泛素连接酶β-TrCP靶向降解CHK1在DNA损伤应答和肿瘤发生中的作用研究

CHK1在DNA损伤应答、细胞周期检查点、维持基因组稳定性等方面发挥重要作用，通常认为其是抑癌蛋白，但是大量研究显示其也有明显促癌特性。在多种人类肿瘤中存在CHK1蛋白水平和活性异常，却很少发现功能缺失突变，提示其通过水平和活性改变参与肿瘤发生。由于CHK1是细胞生存所必须的，全敲或半敲Chk1对体内肿瘤发生的作用恰好相反，这都给理解其抑癌或促癌特性增加了复杂性。我们前期发现DNA损伤能诱导泛素连接酶βTrCP靶向降解CHK1，但机制和生理功能不清楚。为此，我们制备了降解抵抗的突变体敲入鼠，以增强体内CHK1的功能。本项目拟：1）在生理和DNA损伤条件下研究βTrCP靶向降解CHK1的分子机制；2）利用突变敲入鼠研究Chk1降解在DNA损伤应答和体内肿瘤发生中的作用；3）在临床水平研究βTrCP-CHK1轴与乳腺癌患者放疗敏感性和预后的关系，从而帮助确定CHK1在体内肿瘤发生中的作用。

Given the critical roles of CHK1 in DNA damage response, cell cycle checkpoints and genomic stability, CHK1 was initially thought to function as a tumor suppressor. However, accumulating studies suggest that CHK1 also promotes tumor growth. Although the protein levels and activity of CHK1 are found to be dysregulated in a wide range of human tumors, no homozygous loss-of-function mutation in CHK1 has been detected. Thus, dysregulation of CHK1 in its protein levels and activity plays significant roles in tumorigenesis. In addition, CHK1 is essential for cell survival and the studies using conditional knock-out Chk1 mouse tumor models indicated conflicting roles of deletion in one or both Chk1 allele(s) in tumorigenesis, which further renders the complexity of CHK1 function in tumorigenesis. Recently, we found that SCFβ-TrCP (Skp1-Cullin1-βTrCP E3 ubiquitin ligase) targets CHK1 for polyubiquitination and proteasomal degradation in response to DNA damage. However, the mechanism underlying β-TrCP targeting CHK1 for degradation and the roles of CHK degradation by β-TrCP in DNA damage response and tumorigenesis remain unknown. Thus, we generated a Chk1 3A knock-in (KI) mouse model, in which Chk1 3A, a Chk1 mutant resistant to SCFβTrCP-mediated degradation, was expressed to enhance the levels and activity of CHK1 in vivo. In this proposal, we are going 1) to explore the molecular mechanisms by which SCFβTrCP targets CHK1 for degradation under normal and DNA damage stress conditions; 2) to use Chk1 3A KI mouse model, alone or in combination with Pten loss (Pten+/- or Ptenfl/fl) to determine the physiological role of Chk1 degradation in DNA damage response and tumorigenesis; 3) to explore the correlation of βTrCP-CHK1 axis with the prognosis and radiation sensitivity in breast cancer patients. Our study will elucidate the physiological role of Chk1 degradation in DNA damage response and tumorigenesis and help to determine whether CHK1 is a tumor suppressor gene or an oncogene in vivo.