ILKAP催化核仁磷蛋白NPM去磷酸化调控骨肉瘤恶性生物学行为的机制研究

Osteosarcoma is the most common primary malignant bone tumor，with high malignance, fast growing, early metastasis and strong invasion, about 80% of patients had been found the transfer of lung or other parts at diagnosis in clinical work and no definite molecular target point for the cure of osteosarcoma. Nucleophosmin（NPM）is a phosphorus-rich protein localized in nucleolus and closely related with tumors. The over-phosphorylation of NPM causes tumor generation and proliferation especially in human osteosarcoma. The dysregulation of NPM phosphorylation is the main cause of the over-phosphorylation of NPM and the generation and proliferation of osteosarcoma. To discover the mechanism of the dysregulation of NPM is the way to resolve the molecular target point for the cure of osteosarcoma. We found ILKAP is the only known phosphatase localized in the nucleolus and directly catalysis of NPM. We also found ILKAP inactivated mutants widely exist in human osteosarcoma. Those resules suggest that ILKAP maybe regulate the generation, proliferation and metastatic potential in osteosarcoma through dephosphorylation of NPM. We plan to explore the specific sites about ILKAP catalytic of NPM, in order to clarify the mechanism about the dephosphorylation of NPM. We plan to detect the change of NPM phosphorylation and the ability of cell proliferation, invasion, and tumorigenic by overexpression or knockout of ILKAP in human osteosarcoma cell lines. We also plan to explore the impact about ILKAP dephosphorylat of NPM in the generation and malignant behavior of human osteosarcoma with animal models. In this study we can provid the new theory of the molecular mechanism in the generation and malignant behavior of human osteosarcoma and provid now direction and medicine target point for the cure of osteosarcoma

骨肉瘤是一种常见的原发恶性骨肿瘤，具有恶性度高，侵袭力强且易转移的特点，目前尚缺乏明确的分子治疗靶点。近年来研究显示，核仁磷蛋白(NPM)的磷酸化状态与多种肿瘤的增殖与转移密切相关。本课题组在前期研究中发现，一种核仁定位的蛋白磷酸酶ILKAP可以催化NPM的去磷酸化，并抑制骨肉瘤细胞的增殖。同时发现骨肉瘤中ILKAP存在广泛的失活突变；这提示ILKAP可能通过调节NPM的磷酸化影响骨肉瘤的发生、增殖与转移。本项目拟通过探讨ILKAP催化NPM的磷酸化位点,以确定其催化NPM后对应的肿瘤相关信号通路，并利用骨肉瘤模型细胞系检测过量表达或敲除ILKAP后NPM的磷酸化状态，观察骨肉瘤细胞增殖、侵袭、粘附能力的变化。再利用动物模型检测过量表达或敲除ILKAP对骨肉瘤成瘤与转移的影响。阐明ILKAP调控NPM的去磷酸化机制及对骨肉瘤恶性生物学行为的影响，为骨肉瘤的治疗提供新的分子靶点和治疗途径。

首先在对ILKAP亚细胞核定位及功能的探索中，我们发现ILKAP具有明显高的核定位表达，同时我们发现ILKAP通过自身关键氨基酸77-79位K-R-K介导了ILKAP与Importin之间的结合并利用Importin进入细胞核中。.随后我们利用328例骨肉瘤样本发现ILKAP在88.2%的骨肉瘤样本中存在突变，其中两种突变型占总突变数的91.3%，NCBI登录号为FJ491380和FJ491381（我们已于2010年提交至GeneBank)。.随后我们选择了8个Ser以及Thr位点并构建了特异性磷酸化抗体。我们发现ILKAP可以显著的降低NPM蛋白Ser10以及Ser207两个位点的磷酸化状态。我们认为ILKAP是通过调控NPM蛋白Ser10以及Ser207位点的去磷酸化发挥调控NPM蛋白功能的作用（Fig.9）。.在明确ILKAP催化NPM对骨肉瘤恶性生物学行为的影响这一部分中，我们构建了基于骨钙素的骨骼系统特异性敲除ILKAP的转基因小鼠KKX-I品系。KKX-I品系鼠在出生后16-20周可以自发性的形成骨肉瘤（Fig.10）。而利用Tet-on系统，表达ILKAP的多种骨肉瘤细胞系，均发生了克隆增值能力下降，迁移、侵袭能力降低等肿瘤标志行为的丧失。这些结果说明ILKAP在骨肉瘤发生的进程中起到至关重要的作用。