膜联蛋白Annexin II在急性早幼粒细胞白血病中高表达的病理生理效应及分子调控机制

Aberrant annexin II expression and consequent hyperfibrinolysis in acute promyelocytic leukemia (APL) are one of causes for the early death of APL patients. In the past 5 years, the applicant has been focusing on the molecular mechanism of regulation of tissue factor, which triggers disseminated intravascular coagulation in APL bleeding complication, and the transactivation of tissue factor by PML/RAR alpha fusion protein was accepted and published in PNAS, 2010. Annexin II, which aberrantly expressed in APL cells, is a membrane protein as the cofactor for t-PA and plasminogen. High level expression of annexin II overproduces plasmin to induce hyperfibrinolysis, a known major component of APL bleeding complication. The recent findings in our laboratory indicate that annexin II is up-regulated by PML/RAR alpha fusion protein, an oncoprotein specific for the leukemogenesis of APL, but down-regulated by differentiation agents such as all-trans retinoic acid and/or arsenic trioxide (ATRA/AS2O3) for rapid alleviation of bleeding. Therefore, the current project is designed to further investigate: 1) the functional significance of aberrant annexin II expression in APL cells, such as hyperfibrinolysis and cell invasion; 2) the molecular mechanism of annexin II up-regulation by PML/RAR alpha fusion protein using promoter deletion analysis, luciferase activity assay, EMSA, chromatin immunoprecipitation and co-immunoprecipitation; 3) the molecular mechanism of annexin II down-regulation by differentiation agents ATRA/AS2O3 using similar research methods outlined above. Taken together, this project is intended to elucidate the functional significance and underlying molecular basis of aberrant annexin II expression in the pathogenesis of APL coagulopathy. The ramification of our findings has clinical importance, as annexin II may serve as a novel therapeutic target aiming to minimize the fatality caused by coagulopathy and relapse from leukemia infiltration in APL patients.

Annexin II（Ann-II）在急性早幼粒细胞白血病（APL）细胞中异常高表达，所致的纤维蛋白溶解亢进是患者早期出血死亡的关键原因之一。申请者前期阐明了导致APL出血的重要因子Tissue factor的分子调控机制（PNAS 2010），目前又发现另一出血关键因子Ann-II的高表达与APL特征性PML/RARα融合蛋白调控有关；而诱导分化药物可迅速下调Ann-II并改善临床出血症状。故拟在U937/PR9细胞中，研究1）PML/RARα融合蛋白上调Ann-II高表达所致纤溶亢进、白血病细胞侵袭等病理生理效应；2）应用启动子截短分析策略，通过荧光素酶活性分析、EMSA、ChIP、Co-IP等手段，探讨PML/RARα融合蛋白上调Ann-II表达的分子机制。3）应用上述类似的转录调控研究手段，阐明诱导分化药物下调其表达的分子机制。为系统解释并防治出凝血并发症、白血病浸润提供理论基础。

本研究主要采用RT-PCR、western blot、generation、invasion及FCM等技术手段和以Zn2+可控PML/RARα融合蛋白表达的APL细胞株(U937/PR9)模型，发现：病理生理学效应：1、Zn2+诱导PML/RARα融合蛋白表达2小时后，ANXA2及其配体S100A10 (p11)在转录水平及翻译水平的表达均明显增高，其中ANXA2呈逐步递增趋势，而S100A10呈组成性的表达，两者表达呈正相关；2、在Zn2+处理过的U937/PR9细胞中，tPA介导的纤溶酶生成速率(IRPG)较未处理的细胞快2.13倍，同时，细胞侵袭的能力提高了27.6%。而ANXA2或S100A10抗体封闭细胞后，IRPG及细胞侵袭的能力均显著下降；4、维甲酸（ATRA）处理Zn2+诱导后的U937-PR9细胞或APL病人血标本中的原始细胞，均能下调细胞内ANXA2及S100A10的表达，并降低纤溶酶生成速率(IRPG)及细胞侵袭能力。相关分子机制：1、构建完成ANXA2启动子荧光素酶报告质粒，首次阐明ANXA2启动子中的转录起始位点及相应功能域分析；2、DNA pull down实验表明ANXA2启动子-567+120与PML/RARα融合蛋白存在结合，提示PML/RARα融合蛋白调控启动子作用的可能；3、通过电穿孔转染的方式建立相应的U937/PR9稳定细胞株（启动子-2100-16），Zn2+作用后比较相应细胞中的荧光素酶活性变化，但目前尚未筛出有效表达的细胞株(1-3)，故具体的调控位置尚无法精确定位。上述结果初步证实了PML/RARα融合蛋白明显上调了ANXA2表达，并产生明确的病理生理学效应；同时，ANXA2 启动子的某些相关功能域业已明确，具体调控的分子机制亦在进一步的研究中，这些均为防治APL出凝血异常提供了新的理论基础。