The BCA2 Ubiquitin E3 Ligase as a Target in Breast Cancer

描述（由申请人提供）：泛素-蛋白酶体系统调节在细胞周期、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靶点。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致癌环指泛素E3连接酶特征。因此，恢复蛋白质稳态的方法可能会导致新的乳腺癌治疗方法。我们利用减法杂交克隆方法从浸润性乳腺细胞系中分离出新的乳腺癌相关基因2 (BCA2)，发现其具有环指结构域。我们证明了BCA2无名指，一个特殊的锌指，负责其内在的自泛素化活动。与正常组织相比，超过50%的浸润性乳腺癌中BCA2过表达。BCA2过表达增加NIH3T3成纤维细胞的增殖，而小干扰RNA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，表明其具有多种功能。BCA2的胞质结合伙伴Rab7参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的过表达导致EGF降解的抑制。在细胞核中，BCA2与雌激素受体共表达。在er阳性乳腺癌中检测到高核BCA2水平，而er阴性乳腺癌中检测到低核BCA2水平。此外，BCA2是一种雌激素反应基因，表明BCA2和ER可能相互作用。我们已经发现了一种抑制剂双硫仑，它的抗肿瘤活性与BCA2的抑制作用有关，这种抑制作用是通过将锌从其催化环结构域排出来实现的。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，这在乳腺癌中很重要，因此无名指泛素E3连接酶BCA2是治疗干预的靶标。我们进一步假设，从无名指结构域射出锌可以导致其E3连接酶活性的特异性抑制。在这个提议中，我们将重点描述乳腺癌细胞中BCA2的细胞质和核功能，以及基于结构的BCA2抑制剂的设计。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连接酶介导的蛋白泛素化，而不仅仅是在癌症中。

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.