NFkB, tissue polarity and apoptosis resistance

描述(申请人提供)：细胞凋亡抵抗在乳腺的恶性转化和转移中起关键作用。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耐药的机制。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MEC的存活是重要的，并且当MEC组装成极化的三维(3D)组织样结构时，无论MEC的生长或恶性状态，这一途径的效率都显著提高。我们确定3D组织结构中的凋亡抵抗与基因表达的整体抑制、NFkappaB炎症基因的诱导性降低、AP-1活性改变、JNK活性降低以及SMRT/N-COR/HDAC的表达和活性增加有关。由于抑制SAPK诱导SMRT/N-COR/HDAC活性并保护生长在3D中的MEC免受凋亡诱导，而降低3D组织结构中SMRT/N-COR的表达或HDAC活性允许通过外源性凋亡刺激而死亡，我们预测α6beta4整合素通过促进RAC和NFkappaB的激活来指导MEC的生存，但这一凋亡抵抗途径的效率受到SAPK和SMRT/N-COR/HDAC之间的串扰的影响。本研究的目的是明确α6beta4整合素如何通过RAC和NFkappaB引导MEC存活，并探讨SAPK通路和SMRT/N-COR活性作为这一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.α6β4整合素是如何激活NFkappaB和RAC的？我们将使用Beta4整合素突变小鼠的Beta4整合素表达结构和MEC，以及信号中间结构、NFkappaB功能分析和PAK下拉试验来绘制Beta4整合素如何调节NFkappaB和RAC，并使用蛋白质组学的生化操作来表征候选信号。2.RAC是如何激活NFkappaB调节MEC存活的？我们将使用突变的RAC GTP酶和信号中间产物，RNAi策略以及从PAK缺失小鼠分离的MEC来探索和识别与NFkappaB激活相关的RAC候选效应因子，并使用GST构建和蛋白质组学来定位新的RAC细胞靶点。3.NFkappaB如何在2D和3D中介导MEC存活？我们将使用微阵列、芯片、凝胶漂移和荧光拯救分析来确定NFkappaB的靶基因，并表征转录机制，以及表达和RNAi策略来评估与细胞凋亡抵抗相关的功能。4.SAPK-SMRT/N-COR串扰在MEC存活中的作用是什么？我们将使用突变型和野生型表达结构，结合RNAi和生化分析来表征SAPK-SMRT/N-COR串扰在培养的MEC存活中的作用。我们的研究应该有助于开发合理的肿瘤治疗方法。

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.