This proposal will test the hypothesis that 3-phosphoinositide-dependent protein kinase-1 (PDK1) activates and interacts with the ?-catenin/TCF-target gene, peroxisome proliferator-activated receptor-delta (PPARd) 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 ?-catenin/TCF-modulation downstream of PDK1 in proliferation, invasion and stem cell self-renewal. We have reported that PDK1 and its downstream effector, PKCa, increased expression of the ?-catenin/TCF 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 PPARd-dependent transcriptional activity. Thus, the dependency of MCF-7/PDK1 cells on PPARd will be examined in vitro and in nude mice in the presence and absence of treatment with a PPARd agonist. MEC and MCF-7 cell lines stably expressing either PPARd 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 PPARd, will be engineered to express dnTCF, dnPKCo, dnPDKI and dnPPARd 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 PPARd 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 PPARd. We have found that endogenous PDK1 interacts directly with PPARJ in mammary tumor cells, as well as in cells transiently expressing both proteins. To study this interaction in more detail, domain mapping of potential sites of interaction between PDK1 and PPARd will be examined. We will determine if PDK1 functions as a coactivator of PPARd and whether this interaction alters transcriptional activity. We will determine if phosphorylation of PPARd by PDK1, and Ser and/or Tyr phosphorylation of PDK1 are prerequisites for their association. Colocalization of PDK1 with PPARd in response to PPARd agonist or growth factor receptor activation will be studied by fluorescent confocal microscopic imaging of live tumor cells expressing PDK1-GFP and PPARd-RFP.
Aim #3 : Determine the genetic consequences and tumorigenic effects of PDK1 and PPARd expression in the mammary gland. PDK1 and PPARd as tumor initiating and promoting genes will be analyzed in transgenic models in the absence or presence of a PPARd agonist diet. MMTV-PPARd transgenics generated by mammary gland-specific recombination in loxP-Stop-loxP-PPARd mice, will be used to study the role of PPARS in lactation, involution and tumorigenesis. We will evaluate the sensitivity of MMTV-PPARd transgenics to progestin/DMBA carcinogenesis, as well as their response to a PPARd agonist-supplemented diet. The consequences of spatial, temporal and reversible regulation of PDK1 on mammary gland function and pathogenesis will be examined in a new ponasterone A-inducible transgenic model, utilizing our transgenic 'OncoBlue' 'receptor/reporter' mice. The influence of PDK1 and PPARd transgene expression on Sca-1 (+) mammary stem cells will also be evaluated. ? ? ?
