VEGF/VEGFR-2/NRP-1通路促进三阴性乳腺癌干细胞及肿瘤干细胞龛形成的作用及机制研究

Triple-negative breast cancers (TNBC) are defined as tumors that lack expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2(HER2). As a group, patients with triple-negative breast cancers have a relatively poor outcome because of early relapse and metastasis and the lack of effective therapeutic target. Consequently, the treatment of TNBC becomes an obstacle in clinical practice. In many human malignancies, cancer stem cells (CSCs) appear to play a critical role in tumor invasion, metastasis, drug resistance and disease recurrence. It is reported that triple-negative breast cancers display a profile of cell surface markers that is similar to that of breast cancer stem cells, characterized by the phenotype CD44+CD24- and the expression of aldehyde dehydrogenase 1 (ALDH1). Our previous work showed that autocrine VEGF/NRP-1 axis positively regulates the tumorigenesis and metastasis of breast cancer by enhancing the epithelial-mesenchymal transition (EMT) process. NRP-1 is a co-receptor of VEGFR-2 and EMT generates cells with properties of stem cells. Based on these findings, we speculate that autocrine VEGF may involved in the acquisition and maintenance of breast cancer stem cell phenotype through VEGFR-2/NRP-1 pathways in TNBC, thereby promoting tumorigenesis and recurrence. Based on this assumption, our group intends to detect whether and how autocrine VEGF/VEGFR-2/NRP-1 pathways regulate the CSCs in TNBC. CRISPR/Cas9 technology was employed to elucidate the possible roles of autocrine VEGF/VEGFR-2/NRP-1 in the regulation of CSC niche in tumor microenvironment. Our study may provide new ideas for the targeted therapy of triple negative breast cancer.

三阴性乳腺癌是乳腺癌的一个特殊类型，易早期复发和转移，缺乏有效治疗靶点，是临床治疗的难点。肿瘤干细胞是肿瘤发生和复发转移的重要原因，干细胞多定植于微血管龛附近，研究报道三阴性乳腺癌细胞具有乳腺癌干细胞的表面特征，我们发现VEGF/NRP-1通路可能参与了三阴性乳腺癌的迁移、侵袭、EMT转化等生物学行为的调控。NRP-1是VEGFR-2的协同配体，EMT转化能够促进肿瘤干细胞形成及“干性”维持。因此我们推测：VEGF通过VEGFR-2/NRP-1促进三阴性乳腺癌的“干性”获得及干细胞龛的形成，从而促进肿瘤发生及复发转移。本课题拟采用细胞分子生物学方法探索VEGF/VEGFR-2/NRP-1对三阴性乳腺癌细胞“干性”获得及干细胞龛形成的作用，并采用CRISPR/Cas9技术建立荷瘤裸鼠模型，在体内探索VEGFR-2/NRP-1在肿瘤干细胞龛形成中的作用，为三阴性乳腺癌的靶向治疗提供新的思路。

三阴性乳腺癌是乳腺癌的一个特殊类型，易早期复发和转移，缺乏有效治疗靶点，是临床治疗的难点。课题组前期研究发现VEGF/NRP-1通路可能参与了三阴性乳腺癌的迁移、侵袭、EMT转化等生物学行为的调控。EMT转化能够促进肿瘤干细胞形成及“干性”维持。我们推测：VEGF/NRP-1轴可能参与了三阴性乳腺癌肿瘤干细胞的形成，同时VEGF可能通过VEGFR-2调控肿瘤微环境及肿瘤干细胞龛的形成，促进肿瘤发生及复发转移。本研究采用分子生物学、质谱技术、组织芯片技术等探究VEGF/NRP-1轴调控三阴性乳腺癌干细胞及肿瘤干细胞龛形成的作用及其具体机制。结果显示VEGF在三阴性乳腺癌组织中呈高表达，VEGF高表达与乳腺癌预后不良相关。VEGF/NRP-1在三阴性乳腺癌细胞系及微球体中表达升高，与肿瘤干细胞特性呈正相关。VEGF/NRP-1轴的激活能够促进肿瘤干细胞的形成。通过免疫共沉淀联合质谱检测技术初步阐明了NRP-1可能与GAPVD1存在直接相互作用，GAPVD1可调控下游Wnt/β-catenin信号通路，VEGF/NRP-1通过GAPVD1及下游Wnt/β-catenin信号通路参与了三阴性乳腺癌体外迁移、侵袭、成球能力及肿瘤干细胞特性的调控。干细胞定植需要适宜的肿瘤微环境，VEGF/VEGFR-2/NRP-1对三阴性乳腺癌肿瘤微环境的影响我们将在后续的体内实验中进一步阐明。总之，我们的研究证实VEGF/NRP-1轴参与三阴性乳腺癌干细胞的形成，初步阐明了GAPVD1可能作为中间分子激活Wnt/β-catenin信号通路，促进肿瘤干细胞形成，该研究成果有望为三阴性乳腺癌的靶向治疗提供新的思路。