PDK1 and PPARdelta Signaling in Mammary Tumorigenesis

该提案将检验3-磷酸肌醇依赖性蛋白激酶-1（PDK 1）激活并 与转录因子靶基因过氧化物酶体增殖物激活受体-δ（PPARtf）相互作用。促进 乳腺肿瘤发生这将通过以下具体目标进行研究：目标#1：通过以下方式确定机制： PDK 1增加增殖。我们将确定PDK 1下游的jff-catenin/TCF-调节在 增殖、侵袭和干细胞自我更新。我们已经报道了PDK 1及其下游效应物，PKCa， 增加的表达的/？- cateninfi~CF靶基因、细胞周期蛋白D1和c-Myc。我们现在已经 发现PDK 1在ER（+）MCF-7乳腺癌细胞中的稳定表达赋予了雌激素非依赖性生长 并增加了β-连环蛋白/TCF和PPAR 5依赖的转录活性。因此，MCF-7/PDK 1的依赖性 将在体外和在存在和不存在PPARJ治疗的裸鼠中检查PPARJ <5的细胞 激动剂将使用稳定表达PPARJ或PDK 1或这两种基因的MEC和MCF-7细胞系来研究细胞周期。 这些基因在体外对生长、转化和侵袭以及体内对致瘤性的协同作用。 表达PDK 1和PPARrf的MDA-MB-231乳腺癌细胞将被工程化以表达dnTCF、dnPKCo， 在ponasterone A-诱导型启动子下的dnPDKI和dnPPAR？J，以确定生长和 在存在和不存在PPARJ激动剂的情况下对这些信号传导途径的侵袭。MCF-7/PDK 1和MEC/PDK 1 细胞，以及新建立的表现出高干细胞抗原-1（Sca-1）的乳腺癌细胞系（MC细胞）， 表达将用于检查PDK 1信号传导在干细胞自我更新中的作用。目标#2：确定 我们已经发现内源性PDK 1直接与 PPARJ在乳腺肿瘤细胞中，以及在瞬时表达两种蛋白质的细胞中。为了研究这种相互作用， 更详细地，将检查PDK 1和PPAR 5之间相互作用的潜在位点的结构域映射。我们将 确定PDK 1是否作为PPAR α的共激活因子发挥作用，以及这种相互作用是否改变转录活性。我们 将确定PDK 1对PPAR α的磷酸化，以及PDK 1的Ser和/或Tyr磷酸化是否是 他们的协会。响应于PPARj激动剂或生长因子受体的PDK 1与PPARrf的共定位 将通过表达PDK 1-GFP的活肿瘤细胞的荧光共聚焦显微成像来研究活化， PPAR<5-RFP。目的#3：确定PDK 1和PPARrf的遗传后果和致瘤作用 在乳腺中的表达。作为肿瘤起始和促进基因的PDK 1和PPARJ将在 在不存在或存在PPARrf激动剂饮食的情况下的转基因模型。MMTV-PPARrf转基因产生的 在loxP-Stop-loxP-PPARJ小鼠中的乳腺特异性重组，将用于研究PPARS在 哺乳、退化和肿瘤发生。我们将评估MMTV-PPARrf转基因对 DMBA致癌作用，以及它们对补充PPARtf激动剂的饮食的反应。后果 PDK 1对乳腺功能和发病机制的空间、时间和可逆调节将被检查 在一种新的ponasterone A-诱导的转基因模型中，利用我们的转基因“OncoBlue”“受体/报告基因”小鼠。的 还将评价PDK 1和PPARtf transaene表达对Sca-1 M乳腺干细胞影响。

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<J. 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 PPAR<5 will be examined. We will determine if PDK1 functions as a coactivator of PPAR<J and whether this interaction alters transcriptional activity. We will determine if phosphorylation of PPAR<J by PDK1, and Ser and/or Tyr phosphorylation of PDK1 are prerequisites for their association. Colocalization of PDK1 with PPARrf in response to PPARJ agonist or growth factor receptor activation will be studied by fluorescent confocal microscopic imaging of live tumor cells expressing PDK1-GFP and PPAR<5-RFP. Aim #3: Determine the genetic consequences and tumorigenic effects of PDK1 and PPARrf expression in the mammary gland. PDK1 and PPAR<J as tumor initiating and promoting genes will be analyzed in transgenic models in the absence or presence of a PPARrf agonist diet. MMTV-PPARrf transgenics generated by mammary gland-specific recombination in loxP-Stop-loxP-PPARJ mice, will be used to study the role of PPARS in lactation, involution and tumorigenesis. We will evaluate the sensitivity of MMTV-PPARrf transgenics to progestin/DMBA carcinogenesis, as well as their response to a PPARtf 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 PPARtf transaene expression on Sca-1 M mammary stem cells will also be evaluated.