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普遍存在的失调机制，在肿瘤发生中起着重要作用，并赋予传统癌症治疗的抗性。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是抗凋亡蛋白的原型，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Nur77通过与Bcl-2相互作用诱导癌细胞凋亡。在对凋亡刺激的反应中，Nur77定位于线粒体，在那里它与Bcl-2相互作用，导致Bcl-2暴露其BH3结构域，Bcl-2蛋白家族的“杀手”结构域。在人胚胎肾细胞系293T中，过度表达野生型Bcl-2而非BH3 Bcl-2突变体的Nur77可导致细胞凋亡。同样，Nur77与Bcl-2同源物Bcl-B和Bcl2A1共表达可导致细胞凋亡增加。此外，添加含有Nur77 Bcl-2相互作用区域的肽，结合到细胞穿透肽，可以诱导几种人类癌细胞系死亡。在原代正常细胞中，我们的实验室发现，当未成熟的T细胞（胸腺细胞）通过其T细胞受体复合物诱导死亡时，Nur77及其高度同源的家族成员Nor-1暴露于Bcl-2 BH3的机制可以在它们中发现。Nur77/Nor-1线粒体易位依赖于蛋白激酶C （PKC）信号，抑制Nur77/Nor-1的核输出可显著拯救胸腺细胞凋亡。因此，利用这种凋亡机制的生理例子为研究这一途径的体内意义和分子机制以及Bcl-2促凋亡功能如何在体内杀死原发肿瘤细胞提供了一个独特的系统。在Aim 1中，我们将确定能够阻止或组成性地促进Nur77和Nor-1线粒体定位的突变。将评估PKC在通过突变区域调节易位中的作用以及易位对诱导胸腺细胞和癌细胞死亡的重要性。将在T细胞特异性转基因小鼠模型中表达一个组成部分定位于线粒体的Nor-1突变体，以评估该促凋亡途径在体内的意义，并在Bcl-2/Bcl2A1高表达或低表达的癌细胞系中评估其与Bcl-2/Bcl2A1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的治疗潜力。Nur77和no -1肽以bcl2a1依赖的方式诱导细胞凋亡的能力将在人类癌细胞系中进行评估。Bcl2A1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。Nur77和Nor-1肽诱导表达Bcl-2和Bcl2A1的细胞凋亡的能力将在体外的人类癌细胞系、原代正常和恶性T细胞以及体内的异种移植和同种异体移植模型中进行测试。

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.