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 基因座编码两种肿瘤抑制因子：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 有助于抑制其生长抑制功能，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，并激活基因转录，包括 Mdm2 和 p21/CIP1，根据生物学背景诱导细胞周期停滞或细胞凋亡。无论患者年龄或肿瘤类型如何，Rb、p53 和 INK4a/ARF 都是人类癌症中最常被破坏的基因。该提案的目的是通过研究小鼠不同生理环境下 INK4a/ARF 的调节、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。尽管已鉴定出编码相关 cdk4/6 抑制剂的四个 INK4 基因，但只有 p16/INK4a 被强烈认为是人类中的有效肿瘤抑制因子。然而，缺乏 INK4a/ARF 或单独 ARF 的小鼠表现出类似的癌症倾向表型，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-null 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，我希望创建条件性 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，每种蛋白质都会影响另一种蛋白质的活性。我希望利用最近克隆的 Mdm2 同工型（在 Emu-Myc 小鼠淋巴瘤中自然表达，不与 p53 结合）更详细地探索这一特征。此外，虽然单独缺乏p53的细胞对ARF的作用具有抵抗力，但我们现在发现在缺乏Mdm2和p53（有或没有ARF）的细胞中强制表达p19ARF会诱导细胞周期停滞。我提供了一些旨在识别除 Mdm2 之外的新 ARF 目标的策略。总之，我建议关注以下未解决的问题：p16 INK4a 是小鼠的肿瘤抑制因子吗？它在哪些细胞类型中发挥作用？肿瘤发生过程中哪些基因改变与 ARF 缺失相配合？ Mdm2 过度表达是否与 ARF 缺失协同促进肿瘤生长？Mdm2 的异常异构体如何发挥作用？除了 Mdm2 之外，p19ARF 与哪些靶标相互作用以诱导细胞周期停滞？这些研究解决了 INK4a-ARF 信号传导的许多鲜为人知的特征，这些特征应该与多种形式的人类癌症直接相关。