Breast cancer progression may be characterized by a switch from hormone- dependent to hormone-independent growth that involves several cellular alterations. Overexpression of many growth factor receptors, as well as growth factors, has been shown to confer varying degrees of estrogen- independent growth on estrogen receptor (ER) positive breast cancer cells. The protooncogene Raf-1, a serine/threonine kinase, is a key intermediate in the signal transduction pathway of many of these growth factor receptors, and when constitutively activated in fibroblasts is transforming. Expression of a constitutively activated Raf in ER+MCF-7 human breast cancer cells is incompatible with growth in the presence of estrogen; that is cells down-regulate expression of the transfected Raf. Constitutive Raf activity does allow for growth of the cells in the absence of estrogen, suggesting that activation of growth factor signaling pathways through Raf may confer a selective advantage for growth of breast cancer cells under estrogen-deprived conditions. In addition, the high levels of Raf activity induce apoptosis in cells grown under either condition. This is a novel activity for Raf, and may occur because the levels of the constitutive Raf are extremely high in these cells. Therefore, it can be hypothesized that lower levels would mediate growth, while the higher levels result in additional, differential activation of other pathways, such as those involved in inducing apoptosis. We propose to use this model to study the role of Raf signaling in breast cancer. To determine if the growth effects of Raf can be separated from the apoptosis effects, an inducible and regulatable constitutive Raf expression vector will be constructed using the tetracycline repressible expression system. The extent of Raf signaling through the MEK/MAPK (erk) pathways will be assessed. A dominantly acting MEK under the control of the tet-repressible promoter will be transfected into cells to determine whether this pathway accounts for growth, apoptosis, or both. In addition, the interaction of Raf with p53 will be studied to determine the role this plays in apoptosis. Finally, the possibility that Raf is aberrantly signaling through the jun kinase pathway, thus inducing apoptosis in this manner, will be examined by inhibiting the normal activation of this pathway. A dominant negative MEKK will be transfected into cells already containing constitutively active Raf. The effects on apoptosis, as well as the activity of downstream effectors of this pathway, will then be assessed.
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|El-Ashry, D; Miller, D L; Kharbanda, S et al. (1997) Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis. Oncogene 15:423-35|