FUNCTION OF INK4A/ARF IN PEDIATRIC NEOPLASIA

INK4A/ARF 在儿科肿瘤中的作用

• 批准号: 6595011
• 负责人: MARTINE F. ROUSSEL (SHERR)
• 金额: $ 24.14万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6595011
• 关键词: cyclin dependent kinase; cyclins; enzyme inhibitors; gene induction /repression; gene mutation; genetic promoter element; laboratory mouse; ligase; microarray technology; neoplasm /cancer genetics; nucleolus; oncoproteins; pediatric neoplasm /cancer; protein structure function; retinoblastoma protein; transcription factor; tumor suppressor genes; tumor suppressor proteins

The INK4a/ARF locus encodes two tumor suppressors, p165/INK4a and p19ARF (p14ARF in humans) that regulate the functions of the retinoblastoma protein (Rb) and the p53 transcription factor, respectively. p16/INK4A is an inhibitor of the cyclin D-specific kinases, cdk4 and cdk6, whose phosphorylation of Rb in the G1 phase of the cell division cycle helps to cancer its growth suppressive functions, thereby enabling cells to enter S phase. p19ARF binds to the p53 negative regulator Mdm2, inhibiting its E3 ubiquitin ligase activity and sequestering it in the nucleolus. This enables p53 to accumulate in the nucleoplasm where is activates the transcription of genes, including Mdm2 and p21/CIP1, that induce either cell cycle arrest or apoptosis depending on the biologic context. Rb, p53 and INK4a/ARF are the most frequently disrupted genes in human cancer, regardless of patient age or tumor type. The goal of this proposal is to further understand how INK4a/ARF mediates in tumor suppressive functions by studying its regulation, downstream targets and genetic modifiers in different physiologic contexts in mice. Although four INK4 genes encoding related cdk4/6 inhibitors have been identified, only p16/INK4a has been strongly implicated as a potent tumor suppressor in humans. However, mice lacking INK4a/ARF or ARF alone exhibit a similar cancer prone phenotype, suggesting that p16/INK4a may play a subtler role in tumor suppression in this species. I propose to address this question by creating INK4a-null mice that retain ARF function. In turn, I hope to address this question by creating INK4a- null mice that retain ARF function. In turn, I hope to create conditional ARF knock-out strains that more closely model the role of this gene in particular human cancers. Although the p19/ARF-Mdm2-p53 pathway is frequently disrupted in tumors, both p19ARF and Mdm2, are each likely to have other targets. Preliminary data suggest that the ARF-Mdm2 interaction is bi-directional with each protein affecting the activity of the other. I hope to explore this feature in greater detail, taking advantage of recently cloned Mdm2 isoforms, naturally expressed in Emu-Myc mouse lymphomas, which do not bind to p53. Moreover, although cells lacking p53 alone are resistant to ARF's effects, we have now found that enforced expression of p19ARF in cells lacking both Mdm2 and p53 (with our without ARF) induces cell cycle arrest. I offer a number of strategies aimed to identify novel ARF targets, other than Mdm2. In summary, I propose to focus on the following unresolved questions: Is p16 INK4a a tumor suppressor in mice and in what cell types does it act? What genetic alterations cooperate with ARF loss in tumorigenesis? Does Mdm2 over-expression synergize with ARF-loss in promoting tumors, and how do unusual isoforms of Mdm2 contribute? With what targets, apart from Mdm2, does p19ARF interact to induce cell cycle arrest? These studies address many poorly understood features of INK4a-ARF signaling that should have direct relevance to many forms of human cancer.

INK4a/ARF基因座编码两个肿瘤抑制因子p165/INK4a和p19ARF（人类为p14ARF），分别调节视网膜母细胞瘤蛋白（Rb）和p53转录因子的功能。p16/INK4A是细胞周期蛋白d特异性激酶cdk4和cdk6的抑制剂，其在细胞分裂周期G1期磷酸化Rb有助于抑制其生长功能，从而使细胞进入S期。p19ARF与p53负调节因子Mdm2结合，抑制其E3泛素连接酶活性并将其隔离在核仁中。这使得p53在核质中积累，激活基因转录，包括Mdm2和p21/CIP1，根据生物学背景诱导细胞周期阻滞或凋亡。无论患者年龄或肿瘤类型如何，Rb、p53和INK4a/ARF是人类癌症中最常被破坏的基因。本课题的目的是通过研究INK4a/ARF在小鼠不同生理环境下的调控、下游靶点和遗传修饰因子，进一步了解INK4a/ARF如何介导肿瘤抑制功能。虽然已经鉴定出四个INK4基因编码相关的cdk4/6抑制剂，但只有p16/INK4a在人类中被强烈地认为是一种有效的肿瘤抑制因子。然而，缺乏INK4a/ARF或单独缺乏ARF的小鼠表现出类似的癌症易感表型，这表明p16/INK4a可能在该物种的肿瘤抑制中发挥更微妙的作用。我建议通过创建保留ARF功能的ink4a缺失小鼠来解决这个问题。反过来，我希望通过创建保留ARF功能的INK4a- null小鼠来解决这个问题。反过来，我希望创造出有条件的ARF敲除菌株，更接近地模拟该基因在特定人类癌症中的作用。尽管p19/ARF-Mdm2-p53通路在肿瘤中经常被破坏，但p19ARF和Mdm2都可能有其他靶点。初步数据表明，ARF-Mdm2相互作用是双向的，每一种蛋白都会影响另一种蛋白的活性。我希望更详细地探索这一特征，利用最近克隆的Mdm2亚型，在Emu-Myc小鼠淋巴瘤中自然表达，不与p53结合。此外，尽管单独缺乏p53的细胞对ARF的作用具有抗性，但我们现在发现，在缺乏Mdm2和p53的细胞中，p19ARF的强制表达会诱导细胞周期阻滞。我提出了一些旨在确定除Mdm2以外的新的ARF靶点的策略。总之，我建议关注以下未解决的问题：p16 INK4a在小鼠中是肿瘤抑制因子吗？它在哪些细胞类型中起作用？在肿瘤发生过程中，哪些基因改变与ARF丢失有关？Mdm2过表达是否与arf缺失协同促进肿瘤发生？ Mdm2异常异构体是如何起作用的？除了Mdm2， p19ARF还与哪些靶点相互作用以诱导细胞周期阻滞？这些研究解决了许多鲜为人知的INK4a-ARF信号特征，这些特征应该与许多形式的人类癌症直接相关。