骨桥蛋白促乳腺癌干细胞向血管内皮转分化的作用机制研究

Transdifferentiation of cancer stem cells into vascular endothelial cells has proved to be an important process of tumor angiogenesis, but the underlying mechanism remains unclear. Tumor-derived endothelial cell (TDEC) is one of the reasons for resistance of anti-angiogenesis therapy. We have demonstrated for the first time that overexpression of osteopontin (OPN) enhanced tumor neovascularization in a MCF-7 xenograft model (Oncogene 2009). Further studies indicated that MDA-MB-231 xenograft had a mixed vasculature combined with human and murine origins. Anti-OPN antibody significantly inhibited both the mouse and human vasculature in xenograft tumors. In vitro studies showed that exogenous OPN induced the expression of endothelial markers in breast cancer stem cells derived from MCF-7 cell line. Blocking OPN by neutralizing antibody could inhibit the formation of capillary structure in matrigel. Based on these results, we hypothesize that OPN induces angiogenesis by mediating endothelial transdifferentiation of breast cancer stem cell. This study is designed to analyze the effect of OPN in the regulation of endothelial transdifferentiation process by in vitro and in vivo experiments. Furthermore, receptor blocking, integrated analysis of mRNA and microRNA expression microarrays, loss-of-function and gain-of-function experiments will be performed to screen the key effectors and to demonstrate the molecular mechanism and downstream signaling pathway. Our studies may provide insight into a new mechanism of tumor angiogenesis and the evidence for us to consider OPN as a new target for anti-angiogenesis therapy in breast cancer.

肿瘤干细胞转分化为血管内皮细胞是近年来肿瘤血管生成方面的重要发现，是肿瘤抗血管治疗耐受的重要因素。本课题组前期在Oncogene杂志上首次报道了骨桥蛋白（OPN）促进乳腺癌肿瘤血管生成的作用。后续进一步研究发现，人乳腺癌小鼠移植瘤血管为人/鼠混合血管网络，抗OPN抗体对人/鼠源血管生成均有显著抑制作用；体外研究表明OPN可以诱导人乳腺癌干细胞表达血管内皮标志，阻断OPN可抑制管腔样结构形成，提示OPN可能参与乳腺癌干细胞的血管内皮转分化过程。本课题将从体外研究、体内实验和分子机制水平，研究OPN诱导乳腺癌干细胞血管内皮转分化的表型和内皮细胞功能变化；通过受体阻断实验、基因芯片表达谱-microRNA联合分析等手段，筛选关键的效应分子，功能缺失和功能获得实验明确OPN的下游信号通路。研究结果将有助于揭示肿瘤血管生成新的分子机制，可能为肿瘤抗血管治疗提供新的靶点。

研究目的：.探讨外源性OPN促进乳腺癌干细胞转分化为TDEC的生物学功能；以证实OPN是否参与乳腺癌肿瘤干细胞向血管内皮转分化过程的调控。筛选鉴定OPN促进乳腺癌转分化为TDEC的下游信号通路和相关基因。..研究方法：.从体外实验证实外源性OPN促进乳腺癌干细胞转分化为有功能的血管内皮细胞；从动物体内实验证实OPN促进乳腺癌干细胞转分化为TDEC的作用；通过信号通路芯片分析，证实OPN与受体的相互作用，筛选关键的效应分子及信号通路。..研究结果：.本课题研究了OPN在乳腺癌干细胞转分化为血管内皮细胞的作用，以及相应的分子机制。.首先我们利用MDA-MB-231细胞和可诱导型OPN过表达慢病毒，构建可诱导型OPN过表达细胞株。利用诱导剂DOX诱导和成球实验，获得了纯度较高的肿瘤干细胞，并流式鉴定CD44+/CD24-比例确定干细胞质量。.其次，我们研究了OPN在体内外促进乳腺癌干细胞转分化为血管内皮细胞的生物学功能。PCR和WB试验证实，VEGF协同OPN过表达体外诱导乳腺癌干细胞向血管内皮细胞转分化的效果较VEGF单独诱导更加显著。免疫荧光检测结果提示，OPN在VEGF基础上将乳腺癌干细胞诱导成了具有血管内皮细胞特性的细胞类型，CD31标志物表达水平的显著增加。3D成管实验的结果表明，OPN过表达后，乳腺癌干细胞的成管能力较对照相比获得了显著的提升。体内研究结果提示，内源的OPN高表达显著增强了肿瘤的生长；内源性过表达OPN或者外源直接注射OPN重组蛋白都能够显著增加有人乳腺癌干细胞转分化来源的血管生成。.最后，探讨了OPN在乳腺癌干细胞转分化为血管内皮细胞的机制。芯片数据显示：整合素Integrin beta-3（CD61）在Tyr785位点的磷酸化在OPN过表达情况下显著增强。WB实验证实，OPN过表达或外源OPN重组蛋白都能够显著增加CD61-Tyr785的磷酸化。..研究结论：.OPN在体内体外能够促进乳腺癌干细胞向血管内皮细胞的转分化。OPN的过表达促进了皮下移植瘤的血管生成并伴随促进肿瘤的生长。推测由于整合素是VEGF下游激活对象，直接参与血管新生等生物学过程。于是，OPN能够通过激活CD61促进VEGF诱导的肿瘤干细胞向血管内皮细胞的转分化。