The process of mammary carcinogenesis can result in the loss of differentiated cell function, and exposure of primary mammary epithelial cell cultures to mitogens can block lactogenic differentiation. Enhancement of differentiation of transformed cells can allow the reacquisition of some aspects of cellular growth control and susceptibility to apoptosis. Hence, a complete understanding of the regulation of the differentiation process will aid in elucidating the mechanisms leading to mammary carcinogenesis and allow evaluation of therapeutic strategies on the differentiation process. The HC11 mouse mammary epithelial cells differentiate and synthesize beta-casein following stimulation with the lactogenic hormone mix, DIP (dexamethasone, insulin, prolactin), resulting in prolactin-dependent activation of Stat5 a and b. In HC11 cells the activation of Stat5 is not dependent on the Ras-Erk pathway and can be blocked by receptor tyrosine kinase signaling at the time of prolactin addition. Inhibition of HC11 differentiation by an EGF family members occurs through a Ras-Erk and Ras-phosphatidylinositol-3-kinase (PI-3 kinase)-dependent mechanism. Because inhibition of differentiation in HC11 cells appears to be dependent upon Ras, and possibly its association with PI-3-kinase, we propose that the growth factor regulated inhibition of DIP-induced differentiation of HC11 cells results from the activation of Ras effector pathways in addition to Raf-Mek-Erk. Specifically, inhibition may require activation of the Ras-PI-3-kinase pathway and/or the Ras-Rho and -Rac pathways. We will test this. hypothesis by constructing HC11 cell lines carrying: effector mutants of Ras that activate only a subset of effector pathways, and dominant-negative (DN) mutants of Pl-3-kinase, Rac and Rho. In addition, the role of Mek-1 and KSR, two proteins that regulate Ras-induced activation of the Erk pathway, and are down-regulated during DIP-induced differentiation, will be studied. Transfected cell lines will be used to dissect the control of mammary differentiation using a series of markers for differentiation.
The Specific Aims of this study include: 1. Determine the effect of Ras effector mutants and dominant negative mutants of RasGAP, PI-3-kinase, Erk and Rho on differentiation of HC11 mouse mammary epithelial cell lines. 2. Elucidate the role of Mek-1 and KSR in inhibition of mammary differentiation. 3. Determine which Ras effector pathways are required for disruption of differentiation by mitogens in primary mouse mammary epithelial cell cultures and explants. The research proposed here will determine the mechanism by which an important mitogenic signal transduction pathway, which is frequently activated in breast carcinoma, inhibits mammary differentiation and apoptosis.
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