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

描述（由申请人提供）：抗凋亡蛋白的过度表达是癌症中失调的普遍机制，在肿瘤发生中起重要作用，并赋予对常规癌症治疗的抗性。因此，诱导癌细胞凋亡的能力对于肿瘤对治疗的敏感性是必要的。Bcl-2是原型抗凋亡蛋白，已被证明在许多人类癌症中过度表达。然而，最近的证据表明，Bcl-2在某些情况下可以作为促凋亡蛋白。核受体Nur 77通过与Bcl-2相互作用诱导癌细胞凋亡。响应于凋亡刺激，Nur 77定位于线粒体，在那里它与Bcl-2相互作用，导致Bcl-2暴露其BH 3结构域，Bcl-2蛋白家族的“杀伤”结构域。Nur 77与野生型Bcl-2，而不是BH 3 Bcl-2突变体的过表达，可以导致人胚肾细胞系293 T的凋亡。类似地，Nur 77与Bcl-2同源物Bcl-B和Bcl 2A 1的共表达导致细胞凋亡增加。此外，添加与细胞穿透肽缀合的含有Nur 77 Bcl-2相互作用区的肽可以诱导几种人癌细胞系的死亡。在原代正常细胞中，我们的实验室表明，Nur 77及其高度同源的家族成员Nor-1暴露Bcl-2 BH 3的机制可以在未成熟的T细胞（胸腺细胞）中发现，当它们通过T细胞受体复合物被诱导死亡时。Nur 77/Nor-1线粒体转位依赖于蛋白激酶C（PKC）信号传导，抑制Nur 77/Nor-1的核输出可显著挽救胸腺细胞免于凋亡。因此，采用这种凋亡机制的这种生理学实例提供了一种独特的系统，用于检查这种途径的体内意义和分子机制以及Bcl-2促凋亡功能如何用于体内杀死原发性肿瘤细胞。在目标1中，我们将鉴定可以阻止或组成性促进Nur 77和Nor-1的线粒体定位的突变。PKC在调节通过突变区域的易位中的作用以及易位对于诱导胸腺细胞和癌细胞死亡的重要性将被评估。组成性定位于线粒体的Nor-1突变体将在T细胞特异性转基因小鼠模型中表达以评估该促凋亡途径的体内意义，并在Bcl-2/Bcl 2A 1高表达或低表达癌细胞系中表达以评估其与Bcl-2/Bcl 2A 1表达相关的凋亡功能。在目的2中，我们将探索具有细胞穿透能力的Nur 77和Nor-1肽在体外和体内靶向Bcl-2和Bcl 2A 1的治疗潜力。将在人癌细胞系中评估Nur 77和Nor-1肽以Bcl 2A 1依赖性方式诱导细胞凋亡的能力。Bcl 2A 1的促凋亡功能的机制将通过在原代胸腺细胞中的相互作用研究来描述。Nur 77和Nor-1肽诱导表达Bcl-2相对于Bcl 2A 1的细胞凋亡的能力将在体外在人癌细胞系和原代正常和恶性T细胞中以及在体内在异种移植物和同种异体移植物模型中进行测试。

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.