The Mechanism of Nur77 and Nor1 Induced Apoptosis Through Interaction with Bcl2

Nur77 和 Nor1 通过与 Bcl2 相互作用诱导细胞凋亡的机制

• 批准号: 8456487
• 负责人: Megan Linn Burger
• 金额: $ 3.68万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456487
• 关键词: Allografting; Antisense Oligonucleotides; Apoptosis; Apoptotic; Arginine; B-Cell Lymphomas; BCL-2 Protein; BCL2 gene; BH3 Domain; Binding; Cancer cell line; Cell Count; Cell Death; Cell Line; Cell Nucleus; Cells; Cessation of life; Clinical; Complex; Development; Embryo; Employment; Family member; Hela Cells; Hematologic Neoplasms; Homologous Gene; Human; In Vitro; Lead; Malignant - descriptor; Malignant Neoplasms; Mitochondria; Molecular; Mus; Mutate; Mutation; N-terminal; Normal Cell; Nuclear Export; Nuclear Receptors; Oncogenic; Organism; Pathway interactions; Peptides; Phase; Physiological; Play; Primary Neoplasm; Process; Protein Family; Protein Kinase C; Proteins; Regulation; Resistance; Role; Signal Transduction; Stimulus; System; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; Transgenic Mice; Up-Regulation; Xenograft procedure; cancer cell; cancer therapy; cancer type; cell growth; cell killing; chemotherapy; conventional therapy; functional restoration; in vivo; in vivo Model; kidney cell; killings; leukemia/lymphoma; malignant breast neoplasm; mouse model; mutant; neoplastic cell; prevent; pro-apoptotic protein; protein expression; prototype; public health relevance; response; restoration; small molecule; therapeutic target; thymocyte; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Over-expression of anti-apoptotic proteins is a prevalent mechanism of dysregulation in cancer that plays an essential role in tumorigenesis and confers resistance to conventional cancer therapies. The ability to induce apoptosis in cancer cells is therefore necessary for tumor sensitivity to treatment. Bcl-2 is the prototype anti apoptotic protein that has been shown to be over-expressed in many human cancers. However, recent evidence suggests that Bcl-2 can act as a pro-apoptotic protein under certain circumstances. Nuclear receptor Nur77 was shown to induce cancer cell apoptosis through interaction with Bcl-2. In response to apoptotic stimuli, Nur77 localizes to the mitochondria where it interacts with Bcl-2, causing Bcl-2 to expose its BH3 domain, the "killer" domain of the Bcl-2 protein family. Over-expression of Nur77 with wild-type Bcl-2, but not a BH3 Bcl-2 mutant, can lead to apoptosis in a human embryonic kidney cell line 293T. Similarly, co-expression of Nur77 with Bcl-2 homologs Bcl-B and Bcl2A1 causes increased apoptosis. Furthermore, addition of a peptide containing the Nur77 Bcl-2 interacting region, conjugated to a cell penetrating peptide, can induce death in several human cancer cell lines. In primary normal cells, our lab showed that the mechanism of Bcl-2 BH3 exposure by Nur77, as well as its highly homologous family member Nor-1, can be found in immature T cells (thymocytes) when they are induced to die through their T-cell receptor complex. Nur77/Nor-1 mitochondrial translocation is dependent on Protein Kinase C (PKC) signaling and inhibition of nuclear export of Nur77/Nor-1 significantly rescues thymocytes from apoptosis. This physiological example of employment of this apoptotic mechanism thus provides a unique system for the examination of the in vivo significance and molecular mechanism of this pathway and how the Bcl-2 pro-apoptotic function may be used to kill primary tumor cells in vivo. In Aim 1, we will identify mutations that can prevent or constitutively promote mitochondrial localization of Nur77 and Nor-1. PKC's role in regulating translocation through the mutated region and the importance of translocation for induction of thymocyte and cancer cell death will be assessed. A Nor-1 mutant constitutively localized to the mitochondria will be expressed in a T cell-specific transgenic mouse model to assess the in vivo significance of this pro-apoptotic pathway and in Bcl-2/Bcl2A1 high- or low-expressing cancer cell lines to assess its apoptotic function in relation to Bcl-2/Bcl2A1 expression. In Aim 2, we will explore the therapeutic potential of Nur77 and Nor-1 peptides with cell penetrating ability to target Bcl-2 and Bcl2A1 in vitro and in vivo. The abilityof Nur77 and Nor-1 peptides to induce apoptosis in a Bcl2A1-dependent manner will be assessed in human cancer cell lines. The mechanism of Bcl2A1's pro-apoptotic function will be delineated by interaction studies in primary thymocytes. The capacity of Nur77 and Nor-1 peptides to induce apoptosis of cells expressing Bcl-2 versus Bcl2A1 will be tested in human cancer cell lines and primary normal and malignant T cells in vitro and in xenograft and allograft models in vivo.

描述（由申请人提供）：抗凋亡蛋白的过表达是癌症失调失调的普遍机制，在肿瘤发生中起着至关重要的作用，并赋予对常规癌症疗法的耐药性。因此，对于肿瘤对治疗的敏感性，必须诱导癌细胞凋亡的能力。 Bcl-2是原型抗凋亡蛋白，在许多人类癌症中已被证明过表达。但是，最近的证据表明，在某些情况下，Bcl-2可以充当促凋亡蛋白。核受体NUR77显示通过与Bcl-2相互作用诱导癌细胞凋亡。为了响应凋亡刺激，NUR77定位于线粒体与Bcl-2相互作用的线粒体，从而导致Bcl-2暴露其BH3结构域，BH3域是Bcl-2蛋白家族的“杀伤”结构域。用野生型Bcl-2（而不是BH3 BCl-2突变体）对NUR77的过表达可能导致人类胚胎肾细胞系293T的凋亡。同样，NUR77与Bcl-2同源物Bcl-B和Bcl2a1共表达会导致凋亡增加。此外，添加含有NUR77 Bcl-2相互作用区域的肽，结合到穿透性肽的细胞中，可以诱导几种人类癌细胞系中的死亡。在原发性正常细胞中，我们的实验室表明，在未成熟的T细胞（胸腺细胞）通过其T细胞受体复合物死亡时，NUR77及其高度同源的家族成员nor-1的BCl-2 BH3暴露机理可以在未成熟的T细胞（胸腺细胞）中找到。 NUR77/NOR-1线粒体易位取决于蛋白激酶C（PKC）信号传导和NUR77/NOR-1核出口的抑制作用可显着从凋亡中挽救胸腺细胞。因此，这种凋亡机制使用的生理例子为研究该途径的体内意义和分子机制提供了独特的系统，以及如何使用Bcl-2促凋亡功能来杀死原发性肿瘤细胞。在AIM 1中，我们将确定可以预防或组成性促进NUR77和NOR-1的线粒体定位的突变。 PKC在调节突变区域的易位方面的作用以及易位诱导胸腺细胞和癌细胞死亡的重要性。在T细胞特异性的转基因小鼠模型中将表达成组成型的NOR-1突变体，以评估该促凋亡途径的体内重要性以及在BCL2/BCL2A1高或低表达的癌细胞系中的体内重要性，以评估其与BCL-2/BCLCCL2A的表达相关的凋亡功能。在AIM 2中，我们将探索具有细胞穿透能力的NUR77和NOR-1肽在体外和体内靶向Bcl-2和Bcl2a1的能力。 NUR77和NOR-1肽的能力将在人类癌细胞系中评估以Bcl2a1依赖性方式诱导凋亡。 BCL2A1促凋亡功能的机制将通过原发性胸腺细胞的相互作用研究来描述。 NUR77和NOR-1肽诱导表达BCL-2与BCL2A1的细胞凋亡的能力将在人类癌细胞系以及原发性和恶性T细胞体外，异种移植物和同种异体移植模型中进行测试。