NFkB, tissue polarity and apoptosis resistance

NFkB、组织极性和细胞凋亡抵抗

• 批准号: 6822394
• 负责人: VALERIE MARIE WEAVER
• 金额: $ 25.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6822394
• 关键词: AP1 protein; JUN kinase; RNA interference; acinar cell; amidohydrolases; apoptosis; basement membrane; bioinformatics; breast neoplasms; cellular oncology; cellular polarity; chromatin immunoprecipitation; enzyme activity; extracellular matrix; gel mobility shift assay; gene expression; genetic regulation; genetically modified animals; human tissue; integrins; laboratory mouse; mammary epithelium; microarray technology; neoplastic process; nuclear factor kappa beta; protein structure function; proteomics

DESCRIPTION (provided by applicant): Apoptosis resistance plays a key role in malignant transformation and metastasis of the breast. Our goal is to understand mechanisms underlying apoptosis resistance in breast cancer by characterizing the role of integrin-extracellular matrix interactions in mammary epithlelial cell (MEC) survival. We found that laminin ligation of alpha6beta4 integrin and activation of RAC and NFkappaB are important for MEC survival, and that the efficiency of this pathway, irrespective of growth or malignancy status, is significantly enhanced when MECs assemble into polarized 3-dimensional (3D) tissue-like structures. We determined that apoptosis resistance in 3D tissue structures is associated with global repression of gene expression, decreased inducibility of NFkappaB inflammatory genes, altered AP-1 activity, reduced JNK activity and increased expression and activity of SMRT/N-CoR/HDAC. Because inhibiting SAPK induces SMRT/N-CoR/HDAC activity and protects MECs grown in 3D from apoptosis induction, and reducing SMRT/N-CoR expression or HDAC activity in 3D tissue structures permits death via exogenous apoptotic stimuli, we predict that alpha6beta4 integrin directs survival in MECs by facilitating activation of RAC and NFkappaB, but that the efficiency of this apoptosis resistance pathway is tempered by crosstalk between SAPK and SMRT/N-CoR/HDAC. The objective of the research outlined in this proposal is to define how alpha6beta4 integrin directs MEC survival through RAC and NFkappaB, and to explore the role of the SAPK pathway and SMRT/N-CoR activity as critical regulators of this pathway. We will achieve this goal by answering the following questions: 1. How does alpha6beta4 integrin activate NFkappaB and RAC? We will use mutant beta4 integrin expression constructs and MECs from beta4 integrin mutant mice, together with signaling intermediate constructs, NFkappaB functional assays and a PAK pulldown assay to map how beta4 integrin regulates NFkappaB and RAC, and biochemical manipulations with proteomics to characterize signaling candidates. 2. How does RAC activate NFkappaB to regulate MEC survival? We will use mutant RAC GTPases and signaling intermediates, and RNAi strategies as well as MECs isolated from a PAK null mouse to probe the role of and to identify candidate effectors of RAC linked to NFkappaB activation, and GST constructs and proteomics to map novel RAC cellular targets. 3. How does NFkappaB mediate MEC survival in 2D and 3D? We will use microarrays, CHIP, gel shift and fluorescence rescue assays to identify NFkappaB target genes and characterize transcriptional mechanisms, and expression and RNAi strategies to assess functional relevance to apoptosis resistance. 4. What is the role of SAPK-SMRT/N-CoR crosstalk in MEC survival? We will use mutant and wild type expression constructs, together with RNAi and biochemical assays to characterize the role of SAPK-SMRT/N-CoR crosstalk in MEC survival in culture. Our studies should facilitate the development of rational tumor therapies.

描述（申请人提供）：细胞凋亡抵抗在乳腺恶变和转移中发挥关键作用。 我们的目标是通过表征整合素-细胞外基质相互作用在乳腺上皮细胞（MEC）存活中的作用来了解乳腺癌细胞凋亡抵抗的机制。 我们发现，α6β4 整合素的层粘连蛋白连接以及 RAC 和 NFkappaB 的激活对于 MEC 的存活非常重要，并且当 MEC 组装成极化的 3 维（3D）组织时，无论生长或恶性状态如何，该途径的效率都会显着增强类结构。 我们确定，3D 组织结构中的细胞凋亡抵抗与基因表达的整体抑制、NFkappaB 炎症基因的诱导能力降低、AP-1 活性改变、JNK 活性降低以及 SMRT/N-CoR/HDAC 表达和活性增加有关。 因为抑制 SAPK 会诱导 SMRT/N-CoR/HDAC 活性并保护 3D 中生长的 MEC 免受凋亡诱导，并且减少 3D 组织结构中的 SMRT/N-CoR 表达或 HDAC 活性允许通过外源凋亡刺激而死亡，因此我们预测 α6β4 整合素会指导死亡通过促进 RAC 和 NFkappaB 的激活来促进 MEC 的存活，但是这种细胞凋亡抵抗途径的效率受到两者之间的串扰的影响SAPK 和 SMRT/N-CoR/HDAC。 本提案中概述的研究目的是确定 alpha6beta4 整合素如何通过 RAC 和 NFkappaB 指导 MEC 存活，并探索 SAPK 通路和 SMRT/N-CoR 活性作为该通路关键调节因子的作用。 我们将通过回答以下问题来实现这一目标： 1. alpha6beta4 整合素如何激活 NFkappaB 和 RAC？ 我们将使用突变型β4整合素表达构建体和来自β4整合素突变小鼠的MEC，以及信号传导中间构建体、NFkappaB功能测定和PAK下拉测定来绘制β4整合素如何调节NFkappaB和RAC，并通过蛋白质组学进行生化操作来表征信号传导候选物。 2. RAC如何激活NFkappaB来调节MEC存活？ 我们将使用突变的 RAC GTPases 和信号中间体、RNAi 策略以及从 PAK null 小鼠中分离的 MEC 来探测与 NFkappaB 激活相关的 RAC 的作用并识别候选效应子，并使用 GST 构建体和蛋白质组学来绘制新的 RAC 细胞图谱目标。 3. NFkappaB 如何介导 MEC 在 2D 和 3D 中的存活？ 我们将使用微阵列、CHIP、凝胶位移和荧光救援测定来鉴定 NFkappaB 靶基因并表征转录机制，以及表达和 RNAi 策略来评估与细胞凋亡抗性的功能相关性。 4. SAPK-SMRT/N-CoR 串扰对 MEC 存活有何作用？ 我们将使用突变型和野生型表达构建体，以及 RNAi 和生化测定来表征 SAPK-SMRT/N-CoR 串扰在 MEC 培养中存活的作用。 我们的研究应该促进合理的肿瘤疗法的发展。