The long term goal of this work is to determine the mechanism of action of steroid antagonists and the molecular basis for cellular resistance to anti-steroids that frequently develops in breast cancer. This proposal will focus on progesterone receptor (PR) and progesterone antagonists and thus will build on an experimental system that we have developed over several years. Based on our biochemical studies of PR and the biological behavior of current anti-progestins, in particular functional switching from antagonists to agonists by cAMP signaling pathways, we propose the following hypotheses: I) Anti-progestins bound to PR act in part as trans- repressors of receptor in the same cell bound to agonist. 2) Cellular resistance to anti-steroids can be explained by enhancement of the activation potential of the receptor-antagonist complex by alternate signal transduction pathways. This proposal will use combined biochemical, molecular and genetic approaches to investigate these novel mechanisms.
AIM #1 will determine whether anti-progestins can act in vivo as transrepressors by combined mechanisms of heterodimerization and competition for binding to specific DNA. On approach will rely on use of ligand specificity PR mutants (agonist or antagonist specific) for detection and functional analysis of heterodimers in the intact cell. Promoter interference will be used to determine the extent to which ligand specific PRs can compete for binding to DNA in vivo.
AIM #2 will determine whether different classes of anti-progestins promote the same or different conformational changes in PR structure and will correlate these changes with biological properties as either weak or strong transrepressors.
AIM #3 will isolate the cellular gene products involved in mediating the uncovering of the transcriptional potential of the PR-antagonist by alternate signaling pathways. We will use a powerful genomic mutagenesis and selection strategy with a functional PR system reconstituted in yeast.
Aim 3, therefore, will provide important first steps toward proving our hypothesis for cellular resistance to anti-steroids and for dissecting the molecular mechanisms that mediate signal transduction pathway crosstalk. The expectation of this research is to enhance our knowledge of steroid hormone action in general and to uncover novel receptor mechanisms that may explain the biological behavior of anti-progestins. Further, the hope is that mechanistic information derived from these studies can be exploited to improve the efficacy of current steroid antagonists as therapeutic agents or to develop novel antagonists.
Showing the most recent 10 out of 28 publications
