Ser68位点磷酸化调控干性因子Nanog的蛋白稳定性及肿瘤细胞干性的功能和机制研究

The protein level of stemness transcription factor Nanog serves as a hallmark of malignancy and poor prognosis in cancer treatments. However, the regulatory mechanisms underlying the protein level of Nanog remain elusive. Our preliminary data demonstrate that Nanog can interact with E3 ubiquitin ligase SPOP followed by consequent ubiquitination and degradation of Nanog, leading to decreased stemness properties of embryonic stem cells（ESCs）and cancer stem cells (CSCs). In parallel, both Nanog mutations and SPOP mutations that are frequently observed in cancer patients attenuate the interaction between SPOP and Nanog with decreased ubiquitination and degradation of Nanog, which increases the protein level of Nanog and endows cancer cells with stem-like traits and aggravated tumorigenic capability. Recently, Our preliminary data have shown that the phosphorylation of Nanog at Ser68 also interferes the interaction between Nanog and SPOP, thereby enhancing the protein stability of Nanog. Based upon above evidence, we hypothesize that the phosphorylation of Nanog at Ser68 increases Nanog stability and thus contributes to the acquisition of stem-like properties in cancer cells. We will continue to explore the role of the phosphorylation of Nanog at Ser68 in the control of Nanog stability in cancer cells as well as the mechanisms underlying their acquisition of stem-like properties. Our study will shed light on the identification of novel drug targets and new therapeutic strategies for cancer treatments.

肿瘤细胞内干性因子Nanog的表达水平与肿瘤恶性程度正相关。由于Nanog降解非常迅速, 其蛋白稳定性是决定蛋白表达水平的关键因素, 但调控Nanog蛋白稳定性的机制尚不清楚。申请人的前期工作筛选并证明E3泛素连接酶SPOP通过结合Nanog对其进行泛素化降解; 肿瘤相关的SPOP突变体或Nanog突变体皆可减弱两者结合, 促进Nanog蛋白稳定性和肿瘤细胞干性。近期, 申请人发现Nanog Ser68磷酸化亦可减弱其与SPOP的结合, 并提高细胞内Nanog蛋白量。由此,申请人提出肿瘤中Nanog Ser68磷酸化通过影响Nanog蛋白稳定性调控肿瘤细胞干性的科学假设。申请人拟进一步明确介导Nanog Ser68磷酸化的胞内信号蛋白及其调控Nanog蛋白稳定性和肿瘤细胞干性的分子机制, 并评估该磷酸化作为肿瘤复发转移的诊断指标和潜在治疗靶点的可能性，为开发靶向肿瘤细胞干性的新药指出方向。

Nanog在肿瘤细胞中重新获得表达，现已在胶质瘤、前列腺癌、肺癌、肝癌等恶性肿瘤中发现Nanog的表达，而且肿瘤级别越高，Nanog表达越强，临床预后越差。 Nanog在癌细胞增殖、迁移和侵袭，对药物和化疗抵抗方面都具有重要作用。因此，Nanog可作为肿瘤的一个敏感性、特异性的标志物和治疗靶点。目前，诸多临床证据表明Nanog在肿瘤细胞中的高表达可促进肿瘤细胞干性获得，继而导致肿瘤干细胞的产生。. Nanog的表达水平受其蛋白转录翻译及蛋白稳定性调控等方面的影响。由于Nanog在细胞中是一个极不稳定的蛋白，蛋白半衰期非常短暂，导致Nanog蛋白稳定性的改变将在很大程度上影响其在细胞内的表达水平。已有报道证实Nanog可发生磷酸化修饰，从而影响其活性及稳定性。但是肿瘤细胞中Nanog磷酸化对其蛋白稳定性的调控机制仍不清楚。. 申请人团队通过体内体外筛选实验，发现Cullin3依赖的E3泛素连接酶SPOP能通过结合Nanog蛋白而将其泛素化降解。肿瘤相关的SPOP突变体或Nanog突变体皆可使两者之间的相互作用减弱，阻断SPOP对Nanog的泛素化降解，并显著增强Nanog的稳定性和表达水平；Nanog S68磷酸化可阻断SPOP和Nanog的相互作用，并由此诱导Nanog表达水平升高和促进肿瘤细胞干性获得；通过体外磷酸化实验的筛选，申请人发现7种蛋白激酶可磷酸化Nanog S68。其中，BRAF对Nanog S68的磷酸化起主要作用。. 基于申请人自己的工作及相关文献，申请人提Nanog S68磷酸化通过影响SPOP对干性转录因子Nanog的泛素化降解，调控Nanog蛋白稳定性，继而促进肿瘤细胞干性获得的科学假设。本项目进一步以前列腺癌为代表，对Nanog S68磷酸化如何调控SPOP对干性转录因子Nanog的泛素化降解和肿瘤细胞干性获得，以及磷酸化Nanog S68的蛋白激酶和去磷酸化Nanog S68的磷酸酶在其中的作用，展开了深入研究（具体研究内容由于篇幅限制，请见正文）。

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