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强效诱导剂，在大多数人类肿瘤中观察到c-Myc蛋白水平升高。然而，c-Myc过表达也诱导细胞凋亡，这限制了c-Myc的致瘤潜力。存在两个保守的磷酸化位点，ThreeThree 58（T58）和Serine 62（S62），其响应于促有丝分裂刺激而差异调节c-Myc蛋白稳定性，其中S62磷酸化增加c-Myc稳定性，而T58磷酸化降低c-Myc稳定性。最近的证据表明，在这些网站的磷酸化也调节c-Myc的凋亡与致瘤潜力。具体而言，T58处的低磷酸化和S62处的高磷酸化（其是更稳定的c-Myc的特征）似乎抑制c-Myc的凋亡活性并增强其增殖特性。最近的结果表明，较低的T58和较高的S62磷酸化发生在一些测试的人癌细胞系与异常稳定的c-Myc蛋白由于失调的途径，控制c-Myc降解。此外，在原发性乳腺癌样本中也检测到了类似的c-Myc磷酸化改变。这表明癌细胞可能含有更多致癌形式的c-Myc。这项研究的目的是研究T58和S62磷酸化在控制c-Myc细胞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。1）使用独特的小鼠模型在体内检查c-Myc T58和S62磷酸化突变体的活性; 2）研究可能是对c-Myc T58和S62磷酸化突变体表达的不同表型应答的基础的机制;和3）检查人癌细胞中c-Myc在T58和S62处的磷酸化状态以及c-MycWT磷酸化的操作是否可以影响其致癌潜力。这些目标涉及研究在特定组织中表达野生型c-Myc或c-Myc T58或S62磷酸化突变体的新型诱导型c-Myc敲入小鼠，体外和体内测定以研究这些位点如何影响c-Myc活性的潜在机制，以及对人类癌症的分析，以探索这些位点磷酸化改变的相关性，以及细胞转化是否会受到c-Myc T58和S62磷酸化的非遗传操作。这项研究具有重要的治疗意义，因为靶向调节T58和S62磷酸化的途径可能会影响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，这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的表达升高，因此了解增加或减少c-Myc致癌潜力的机制对于开发靶向这种强效癌蛋白的未来疗法至关重要。

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.