EGFR/TRPV1信号通路在肿瘤发生发展中的作用及其机制研究

EGFR is a member of epidermal growth factor receptor family, which is overexpressed and activated in many human epithelial cancers and is associated with tumor development. The transient receptor vanilloid 1 (TRPV1) is a non-selective cation channel.It was reported that TRPV1 inhibited tumor development, while the detailed mechanism remained unclear. We previously reported that EGFR interacted with TRPV1. The absence of TRPV1 in mice or the topical application of TRPV1 antagonist resulted in a striking increase in skin carcinogenesis mediated through a significant increase in the expression level of EGFR and the activity of its downstream signaling pathway. Here，we found that EGFR phosphorylated TRPV1 in vitro. However, further studies are required to investigate the phosphorylation level of TRPV1 and the activity of phosphorlyated TRPV1 channel as well as its role in tumor development in the tumors with high activity of EGFR. In this study, we propose to utilize different experimental methods including co-immunoprecipitation, GST pull- down, immunofluorescence, point mutation, kinase assay, patch clamp, intracellular calcium fluorescence imaging, siRNA, foci formation, Annexin V and propidium iodide staining, Tunel assay, Transwell assay, tumor xenografts and so on, to study the phosphorylation of TRPV1 by EGFR, to identify the effect on TRPV1 channel, and to investigate the relationship between EGFR and TRPV1 signaling in tumor development. Our research will present important evidences to reveal the crosstalk of EGFR and TRPV1 signaling pathway, and the molecular mechanism and the function of EGFR/TRPV1 signaling in tumor development. These might provide significant theoretical and experimental supports to the study on EGFR/TRPV1 related tumorigenesis, and develop potential targets in cancer prevention and therapy.

EGFR基因突变、过量表达或活性异常增高，与肿瘤发生密切相关。TRPV1是一种阳离子通道，研究显示具有肿瘤抑制作用，但作用机制不明。我们曾首次报道TRPV1结合并调节EGFR；敲除TRPV1基因或使用拮抗剂，促进小鼠皮肤癌发生。 我们新近又发现EGFR使TRPV1磷酸化。然而，在EGFR高活性的肿瘤中，TRPV1的磷酸化水平、离子通道活性的改变、以及在肿瘤发生发展中的作用和分子机制不明，需深入研究。 本项目拟采用GST pull-down、免疫荧光染色、点突变、激酶实验、膜片钳、钙离子荧光标记、siRNA、平板集落形成、AnnexinV/PI双染法、Tunel法、Transwell小室、荷瘤小鼠模型等方法，研究EGFR对TRPV1的磷酸化作用，阐明该作用对TRPV1通道门控及通道功能的影响，探索EGFR与TRPV1通路间的串话机制，以及在肿瘤发生发展中的作用，为肿瘤预防和治疗提供新靶点。

EGFR基因突变、过量表达或活性异常增高，与肿瘤发生密切相关。TRPV1是一种阳离子通道，研究显示具有肿瘤抑制作用，但作用机制不明。我们曾首次报道TRPV1结合并调节EGFR；敲除TRPV1基因或使用拮抗剂，促进小鼠皮肤癌发生。我们新近又发现EGFR使TRPV1磷酸化。然而，在EGFR高活性的肿瘤中，TRPV1的磷酸化水平、离子通道活性的改变、以及在肿瘤发生发展中的作用和分子机制不明，需深入研究。.本项目利用免疫共沉淀、GST pull-down技术，发现TRPV1与EGFR存在相互作用，并且确定TRPV1的C端与EGFR相互作用。利用TRPV1 的C端GST 融合蛋白，并构建磷酸化位点突变体，通过激酶实验，发现EGFR 可以磷酸化大鼠TRPV1 的Y738位点，相当于人TRPV1 的Y739位点；通过构建大鼠TRPV1野生型、磷酸化位点失活突变体 Y738F及磷酸化位点模拟活化突变体 Y738E真核表达质粒，采用全细胞膜片钳技术检测TRPV1通道特性，发现EGFR对TRPV1的磷酸化影响TRPV1通道瞬时门控和电位变化，EGFR磷酸化TRPV1 的Y738位点导致TRPV1通道对辣椒素更敏感；采用QPCR和Western Blot，发现TRPV1在皮肤癌、鼻咽癌、结肠癌、乳腺癌、肝癌等多种肿瘤细胞中表达。TRPV1在鼻咽癌组织、肺癌组织中表达下调，并且TRPV1高表达有利于肺癌病人的存活； 研究发现TRPV1促进鼻咽癌细胞SUNE1和CNE1增殖和迁移， 并且抑制CNE1细胞IL6、 IL8、IL11的表达水平。利用磷酸化位点突变体，发现TRPV1野生型抑制肺癌细胞增殖，TRPV1激活突变体逆转抑制作用；发现TRPV1激活剂辣椒素在鼻咽癌细胞CNE2、SUNE1中，促进细胞凋亡、抑制细胞增殖、迁移和荷瘤小鼠肿瘤形成，并发现P38及上游激酶介导了辣椒素对鼻咽癌的抑癌作用。.本项目的研究初步揭示了TRPV1及EGFR磷酸化TRPV1在肿瘤发生发展中的作用和相关的下游信号通路，为相关的肿瘤诊断与靶向治疗提供了新的重要实验证据。至今，已发表标注本项目资助的SCI论文7篇，培养博士后2名、研究生3名。完成项目预期的目标和任务。