PEDF抑制乳腺癌异质血管生成的作用及机制

Sorafenib, a typical anti-angiogenic drug targeting vascular endothelial cells, has been suspended in the III phase clinical trial of breast cancer because of the poor efficacy. It is urgent to elucidate the reason behind failure and to find new effective target drugs. Our preliminary experiments found that tumor cells participated in the constitution of heterogeneous vascularity (vascular mimicry and mosaic vessels) in human breast cancer samples. Further, only the stem-like cells sorted from MDA-MB-231 cells have tube-formation ability in vitro. PEDF, an endogenous angiogenesis inhibitor which was studied a lot in my lab, could inhibit the blood vessels from both endothelial cells and tumor cells in vivo and in vitro, whereas sorafenib could inhibit the tube formation from endothelial cells rather than breast cancer stem-like cells. Moreover, sorafenib treatment appeared early effective and late resistance in breast cancer nude mice xenograft model and increased tumor heterogeneous vascularity. These results suggested that the noneffective on tumor cells in heterogeneous vascularity may be crucial for the failure of typical angiogenesis-targeting drugs such as sorafenib. This project aims to clarify: ① whether the breast cancer cells in the heterogeneous vascularity possess the stem cell properties; ②the key molecules in breast cancer-like stem cells confer the ability of vasculogenesis; ③ whether the sensitivity or resistance of PEDF / sorafenib is due to their different regulation on the key molecules in breast cancer-like stem cells. The purpose of this study is to reveal the mechanism of failure for the typical angiogenesis-targeting drugs and provide a theoretical basis for developing novel anti-heterogeneous vascularity drugs.

靶向血管内皮细胞的抗新生血管药物如索拉菲尼等由于疗效不佳在乳腺癌临床试验阶段被终止，阐明其失败机制并寻找新的有效药物十分迫切。预实验发现在人乳腺癌组织标本中存在肿瘤细胞参与的异质血管（拟态血管和马赛克血管），从乳腺癌MDA-MB-231细胞中分选出的干细胞才有成管能力；我们长期研究的血管新生抑制因子PEDF对231干细胞体外成管和体内异质血管均具有抑制作用；索拉菲尼对231干细胞体外成管无效，体内治疗早期有效后期抵抗及肿瘤异质血管比例增加。上述结果提示对参与异质血管的肿瘤细胞是否有效可能是治疗成功或失败的关键。本项目拟明确:①异质血管中的乳腺癌细胞是否必需具有肿瘤干细胞性质；②赋予乳腺癌（干）细胞血管新生能力的关键分子；③PEDF/索拉菲尼是否因为对乳腺癌（干）细胞关键分子调控的差异表现出敏感或抵抗。旨在揭示经典新生血管靶向药物治疗失败的原因和机制，为研发新的抗异质血管药物提供理论依据。

近年来，抗新生血管治疗在乳腺癌等肿瘤中的运用遭遇瓶颈，阐明其失败机制并寻找新的有效药物十分迫切。预实验发现在人乳腺癌组织标本中存在肿瘤细胞参与的异质血管（拟态血管和马赛克血管），三阴性乳腺癌组织中存在ALDH1+的肿瘤干细胞直接参与形成的异质血管，只有ALDH1+的乳腺癌干细胞才具有体外成管能力；乳腺癌干细胞表达αSMA是参与成管的关键分子。我们检测了乳腺癌干细胞中血管周细胞分子标记物（αSMA、PDGFR-β、Desmin）和血管内皮细胞分子标志物（CD31、 VE-cadherin），发现乳腺癌干细胞表达周细胞分子标记物α-平滑肌肌动蛋白（αSMA）却不表达内皮细胞标记物。进一步干扰或过表达乳腺癌细胞中的αSMA可以显著影响细胞成管能力。HIF1α直接结合在αSMA的启动子上促进αSMA转录。干性强的MDA-MB-231细胞比干性弱的MCF7细胞中HIF1α和αSMA表达增高。我们在MDA-MB-231细胞中抑制HIF1α，而在MCF7细胞中通过低氧培养上调HIF1α，从而正反向证明HIF1α可以在转录和蛋白水平调控乳腺癌细胞中αSMA的表达。并进一步通过荧光素酶报告基因和染色质免疫共沉淀技术证明αSMA基因的启动子调节区域存在HIF1α的结合元件（HRE）。HIF1α上调乳腺癌细胞系中肿瘤干细胞比例进而促进血管形成。我们在MDA-MB-231细胞中抑制HIF1α，可以显著下调ALDH1+细胞比例并抑制体外成管能力；而在MCF7细胞中上调HIF1α，可以促进MCF7中ALDH1+细胞数并诱导MCF7细胞体外成管。PEDF抑制HIF1α-αSMA信号抑制MDA-MB-231细胞体外成管。PEDF能够抑制乳腺癌细胞上的HIF1α和αSMA的表达从而抑制乳腺癌MDA-MB-231细胞单独成管，而传统药物索拉菲尼不能抑制HIF1α和αSMA的表达，也对MDA-MB-231细胞单独成管无效。本研究证明了乳腺癌干细胞通过拟态血管和马赛克血管的形式直接参与肿瘤异质血管的形成；发现αSMA是异质血管形成的关键分子；而HIF1α一方面可以促进乳腺癌细胞中干性样细胞比例，另一方面可以直接结合在αSMA基因启动子上促进αSMA转录表达。而PEDF通过抑制HIF1α-αSMA信号抑制乳腺癌中异质血管生成。本研究为理解乳腺癌中异质血管特性提供理