PDK1 and PPARdelta Signaling in Mammary Tumorigenesis

PDK1 和 PPARdelta 信号转导在乳腺肿瘤发生中的作用

• 批准号: 7090940
• 负责人: ROBERT I. GLAZER
• 金额: $ 27.45万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7090940
• 关键词: athymic mouse; breast neoplasms; carcinogenesis; cell line; cyclins; diet; estrogens; gene targeting; genes; mammary gland; model; neoplasm /cancer; neoplastic cell; phosphorylation; progestins; proteins; receptor; receptor expression; role; stem cells; transfection

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.

描述(由申请人提供)：这项建议将检验这一假设，即3-磷酸肌醇依赖的蛋白激酶-1(PDK1)激活并与连环蛋白/TCF-靶基因--过氧化物酶体增殖物激活受体-β(PPARd)相互作用，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：确定PDK1促进增殖的机制。我们将确定PDK1下游的连环蛋白/TCF调节因子在增殖、侵袭和干细胞自我更新中的作用。我们已经报道，PDK1及其下游效应蛋白PKCA在转化过程中增加了目的基因-连环蛋白/TCF的表达，即细胞周期蛋白D1和c-Myc。我们现在已经发现，在ER(+)的MCF-7乳腺癌细胞中稳定表达PDK1可以促进雌激素非依赖性的生长，并增加β-catenin/TCF和PPARd依赖的转录活性。因此，在存在和不存在PPARd激动剂的情况下，将在体外和裸鼠体内检测MCF-7/PDK1细胞对PPARd的依赖性。稳定表达PPARd或PDK1或两种基因的MEC和MCF-7细胞株将被用来研究这些基因在体外对生长、转化和侵袭的协同作用以及对体内致瘤性的影响。表达PDK1和PPARd的乳腺癌细胞MDA-MB-231将在PPARd激动剂存在和不存在的情况下，在可诱导启动子的作用下表达dnTCF、dnPKCo、dnPDKI和dnPPARd，以确定这些信号通路对生长和侵袭的依赖性。利用McF-7/PDK1和MEC/PDK1细胞，以及新建立的高表达干细胞抗原-1(SCA-1)的乳腺癌细胞系(MC细胞)，研究PDK1信号在干细胞自我更新中的作用。目的#2：确定PDK1与PPARd相互作用的机制。我们发现，在乳腺肿瘤细胞中，内源性PDK1与PPARJ直接相互作用，在瞬时表达这两种蛋白的细胞中也是如此。为了更详细地研究这种相互作用，将检查PDK1和PPARd之间潜在相互作用位点的结构域图。我们将确定PDK1是否作为PPARd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对PPARd的磷酸化，以及PDK1的Ser和/或Tyr磷酸化是否是它们结合的先决条件。通过表达PDK1-GFP和PPARd-RFP的活肿瘤细胞的荧光共聚焦显微镜成像，将研究PDK1和PPARd在PPARd激动剂或生长因子受体激活反应中的共定位。目的#3：确定乳腺中PDK1和PPARd表达的遗传后果和致癌作用。作为肿瘤启动和促进基因的PDK1和PPARd将在没有或存在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转基因将被用于研究PPAR在哺乳、退化和肿瘤发生中的作用。我们将评估MMTV-PPARd转基因基因对孕激素/DMBA致癌的敏感性，以及它们对添加PPARd激动剂的饮食的反应。我们将利用我们的转基因‘OncoBlue’受体/报告小鼠，在一种新的可诱导的转基因模型中，研究PDK1对乳腺功能和发病机制的空间、时间和可逆调节的后果。此外，还将评估PDK1和PPARd转基因表达对SCA-1(+)乳腺干细胞的影响。