Involvement of leptin and interleukin-1 signaling in mammary cancer progression

描述（由申请人提供）：通知编号：NOT-OD_09-058通知标题：NIH宣布为竞争性修订申请提供恢复法案资金。在子宫内膜癌和乳腺癌（BC）细胞中发现的瘦素和IL-1信号之间的特异性串扰可能参与其促血管生成作用。据推测，瘦素诱导的乳腺癌效应可能发生在瘦素信号传导MAPK和NFkB的激活以及VEGF/VEGFR 2表达增加后，这可能与IL-1信号传导有关或部分受IL-1信号传导调节。为了检验这一假设，将开发涉及小鼠（4 T1、EMT 6和MMT）和人BC细胞（MCF-7和MDA-MB 231）体外研究的三个目的。目的1：在小鼠和人BC细胞中研究瘦素参与IL-1系统表达的调节机制（配体、受体和拮抗剂）。为了确定IL-1和IL-1 R启动子-荧光素酶报告基因（不同转录因子的完整和定点突变）的瘦素转录调控机制，将使用。将在小鼠和人BC细胞中分析ER表达和活化状态与瘦素- IL-1串扰的关系。在目的2中，IL-1信号传导中介物的瘦素调节（即，MyD 88、IRAK 1、IRAK 4和TRAF 6）以及瘦素介导的MAPK和MAP 3激酶（TAK 1和MEKK 1）以及与IL-1信号传导相关的JNK和P38激酶的活化之间的关系。将使用不同的载体和ChIP测定来研究MyD 88的基因调控。ChIP试验和免疫共沉淀试验将用于鉴定瘦素激活的转录因子与MyD 88基因的特异性结合，并确定MyD 88同源二聚化的程度。目的3探讨瘦素对乳腺癌细胞VEGF和VEGFR 2表达的调控机制。在保留转录因子的其他结合区域的同时，将使用一系列主要顺式作用元件单独缺失的VEGF启动子-报告基因构建体。为了研究含有全长VEGFR 2启动子和AP 2的截短形式的VEGR 2荧光素酶构建体的瘦素调节机制，将使用NFkB结合缺失。来自这些研究的数据将提供强有力的科学依据，以了解瘦素-IL-1串扰，并评估是否瘦素信号转导的中断可以作为一种新的方法，用于预防/治疗BC。 公共卫生相关性：这些研究的结果将使我们更接近于潜在的瘦素信号传导（PEG-LPrA）的翻译用途，用于预防和/或治疗BC，这对肥胖和绝经后妇女非常重要。PEG-LPrA可能是一种有用的生物制剂或佐剂，可以提高BC预防/治疗方案的功效。

DESCRIPTION (provided by applicant): Notice Number:NOT-OD_09-058 Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. Specific cross-talk between leptin and IL-1 signaling found in endometrial and breast cancer (BC) cells could be involved in their pro-angiogenic effects. It is hypothesized that the leptin-induced effects on breast cancer could occur upon leptin signaling the activation of MAPK and NFkB and, an increased expression of VEGF/VEGFR2, which may be linked to, or regulated, in part by IL-1 signaling. To test this hypothesis three Aims involving in vitro investigations in mouse (4T1, EMT6 and MMT) and human BC cells (MCF-7 and MDA-MB231) will be developed. In Aim 1, leptin's regulatory mechanisms involved in IL-1 system expression (ligand, receptor and antagonist) will be investigated in mouse and human BC cells. To determine mechanisms of leptin-transcriptional regulation of IL-1 and IL-1R promoter-luciferase reporters (intact and site-directed mutagenesis for different transcription factors) will be used. ER expression and activation status relationships to leptin- IL-1 crosstalk will be analyzed in mouse and human BC cells. In Aim 2, leptin regulation of IL-1 signaling intermediaries (i.e., MyD88, IRAK1, IRAK4 and TRAF6) and relationships between leptin-mediated activation of MAPK and MAP3 kinases (TAK1 and MEKK1) and JNK and P38 kinases related to IL-1 signaling will be investigated. Different vectors and ChIP assays will be used to study gene regulation of MyD88. ChIP assays and co-immunoprecipitation tests will be used to identify specific binding of leptin-activated transcription factors to MyD88 gene and to determine the extent of MyD88 homodimerization. Aim 3 will investigate the molecular mechanism(s) involved in the leptin regulation of VEGF and VEGFR2 in breast cancer cells. While retaining the other binding regions for transcription factors, a series of VEGF promoter-reporter constructs with major cis-acting elements individually deleted will be used. To investigate the mechanisms of leptin regulation of VEGR2 luciferase constructs containing full-length of VEGFR2 promoter and truncated versions for AP2, NFkB binding deletions will be used. Data from these investigations will provide strong scientific basis to understand leptin-IL-1 crosstalk and to assess whether disruption of leptin-signaling could serve as a novel method for prevention/treatment of BC. PUBLIC HEALTH RELEVANCE: Results from these investigations would allow us to be a step closer to the potential translational use of inhibitoes [sic] of leptin signaling (PEG-LPrA) for prevention and/or treatment of BC that is highly important for obese and post-menopausal women. PEG-LPrA could be a useful biological agent or adjuvant to improve the efficacy of preventive/therapeutic regimens for BC.