Aurora-A经由p53/Mcl-1通路调控白血病干细胞自我更新的机制研究

Leukemia Stem cells (LSCs) are the root of leukemia development and relapse. We previously showed blockade of Aurora-A by a short hairpin (sh)RNA against Aurora-A significantly impaired self-renewal capability of LSCs, however, the mechanisms by which Aurora-A regulated the self-renewal capability of LSCs were unclear. Our recent results showed that down-regulation of Aurora-A by a shRNA potently decreased the phosphorylated forms of p53. In further study we found that silencing of p53 by a shRNA up-regulated the expression of Mcl-1 and attenuated the ability of MLN8237, a selective inhibitor of Aurora-A, to inhibit self-renewal capability of LSCs. Importantly, blockade of Aurora-A did not significantly impair self-renewal capability of LSCs when Mcl-1 was forced expressed in those cells. On the basis of these findings, we proposed the hypothesis that Aurora-A inhibited the activity of p53 via phosphorylation, which up-regulated the expression of Mcl-1 and enhanced the capacity of self-renewal of LSCs. This project will provide theoretical and experimental evidence for the development mechanism of leukemia and targeted clinical treatment.

白血病干细胞（Leukemia Stem Cell，LSCs）是白血病发生、发展和复发的根源。申请者完成日本学术振兴会资助项目时发现：基因沉默Aurora-A能够抑制LSCs的自我更新能力。 然而，Aurora-A调控该细胞自我更新能力的分子机制尚不清楚。前期研究发现：阻断 Aurora-A能够下调p53磷酸化。 进一步研究发现：基因沉默p53能够上调LSCs 中Mcl-1的表达，同时明显减弱Aurora-A抑制剂对该细胞自我更新能力的影响。此外，当LSCs转染外源性Mcl-1后，阻断Aurora-A并未明显抑制该细胞的自我更新能力。据此申请人提出以下科学假说：Aurora-A通过磷酸化作用下调p53功能活性，从而增强Mcl-1的表达，进而促进LSCs的自我更新能力。本项目的开展能够为研究白血病的发生发展机理、探寻临床更有针对性的治疗方法提供了新的思路。

Aurora-A是调控细胞有丝分裂的一类重要的丝氨酸/苏氨酸蛋白激酶---Aurora激酶家族中一员，其主要参与中心体的形成、纺锤体的组装和维持、胞质分裂等过程。Aurora-A在多种肿瘤组织中高表达，包括结直肠癌、乳腺癌、神经胶质瘤、胰腺癌等，其通过调控肿瘤细胞增殖、耐药、以及肿瘤干细胞干性等参与肿瘤的发展及复发。此外，该基因异常高表达并且与患者预后密切相关。Alisertib（MLN8237）是一种高度选择性抑制Aurora-A激酶靶向药物，在体内和体外实验中证实alisertib对多种肿瘤有明显治疗效果。然而部分临床试验发现alisertib未能达到预期的疗效。因此我们提出科学假说：在体内，Aurora-A可能与其他蛋白分子相互作用，利用alisertib抑制Aurora-A功能可能会直接或间接地影响Aurora-A与其他蛋白分子的相互作用，进而影响alisertib的治疗效果。本研究利用人结肠癌HCT-8荷瘤裸鼠模型发现：1）与对照组相比，给予小鼠alisertib后，促进信号转导及转录激活蛋白1 (Signal transducer and activator of transcription 1， STAT1)的扩增和活化，且呈干扰素非依赖性；2）STAT1异常高表达是导致肿瘤细胞对alisertib产生抵抗的主要因素之一；3）进一步研究发现：Alisertib干扰Aurora-A-泛素样含PHD和环指域1(ubiquitin-like with PHD and ring finger domains 1，UHRF1)结合、加速DNA甲基转移酶1（DNA methyltransferase 1，DNMT1）泛素化，最终导致STAT1表观活化；4）Aurora-A及UHRF1在结肠癌中均呈现高表达，双干扰Aurora-A和UHRF1抑制结肠癌细胞的增殖。本研究通过一系列实验阐明肿瘤细胞对alisertib抵抗的分子机制，为提高临床治疗效果提供思路。