GSK-3β激酶磷酸化ULK1调控自噬及其在肿瘤治疗中的作用

Increasing evidence indicates the importance of autophagy in human cancers. ULK1 is the furthest upstream kinase involved in autophagosome formation. The identification of intimate regulatory pathways and their functions in cancer may prove useful for cancer treatment. In our prelimilary study, we identified that ULK1 shared a structurally conserved domain with the phosphorylation substrate of GSK-3β. GSK-3β could interact with ULK1 and phosphorylate ULK1 on Ser409， and the expression level of ULK1 was negatively regulated by GSK-3β. Additionally, constitutively activated AKT transfection could up-regulate ULK1 expression through phosphorylating GSK-3β and inhibiting its activity. In esophagus squamous cell carcinoma, intensive ULK1 expression was correlated with poor prognosis. In this proposal, we will further extend our study to demonstrate the molecular mechanism that GSK-3β can phosphorylate Ser409 of ULK1 and subsequently regulate autophagy through promoting ubiquitin-mediated ULK1 proteolysis. Moreover, we will explore whether ULK1 knockdown can promote abnormal mitochondria potential and sensitize cancer cells to apoptotic inducers particularly during GSK-3β inactivation by hyperactivated AKT. The correlation of p-AKT, p-GSK-3β and ULK1 expression and their clinical significance in human tumor tissue will be investigated. The study will provide a comprehensive understanding of relationship between autophagy and cancer, and identify a previously unrecognized cancer cell vulnerability, which may provide a new therapeutic target in malignancies with dysregulated ULK1.

自噬与肿瘤发生发展密切相关，ULK1是自噬途径的核心蛋白，对其调控机制的研究是关注的焦点。我们研究发现ULK1结构域上具有GSK-3β磷酸化底物的共同保守序列；GSK-3β能与ULK1结合，负性调控ULK1蛋白的表达,并在体内外均能磷酸化ULK1的S409位点；细胞内激活的AKT可以通过磷酸化并灭活GSK-3β上调ULK1的表达；食管癌组织中ULK1高表达与预后负相关。本课题拟深入研究GSK-3β通过磷酸化ULK1(S409)致其泛素化降解，进而调控自噬的分子机制；研究激活的AKT通过上调ULK1来维持肿瘤细胞线粒体稳定的机制，探讨体内外靶向ULK1能否增强AKT高表达的肿瘤细胞对化疗的敏感性；并在多种肿瘤组织芯片标本中研究p-AKT, p-GSK-3β及ULK1表达的相关性及其对肿瘤发展进程及预后的影响。为深入理解自噬在肿瘤中的作用、靶向ULK1药物研究及其在肿瘤防治中的应用提供指导。

自噬与肿瘤发生发展密切相关，ULK1 是自噬途径的核心蛋白，对其调控机制的研究是关注的焦点。在本项目中我们重点探讨了GSK-3β磷酸化ULK1致其泛素化降解的分子机制；研究ULK1 对维持肿瘤细胞线粒体稳态的重要作用；并探讨GSK-3β磷酸化ULK1在肿瘤治疗中的意义。重要结果及关键数据如下：① 阐明GSK-3β 激酶在 ERK1 激酶的协助下磷酸化 ULK1导致其泛素化降解的分子机制：发现 ULK1 结构域上具有 GSK-3β 磷酸化底物的共同保守序列；阐明GSK-3β能与ULK1相互结合并磷酸化 ULK1的S409位点，且GSK-3β磷酸化ULK1需要ERK预磷酸化ULK1 S413位点；揭示GSK-3β/ERK1激酶介导的ULK1磷酸化促进ULK1发生泛素化，并促使其发生蛋白酶体途径降解。② 明确GSK-3β激酶介导的ULK1蛋白酶体降解对维持肿瘤细胞线粒体自噬的重要作用：发现ULK1的低表达使乳腺癌细胞在缺氧环境下线粒体自噬能力下降；阐明这种线粒体自噬能力缺失，导致NLRP3炎症体激活，促进炎症因子的分泌，从而显著增强肿瘤细胞的侵袭转移能力，促进乳腺癌骨转移和肝转移的发生。③ 阐明GSK-3β/ ERK磷酸化ULK1在肿瘤治疗中的意义：发现在肿瘤组织标本中p-GSK-3β 、p-ERK及 ULK1 表达有显著的相关性。在乳腺癌患者中ULK1表达与GSK-3β活化呈负相关，在宫颈癌患者中ULK1表达与p-ERK表达呈负相关。以上研究提示对于ULK1表达失调的肿瘤，GSK-3β和ERK激酶可作为治疗的靶标。本研究为深入理解自噬在肿瘤中的作用、靶向自噬药物研究及其在肿瘤防治中的应用提供理论依据和实验指导。.另外受该基金资助，我们发现蛋白激酶CK1γ通过磷酸化 Beclin 1，促进Beclin 1与p300结合并发生乙酰化，导致Beclin 1与Rubicon的结合增多，抑制了自噬体成熟和胞吞运输，阻断自噬过程，最终促进肿瘤生长；发现DNA 损伤类抗肿瘤药物能激活ATM，促进PTEN的磷酸化以及细胞核转位，继而通过激活p-JUN-SESN2/AMPK 信号通路诱导自噬适应性反应的发生，以此影响肿瘤细胞的药物敏感性。