Structure and function of BCAR1 and BCAR3 association in breast cancer malignancy

描述(申请人提供)：乳腺癌是女性癌症死亡的第二大原因，尽管有许多治疗选择。大多数乳腺癌是雌激素受体阳性的，通常使用抗雌激素治疗，而抗体和激酶抑制剂用于治疗过度表达HER2受体酪氨酸激酶的癌症。不幸的是，由于特定基因的过度表达，肿瘤往往具有内在的耐药性，或者对这些药物产生耐药性。这些基因之一是支架蛋白BCAR1(乳腺癌抗雌激素抵抗1或p130Cas)，它是整合素黏附受体下游调控细胞增殖、生存和迁移/侵袭的信号通路的关键组成部分。BCAR1也是HER2致癌信号通路的关键节点和对阿霉素耐药的中介，阿霉素是一种经常用于治疗对抗雌激素或HER2靶向治疗无效的乳腺癌的药物。因此，BCAR1是控制乳腺癌恶性的信号网络中的中心角色。另一个重要但特性不佳的抗雌激素抵抗因子是SH2结构域蛋白BCAR3，它是一个包含SHEP1的三种蛋白家族的成员，我们在Eph受体酪氨酸激酶结合分子的筛选中发现了SHEP1。BCAR1和BCAR3家族的成员通过其C-末端区域的直接结合而以一致的方式发挥作用，但他们的复合体的结构一直没有得到研究人员的了解。为了深入了解这些关键而神秘的组装，我们解决了BCAR1-BCAR3家族复合体的第一个晶体结构，即BCAR1和SHEP1的晶体结构，揭示了一种新型的蛋白质相互作用。SHEP1的C末端区域总体上类似于CDC25型鸟核苷酸交换因子结构域。然而，关键区域被BCAR1结合改变，导致SHEP1的“封闭”构象，不能与RAS GTP酶结合。我们现在试图通过BCAR1和BCAR3的联合作用来揭示乳腺癌恶性和化疗耐药的机制基础，BCAR3是一种与SHEP1相关的蛋白质，但在关键功能上有明显的差异。因此，我们提出了一种多学科的方法，整合了一系列有效的工具，从高分辨率的结构和生化分析到在培养和小鼠乳腺癌异种移植模型中对工程癌细胞的功能研究。我们的目标将通过(1)解开BCAR1-BCAR3信号关联的分子细节以准确定义复杂形成时发生的调节修饰；以及(2)阐明BCAR-BCAR3关联在乳腺癌生长、侵袭性和化疗耐药性中的作用。我们的研究将使我们对BCAR1-BCAR3信号节点以及相关蛋白质的复合体有一个新的理解，并阐明乳腺癌细胞如何获得使其能够转移和应对化疗侮辱的恶性特征。因此，这项工作最终将为开发克服癌症耐药性的治疗方案提供新的基础。

DESCRIPTION (provided by applicant): Breast cancer is the second leading cause of cancer deaths in women, despite a number of treatment options. Most breast cancers are estrogen receptor-positive and commonly treated with anti-estrogens, while antibodies and kinase inhibitors are used to treat cancers that overexpress the HER2 receptor tyrosine kinase. Unfortunately, tumors are often intrinsically resistant or develop resistance to these drugs due to overexpression of specific genes. One of these genes is the scaffolding protein BCAR1 (breast cancer anti- estrogen resistance 1 or p130Cas), which is a well-known key component of the signaling pathways that regulate cell proliferation, survival and migration/invasion downstream of integrin adhesion receptors. BCAR1 is also a critical node in HER2 oncogenic signaling pathways and mediator of resistance to adriamycin, a drug frequently used to treat breast cancers that do not respond to anti-estrogens or HER2-targeted therapies. Thus, BCAR1 is a central player in the signaling networks that control breast cancer malignancy. Another important but poorly characterized factor in anti-estrogen resistance is the SH2 domain-containing protein BCAR3, a member of a family of three proteins that also includes SHEP1, which we identified in a screen for Eph receptor tyrosine kinase-binding molecules. Members of the BCAR1 and BCAR3 families function in a concerted manner through direct association of their C-terminal regions, but the structure of their complexes has eluded investigators. To gain insight into these crucial yet enigmatic assemblies, we have solved the first crystal structure of a BCAR1-BCAR3 family complex, that of BCAR1 and SHEP1, revealing a novel type of protein interaction. The SHEP1 C-terminal region overall resembles a Cdc25-type guanine nucleotide exchange factor domain. However, crucial regions are altered by BCAR1 binding, resulting in a "closed" conformation of SHEP1 that cannot bind Ras GTPases. We now seek to unravel the mechanistic basis of breast cancer malignancy and resistance to chemotherapy mediated by BCAR1 association with BCAR3, a protein related to SHEP1 but with distinct differences in key features. Thus, we propose a multidisciplinary approach that integrates a spectrum of effective tools ranging from high resolution structural and biochemical analysis to functional studies in engineered cancer cells in culture and mouse breast cancer xenograft models. Our goals will be accomplished by (1) unraveling the molecular details of the BCAR1-BCAR3 signaling association to precisely define the regulatory modifications occurring upon complex formation; and (2) elucidating the role of the BCAR-BCAR3 association in breast cancer growth, invasiveness and resistance to chemotherapy. Our studies will lead to a new understanding of the BCAR1-BCAR3 signaling node as well as complexes of related proteins, and shed light on how breast cancer cells acquire the malignant characteristics that enable them to metastasize and cope with chemotherapeutic insults. Thus, this work will ultimately provide a new basis for developing treatment options to overcome cancer drug resistance.