by dying, whereas in patients the same responses are often not recapitulated. Also, normal cells also die in 2D when they are used but often are not. In vivo, cells do not exist in 2D, and tumors arise within the context of an organ and not from a mass of cells injected with a syringe. Accordingly, the research community has become increasingly aware of the importance of a tissue?s 3-dimensional architecture (3D) as a better illustration of the reality. Tissue organization as well as function is lost when cells are explanted onto flat 2D substrata but both can be restored and maintained if cells are placed in or on appropriate 3D conditions. Some of the elements that are important in building a tissue, a process called morphogenesis, are: cell polarity, which tells cells and tissues which direction to secrete; organization, which allows compartmentalization and specialization within a tissue and organ; and morphology, the overall form of the tissue that appears to additionally orchestrate the interactions between cells within and between tissues. Cancer occurs when these properties that we refer as tissue/organ specificity go awry. Owing to their physiological relevance, 3D cellular models are becoming a fundamental research tool in cell biology and cancer research. Much of these concepts and models come from the laboratory of Mina Bissell who is my mentor.
I am proposing to investigate the roles played by the Eph receptors, molecules which are known to direct tissue morphogenesis by regulating a broad range of developmental processes in normal branching glandular tissues, such as kidney, salivary and mammary glands. In a normal context, these receptors mold the behavior of cells by binding their ligands, the ephrins, at sites of cell-cell contact. Interactions between the receptors and their ligands produce a plethora of different signals responsible for the phenotypes observed. To date, molecular mechanisms by which two of these receptors, EphA2 and EphB4, which are found in the mammary gland, are not well understood within this organ but much is known in other branching organs. It is, however, clear that these two proteins are deeply involved in managing the morphogenesis of the mammary gland. My belief is that Eph receptors tightly coordinate proliferation, differentiation, polarity, migration, and invasion during mammary branching morphogenesis in a 3D context. Importantly, the two receptors are found to be deregulated in many breast cancers, and the data suggest that such deregulation may be a cause of the disease and not merely a consequence. Specifically, our preliminary analysis shows that Eph expression is significantly higher in invasive breast cancers cells in 3D. Because all of the properties attributed to Eph receptors are frequently misregulated in cancers, I deem it worthwhile to understand their role in the normal context of the mammary gland, which will then enhance our understanding of their role in the malignant state. To do so, I will use relevant physiologically 3D models of both mouse and human cells which would allow manipulation of pathways, crucial to investigate the function of these receptors in normal and tumor contexts. This study will shed light on how Eph receptors regulate mammary branching morphogenesis, will provide insights on the possible consequences of deregulation of these pathways in breast cancer, and will thus suggest appropriate intracellular targets for effective inhibition of invasive cancers of the mammary gland.