Regulation and Role of ODC in Apoptosis and Cancer

描述（由申请人提供）：MYC癌基因家族是大多数癌症的共同靶点，其选择性激活反映了它们促进细胞持续增殖、诱导转化和肿瘤血管生成的能力。c-Myc如何触发这些过程尚不清楚，尽管它们都需要Myc的转录功能。我们的工作证实，鸟氨酸脱羧酶（ODC）是多胺生物合成的关键调节因子，是Myc的转录靶点，Myc在癌症中的激活导致ODC水平的高度升高。然而，c-Myc如何激活ODC转录尚未解决。c-Myc是一种碱性螺旋-环-螺旋-亮氨酸拉链（bHLH-Zip）转录因子，传统模型假设Myc与另一种bHLH-Zip蛋白Max二聚体结合，Myc:Max二聚体与靶基因中一致的CACGTG元件结合并激活转录。我们在小鼠中创建了ODC的敲入等位基因，其中Myc:Max结合位点都发生了突变，这表明Myc如何调节其靶基因的模式发生了转变。这些研究表明，这些CACGTG元件对转录抑制相当重要，并支持c-Myc通过从Mnt:Max复合体中分离Max来激活ODC的模型，Mnt:Max复合体积极抑制ODC转录。因此，在Aim 1中，我们提出了实验来确定c-Myc调节ODC的机制，通过创建额外的敲入小鼠来确认ODC内含子1 CACGTG元件与转录调控的相关性，并再次使用小鼠模型来确定Mnt在调节ODC中的作用。正常细胞中的Myc激活是通过触发凋亡程序来控制的。我们已经确定这涉及到ARF-p53检查点的诱导和抗凋亡基因bcl-2或bcI-X的抑制，并且这两种途径在myc诱导的肿瘤发生过程中都被破坏。我们的研究证实，ODC缺失或过表达可诱导细胞凋亡，ODC过表达也可抑制bcl-2的表达。因此，提出的研究的第二个目标是确定ODC在myc诱导的细胞凋亡和肿瘤发生中的生理作用。在Aim #2中，我们将评估ODC是否足以触发“Myc”凋亡途径，以及使用有条件的ODC敲除ODC是否会损害Myc诱导的凋亡。最后，在Aim #3中，我们通过评估ODC损失对Emu-myc转基因小鼠中myc诱导的淋巴瘤发生的影响，以及ODC在ODC表达缺陷的c-myc-/-胚胎干细胞中的过表达和肿瘤血管生成的影响，来解决ODC作为myc诱导的肿瘤发生和血管生成的调节剂是否必要。条件ODC基因敲除的创建提供了一个重要的临床前小鼠模型来测试靶向ODC在肿瘤发展、维持和血管生成中的功效。

DESCRIPTION (provided by applicant): The MYC family of oncogenes is common targets of most cancers and their selective activation reflects their ability to promote continuous cell proliferation, and to provoke transformation and tumor angiogenesis. How c-Myc triggers these processes is not clear, although they all require Myc's transcription functions. Our work established that ornithine decarboxylase (ODC), the key regulator of polyamine biosynthesis, is a Myc transcription target and that MYC activation in cancer results in highly elevated levels of ODC. However, how c-Myc activates ODC transcription is not resolved. c-Myc is a basic-helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor and the conventional model posits that Myc dimerizes with Max, another bHLH-Zip protein, and that Myc:Max dimers bind to consensus CACGTG elements in target genes and activate transcription. Our creation of a knockin allele of ODC in mice, in which both Myc:Max binding sites are mutated, suggest a paradigm shift in how Myc may regulate its target genes. These studies indicate that these CACGTG elements are rather important for transcriptional repression, and support a model whereby c-Myc activates ODC by sequestering Max from the Mnt:Max complex, which actively represses ODC transcription. Therefore, in Aim 1 experiments are proposed to determine the mechanism by which c-Myc regulates ODC, to confirm the relevance of the ODC intron 1 CACGTG elements to transcriptional regulation by creating additional knock-in mice, and to define the role of Mnt in regulating ODC, again using mouse models. Myc activation in normal cells is held in check by triggering the apoptotic program. We have established this involves the induction of the ARF-p53 checkpoint and the suppression of the anti-apoptotic genes, bcl-2 or bcI-X, and that both of these pathways are disrupted during Myc-induced tumorigenesis. Our studies have established that ODC loss or overexpression induces apoptosis and that ODC overexpression also suppresses the expression of bcl-2. Therefore, a second goal of the proposed studies is to determine the physiological role of ODC in Myc-induced apoptosis and tumorigenesis. In Aim #2 we will evaluate whether ODC is sufficient to trigger the "Myc" apoptotic pathways, and whether ODC loss, using a conditional ODC knockout, impairs Myc-induced apoptosis. Finally, in Aim #3 we address whether ODC is necessary as a regulator of Myc-induced tumorigenesis and angiogenesis by evaluating effects of ODC loss on Myc-induced lymphomagenesis in Emu-myc transgenic mice, and of ODC overexpression in c-myc-/- embryonic stem cells, which are defective in ODC expression, and tumor angiogenesis. The creation of the conditional ODC knockout provides an important pre-clinical mouse model to test the efficacy in targeting ODC in tumor development, maintenance and angiogenesis.