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（由申请方提供）：本提案将检验3-磷酸肌醇依赖性蛋白激酶-1（PDK 1）激活并与<$-连环蛋白/TCF靶基因过氧化物酶体增殖物激活受体-δ（PPARd）相互作用以促进乳腺肿瘤发生的假设。这将通过以下具体目的进行研究：目的#1：确定PDK 1增加增殖的机制。我们将确定PDK 1下游的连环蛋白/TCF调节在增殖、侵袭和干细胞自我更新中的作用。我们已经报道了PDK 1及其下游效应物PKCa在转化过程中增加了<$-catenin/TCF靶基因，细胞周期蛋白D1和c-Myc的表达。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<$-catenin/TCF和PPARd依赖性转录活性。因此，在存在和不存在PPARd激动剂处理的情况下，将在体外和裸鼠中检查MCF-7/PDK 1细胞对PPARd的依赖性。将使用稳定表达PPARd或PDK 1或这两种基因的MEC和MCF-7细胞系来研究这些基因之间对体外生长、转化和侵袭以及体内致瘤性的协同作用。表达PDK 1和PPARd的MDA-MB-231乳腺癌细胞将被工程化以在ponasterone A诱导型启动子下表达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传导途径的依赖性。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）表达的乳腺癌细胞系（MC细胞）将用于检查PDK 1信号传导在干细胞自我更新中的作用。目的#2：确定PDK 1与PPARd相互作用的机制。我们已经发现，内源性PDK 1直接与乳腺肿瘤细胞中的PPARJ相互作用，以及在瞬时表达这两种蛋白质的细胞中。为了更详细地研究这种相互作用，将检查PDK 1和PPARd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PPARd的共激活因子发挥作用，以及这种相互作用是否会改变转录活性。我们将确定PDK 1对PPARd的磷酸化，以及PDK 1的Ser和/或Tyr磷酸化是否是它们结合的先决条件。将通过表达PDK 1-GFP和PPARd-RFP的活肿瘤细胞的荧光共聚焦显微成像来研究响应于PPARd激动剂或生长因子受体活化的PDK 1与PPARd的共定位。目的#3：确定乳腺中PDK 1和PPARd表达的遗传后果和致瘤作用。将在不存在或存在PPARd激动剂饲料的转基因模型中分析作为肿瘤起始和促进基因的PDK 1和PPARd。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、退化和肿瘤发生中的作用。我们将评估MMTV-PPARd转基因对Streptestin/DMBA致癌作用的敏感性，以及它们对PPARd激动剂补充饮食的反应。PDK 1对乳腺功能和发病机制的空间、时间和可逆调节的后果将在新的ponasterone A诱导的转基因模型中进行检查，使用我们的转基因“OncoBlue”“受体/报告”小鼠。还将评价PDK 1和PPARd转基因表达对Sca-1（+）乳腺干细胞的影响。