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: 7575766
• 负责人: ROBERT I. GLAZER
• 金额: $ 26.65万
• 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/7575766
• 关键词: Accounting; Address; Agonist; Anchorage-Independent Growth; Animal Model; Animals; Antigens; Breast Adenocarcinoma; Breast Cancer Cell; Breast Carcinoma; CCND1 gene; Cancer cell line; Cell Line; Cells; Collaborations; Cooperative Breast Cancer Tissue Resource; Cyclin D1; Data; Dependency; Diet; Dominant-Negative Mutation; Eating; Engineering; Epithelial Cells; Estrogens; Event; Exhibits; Figs - dietary; Gene Targeting; Genes; Genetic; Genetic Recombination; Goals; Growth; Growth Factor Receptors; Human; Image; In Vitro; Isogenic transplantation; Laboratories; Lactation; Life; MCF7 cell; Malignant Epithelial Cell; Mammary Neoplasms; Mammary Tumorigenesis; Mammary gland; Microscopic; Modeling; Mus; Nude Mice; Oncogenic; PPAR delta; Pathogenesis; Pathway interactions; Phosphorylation; Physiological; Progestins; Protein Kinase; Proteins; Publishing; Receptor Activation; Regulation; Reporter; Reporter Genes; Reporting; Research Personnel; Response Elements; Role; Signal Pathway; Signal Transduction; Site; Stem cells; Testing; Transactivation; Transgenic Animals; Transgenic Mice; Transgenic Model; Transgenic Organisms; Tumor Promoters; Tumorigenicity; United States National Institutes of Health; Xenograft procedure; bcl-1 Genes; c-myc Genes; carcinogenesis; dietary supplements; dimethylbenzanthracene; domain mapping; ecdysone receptor; human disease; in vitro activity; in vivo; keratinocyte; malignant breast neoplasm; mammary epithelium; neoplastic cell; ponasterone A; programs; promoter; receptor; response; self-renewal; transgene expression; tumor; tumor initiation; tumor progression; tumorigenesis; tumorigenic

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.

该提案将检验以下假设：3-磷6inbsitide依赖性蛋白激酶-1（PDK1）激活和 与/tcf-target基因中的FF摄入的过氧化物组增殖者激活受体 - 戴尔塔（PPARTF）相互作用。促进 乳腺肿瘤发生。这将通过以下特定目的研究：目标＃1：确定机制 哪个PDK1增加了增殖。我们将确定pdk1在 增殖，入侵和干细胞自我更新。我们报告说PDK1及其下游效应器PKCA， 转化过程中 /？ - cateninfi〜cf靶基因，细胞周期蛋白D1和C-MYC的表达升高。我们现在有 发现PDK1在ER（+）MCF-7乳腺癌细胞中的稳定表达承认雌激素非依赖性生长 并增加� -Catenin/TCF和PPAR5依赖性转录活性。那，MCF-7/PDK1的依赖性 在存在和不使用PPARJ治疗的情况下，将在体外和裸鼠中检查PPAR <5的细胞 MEC和MCF-7细胞系稳定地表达PPARJ或PDK1或两个基因将用于研究 这些基因在体外的生长，转化和侵袭以及体内的肿瘤性之间的协同作用。 表达PDK1和PPARRF的MDA-MB-231乳腺癌细胞将被设计为表达DNTCF，DNPKCO， DNPDKI和DNPPAR j在Ponasterone A-诱导启动子下，以确定生长的依赖性和 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 细胞以及新建立的乳腺癌细胞系（MC细胞），表现出高干细胞抗原1（SCA-1） 表达将用于检查PDK1信号在干细胞自我更新中的作用。目标＃2：确定 PDK1与PPAR <J相互作用的机制。我们发现内源性PDK1与 乳腺肿瘤细胞以及瞬时表达这两种蛋白质的细胞中的PPARJ。研究这种相互作用 将检查更详细的是，将检查PDK1和PPAR <5之间的潜在相互作用位点的域映射。我们将 确定PDK1是否充当PPAR <J的共激活因子，并且这种相互作用是否会改变转录活性。我们 将确定PDK1对PPAR的磷酸化以及PDK1的Ser和/或Tyr磷酸化是否是前提条件 他们的协会。 PDK1与PPARRF的共定位响应PPARJ激动剂或生长因子接收器 激活将通过表达PDK1-GFP和 PPAR <5-RFP。 AIM＃3：确定PDK1和PPARRF的遗传后果和致瘤作用 在乳腺中的表达。将在 在不存在或存在PPARRF激动剂饮食的情况下转基因模型。 MMTV-PPARRF TRONSSICES生成 Loxp-Stop-Stop-loxp-Pparj小鼠中的乳腺特异性重组将用于研究PPAR在PPAR中的作用 哺乳，涉及和肿瘤发生。我们将评估MMTV-PPARRF Transformics对 孕激素/DMBA癌变及其对PPARTF激动剂补充饮食的反应。后果 将检查PDK1对乳腺功能和发病机理的空间，临时和可逆调节 在新的Ponasterone A诱导的转基因模型中，使用我们的转基因“ Coblue”“受体/报告基因”小鼠。这 还将评估PDK1和PPARTF Transaene表达对SCA-1 M乳腺干细胞的影响。