AURKA调控乳腺癌干细胞形成血管拟态的机制研究

Growth, proliferation, and metastasis of tumor have been thought to be dependent on the establishment of tumor vascular network, which provides nutrients required for cancer cell expansion and drains away wastes produced by cancer cells. Recent studies have suggested that tumor cells might be the progenitor for tumor vasculature. Endothelial cells could form blood vessels, which were called angiogenesis. However, a non-angiogenesis-dependent pathway, in which tumors can feed themselves, has also been reported. The process by which a vessel is formed from tumor cells is called vasculogenic mimicry (VM). .Cancer stem cells (CSCs) have been highlighted lately, due to the opinion that these CSCs are the culprits for therapeutic resistance, metastasis, and recurrence of the tumors, and therefore, might be selected as the target of treatment. The research in melanoma, glioma and renal carcinomas showed that CSCs can transdifferentiate into different phenotype, they express angiogenic and vasculogenic markers and they are able to organize pseudovascular network. .AURKA is frequently altered in epithelial malignancies. Overexpressing AURKA induces centrosome amplification and G2/M cell cycle progression. AURKA overexpression and VM existed in human tumors and was related to tumor invasiveness and metastasis. Cancer stem cells (CSCs), including breast CSCs, have the ability of transdifferentiation and could form VM. The mechanism of VM formation by breast CSCs is not studied until now. Our previous studies showed that holoclone cells separated from MDA-MB-231 showed CSCs character, AURKA overexpression and VM formation. This study will inhibit AURKA expression in holoclone cells; will observe the activation of NF-κB pathway, EMT phenotype, VM and VM mediators expression with AURKA knockdown in vitro and in vivo; will detect EMT phenotype, VM and VM mediators expression in holoclone cells with NF-κB pathway activation or inhibition in vitro. Our studies will demonstrate AURKA regulate EMT and VM formed by breast CSCs through NF-κB pathway.

癌基因AURKA高表达及血管生成拟态（VM）现象存在于人类多种类型肿瘤中，与肿瘤的侵袭和浸润密切相关。肿瘤干细胞（CSCs）包括乳腺CSCs，具有高度的横向分化能力，在VM形成中起重要作用。但是调控CSCs形成VM的分子机制尚未阐明。本课题组的前期体外实验发现MDA-MB-231的holoclone细胞具有CSCs特征，呈现AURKA高表达，能够形成VM。本研究将抑制holoclone细胞的AURKA表达，通过体外和体内实验观察抑制AURKA表达后NF-κB信号通路的激活状况、上皮间质转换（EMT）表型、VM状况及VM相关分子表达情况；将观察holoclone细胞NF-κB通路激活或抑制状态时的EMT表型、VM状况及VM相关分子的表达情况。阐明AURKA通过NF-κB通路调控EMT，从而调控乳腺CSCs形成VM。

项目的背景：血管生成拟态（VM）指侵袭性高的肿瘤细胞通过自身基因型的改变和细胞外基质的重塑产生血管样通道，有利于肿瘤的侵袭和转移。肿瘤干细胞（CSCs）包括乳腺CSCs，具有高度的横向分化能力，在VM形成中起重要作用。中心体激酶AURKA过表达存在于许多类型肿瘤中，可导致中心体扩增和染色体异常。泛素特异性蛋白酶44 （USP44）是重编程因子及干细胞相关基因OCT4的下游信号通路分子，USP44和RNF20协同作用可以调节干细胞分化。.主要研究内容、重要结果及关键数据：本课题随机选取92例人乳腺浸润性导管癌组织并获得临床病理资料，同时采用流式细胞仪、分子克隆技术、免疫荧光及免疫组织化学CD31/PAS双染实验、western实验、三维培养、动物模型等体内体外实验阐明了AURKA及USP44在乳腺CSCs形成VM中的作用及分子机制。结果表明：①人乳腺癌组织中存在AURKA过表达导致的中心体扩增表型和VM，二者存在统计学上的相关性。中心体扩增及VM形成与乳腺癌转移存在直接的相关性，中心体扩增及VM形成病例显示较高的淋巴结转移、较晚的临床分期和较短的生存时间。②癌干细胞标记物ALDH1及OCT4、USP44、IL-6和IL-8的表达与VM形成密切相关。ALDH1、OCT4及USP44阳性病例生存时间显著缩短，可作为显示乳腺癌病人预后差的指标。③ 利用干细胞超低吸附培养板无血清悬浮培养可获得乳腺癌干细胞球，其干细胞转录因子c-myc、sox-2表达高于常规培养细胞。流式细胞术显示ALDH1阳性表达率显著增加，验证了癌干细胞球的干性特征。同时癌干细胞球显示AURKA高表达，而AURKA过表达通过诱导USP44的高表达有助于中心体扩增表型的癌干细胞建立两极纺锤体及VM的形成。④接种乳腺CSCs于裸鼠形成移植瘤，AURKA蛋白激酶抑制剂MLN8237能明显抑制人乳腺癌裸鼠体内移植瘤的生长和VM的形成。.科学意义：① 本项目进一步丰富了肿瘤血管生成理论，揭示了乳腺癌组织中存在着一种特殊的血液供应模式（VM），而且形成VM的肿瘤细胞可以通过细胞分裂进入微循环，从而为肿瘤的转移创造了条件。② 体外实验、动物模型和人体乳腺癌组织中的实验结果阐明了AURKA促进了乳腺癌干性的发展、USP44高表达及VM的形成。③ AURKA蛋白激酶抑制剂有望成为治疗进展期乳腺癌的特异性分子靶向药物。