TRPV1及Y739磷酸化在非小细胞肺癌发生发展中的作用及其分子机制研究

Lung cancer is the most common malignancy in terms of both incidence and mortality. Lung cancer are categorized into small cell lung cancer and non-small cell lung cancer (NSCLC). Nearly 85% of lung cancers are non-small cell lung cancer. ErbB1 is a member of epidermal growth factor receptor family, whose overexpression, mutation and activation is associated with NSCLC. The transient receptor vanilloid 1 (TRPV1) is a non-selective cation channel. It has reported that TRPV1 plays role in tumor inhibition. There is no report about the association between TRPV1 and NSCLC. We previously reported that ErbB1 interacts with TRPV1. Here, we found that ErbB1 phosphorylated TRPV1 at Y739. Furthermore, we found that overexpression of TRPV1 inhibited NSCLC cell proliferation while promoted NSCLC cell migration. Compared with TRPV1, overexpression of TRPV1-Y739E which is the phosphorylation constitutively active mutant promoted NSCLC cell proliferation while inhibited NSCLC cell migration. However, further studies are required to investigate the role of TRPV1 and TRPV1-Y739 phosphorylation in tumorigenesis and development in NSCLC with high activity of ErbB1.. In this study, we will use patch clamp and intracellular calcium fluorescence imaging to study the effect of TRPV1-Y739 phosphorylation on TRPV1 channel activity. A series of approaches including immunohistochemistry, MTS assay, Annexin V and propidium iodide staining, Transwell assay, tumor xenografts and so on will be used to study the role of TRPV1 and TRPV1-Y739 phosphorylation in NSCLC tumorigenesis and development. This study will provide essential evidences to reveal the function and molecular mechanism of ErbB1/TRPV1 signaling pathway in NSCLC tumorigenesis and development. It will identify a novel potential therapeutic target for NSCLC.

ErbB1过表达、基因突变或活性异常增高，与非小细胞肺癌发生密切相关。TRPV1是一种阳离子通道，具有抑制肿瘤作用，但与非小细胞肺癌的相关性未有报道。我们发现ErbB1磷酸化TRPV1，且磷酸化位点是Y739。在非小细胞肺癌细胞中，过表达TRPV1抑制细胞增殖，促进细胞迁移；与之相比，过表达该位点磷酸化的激活突变体促进细胞增殖，抑制细胞迁移。ErbB1高活性的非小细胞肺癌中，TRPV1-Y739磷酸化水平、离子通道活性变化，及在非小细胞肺癌发生发展中的作用和分子机制不明，需深入研究。..本项目拟采用膜片钳、钙离子荧光标记研究TRPV1-Y739磷酸化对TRPV1通道门控及功能的影响；采用免疫组化、MTS、Annexin V/PI染色、Transwell、荷瘤小鼠模型等方法研究TRPV1及Y739磷酸化在非小细胞肺癌发生发展中的作用及分子机制，有望为非小细胞肺癌预防和治疗提供新靶点。

据2020年全球癌症统计报告显示：在全球范围内，肺癌的发病率和死亡率均排名第二。大约85％的肺癌病例是非小细胞肺癌。化疗药耐受是非小细胞肺癌治疗失败的重要原因之一。ErbB1过表达、基因突变或活性异常增高，与非小细胞肺癌发生密切相关。瞬时感受器电位香草酸受体（Transient receptor potential cation channel subfamily V, member 1, TRPV1)，是一种非选择性阳离子通道，可被各种物理和化学刺激激活，活化后介导钙离子和钠离子等阳离子内流，使细胞内阳离子浓度升高，引起相应的生理和病理变化。TRPV1在肿瘤发生发展中起多种作用，包括肿瘤细胞增殖、死亡和转移以及对治疗的反应。在这项研究中，我们发现ErbB1磷酸化TRPV1，且磷酸化位点是Y739。TRPV1在非小细胞肺癌中高表达，并且预后不良。TRPV1的过表达促进非小细胞肺癌细胞迁移；通过下调CEACAM6，HBEGF和ATF3的表达来抑制非小细胞肺癌细胞增殖；对凋亡不产生影响。与野生型TRPV1相比，模拟活化突变体Y739E过表达促进细胞增殖，抑制迁移，而失活突变体Y739F过表达抑制细胞增殖，促进迁移。另外，我们发现TRPV1过表达诱导非小细胞肺癌细胞A549对化疗药顺铂（Cisplatin，DDP）和氟尿嘧啶（Fluorouracil，5-FU）耐受，且此表型不依赖于其通道功能。之后我们建立了A549-DDP和A549-5-FU耐药株，发现TRPV1的mRNA和蛋白水平在A549-DDP和A549-5-FU耐药细胞中皆上调，敲低TRPV1恢复了DDP和5-FU的敏感性。进一步实验证明TRPV1过表达通过上调药物转运蛋白基因ABCA5表达，从而增加药物外排量来介导DDP和5-FU耐药性；同时增强同源重组DNA修复，减轻化疗药诱导的细胞凋亡；并且增加IL-8表达，促进细胞存活。这些发现表明TRPV1在非小细胞肺癌的化疗药耐受中起着重要作用，并暗示TRPV1可作为潜在的化学治疗靶标。