PDK1 and PPARdelta Signaling in Mammary Tumorigenesis

这一提议将检验3-磷酸6-吲哚依赖的蛋白激酶-1(PDK1)激活和 与失活的in/Tcf靶基因--PPARtf相互作用。为了促进 乳腺肿瘤的发生。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 其中PDK1促进细胞增殖。我们将确定PDK1下游的Jff-catenin/Tcf-调制在 增殖、侵袭和干细胞自我更新。我们已经报道了PDK1及其下游效应蛋白PKCA， 转化过程中/？-cateninfi~cF靶基因、细胞周期蛋白D1和c-Myc的表达增加。我们现在有了 发现PDK1在ER(MCF-7)乳腺癌细胞中稳定表达可促进雌激素非依赖性生长 并增加β-连环蛋白/TCF和PPAR5依赖的转录活性。因此，MCF-7/PDK1的依赖性 PPAR&lt；5上的细胞将在有无PPARJ处理的情况下进行体外和在裸鼠体内的检测 激动剂。稳定表达PPARJ和/或PDK1基因的MEC和MCF-7细胞株将用于研究 这些基因在体外对生长、转化和侵袭以及对体内致瘤性的协同作用。 表达PDK1和PPARrf的乳腺癌细胞MDA-MB-231将被改造成表达dnTCF，dnPKCo， DnPDKI和dnPPAR？J，以确定生长和生长的依赖性 在存在和不存在PPARJ激动剂的情况下对这些信号通路的侵袭。MCF-7/PDK1和MEC/PDK1 细胞，以及新建立的表达高干细胞抗原-1(SCA-1)的乳腺癌细胞系(MC细胞)。 表达将被用来研究，PDK1信号在干细胞自我更新中的作用。目标2：确定 PDK1与PPAR的相互作用机制我们发现内源性PDK1与PPAR和PPAR直接相互作用 PPARJ在乳腺肿瘤细胞中表达，以及在瞬时表达这两种蛋白的细胞中表达。要在中研究这种相互作用 更详细地，将研究PDK1和PPAR&lt；5之间潜在相互作用位点的结构域图。我们会 确定PDK1是否作为PPAR&J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PDK1是否将PPAR和J的磷酸化，以及PDK1的丝氨酸和/或酪氨酸磷酸化是否为 他们的关系。PDK1与PPARrf共定位对PPARJ激动剂或生长因子受体的影响 将通过表达PDK1-GFP和PDK1-GFP的活肿瘤细胞的荧光共聚焦显微镜成像来研究激活 PPAR&lt；5-RFP。目的#3：确定PDK1和PPARrf的遗传后果和致癌作用 在乳腺中的表达。作为肿瘤启动和促进基因的PDK1和PPAR；J将在 没有或存在PPARrf激动剂饮食的转基因模型。MMTV-PPARrf转基因产生 在loxP-Stop-loxP-PPARJ小鼠中，乳腺特异性重组将被用来研究PPAR在 哺乳、复旧和肿瘤的发生。我们将评估MMTV-PPARrf转基因对 孕激素/DMBA致癌，以及它们对添加PPARtf激动剂的饮食的反应。后果是什么？ 将研究PDK1对乳腺功能和发病机制的空间、时间和可逆调节。 在一种新的由倍他司酮A诱导的转基因模型中，利用我们的转基因‘OncoBlue’受体/报告‘小鼠。这个 还将评估PDK1和PPARtf反式表达对SCA-1M乳腺干细胞的影响。

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.