三阴乳腺癌Aurora-A调控FOXC1抑制分化的机制研究及干预

Poor differentiation is an obvious characteristic of triple-negative breast cancer and is the key reason for its aggressive behavior and poor prognosis. Previously we showed that Aurora-A was related to differentiational phenotype of breast cancer (Mol. Cancer. Ther., 2014；Nat Commun., 2016). Recently we find that Aurora-A is over-expressed in triple-negtive breast cancer. Inhibition of Aurora-A kinase activity induces differentiation of triple-negative breast cancer cells. We also find that the key differentiational transcription factor FOXC1 is phosphorylated by Aurora-A. Thus we hypothesize that Aurora-A phosphorylates and transcriptional activites FOXC1 to inhibit the differentiation of triple-negtive breast cancer which contributes to tumorigenesis and progress of triple-negtive breast cancer. In this study we will identify the Aurora-A phosphorylation sites on FOXC1 and investigate its influence on FOXC1 nuclear localization, transcription activity and down-stream gene expression. We will also investigate Aurora-A-FOXC1 signaling pathway on the tumorigenesis and progress of triple-negtive breast cancer. Finally we will perform the animal experiment and analyze the clinical sample. Our study will provide the evidence for developing the therapeutic method and molecular marker of triple-negtive breast cancer.

细胞分化差是三阴乳腺癌的显著特点，也是恶性程度高和预后差的关键原因。本课题组前期研究发现Aurora-A与乳腺癌分化表型密切相关（Mol. Cancer. Ther., 2014, Nat Commun., 2016）。最近我们发现Aurora-A在三阴乳腺癌高表达，抑制Aurora-A激酶活性后诱导三阴乳腺癌细胞分化，并发现分化关键转录因子FOXC1受到Aurora-A磷酸化。因此我们推测Aurora-A通过磷酸化激活FOXC1，促进其下游基因表达调控，从而抑制三阴乳腺癌分化并促进其发生发展。本课题将围绕以上科学假设，阐明Aurora-A磷酸化FOXC1的位点，研究该位点对FOXC1核内定位、转录活性及下游基因表达调控的影响，从分化的角度探讨Aurora-A-FOXC1信号通路在三阴乳腺癌发生发展的作用，最后通过动物实验与临床标本进行验证，为发现三阴乳腺癌治疗靶点和分子标记提供科学依据。

细胞分化差是三阴乳腺癌恶性程度高和预后差的关键原因，本项目从分化的角度探讨三阴乳腺癌发生发展的分子机制，主要研究内容为：① 研究Aurora-A和FOX对三阴乳腺癌细胞分化状态和生长增殖的影响；② 研究Aurora-A调控FOX并抑制三阴乳腺癌分化的分子机制；③ 临床标本和动物实验验证Aurora-A/FOX信号通路在三阴乳腺癌发生发展的作用。重要结果和关键数据如下：首先，Aurora-A和FOX在体内和体外明显促进三阴乳腺癌细胞生长增殖并维持乳腺癌干细胞“干性”，干扰了Aurora-A和FOX的三阴乳腺癌细胞在小鼠体内生长受到明显抑制，CD44+/CD24-的乳腺癌干细胞群比例明显下降，干性相关基因Nanog、Oct4和C-myc明显下调；机制实验显示Aurora-A与FOX共同定位于细胞核，并发生直接相互作用，Aurora-A结合并激活FOX的启动子，以非激酶依赖的方式转录激活FOX的表达，同时，FOX也直接结合于Aurora-A的启动子区，促进Aurora-A的表达，因此得出结论Aurora-A与FOX在转录水平互相激活各自基因的表达；其次，Aurora-A还通过降低FOX泛素化，在细胞周期G1/S期抑制FOX降解促进三阴乳腺癌对紫杉醇耐药，在三阴乳腺癌细胞系干扰Aurora-A表达后，FOX的泛素化水平增高、降解加速；最后，Aurora-A与FOX的抑制剂在小鼠体内联合用药明显抑制三阴乳腺癌生长并克服紫杉醇耐药。TCGA数据库分析显示Aurora-A与FOX在三阴乳腺癌中比其他类型的乳腺癌明显增高，并且两者在三阴乳腺癌临床样本中共同表达升高，呈正相关。综上所述，我们的研究结论是Aurora-A通过转录激活和抑制降解两种方式上调FOX的水平，此调控作用不依赖Aurora-A的激酶活性，并且两者形成正反馈调控方式以维持两者在三阴乳腺癌细胞中的高水平表达。本研究的科学意义是：发现了Aurora-A与FOX形成正反馈调控环路促进三阴乳腺癌发生发展，验证了以Aurora-A与FOX为靶点的三阴乳腺癌治疗新策略，揭示了Aurora-A在细胞核内新的生物学功能，即作为转录激活因子以非激酶依赖的方式促进下游基因的表达。提示了在以Aurora-A为靶点的临床治疗中单独抑制Aurora-A激酶活性并不能完全抑制其致癌活性，应该同时抑制Aurora-A的非激酶活性。