FUNCTION OF INK4A/ARF IN PEDIATRIC NEOPLASIA

修订摘要：INK 4a/ARF基因座编码两个肿瘤抑制因子，p165/INK 4a和p19 ARF（人类中的p14 ARF），其分别调节视网膜母细胞瘤蛋白（Rb）和p53转录因子的功能。p16/INK 4A是细胞周期蛋白D特异性激酶cdk 4和cdk 6的抑制剂，其在细胞分裂周期的G1期的Rb的磷酸化有助于癌症的生长抑制功能，从而使细胞进入S期。 p19 ARF与p53负调节因子Mdm 2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能够在核质中积累，在核质中激活包括Mdm 2和p21/CIP 1的基因的转录，其根据生物学背景诱导细胞周期停滞或凋亡。Rb、p53和INK 4a/ARF是人类癌症中最常被破坏的基因，无论患者年龄或肿瘤类型如何。本研究的目的是通过研究INK 4a/ARF在小鼠不同生理环境中的调控、下游靶点和遗传修饰因子，进一步了解INK 4a/ARF如何介导肿瘤抑制功能。虽然已经鉴定了编码相关cdk 4/6抑制剂的四种INK 4基因，但只有p16/INK 4a强烈暗示为人类中的有效肿瘤抑制剂。然而，缺乏INK 4a/ARF或仅ARF的小鼠表现出类似的癌症倾向表型，表明p16/INK 4a可能在该物种的肿瘤抑制中发挥更微妙的作用。我建议通过创造保留ARF功能的INK 4a-null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INK 4a缺失小鼠来解决这个问题。反过来，我希望创造条件性ARF敲除菌株，更密切地模拟该基因在特定人类癌症中的作用。虽然p19/ARF-Mdm 2-p53通路在肿瘤中经常被破坏，但p19 ARF和Mdm 2都可能有其他靶点。初步数据表明，ARF-Mdm 2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Mdm 2同种型更详细地探索这一特征，Mdm 2同种型在Emu-Myc小鼠淋巴瘤中天然表达，不与p53结合。此外，虽然细胞缺乏p53单独耐ARF的影响，我们现在已经发现，在缺乏Mdm 2和p53的细胞（有或没有ARF）p19 ARF的强制表达诱导细胞周期停滞。我提供了一些策略，旨在确定新的ARF目标，而不是Mdm 2。总之，我建议集中在以下未解决的问题：p16 INK 4a是小鼠的肿瘤抑制因子吗？它在哪些细胞类型中起作用？什么样的基因改变与肿瘤发生中的ARF丢失有关？Mdm 2过度表达是否与ARF缺失协同促进肿瘤发生，以及Mdm 2的不寻常亚型如何起作用？除了Mdm 2，p19 ARF与哪些靶点相互作用以诱导细胞周期停滞？这些研究解决了INK 4a-ARF信号传导的许多知之甚少的特征，这些特征应该与许多形式的人类癌症直接相关。

Revised Abstract: 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.