The two principal hallmarks of breast cancer are uncontrolled cell growth and loss of organized tissue structure. Although the exact mechanism leading to breast cancer is unknown, it has been established that cancer cells have highly active pro-growth signaling and that blocking this signaling greatly decreases cancer cell growth. But how cells lose tissue organization during the cancer development and whether or not inhibition of progrowth signaling could restore the normal tissue structure are not clear. We have used a human mammary epithelial cell progression series to study the development of breast cancer. The cells were isolated from breast reduction surgery. The cells of early passage are ¿normal¿ (S1 cells), and a subset of S1 cells becomes tumorigenic (T4-2 cells). T4-2 cells were found to have significantly high levels of epidermal growth factor receptor (EGFR), a receptor promoting cell growth, and b1-integrin, a receptor important for cell survival. It is well established that breast epithelial cells live in an environment called extracellular matrix (ECM) inside our bodies; ECM is required for breast cells to form the appropriate tissue structures for proper functioning. In order to study the mechanisms of breast cancer development in an environment that mimics the conditions inside the body, we have developed a system called three-dimensional (3D) cell culture system in which cells are grown inside a reconstituted extracellular matrix called rBM. Once grown inside rBM, normal S1 cells develop into a normal tissue structure, but T4-2 cells form invasive, disorganized tumors that can be reverted to the normalized mammary tissue when EGFR and b1-integrin are reduced to the levels of normal S1. PI3 kinase, an enzyme promoting cell growth and affecting cell shape, is the downstream target of EGFR and b1-integrin and has been found to be affected by these two receptors. I propose to study if PI3 kinase and its downstream signaling pathway could play a role in determining the mammary tissue organization in breast cancer and the mechanisms by which this enzyme mediates tissue organization. I propose to accomplish this goal by introducing PI3 kinase and its downstream genes into S1 and T4-2, using a technique called transfection. These genes can make cells overproduce their protein products. I will manipulate the gene expression that activates PI3 kinase signaling pathways in normal S1 cells and block PI3 kinase signaling in T4-2 tumor cells. Then, by using a technique called immunostaining, I will identify the location of tissue organization markers after the manipulation, and define the effects of PI3 kinase pathway on the breast tissue organization. The results generated from this proposal will help us to better understand the mechanisms involved in how the mammary tissue is disorganized and how the signals are relayed during the breast cancer development. The elucidation of this mechanism might be used to design more specific anti-breast cancer therapeutic approaches.