NFkB, tissue polarity and apoptosis resistance

描述（由申请人提供）：细胞凋亡抵抗在乳腺恶性转化和转移中起关键作用。 我们的目标是通过描述整合素-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潜在机制。 我们发现，层粘连蛋白连接α 6 β 4整联蛋白和激活RAC和NF κ B是重要的MEC生存，这一途径的效率，无论生长或恶性状态，显着增强时，MEC组装成极化的三维（3D）组织样结构。 我们确定了三维组织结构中的凋亡抗性与基因表达的整体抑制、NF κ B炎性基因的诱导减少、AP-1活性改变、JNK活性降低以及SMRT/N-CoR/HDAC的表达和活性增加相关。 因为抑制SAPK诱导SMRT/N-CoR/HDAC活性并保护在3D中生长的MEC免于凋亡诱导，并且降低3D组织结构中的SMRT/N-CoR表达或HDAC活性允许通过外源性凋亡刺激死亡，我们预测α 6 β 4整联蛋白通过促进RAC和NF κ B的激活来指导MEC的存活，但是这种抗凋亡途径的效率受到SAPK和SMRT/N-CoR/HDAC之间的串扰的影响。 本提案中概述的研究目的是确定α 6 β 4整合素如何通过RAC和NF κ B指导MEC存活，并探索SAPK途径和SMRT/N-CoR活性作为该途径的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。α 6 β 4整合素如何激活NF κ B和RAC？ 我们将使用突变的β 4整联蛋白表达构建体和来自β 4整联蛋白突变小鼠的MEC，连同信号传导中间体构建体、NF κ B功能测定和PAK下拉测定来绘制β 4整联蛋白如何调节NF κ B和RAC，以及使用蛋白质组学的生化操作来表征信号传导候选物。 2. RAC如何激活NF κ B以调节MEC存活？ 我们将使用突变的RAC GTP酶和信号中间体，RNAi策略以及从PAK敲除小鼠中分离的MEC来探测RAC的作用并鉴定与NF κ B激活相关的RAC的候选效应子，以及GST构建体和蛋白质组学来绘制新的RAC细胞靶点。 3. NF κ B如何在2D和3D中介导MEC存活？ 我们将使用微阵列，芯片，凝胶移位和荧光救援分析，以确定NF κ B的靶基因和转录机制的特点，表达和RNAi策略，以评估功能相关的抗凋亡。 4. SAPK-SMRT/N-CoR串扰在MEC存活中的作用是什么？ 我们将使用突变体和野生型表达构建体，以及RNAi和生化测定来表征SAPK-SMRT/N-CoR串扰在培养的MEC存活中的作用。 我们的研究将促进合理的肿瘤治疗的发展。

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.