速激肽受体Neurokinin-1功能选择性及其在肿瘤细胞增殖迁移中的作用

The tachykinin receptor neurokinin-1 (NK1) belongs to GPCR superfamily. In the past decade, NK1 receptor has received a lot of attention as a new potential target in cancer therapy. A growing body of studies demonstrated that tumor issues expressed functional NK1 receptor and the expression level of NK1 receptor seemed to be related with the malignant degree of tumors. Tumors expressing NK1 receptor can misuse the tachykinin-induced signaling to promote the proliferation and survival of cancer cell. In addition, tachykinins may favor cancer metastasis progression by influencing neovascularization as well as inducing immunesuppression mediated by neurogenic inflammation. The pharmacological behavior of NK1 receptor is unique. The receptor mediates multiple signaling pathways via the interaction with Gs, Gq and β-arrestin. The signaling patterns of NK1 receptor can be ligand-specific and tissue-specific. That means NK1 receptor may exert its biofunctions by specific signaling pathway set in different physiological and pathological condition. Previously we demonstrated that human HK-1 was a NK1 agonist with functional selective property and that human HK-1 stimulated the NK1 receptors in glioblastoma U-251, directly promoting cell migration by Gq pathway. Based on the preliminary studies, we'll investigate the functional selective properties of NK1 receptor in tumor cells proliferation and migration; we'll study the structure-activity relationship of human HK-1 and NK1 receptor by classical Ala and D-scan based on functional selectivity of NK1 receptor. Our aim is to provide a new perspective to design new type of anticancer drugs targeted NK1 receptor.

速激肽受体NK1在肿瘤组织中异常表达，参与肿瘤细胞增殖、血管生成，帮助肿瘤细胞逃避免疫监视，是新一代抗肿瘤药物的理想靶点。NK1受体的药理学性质独特，可介导包括Gs、Gq、β-arrestin 在内的多条信号通路。申请人前期研究发现人HK-1是具有功能选择性的NK1受体激动剂；在恶性胶质瘤中，人HK-1可激活NK1受体，通过Gq通路直接参与细胞迁移。本项目将以NK1受体功能选择性为重点，研究NK1受体引起的肿瘤细胞增殖迁移与受体功能选择性的关系，阐明NK1受体的肿瘤药理学作用机制；通过Ala扫描、手性氨基酸替换等方法分析人HK-1上决定受体功能选择性的关键位点，建立计算机模拟的NK1受配体分子对接模型，为设计合成以NK1为靶点的具有功能选择性的新型抗肿瘤药物提供理论和实验基础。

NK1速激肽系统在肿瘤组织中异常表达，在肿瘤发展过程中扮演重要角色。尽管 NK1 受体拮抗剂的抗肿瘤作用已在多个细胞和动物水平模型上得到验证,其具体作用机制还未系统研究。我们选取了乳腺癌、黑色素瘤以及恶性胶质瘤等高表达NK1受体的肿瘤细胞模型，通过QPCR、Westernblot等方法验证比较了NK1受体在细胞株及临床样品中的表达分布，发现NK1受体在肿瘤中以一定比例表达，特别是在恶性胶质瘤中，表达阳性比例非常高。内源性配体hHK-1可通过激活NK1受体提高细胞中基质金属蛋白酶MMP-2、MMP-14活性，促进乳腺癌、黑色素瘤以及恶性胶质瘤细胞迁移。在分子机制上，我们证明激活的NK1受体可活化Gq或Gs蛋白，引起ERK1/2、JNK、p38、Akt等激酶分子磷酸化，增加转录因子NFκB、AP-1等的活性，进而提高肿瘤细胞中基质金属蛋白酶MMP-2、MMP-14的表达水平及活性，促进肿瘤细胞迁移。在恶性胶质瘤细胞中，β-arrestin-1参与介导NK1受体信号通路，这一过程参与调节细胞增殖，敲减β-arrestin-1可导致细胞阻滞在G2/M期，同时周期蛋白cyclin B1表达下调、CDK1、CDC25C活性下降，hHK-1引起的恶性胶质瘤细胞增殖也被抑制。.我们通过Ala扫描、D型氨基酸替换、氮端氨基酸切除等方法，研究了配体hHK-1结构与NK1相关信号通路活化的关系。氮端结构域长度对多肽激动剂活性有显著影响，其中第3位Lys和第6位Gln对于配体的功能选择性起重要作用。