PAG1诱导整合素β1活化介导喉癌原发性放射抗拒的机制

Radiotherapy is an important treatment for laryngeal carcinoma, and its poor curative effects are mainly associated with radioresistance of laryngeal carcinoma cells. The premise of overcoming radioresistance is based on the fact that the molecular mechanism of radioresistance has been revealed. In our previous study, we found that lipid raft-associated protein PAG1 could mediate inherent radioresistance of laryngeal carcinoma, but its mechanism was unknown. It has been reported that the up-regulated expression of PAG1 was able to increase integrin β1 activity. We also confirmed that integrin β1 was a potential combination protein of PAG1 in the inherent radioresistance of laryngeal carcinoma cells. Accordingly, we thought that PAG1 could induce the activation of integrin β1 in lipid rafts so as to regulate the inherent radioresistance of laryngeal carcinoma. In this study, the laryngeal carcinoma clinical tissues were used to investigate the relationship between the expression levels of PAG1 and activated integrin β1 and the radiosensitivity of laryngeal carcinoma patients. Furthermore, the expression of PAG1 was interfered by gene transfection and CRISPR/Cas9 technique in vivo and in vitro. Then the change in the radioresistant property of laryngeal carcinoma, the activation and location of integrin β1 was detected to identify the regulation effect of PAG1 on integrin β1 activation. In addition, lack of cholesterol, truncated mutations and GST pull down assays were carried out to parse the interaction pattern of PAG1 and integrin β1.The results of this study not only clarify the molecular mechanism of PAG1-mediated inherent radioresistance in laryngeal carcinoma, but also provide a reliable intervention target to overcome the radioresistance of laryngeal carcinoma.

放疗是喉癌的重要治疗手段，其疗效不佳主要与喉癌细胞放射抗拒有关，克服放射抗拒前提是揭示其分子机制。我们前期发现脂筏相关蛋白PAG1介导喉癌原发性放射抗拒，但机理不明。研究表明过表达PAG1可增强整合素β1活性，而我们证实原发性放射抗拒喉癌细胞中整合素β1是PAG1的潜在结合蛋白，故推测PAG1通过调控整合素β1在脂筏内活化从而介导喉癌原发性放射抗拒。本课题拟首先采用喉癌临床标本分析PAG1、活化型整合素β1的表达与喉癌患者放疗敏感性关系；进而在体内外通过基因转染和CRISPR/Cas9技术干预PAG1表达，检测喉癌放射抗拒性状改变、整合素β1的活化状态及定位，明确PAG1对整合素β1活化的调控作用；并采用胆固醇缺失、截短突变、GST pull down解析PAG1与整合素β1的相互作用模式。本研究内容将阐明PAG1介导喉癌原发性放射抗拒的分子机制，并为克服喉癌放射抗拒提供可靠的干预靶点。

原发性放射抵抗是导致喉癌患者放疗失败的主要因素。PAG1介导喉癌原发性放射抵抗，整合素β1是其潜在结合蛋白。因此，本项目主要探讨PAG1对整合素β1的调控作用、机制及生物学意义。本项目通过对临床数据分析、细胞学实验和动物体内实验，证明：① PAG1、整合素β1在喉癌组织中高表达，二者表达水平均与淋巴结转移、TNM分期正相关，与患者预后负相关。②PAG1在放射抵抗组织中表达升高，在放射敏感组织中表达下降，但整合素β1在不同敏感性组织中的表达无差异。另外，PAG1在接受单纯放疗的喉癌病理标本中的高表达预示着患者预后不良。③下调放射抵抗细胞中的PAG1，可抑制增殖、迁移、侵袭并提高放射敏感性；上调放射敏感细胞中的PAG1，可促进增殖、迁移、侵袭并增加放射抗性。④通过建立喉癌原发性放射抵抗荷瘤裸鼠模型，发现抑制PAG1表达可导致肿瘤体积-时间曲线变缓，肿瘤体积减小和重量变轻，且裸鼠放射敏感性增加。⑤PAG1可与整合素β1相互结合，结合位点位于PAG1胞浆结构域的Pro216-Arg232和Asn356-Gly377。二者的共结合和共定位依赖于细胞膜脂筏结构的完整性，破坏脂筏或阻断整合素β1均可使二者的相互作用减弱并增加细胞的放射敏感性。⑥PAG1与整合素β1结合后，激活STAT3，进而影响FAK/cortactin通路，抑制STAT3的活化可逆转PAG1介导的放射抵抗。⑦PAG1参与调控整合素β1的活性。更为重要的是，整合素β1的糖基化修饰直接决定其活性状态，其修饰程度与喉癌放射抵抗密切相关，并在食管癌和结肠癌中得到验证，开辟了肿瘤治疗抵抗机制研究的新方向。通过以上工作，本项目阐明了PAG1介导喉癌原发性放射抵抗的分子机理，为克服喉癌放射抵抗提供了新靶点，具有重大临床应用价值。本项目已发表论文9篇，其中SCI期刊收录6篇；参加国内学术会议交流3次。