Aurora激酶介导染色质三维重构促进乳腺癌干细胞干性的机制研究

Molecularly targeted cancer therapy has achieved tremendous advances in the past decade. However, recent studies suggest that cancer stem cells (CSCs) lead to therapy resistance, tumor progression, and recurrence. Based on AURKA-targeted therapy resistance model and AURKA nuclear translocation model, we have found that oncogenic kinase AURKA translocated into nucleus and lead to drug resistance through promoting cancer stemness (Oncogene 2017; Nat Commun 2016). Recently, we performed stable isotope labeling with amino acids in cell culture (SILAC) assays and chromatin immunoprecipitation-sequencing (ChIP-seq) assays to get the interacting proteins and genomic DNA binding sites of nuclear AURKA, respectively. We found that AURKA bind to genome organization protein RAD21/p300 and its binding sites frequently occurred H3K27ac modification. We further performed chromatin conformation capture assay (3C) and found the space interaction of AURKA binding sites. These results suggest that AURKA translocated to nucleus and remodeled three-dimensional genome organization under chemotherapy or AURKA-targeted therapy, thus promoting the formation of (super) enhancer, enhancing cancer stemness, and leading to drug resistance. This project aims to exploit molecular mechanisms of AURKA-dependent 3D genome organization in BCSCs, determine co-factors, illustrate the key target genes, and investigate the clinical significance of AURKA nuclear translocation. Therefore, this project will provide novel target for cancer stemness, and present new rationale for oncogenic kinase targeting.

靶向治疗在乳腺癌中取得突破，但肿瘤干细胞导致的耐药是制约疗效的瓶颈。我们前期发现细胞周期激酶AURKA核转位激活FOXM1/Myc，促进乳腺癌干性，抵抗激酶靶向治疗(Oncogene 2017；Nat Commun 2016)。申请人筛选AURKA胞核互作蛋白发现其与染色质重构蛋白RAD21/p300结合；ChIP-seq发现其基因组结合位点多伴随H3K27ac修饰；染色质构象捕获实验证实其参与了染色质构象重塑。上述结果提示AURKA介导染色质重塑，促进（超级）增强子形成，增强干性基因表达，进而导致耐药。本项目拟在已建立的耐药和核转位模型中，明确AURKA调控染色质三维重构的共作用因子，揭示其复合物形成及作用的机制；阐明其促干性关键靶基因；探寻靶向AURKA核功能的干预靶点及临床意义。本项目是靶向AURKA抗癌领域的远瞻课题，有望揭示AURKA全新的促癌机制，为靶向乳腺癌干细胞提供新靶点。

蛋白激酶基因的突变或扩增引起的激酶异常激活是肿瘤恶性转化、转移和复发的驱动事件。其中，Aurora-A (AURKA)蛋白激酶在多种肿瘤中高表达，并提示不良预后。多个靶向AURKA激酶活性的化合物在全球多中心开展了Ⅱ期或Ⅲ期临床试验。已经公开的临床试验数据表明，其激酶抑制剂在临床试验中具有抗癌效果，但仍面临耐药和复发难题。本项目以课题组前期发现的AURKA核转位促进肿瘤干细胞干性为切入点，探究核转位AURKA调控染色质重塑促进肿瘤干性引发耐药的机制。项目严格按照任务书开展研究，完成了既定的研究目标，取得以下几点研究成果：1）明确了AURKA核转位是导致乳腺癌干性及耐药的驱动因素。2）通过ATAC-seq、ChIP-seq等多组学手段明确了肿瘤细胞向肿瘤干细胞转变过程中染色质发生了广泛的重构。3）通过蛋白质组学筛选及CRISPR文库筛选，发现AURKA相互作用蛋白HsHPK是调控染色质重构及肿瘤干细胞可塑性的关键基因，还发现GSG2等基因是AURKA激酶抑制剂的耐药基因。4）通过活细胞染色质动态追踪、ATAC-seq、原位蛋白质组学等技术明确HsHPK调节染色质动态重构的作用及信号传递机制。5）在动物模型及临床标本中明确了靶向AURKA-HsHPK等关键基因杀伤肿瘤干细胞，或抑制肿瘤干细胞可塑性的临床相关性及抑癌效果。6）设计合成了多个具有自主知识产权的AURKA蛋白质PROTAC降解剂，并初步验证了其抑癌效果。本项目研究结果从染色质三维重构这一全新角度揭示了AURKA激酶促癌及耐药机制，为靶向AURKA核功能提供理论依据，为靶向乳腺癌干细胞提供潜在的新靶点，开拓靶向促癌激酶研究和治疗的新思路。项目执行期间在Cell Death Di、Signal Transduct Target Ther、Clin Cancer Res、Cancer Commun、Mol Neurobiol、EBioMedicine、Nat Med等杂志发表标注本项目资助论文8篇；其中，项目主持人作为第一作者或通讯作者（含共同）论文共6篇。申请专利3项，获得AURKA降解剂等授权专利2项。