FUNCTION OF INK4A/ARF IN PEDIATRIC NEOPLASIA

INK4a/ARF基因编码两个肿瘤抑制基因p165/INK4a和p19ARF(在人类中为p14ARF)，它们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特异性蛋白激酶CDK4和CDK6的抑制因子，其在细胞分裂周期G1期对Rb的磷酸化有助于肿瘤细胞的生长抑制功能，从而使细胞进入S期。P19ARF与P53负调控因子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能够在核质中积聚，在那里它激活包括MDM2和p21/CIP1在内的基因的转录，这些基因可以诱导细胞周期停滞或凋亡，具体取决于生物背景。Rb、P53和INK4a/ARF是人类癌症中最常见的干扰基因，无论患者的年龄或肿瘤类型。该方案的目的是通过研究INK4a/ARF在不同生理环境中的调节、下游靶点和遗传修饰物，进一步了解INK4a/ARF如何在肿瘤抑制功能中发挥作用。虽然已鉴定出四个编码相关CDK4/6抑制物的INK4基因，但只有p16/INK4a在人类中被认为是一个有效的肿瘤抑制因子。然而，缺乏INK4a/ARF或ARF的小鼠表现出类似的癌症倾向表型，这表明p16/INK4a在该物种的肿瘤抑制中可能起到更微妙的作用。我建议通过创造保留ARF功能的INK4a缺失小鼠来解决这个问题。反过来，我希望通过创造保留ARF功能的INK4a缺失小鼠来解决这个问题。反过来，我希望创造有条件的ARF基因敲除株，更接近地模拟这种基因在特定人类癌症中的作用。虽然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的相互作用是双向的，每个蛋白质影响另一个蛋白质的活性。我希望利用最近克隆的MDM2亚型来更详细地探索这一特征，MDM2亚型在Emu-Myc小鼠淋巴瘤中自然表达，不与p53结合。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的细胞(没有ARF)中，p19ARF的强制表达诱导细胞周期停滞。我提出了一些战略，旨在确定除MDM2之外的新的ARF目标。综上所述，我建议将重点放在以下悬而未决的问题上：p16INK4a是小鼠的肿瘤抑制因子吗？它在哪些细胞类型中起作用？在肿瘤发生中，哪些基因改变与ARF缺失有关？MDM2的过度表达与ARF缺失在促进肿瘤方面是否有协同作用，以及不寻常的MDM2亚型是如何起作用的？除了MDM2，p19ARF还与哪些靶点相互作用而导致细胞周期停滞？这些研究解决了许多知之甚少的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。

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.