Re-activation of maspin tumor suppressor gene by designed transcription factors

设计的转录因子重新激活maspin抑癌基因

基本信息

批准号：
7569482
负责人：
PILAR BLANCAFORT
金额：
$ 29.3万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7569482
关键词：
Affect Affinity Animal Model Apoptosis Apoptotic Base Pairing Binding Bioinformatics Biological Assay Bioluminescence Breast Breast Cancer Cell Breast Cancer Model Cancer Patient Cancer cell line Cell Line Cells Chromatin Chromatin Structure Complementary DNA Critical Pathways DNA DNA Microarray Chip Deoxycytidine Dependovirus Disease Progression Down-Regulation Engineering Epigenetic Process Epithelial Extracellular Matrix Extracellular Matrix Proteins Fatty acid glycerol esters Figs - dietary Foundations Gene Silencing Genes Genetic Transcription Histone Acetylation Histone Deacetylase Inhibitor Histones Hormone Receptor Hydroxamic Acids Image Implant In Vitro Lead Limes Link Luciferases MCF7 cell Malignant Neoplasms Mammary gland Maps Measures Mediating Metastatic Lesion Methylation Methyltransferase Modeling Molecular Monitor Mutate Mutation Neoplasm Metastasis Nude Mice Organ Peptide Hydrolases Pharmaceutical Preparations Phase II Clinical Trials Primary Neoplasm Procedures Process Prostate Prostatic Neoplasms Protease Inhibitor Proteins Regulation Relaxation Reporter Research Personnel Retroviral Vector Role SERPINB5 gene Satellite Viruses Serine Proteinase Inhibitors Signal Pathway Signal Transduction Site Specificity TP53 gene Therapeutic Time Tissues Transactivation Transcription Coactivator Transcription Factor AP-1 Transcriptional Regulation Trichostatin A Tumor Cell Invasion Tumor Cell Line Tumor Suppression Tumor Suppressor Genes Tumor Suppressor Proteins Western Blotting Work Xenograft Model Xenograft procedure Zinc Fingers activating transcription factor angiogenesis annexin A5 base biological adaptation to stress cancer cell cancer therapy cancer type cell motility chromatin immunoprecipitation chromatin remodeling design design and construction genome wide association study genome-wide histone methyltransferase inhibitor/antagonist malignant breast neoplasm maspin matrigel methyl group migration mouse model neoplastic neoplastic cell novel novel strategies novel therapeutics overexpression programs promoter small molecule therapeutic target time use transcription factor tumor tumor growth tumor progression

项目摘要

DESCRIPTION (provided by applicant): During tumor progression, cancer cells undergo dynamic transformations, including tumor growth, angiogenesis, and metastatic dissemination. There is a crucial need in cancer therapeutics to develop novel approaches that target critical, ideally multiple steps, during tumor progression. In this proposal, we have designed artificial transcription factors (ATFs) made of zinc finger (ZF) domains as novel therapeutic strategy to inhibit multiple processes during tumor progression. We have targeted the mammary serine protease inhibitor (maspin) gene (SERPINB5), Maspin is an important therapeutic target since its overexpression is associated with tumor suppression, decreased angiogenesis, motility and metastasis in breast and prostate tumor models. Metastatic cells have developed several mechanisms to down-regulate maspin. Maspin is rarely mutated in aggressive tumors. Instead, silencing of maspin involves both, transcriptional regulation and aberrant promoter methylation. Our objective is to construct ATFs able to specifically re-activate maspin in metastatic breast cell lines, overcoming epigenetic silencing. Our hypothesis is that ATFs up-regulating maspin, by themselves or in combination with drugs that increase chromatin accessibility (methyltransferase and histone deacetylase inhibitors), will be able to re-activate maspin functions in tumor cells, reduce tumor growth and metastatic spread in nude mice. First, we have engineered highly specific ATFs made of 6ZF domains targeting 18-base pairs (bp) sites in the maspin promoter. The ZFs are linked to a potent transcriptional activator domain and expressed in tumor cells using retroviral vectors. We will investigate if the ATFs are able to specifically reactivate maspin in several breast cancer cells comprising a methylated and silenced maspin promoter. We will study if the ATFs synergize with methyltransferase and histone deacetylase inhibitors, to up-regulate maspin. We will assess if these ATFs are able to down-regulate cell invasion and to induce apoptosis in metastatic cell lines. Finally, the ATFs will be expressed using Adeno- Associated Viruses (AAVs) and we will study their capability to down-regulate tumor growth and metastasis in an orthotopic xenograft model of breast cancer in nude mice. Metastatic lesions will be monitored in real time using Bioluminescence Imaging (BLI). This work should lead to the characterization of novel anti-cancer regulators, able to reprogram the aberrant epigenetic silencing of tumor suppressors

描述（由申请人提供）：在肿瘤进展过程中，癌细胞进行动态转化，包括肿瘤生长，血管生成和转移性传播。癌症治疗学对肿瘤进展过程中靶向关键的多个步骤的新方法至关重要。在此提案中，我们设计了由锌指（ZF）结构域制成的人工转录因子（ATF）作为新的治疗策略，以抑制肿瘤进展过程中多个过程。我们针对乳腺丝氨酸蛋白酶抑制剂（MASPIN）基因（SERPINB5），Maspin是一个重要的治疗靶标，因为其过表达与肿瘤抑制作用有关，血管生成，血管生成，乳房和前列腺肿瘤模型的降低，血管生成，运动和转移。转移细胞已开发出多种机制来下调maspin。 Maspin在攻击性肿瘤中很少突变。取而代之的是，Maspin的沉默涉及转录调节和异常启动子甲基化。我们的目标是构建能够在转移性乳腺细胞系中特异性重新激活maspin的ATF，从而克服表观遗传沉默。我们的假设是，ATF本身或与增加染色质访问性的药物（甲基转移酶和组蛋白脱乙酰基酶抑制剂）的上调，将能够重新激活Maspin在肿瘤细胞中的功能，从而减少肿瘤生长和转移性在nude小鼠中的扩散。首先，我们设计了高度特异性的ATF，由MASPIN启动子中的18个基准对（BP）位点的6ZF域制成。 ZF与有效的转录激活域相关，并使用逆转录病毒载体在肿瘤细胞中表达。我们将研究ATF是否能够在包含甲基化和沉默的Maspin启动子的几个乳腺癌细胞中特异性重新激活Maspin。我们将研究ATFS是否与甲基转移酶和组蛋白脱乙酰基酶抑制剂协同化，以上调maspin。我们将评估这些ATF是否能够下调细胞浸润并诱导转移性细胞系中的凋亡。最后，将使用腺相关病毒（AAV）表达ATF，我们将研究它们在裸鼠乳腺癌的原位异种移植模型中下调肿瘤生长和转移的能力。使用生物发光成像（BLI）实时监测转移性病变。这项工作应导致新型抗癌调节剂的表征，能够重新编程肿瘤抑制剂的异常表观遗传沉默