RIG-I调节自噬促进口腔鳞状细胞癌转移的效应与分子机制研究

Oral squamous cell carcinoma (OSCC) patients with metastasis have extremely poor prognosis and low 5-year survival rate. Autophagy has dual role on tumor acting as both a tumor suppressor by preventing the accumulation of damaged proteins and organelles and as a mechanism of cell survival that can promote the growth of established tumors. The role of autophagy on OSCC remains elusive. Our previous study demonstrated that retinoic-acid-inducible gene-I (RIG-I) is abundantly expressed in metastasis OSCC cell than the non-metastasis cell and adjacent normal tissue, suggesting activation of RIG-I is related to OSCC metastasis. Our study also found RIG-I had roles of both promotion and suppression on OSCC. The dual effect of RIG-I on OSCC shares similarity of the dual regulation of autophagy on cancer cell. In vitro study in OSCC cell lines showed the low-affinity activation of RIG-I activated expression of LC3, one of the autophagosome markers, as well as promoted proliferation, migration and invasion of OSCC cell. These results suggest RIG-I may promote progression and metastasis of OSCC through the regulation of autophagy. In this study, clinical samples and information are to be studied in large scale. Primary OSCC cells are to be cultured in a three dimensional cell culture system for further study. The methods of overexpression, RNAi, establishment of xenograft mice model, co-immunoprecipitation and mass spectrometry identification will be applied. The relationships among RIG-I, autophagy and metastasis of OSCC will be investigated, focus on the key procedure of the metastasis, the epithelial-to-mesenchymal transitions (EMT). The understanding of the role of RIG-I on metastasis of OSCC may lead to development of novel anti-cancer therapeutics in OSCC.

口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）的转移显著降低了患者的5年生存率。自噬有促癌和抑癌的双重调控作用，对OSCC的效应与机制尚未明晰。课题组前期研究发现，相比未发生转移的肿瘤细胞和周围正常组织，维甲酸诱导基因I（RIG-I）在转移的OSCC细胞中显著高表达，提示转移可能和RIG-I的活化有关。RIG-I对肿瘤的双重作用与自噬对肿瘤的调控类似。进一步体外实验证明低亲和力活化RIG-I在促进OSCC生长、侵袭、转移的同时，活化了自噬标志物LC3，提示RIG-I促进肿瘤生长和转移的效应可能是通过自噬机制起作用。课题组拟进一步扩大临床样本数量、运用3D细胞培养、免疫共沉淀后质谱鉴定等多种技术，着眼于从肿瘤转移关键步骤上皮-间充质转化（EMT）的角度研究RIG-I表达与自噬、OSCC转移的联系，解释RIG-I促进OSCC转移的效应及其分子机制。

Ⅰ型维甲酸诱导基因（retinoic-acid inducible gene I，RIG-I）是重要的模式识别受体，不仅对于检测病毒和启动天然免疫反应至关重要，RIG-I对多种肿瘤细胞也有不同的作用。我们的前期实验发现活化的高水平RIG-I能诱导口腔鳞癌（oral squamous cell carcinoma，OSCC）细胞凋亡，需要进一步明确RIG-I是否参与了抗肿瘤治疗的潜在机制，以及RIG-I是否可以作为OSCC对抗肿瘤治疗敏感性的预测因子。自噬对肿瘤的发生发展具有抑瘤和促瘤的双重作用，近期的实验表明自噬在肿瘤耐药中扮演重要的作用，细胞自噬或许是OSCC对于抗肿瘤治疗响应率低的关键因素。因此，探索OSCC中抗肿瘤治疗是否可以活化自噬，以及阻断自噬流是否可以增强其抗肿瘤效应具有较大的临床意义。第一部分研究中，课题组确定了RIG-I在肿瘤发生发展，以及RIG-I在EGFR靶向药物和干扰素α（interferon-alpha，IFNα）联合抗肿瘤治疗中所起到的作用。RIG-I的表达受到I型干扰素的调控，并能被STAT1所活化。磷酸化的STAT1入核激活RIG-I相关基因，诱导RIG-I表达。而表皮生长因子可以通过泛素化来降解RIG-I。此外，RIG-I高表达能显著抑制肿瘤生长，而RIG-I低表达的OSCC细胞对抗肿瘤治疗的敏感性显著降低。第二部分研究中，课题组研究了自噬对口腔鳞癌细胞发生发展的影响，明确了细胞自噬在干扰素对抗实体肿瘤响应率低的现象中发挥的作用。课题组发现细胞自噬能被IFNα活化，而沉默自噬相关基因（autophagy-related protein 5，ATG5）和STAT1能抑制自噬流。在OSCC细胞和裸鼠移植瘤模型中IFNα与自噬抑制剂具有协同效应，抑制细胞增殖并促进细胞凋亡。综合上述结果，课题组提出RIG-I的表达水平可以作为包括IFNα在内的联合治疗效率的预测指标；此外，在OSCC中IFNα诱导的自噬起到肿瘤细胞保护作用，封闭自噬流能促进IFNα介导的细胞凋亡作用，IFNα与自噬抑制剂联合应用可以作为OSCC治疗的候选策略。