PDK1 and PPARdelta Signaling in Mammary Tumorigenesis

描述（由申请人提供）：该提案将验证3-磷酸肌苷依赖性蛋白激酶-1 （PDK1）激活并与¿-catenin/ tcf靶基因，过氧化物酶体增殖物激活受体- δ (PPARd)相互作用以促进乳腺肿瘤发生的假设。这将通过以下具体目标进行研究：目标1：确定PDK1增加增殖的机制。我们将确定PDK1下游的¿-catenin/ tcf调节在增殖、侵袭和干细胞自我更新中的作用。我们已经报道了PDK1及其下游效应物PKCa在转化过程中增加了¿-catenin/TCF靶基因、cyclin D1和c-Myc的表达。我们现在已经发现，在ER (+) MCF-7乳腺癌细胞中，PDK1的稳定表达赋予了不依赖雌激素的生长，并增加了¿-catenin/TCF和ppar依赖的转录活性。因此，MCF-7/PDK1细胞对PPARd的依赖性将在体外和裸鼠中进行检测，无论是否使用PPARd激动剂。MEC和MCF-7细胞系稳定表达PPARd或PDK1或两种基因，将用于研究这些基因在体外生长、转化和侵袭以及体内致瘤性方面的协同作用。表达PDK1和PPARd的MDA-MB-231乳腺癌细胞将在ponasterone a诱导的启动子下表达dnTCF、dnPKCo、dnPDKI和dnPPARd，以确定在存在和不存在PPARd激动剂的情况下，生长和侵袭对这些信号通路的依赖性。MCF-7/PDK1和MEC/PDK1细胞，以及新建立的具有高干细胞抗原-1 （Sca-1）表达的乳腺癌细胞系（MC细胞）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PPARd相互作用的机制。我们发现内源性PDK1在乳腺肿瘤细胞以及瞬时表达这两种蛋白的细胞中直接与PPARJ相互作用。为了更详细地研究这种相互作用，我们将研究PDK1和PPARd之间相互作用的潜在位点的结构域映射。我们将确定PDK1是否作为PPARd的共激活因子，以及这种相互作用是否会改变转录活性。我们将确定PDK1对PPARd的磷酸化，以及PDK1的Ser和/或Tyr磷酸化是否是它们关联的先决条件。通过对表达PDK1- gfp和PPARd- rfp的活肿瘤细胞进行荧光共聚焦显微镜成像，研究PDK1与PPARd共定位对PPARd激动剂或生长因子受体激活的响应。目的3：确定乳腺中PDK1和PPARd表达的遗传后果和致瘤作用。PDK1和PPARd作为肿瘤启动和促进基因将在缺乏或存在PPARd激动剂饮食的转基因模型中进行分析。在loxP-Stop-loxP-PPARd小鼠中通过乳腺特异性重组产生的MMTV-PPARd转基因，将用于研究PPARS在泌乳、复归和肿瘤发生中的作用。我们将评估MMTV-PPARd转基因对黄体酮/DMBA致癌的敏感性，以及它们对PPARd激动剂补充饮食的反应。PDK1的时空和可逆调控对乳腺功能和发病机制的影响将在一个新的ponasterone a诱导的转基因模型中进行研究，利用我们的转基因“OncoBlue”“受体/报告”小鼠。PDK1和PPARd转基因表达对Sca-1（+）乳腺干细胞的影响也将被评估。

DESCRIPTION (provided by applicant): 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.