Treatment of hormone dependent breast cancer induces regressions lasting 12-18 months in women but relapses invariably occur. Additional responses to subsequent hormonal therapies may occur but reversion later to a hormone independent state ultimately results. We postulate that these sequential responses reflect adaptive changes in tumors whereby hypersensitivity to both the proliferative and pro-apoptotic effects of estradiol occurs in response to up-regulation of growth factor pathways. The current proposal examines why growth factor pathway up-regulation occurs and what mechanistic changes ensue. Our recent data suggest that enhanced non-genomic effects of estradiol acting at the level of the cell membrane might be responsible. Membrane related estrogen receptors co-opt growth factor pathways and utilize the IGF-1 and EGF receptors as well as Shc, Grb-2, and SOS to stimulate MAP kinase, PI-3-kinase and cell motility. In this proposal, we will pursue four Specific Aims #1: confirm the hypothesis that the non-genomic actions of ER alpha are responsible for up-regulation of the MAP kinase and PI-3 kinase pathways in adapted breast cancer cells. We will first validate three direct methods for distinguishing non-genomic effects of estradiol from those which directly stimulate gene transcription: (1) use of a membrane impermeable biotinylated estradiol (2) use of a novel knock out/rescue technique, and (3) use of a dominant negative construct directly exclusively against membrane ERalpha. We will utilize these methods to examine the role of non-genomic actions in hypersensitivity and compare the degree of utilization of non-genomic pathways in adapted and nonadapted cell lines. #2 Determine the mechanisms causing hypersensitivity to estradiol in adapted cells with respect to apoptosis. We will test the hypothesis that estradiol triggers apoptosis predominately through non-genomic estrogen receptoralpha effects by activating the stress kinase (JNK), p38 kinase and the death receptor Fas/FasL pathways as well as the Type II apoptotic, mitochondrial pathways. The strategies to establish causality include the induction of hypersensitivity to estradiol in non-adapted cells, and reversion of hypersensitivity in adapted cells using dominant negative and siRNA techniques. #3 Role of the membrane ER in dynamic membrane changes in adapted cells. We will test the hypothesis that non-genomic effects of estradiol act through the Rac/PAK1 pathways to enhance the formation of membrane changes to increase motility and invasiveness of breast cancer cells. Our working model includes a nodal point consisting of an EGF-R/FAK complex; input through non-genomic ER related pathways and output through PI-3-kinase, c-SRC and p130CAS to Rac/PAK1. #4 Effect of Blockade of mTOR on apoptosis. Determination of the mechanisms involved in apoptosis allows development of new experimental strategies for enhancement of estradiol induced apoptosis. FTS, a novel mTOR inhibitor will be used in vitro to activate the stress kinase pathway and apoptosis. In vivo, FTS will be used to enhance estradiol induced apoptosis in nude mice.
Showing the most recent 10 out of 18 publications
