c-Myc Phosphorylation Sites Regulate Its Apoptotic and Tumorigenic Potential

描述（由申请人提供）：c-Myc转录因子是细胞增殖和转化的有效诱导剂，在大多数人类肿瘤中观察到c-Myc蛋白水平升高。然而，c-Myc过表达也会诱导细胞凋亡，这限制了c-Myc的致瘤潜能。有两个保守的磷酸化位点，苏氨酸58 （T58）和丝氨酸62 (S62)，在有丝分裂刺激下对c-Myc蛋白的稳定性有差异调节，其中S62磷酸化增加c-Myc的稳定性，而T58磷酸化降低c-Myc的稳定性。最近的证据表明，这些位点的磷酸化也调节c-Myc的凋亡与致瘤潜能。具体来说，T58位点的低磷酸化和S62位点的高磷酸化是更稳定的c-Myc的特征，似乎抑制了c-Myc的凋亡活性并增强了其增殖特性。最近的研究结果表明，由于控制c-Myc降解的途径被解除管制，在一些测试的c-Myc蛋白异常稳定的人类癌细胞系中出现了较低的T58和较高的S62磷酸化。此外，在原发性乳腺癌样本中也检测到类似的c-Myc磷酸化改变。这表明癌细胞可能含有一种更致癌的c-Myc。这项资助的目的是研究T58和S62磷酸化在控制c-Myc的凋亡与增殖活性中的作用，以及这种机制是否有助于c-Myc在人类癌症中的转化活性。以下具体目标将追求：1)使用独特的小鼠模型检测c-Myc T58和S62磷酸化突变体在体内的活性；2)研究c-Myc T58和S62磷酸化突变体表达不同表型反应的机制；3)检测人类癌细胞中c-Myc在T58和S62位点的磷酸化状态，以及操纵c-MycWT磷酸化是否会影响其致癌潜能。这些目标包括小说研究诱导原癌基因敲入小鼠表达野生型原癌基因或原癌基因T58或S62磷酸化突变体在特定组织,在体外和体内分析探讨这些网站如何影响原癌基因活动的潜在机制,和人类癌症的分析探索改变磷酸化在这些网站的相关性和操纵细胞转换是否可以受遗传因素影响的原癌基因T58 S62磷酸化。这项研究具有重要的治疗意义，因为靶向调节T58和S62磷酸化的途径可能潜在地影响c-Myc表达水平和致瘤活性。公共卫生相关性：拟议的研究重点是了解转录因子c-Myc的致癌潜力如何受到其在两个高度保守位点的磷酸化状态的影响，磷酸化状态也调节其蛋白质稳定性。重要的是，在许多不同的人类肿瘤中广泛观察到c-Myc表达升高，因此了解增加或减少c-Myc致癌潜能的机制对于开发针对这种强效癌蛋白的未来治疗方法至关重要。

DESCRIPTION (provided by applicant): The c-Myc transcription factor is a potent inducer of cell proliferation and transformation, and elevated levels of c-Myc protein are observed in most human tumors. However, c-Myc overexpression also induces apoptosis, which limits c-Myc's tumorigenic potential. There are two conserved phosphorylation sites, Threonine 58 (T58) and Serine 62 (S62) that differentially regulate c-Myc protein stability in response to mitogenic stimulation, where S62 phosphorylation increases c-Myc stability, while T58 phosphorylation decreases c-Myc stability. Recent evidence suggests that phosphorylation at these sites also regulates c-Myc's apoptotic versus tumorigenic potential. Specifically, low phosphorylation at T58 and high phosphorylation at S62, which is the signature for more stable c-Myc, appears to suppress c-Myc's apoptotic activity and enhance its proliferative properties. Recent results indicate that lower T58 and higher S62 phosphorylation occurs in some tested human cancer cell lines with aberrantly stabilized c-Myc protein due to deregulation of the pathway that controls c-Myc degradation. Moreover, similarly altered phosphorylation of c-Myc has been detected in primary breast cancer samples. This suggests that cancer cells may contain a more oncogenic form of c-Myc. The objective of this grant is to examine the role of phosphorylation at T58 and S62 in controlling c-Myc's apoptotic versus proliferative activity, and whether this mechanism contributes to c-Myc's transforming activity in human cancer. The following specific aims will be pursued: 1) Examine the activity of c-Myc T58 and S62 phosphorylation mutants in vivo using a unique mouse model; 2) Investigate mechanisms that could underlie the different phenotypic responses to expression of c-Myc T58 and S62 phosphorylation mutants; and 3) Examine the phosphorylation status of c-Myc at T58 and S62 in human cancer cells and whether manipulation of c-MycWT phosphorylation can affect its oncogenic potential. These aims involve the study of novel inducible c-myc knock-in mice that express either wild-type c-Myc or c-Myc T58 or S62 phosphorylation mutants in specific tissues, in vitro and in vivo assays to investigate the underlying mechanisms of how these sites affect c-Myc activity, and an analysis of human cancer to explore the relevance of altered phosphorylation at these sites and whether cell transformation can be affected by non-genetic manipulation of c-Myc T58 and S62 phosphorylation. This research has important therapeutic implications, since targeting the pathway that regulates T58 and S62 phosphorylation could potentially affect both c-Myc expression levels and tumorigenic activity. PUBLIC HEALTH RELEVANCE: The proposed research focuses on understanding how the oncogenic potential of the transcription factor c-Myc is affected by its phosphorylation status at two highly conserved sites, which also regulate its protein stability. Importantly, elevated expression of c-Myc is widely observed in many different human tumors and therefore understanding mechanisms that increase or decrease c-Myc's oncogenic potential are critical to the development of future therapies targeting this potent oncoprotein.