The BCA2 Ubiquitin E3 Ligase as a Target in Breast Cancer

BCA2 泛素 E3 连接酶作为乳腺癌的靶标

基本信息

批准号：
7848072
负责人：
QING PING DOU
金额：
$ 23.72万
依托单位：
WAYNE STATE UNIVERSITY
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：
2007-07-18 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7848072
关键词：
Affect Alcohol dehydrogenase Apoptosis BRCA2 gene Binding Biological Assay Breast Breast Cancer Cell Breast Cancer Treatment Cancer cell line Cell Cycle Cell Line Cell Nucleus Cell Proliferation Cells Cloning Collection Complex Cytoplasm Development Disulfides Disulfiram Drug Kinetics Endocytosis Epidermal Growth Factor Epidermal Growth Factor Receptor Estrogen Receptor 2 Estrogen Receptors Estrogens Event Fibroblasts Fingers Genes Growth Homeostasis Immunofluorescence Immunologic Immunoprecipitation In Vitro Lead Ligase Link Malignant Neoplasms Mediating Methods Molecular Weight Mutation Normal tissue morphology Nuclear Nude Mice Oncogenic Pathway interactions Pharmacodynamics Phosphorylation Phosphorylation Site Proteins Proto-Oncogene Proteins c-akt Proto-Oncogenes Recycling Research Personnel Role Signal Pathway Small Interfering RNA Specificity Structure System Testing Therapeutic Intervention Tissue Microarray Transfection Tumor Suppressor Proteins Ubiquitin Ubiquitination Xenograft Model Zinc Zinc Fingers analog base chemical genetics chromatin immunoprecipitation design drug discovery guanine nucleotide binding protein in vitro activity in vivo inhibitor/antagonist kinase inhibitor malignant breast neoplasm multicatalytic endopeptidase complex mutant novel overexpression programs promoter protein degradation protein transport receptor receptor-mediated signaling repaired trafficking ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): The ubiquitin-proteasome system regulates the turnover of proteins that have essential roles in the cell cycle, apoptosis, DMA damage repair, and in protein trafficking, which makes this pathway a target for oncogenic events. Several ubiquitin E3 ligases are proto-oncogenes that degrade tumor suppressor proteins. Breast cancer has an unbalanced proto-oncogenic RING-finger ubiquitin E3 ligase signature. Thus, approaches to restore protein homeostasis could lead to novel breast cancer treatments. We isolated the novel breast cancer associated gene 2 (BCA2) by subtractive hybridization cloning from an invasive breast cell line, and found that it has a RING-finger domain. We demonstrated that the BCA2 RING-finger, a specialized zinc- finger, is responsible for its intrinsic autoubiquitination activity. BCA2 is overexpressed in more than 50% of invasive breast cancers compared to normal tissues. Overexpression of BCA2 increases proliferation of NIH3T3 fibroblasts, whereas small interfering RNA inhibits growth of BCA2-expressing breast cancer cells. BCA2 is expressed in the nucleus and cytoplasm of breast cancer cells suggesting multiple functions. A cytoplasmic binding partner of BCA2 is Rab7, which is involved in receptor endocytosis and recycling. Rab7 was shown to regulate endocytic trafficking of the epidermal growth factor receptor complex. Overexpression of BCA2 leads to inhibition of EGF degradation. In the nucleus, BCA2 appears co-expressed with estrogen receptor. High nuclear BCA2 levels are detected in ER-positive breast cancers, while ER-negative breast cancers have low nuclear BCA2. Moreover, BCA2 is an estrogen responsive gene, suggesting that BCA2 and ER might crosstalk. An inhibitory agent disulfiram, has been identified by us and shows antitumor activity that is related to BCA2 inhibition via ejection of zinc from its catalytic RING domain. Only BCA2-expressing breast cancer cell lines respond to treatment with disulfiram. We hypothesize that BCA2 is a regulator of receptor-mediated signaling pathways that are important in breast cancer and thus that the RING-finger ubiquitin E3 ligase BCA2 is a target for therapeutic intervention. We further hypothesize that ejection of zinc from the RING-finger domain can lead to specific inhibition of its E3 ligase activity. In this proposal we will focus on delineating the cytoplasmic and nuclear functions of BCA2 in breast cancer cells as well as on the structure-based design of BCA2 inhibitory agents. Our Specific Aims are: To use genetic and chemical inhibition for delineating the functional relationships between BCA2 and Rab7, and for studying crosstalk between BCA2 and ER. We will further develop zinc ejecting compounds that can specifically inhibit BCA2 E3 ligase activity. This project could have a major impact on mechanism-based drug discovery for modulation of RING-finger E3 ligase-mediated protein ubiquitination, not only in cancer.

描述（由申请人提供）：泛素-蛋白酶体系统调节在细胞周期、细胞凋亡、DMA 损伤修复和蛋白质运输中起重要作用的蛋白质的周转，这使得该途径成为致癌事件的目标。几种泛素 E3 连接酶是降解肿瘤抑制蛋白的原癌基因。乳腺癌具有不平衡的原癌环指泛素 E3 连接酶特征。因此，恢复蛋白质稳态的方法可能会带来新的乳腺癌治疗方法。我们通过消减杂交克隆从侵袭性乳腺细胞系中分离出新型乳腺癌相关基因 2 (BCA2)，并发现它具有环指结构域。我们证明了 BCA2 环指（一种特殊的锌指）负责其内在的自动泛素化活性。与正常组织相比，BCA2 在超过 50% 的浸润性乳腺癌中过度表达。 BCA2 的过度表达会增加 NIH3T3 成纤维细胞的增殖，而小干扰 RNA 会抑制表达 BCA2 的乳腺癌细胞的生长。 BCA2 在乳腺癌细胞的细胞核和细胞质中表达，表明具有多种功能。 BCA2 的细胞质结合伴侣是 Rab7，它参与受体内吞作用和再循环。 Rab7 被证明可以调节表皮生长因子受体复合物的内吞运输。 BCA2 的过度表达会抑制 EGF 降解。在细胞核中，BCA2 似乎与雌激素受体共表达。 ER 阳性乳腺癌的细胞核 BCA2 水平较高，而 ER 阴性乳腺癌的细胞核 BCA2 水平较低。此外，BCA2 是一种雌激素反应基因，表明 BCA2 和 ER 可能会相互影响。我们已鉴定出一种抑制剂双硫仑，它显示出抗肿瘤活性，该活性与通过从其催化 RING 结构域中喷射锌来抑制 BCA2 相关。只有表达 BCA2 的乳腺癌细胞系对双硫仑治疗有反应。我们假设 BCA2 是受体介导的信号通路的调节剂，该通路在乳腺癌中很重要，因此环指泛素 E3 连接酶 BCA2 是治疗干预的靶点。我们进一步假设锌从环指结构域的喷射可以导致其 E3 连接酶活性的特异性抑制。在本提案中，我们将重点描述乳腺癌细胞中 BCA2 的细胞质和核功能以及 BCA2 抑制剂的基于结构的设计。我们的具体目标是：利用遗传和化学抑制来描述 BCA2 和 Rab7 之间的功能关系，并研究 BCA2 和 ER 之间的串扰。我们将进一步开发能够特异性抑制BCA2 E3连接酶活性的锌喷射化合物。该项目可能会对基于机制的药物发现产生重大影响，以调节环指 E3 连接酶介导的蛋白质泛素化，而不仅仅是在癌症中。