基于VASP分子靶标的黄连素抗Basal-like型乳腺癌转移的机制研究

Basal-like breast carcinoma is an invasive form of breast cancer, which is associated with aggressive behavior and poor prognosis, they are characterized by the absence of oestrogen receptor, progesterone receptor and HER2 expression. Therefore, patients with basal-like cancers are unlikely to benefit from currently available targeted systemic therapy. In almost all steps of metastatic spread, the reorganization and reassembly of the actin cytoskeletonis absolutely necessary for invasive cell behaviour. Cytoskeleton remodeling mediated by the cytoskeleton regulatory protein VASP (vasodilator-stimulated phosphoprotein) is a key step in migration and invasion. In contrast to the chemotherapeutic agents that target microtubules, which have been used in anticancer treatments for decades, drugs specifically targeting actin have not yet been reported. Through experiments conducted on cell and animal model, we draw the conclusion that of various compounds, low doses of berberine could obviously inhibit the cell migration of basal-like subtype breast cancer. In vitro experiment shows that using 0.05μM berberine will change the spacial structure of VASP remarkably. Besides, software prediction makes it clear that the berberine can combine with the EVH1 area. Our project firstly explores the pharmacological action of berberine's inhibiting the cell migration of basal-like subtype breast cancer through affecting cytoskeleton remodeling by means of experiment on cell and animal model. Then, it tries to get VASP as the target protein of the berberine. On this basis, through vivo and in vitro experiments, we will studies the molecular mechanism of the inhibiting function of berberine through VASP, aiming to obtain future targets and medicine for the treatment of basal-like subtype breast cancer.

Basal-like型乳腺癌是一种高转移且预后较差的恶性肿瘤，骨架调节蛋白VASP（vasodilator-stimulated phosphoprotein，血管扩张刺激磷蛋白）介导的细胞骨架重构是肿瘤侵袭转移的关键环节，目前尚缺乏靶向于微丝肌动蛋白及其相关蛋白的治疗药物。本课题组前期筛选发现：在细胞和动物模型中，低剂量的黄连素对Basal-like型乳腺癌细胞迁移具有明显抑制作用，体外实验显示0.05μM的黄连素即可显著改变VASP的空间构象，软件预测结果表明它可以与VASP的EVH1区域结合。本项目拟在细胞和动物模型中检测黄连素通过影响细胞骨架重构抑制Basal-like型乳腺癌细胞迁移的药理作用，解析出其功能发挥的分子靶标- - VASP蛋白，通过体外及体内实验探究黄连素通过VASP蛋白拮抗乳腺癌细胞迁移的分子机制，为Basal-like型乳腺癌的综合治疗提供新的靶点和候选药物。

Basal-like 型乳腺癌具有瘤体大、低分化、容易出现淋巴结及远端转移，缺少相关治疗靶点等特点。为探寻治疗Basal-like 型乳腺癌的有效方式，基于前期对VASP抑制肿瘤机制的相关研究，本项目从小分子药物黄连素出发，从蛋白质空间结构和大分子相互作用的层面重点研究黄连素与骨架相关蛋白的VASP作用机制，即验证黄连素通过靶向于 VASP 的 EVH1 区域，与关键位点 Tyr16，Trp23，Phe79 相结合，抑制 VASP促肌动蛋白纤维组装功能的发挥，进而抑制 Basal-like 型乳腺癌细胞转移。项目实施过程中具体阐释内容如下：1）黄连素能显著抑制Basal-like 型乳腺癌细胞增殖、迁移和侵袭能力。2）黄连素并不影响Basal-like 型乳腺癌细胞中VASP的mRNA和蛋白表达水平，但是黄连素通过与 VASP蛋白 EVH1区域的 Tyr16，Trp23，Phe79 结合影响其蛋白的空间结构。3）黄连素抑制VASP蛋白介导的F-actin聚合的同时影响VASP的亚细胞分布，并诱导肌动蛋白应力纤维的解聚。4）在乳腺癌MDA-MB-231细胞裸鼠移植瘤模型中，黄连素可显著抑制肿瘤的生长。综上，本项目的实施从抗细胞骨架重排的角度解析分子靶标，为 Basal-like 型乳腺癌的综合治疗提供新的靶点和药物。