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）：该提案将测试 3-磷酸肌醇依赖性蛋白激酶-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-δ (PPARd) 促进乳腺肿瘤发生 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。的角色 ¿ -连环蛋白/TCF 调节 PDK1 下游增殖、侵袭和干细胞自我更新我们报道，PDK1 及其下游效应子 PKCa 增加了 ¿ -catenin/TCF 靶基因、细胞周期蛋白 D1 和 c-Myc 在转化过程中发现，PDK1 在 ER (+) MCF-7 乳腺癌细胞中的稳定表达可实现雌激素非依赖性生长并增加 ¿ -连环蛋白/TCF和PPARd依赖性转录活性因此，将在体外和在存在或不存在PPARd激动剂治疗的裸鼠中检查MCF-7/PDK1细胞对PPARd的依赖性。稳定表达 PPARd 或 PDK1 或两种基因的细胞系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 乳腺癌细胞将被改造为在松甾酮 A 诱导型启动子下表达 dnTCF、dnPKCo、dnPDKI 和 dnPPARd，以确定生长和侵袭对这些信号通路的依赖性。缺乏 PPARd 激动剂和 MCF-7/PDK1 和 MEC/PDK1 细胞，以及新建立的乳腺癌。表现出高干细胞抗原 1 (Sca-1) 表达的细胞系（MC 细胞）将用于检查 PDK1 信号传导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，我们对 PDK1 和 PPARJ 之间潜在的相互作用位点进行了域图谱分析。我们将检查 PPARd 是否作为 PPARd 的共激活剂发挥作用，以及这种相互作用是否会改变转录活性。我们将确定 PDK1 的 PPARd 磷酸化以及 PDK1 的 Ser 和/或 Tyr 磷酸化是否是它们共定位的先决条件。将通过荧光共焦显微成像研究表达 PPARd 的活体肿瘤细胞对 PPARd 激动剂或生长因子受体激活的 PDK1 与 PPARd 的反应。 PDK1-GFP 和 PPARd-RFP。 目标 #3：确定乳腺中 PDK1 和 PPARd 表达的遗传后果和致瘤效应，因为将在转基因模型中分析不存在或存在的肿瘤启动和促进基因。 LoxP-Stop-loxP-PPARd 中乳腺特异性重组产生的 PPARd 激动剂饮食。小鼠，将用于研究 PPARS 在哺乳、退化和肿瘤发生中的作用，我们将评估 MMTV-PPARd 转基因对孕激素/DMBA 致癌的敏感性，以及它们对补充 PPARd 激动剂的饮食的反应。 PDK1 对乳腺功能和发病机制的空间、时间和可逆调节将在新的 ponasterone A 诱导转基因模型中进行检查，利用我们的转基因“OncoBlue”“受体/报告基因”小鼠还将评估 PDK1 和 PPARd 转基因表达对 Sca-1 (+) 乳腺干细胞的影响。