This proposal will test the hypothesis that 3-phosph6inbsitide-dependent protein kinase-1 (PDK1) activates and interacts with the ff-eaten in/TCF-target gene, peroxisome proliferator-activated receptor-delta (PPARtf). to promote mammary tumorigenesis. This will be studied by the following Specific Aims:
Aim #1 : Determine the mechanism by which PDK1 increases proliferation. We will determine the role of jff-catenin/TCF-modulationdownstream of PDK1 in proliferation, invasion and stem cell self-renewal. We have reported that PDK1 and its downstream effector, PKCa, ncreased expression of the /?-cateninfi~CF target genes, cyclin D1 and c-Myc during transformation. We have now discovered that stable expression of PDK1 in ER (+) MCF-7 breast cancer cells confers estrogen-independent growth and increases ?-catenin/TCF and PPAR5-dependent transcriptional activity. Thus, the dependency of MCF-7/PDK1 cells on PPAR<5 will be examined in vitro and in nude mice in the presence and absence of treatment with a PPARJ agonist. MEC and MCF-7 cell lines stably expressing either PPARJ or PDK1 or both genes will be used to study the synergy between these genes on growth, transformation and invasion in vitro, as well as on tumorigenicity in vivo. MDA-MB-231 breast cancer cells, which express PDK1 and PPARrf, will be engineered to express dnTCF, dnPKCo, dnPDKI and dnPPAR?J under a ponasterone A-inducible promoter, to determine the dependency of growth and invasion on these signaling pathways in the presence and absence of a PPARJ agonist. MCF-7/PDK1 and MEC/PDK1 cells, as well as a newly established mammary carcinoma cell line (MC cells) exhibiting high stem cell antigen-1 (Sca-1) expression will be used to examine the role of, PDK1 signaling in stem cell self-renewal.
Aim #2 : Determine the mechanism by which PDK1 interacts with PPAR
