Nuclear Accumulation of D-type Cyclins and Oncogenesis

虽然细胞周期蛋白D1在癌症中过度表达，但野生型细胞周期蛋白D1的致癌性很低。穿过 在这笔赠款的支持下，我们率先解决了这一悖论。重要的是，我们的 工作揭示了一种范式，即细胞周期蛋白D1在G1期在细胞核内积聚； 然而，在G1/S边界，单个苏氨酰残基Thr-286的磷酸化通过 GSK-3b触发细胞周期蛋白D1的核输出及其胞浆泛素介导的蛋白降解。一个 非磷酸化突变体D1T286A是结构性核的，对多聚体不敏感。 泛素化，并具有强大的致癌特性。在上一次调查中的一个工具性发现 四年来证明了活跃的核细胞周期蛋白D1/CDK4激酶的积累 在S期，触发DNA的重新复制，导致基因组的不稳定和P53的失活； 这一切都是由于其激活精氨酸甲基转移酶PRMT5的能力所致。现在是时候了 显然，细胞周期蛋白D1受到直接抑制Thr-286的频繁突变的影响 磷酸化，从而证明细胞周期蛋白D1是一个驱动癌基因。示例包括 子宫癌、食道癌、黑色素瘤和多发性骨髓瘤。由于当前模型依赖于 异位启动子突变等位基因的过度表达，我们已经产生了一个新的模型 允许突变的D1T286A从其内源启动子诱导表达。我们 假设来自其内源启动子的细胞周期蛋白D1T286A的表达将驱动 肿瘤生长，并将允许产生模仿人类癌症的小鼠模型 细胞周期蛋白D1的体细胞突变。此外，虽然很多研究都集中在细胞周期蛋白D1上，但由于 由于其在癌症中频繁的调节失调，现在很明显，细胞周期蛋白D3受到类似的 Burkitt淋巴瘤中保守的c-末端磷酸化位点Thr-283的突变 D3突变为非磷酸化残基。这种突变与myc- 易位的频率接近50%。这一发现突显了知识上的差距 关于D3的磷酸化如何有助于其调节和细胞动态平衡。我们 因此，假设Thr-283磷酸化引导细胞周期蛋白D3输出到细胞质 在那里它被泛素化和降解。这些知识上的差距将直接得到解决 通过三个凝聚目标：目标1将确定细胞周期蛋白D1T286A的肿瘤活性 使用可诱导的敲击小鼠模型，允许其内源性启动子表达； 将确定Fbx18在Thr-283磷酸化细胞周期蛋白D3泛素化中的作用； 3将确定Fbx18的肿瘤抑制功能以及是否非磷酸化 Cyclin D3T283A显示出增强的驱动淋巴瘤的能力。

While cyclin D1 is overexpressed in cancer, wild type cyclin D1 is poorly oncogenic. Through work supported by this grant, we have pioneered the resolution to this paradox. Importantly, our work has revealed a paradigm wherein cyclin D1 accumulates in the nucleus during G1 phase; however, at the G1/S boundary, phosphorylation of a single threonyl residue, Thr-286, by GSK-3b triggers cyclin D1 nuclear export and its cytoplasmic ubiquitin-mediated proteolysis. A non-phosphorylatable mutant, D1T286A, is constitutively nuclear, refractory to poly- ubiquitination and has potent oncogenic properties. An instrumental finding during the previous four years was the demonstration that accumulation of active nuclear cyclin D1/CDK4 kinase during S-phase triggers DNA re-replication, induces genomic instability and inactivation of p53; all a consequence of its capacity to activate the arginine methyltransferase PRMT5. It is now evident that cyclin D1 is subject to frequent mutations that directly inhibit Thr-286 phosphorylation, thereby demonstrating that cyclin D1 as a driver oncogene. Examples include uterine cancer, esophageal, melanoma and multiple myeloma. Since current models depend on overexpression of mutant alleles from ectopic promoters, we have generated a new model that allows inducible expression of the mutant D1T286A from its endogenous promoter. We hypothesize that expression of cyclin D1T286A from its endogenous promoter will drive neoplastic growth and will permit the generation of mouse models of human cancer that mimic somatic mutation of cyclin D1. In addition, while much effort has been focused on cyclin D1 due to its frequent dysregulation in cancer, it is now evident that cyclin D3 is subject to an analogous mutation in Burkitt’s lymphoma where the conserved c-terminal phosphorylation site, Thr-283 in D3, is mutated to a non-phosphorylatable residue. Such mutations co-occur with myc- translocations at a frequency approaching 50%. This revelation highlights a gap in knowledge regarding how phosphorylation of D3 contributes to its regulation and cell homeostasis. We therefore hypothesize that Thr-283 phosphorylation directs cyclin D3 for export to the cytoplasm where it is ubiquitylated and degraded. These gaps in knowledge will be directly addressed through three cohesive aims: Aim 1 will determine the neoplastic activity of cyclin D1T286A using an inducible knockin mouse model that permits expression from its endogenous promoter; Aim 2 will determine the role of Fbxl8 in ubiquitylation of Thr-283 phosphorylated cyclin D3; Aim 3 will determine the tumor suppressive function of Fbxl8 and whether non-phosphorylatable cyclin D3T283A exhibits enhanced ability to drive lymphoma.