缺氧时nEGFR磷酸化Aurora-A促进结直肠癌转移的机制研究

Our previous studies identified that upregulation of HIF-1α and Aurora-A (Aur-A) promoted the tumor metastases through epithelial-mesenchymal transition (EMT) pathway, however, the underlying mechanism is still unclear. Our recent mass spectrometric analysis found that the epidermal growth factor receptor (EGFR) might interact with Aur-A. Moreover, the functional assay confirmed that HIF-1α-induced activation of Aur-A was mediated by EGFR. That is HIF-1α firstly promoted EGFR internalization from cellular membrane to nucleus, and secondly the nucluear EGFR (nEGFR) phosphorylated Aur-A. Therefore, we propose that HIF-1α and Aurora-A (Aur-A) induced cancer metastases might be mediated by internalization of nEGFR, raced by phosphorylation of Aur-A, and finally enhance tumor cell metastatic ability via EMT pathway. The present study will identify the specific Aur-A phosphoryaltion site by mass spectrometric analysis, and the EMT and metastasis related cascade molecules by RNA Sequencing of Crisp/Cas9-Aur-A knocked out colorectal cancer cell lines. Furthermore, we will validate the above proposed mechanism using three-dimensional cell culture, animal model and clinical specimen. This study will elucidate the novel molecular mechanism that HIF-1α increases nEGFR internalization, racing by Aur-A phosphorylation to upregulates colorectal cancer cell EMT and metastases. This study will provide a novel insight into HIF-1α/nEGFR/Aur-A based target therapy for metastatic colorectal cancer.

申请人已证实缺氧时HIF-1α与Aurora-A（Aur-A）表达上调，通过上皮间质转化（EMT）促进肿瘤转移，具体的调控机制尚不清楚。申请人新近基于蛋白质质谱分析的研究发现：表皮生长因子受体（EGFR）可与Aur-A结合；功能分析确认HIF-1α促进EGFR自胞膜内化至胞核，胞核EGFR（nuclear EGFR，nEGFR）可磷酸化Aur-A。由此，我们提出了缺氧时HIF-1α与Aur-A上调促进肿瘤细胞转移的深层机制：缺氧时HIF-1α上调nEGFR，后者磷酸化Aur-A，藉由EMT促进转移。本项目拟通过质谱分析明确Aur-A被磷酸化的位点，Crisp/Cas9敲除Aur-A后RNA Seq筛选EMT及转移关键通路分子，并在三维转移模型、动物及临床水平进行机制验证，最终获得缺氧时nEGFR磷酸化Aur-A，调控结直肠癌EMT及转移的具体分子机制，从而为基于此的靶向抗转移治疗提供依据。

结直肠癌是全世界范围内发病率和死亡率最高的恶性肿瘤之一。转移是晚期结直肠癌患者死亡的首要原因。然而，结直肠癌转移的分子机制尚不十分清楚。项目负责人的前期研究结果表明：在缺氧环境中HIF-1α与细胞周期激酶Aurora-A高表达，通过上皮间质转化（EMT）促进肿瘤转移。本项目通过利用建立的三维细胞模型作为结直肠癌细胞迁徙转移的研究平台，发现表皮生长因子受体（EGFR）可与Aurora-A结合；HIF-1α促进前者自细胞膜转移至细胞核内，胞核EGFR（nEGFR）磷酸化Aurora-A，进而促进肿瘤转移。我们的实验结果表明，缺氧环境中EGFR可与Aurora-A特异性结合使其T287/T288位点磷酸化，进而通过EMT途径促进结直肠癌细胞转移，阐明此分子机制有望为未来基于此的靶向抗转移治疗提供理论依据。在本项目的资助下，我们已发表研究论文2篇；构建了新型三维转移细胞培养模型并获得相关的发明专利申请2项。