c-Myc Phosphorylation Sites Regulate Its Apoptotic and Tumorigenic Potential

描述(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的过表达也会诱导细胞凋亡，从而限制了c-Myc的致瘤潜能。有两个保守的磷酸化位点，即苏氨酸58(T58)和丝氨酸62(S62)，它们在有丝分裂刺激下不同地调节c-Myc蛋白的稳定性，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，这些位点的磷酸化也调节c-Myc的凋亡和致瘤潜能。特别是，T58位的低磷酸化和S62位的高磷酸化是更稳定的c-Myc的标志，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，在一些c-Myc蛋白异常稳定的人癌细胞系中，由于控制c-Myc降解的途径的解除调控，出现了较低的T58和较高的S62磷酸化。此外，在原发乳腺癌样本中也检测到了类似的c-Myc磷酸化改变。这表明癌细胞中可能含有致癌性更强的c-Myc。这项资助的目的是研究T58和S62的磷酸化在控制c-Myc的凋亡和增殖活性中的作用，以及这一机制是否有助于c-Myc在人类癌症中的转化活性。我们将致力于以下具体目标：1)利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磷酸化突变体表达的不同表型反应的机制；3)检测c-Myc在T58和S62处的磷酸化状态，以及c-MycWT磷酸化操作是否会影响其致癌潜力。这些目标包括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可诱导c-myc敲入小鼠，体外和体内试验以探讨这些位点如何影响c-Myc活性的潜在机制，以及分析人类癌症以探索这些位点磷酸化改变的相关性以及c-Myc T58和S62磷酸化的非遗传操作是否会影响细胞转化。这项研究具有重要的治疗意义，因为靶向调节T58和S62磷酸化的途径可能会潜在地影响c-Myc的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两个高度保守的位点的磷酸化状态的影响，这也调节其蛋白质的稳定性。重要的是，c-Myc的高表达在许多不同的人类肿瘤中被广泛观察到，因此，了解增加或降低c-Myc的致癌潜力的机制对于未来针对这种有效的癌蛋白的治疗的发展至关重要。

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.