Annexin A1在头颈鳞癌EGF相关ERK/MAPK信号通路中的作用机制

Head and neck squamous cell carcinoma represents the 6th common cancer worldwide, with a poor prognosis of 5-year survival rate about 50%-60%. Annexin A1 is originally identified and involved for a long time to function as a cellular mediator of the anti-inflammatory effects. However, since its discovery as a major cellular substrate for tyrosine phosphorylation by the epidermal growth factor receptor (EGFR), Annexin A1 is implicated in various pathways known to be involved in cancer through the modulation of mitogen-activated protein kinase (MAPK) signaling, calcium ion mobilization, apoptosis, cell proliferation, and inhibition of cell growth. Our previous studies have established an in vitro cellular carcinogenesis model from normal oral mucosal epithelia to cancerous squamous cells, and comparative proteomic technique has been used to screen the differential proteomic profiles between the cells at different stages of carcinogenesis. Annexin A1 is one of the proteins, which is expressed in a low level accompanying with the carcinogenesis. Further validation confirms in the cellular level and tissue level. Significant correlation is also found between the Annexin A1 expression level and pathological differentiation grade of cancerous tissues, a poorer differentiation grade indicating a lower level of Annexin A1 expression. When the Annexin A1 expression is overexpressed in the cancerous cells by gene transferring with the total Annexin A1 gene, decreased cellular proliferative ability and tumorigenesis ability is found in vitro and in vivo, and the differentiation grade of cancerous tissue becomes better from moderately differentiation grade to well differentiation grade. In the cancerous cells with Annexin A1 overexpression, the phosphoration level of extracellular signal-regulated kinase (ERK) is higher than those without Annexin A1 overexpression. The phosphoration level of Annexin A1 is also affected by adding EGFR into the cell culture media. From the literatures, decreased expression of Annexin A1 has been found in many kinds of cancers, and the molecular intervention with Annexin A1 overexpression could increase the phosphoration level of ERK, suggesting the Annexin A1 might function as a suppressor gene and inhibit the ERK/MAPK signal pathway; however, there are several unknown issues on the molecular mechanism how Annexin A1 plays a role in the EGF/ERK/MAPK signal pathway, which needs deeper studies. Based on our previous results, the aim of this project is to study the potential inhibition role of Annexin A1 in the EGF/ERK/MAPK signal pathway using the molecular intervention, in vitro and in vivo methods in the head and neck squamous cell carcinoma, and also the clinical usefulness of Annexin A1 detection for prognostic prewarning and as potential treatment target, which will provide scientific evidence for personalized treatment in head and neck squamous cell carcinoma.

本课题组先前建立了口腔黏膜上皮细胞体外癌变模型，再用比较蛋白质组学的方法发现Annexin A1随着细胞癌变而表达下降，获得体内外细胞和组织水平的验证；并发现其表达高低与口腔鳞癌的分化程度相关，分化程度越差，Annexin A1表达越低；经Annexin A1过表达干预可降低癌细胞的增殖和成瘤能力，癌细胞分化变好，并使ERK磷酸化升高；Annexin A1的磷酸化也受到EGFR干预的影响。但经文献复习，发现Annexin A1在肿瘤EGF/ERK/MAPK信号通路中有许多未知之处，具有深入研究的价值。本研究旨在采用分子干预和体内外实验相结合的手段，以EGF相关的ERK/MAPK信号通路为切入点，探索Annexin A1在头颈鳞癌发展中可能抑制EGF/ERK/MAPK信号通路的作用机制，并评价Annexin A1在头颈鳞癌中的预后预警作用及作为药物靶标的潜能，为头颈鳞癌的个体化治疗提供依据。

本项目采用比较蛋白质组学的方法，筛选出口腔黏膜上皮细胞体外癌变模型中，随着细胞癌变表达下降的Annexin A1，通过体内外细胞和组织学水平的验证，证实Annexin A1在口腔鳞癌中表达显著下降，并且与口腔鳞癌的病理分化程度显著相关，病理分化越差，Annexin A1的表达越低。同时结合临床随机对照试验，回顾性发现病理中低分化的低表达Annexin A1患者，可以从TPF诱导化疗中生存获益。通过体外干预Annexin A1的表达，发现干扰Annexin A1可促进口腔鳞癌细胞的增殖，过表达Annexin A1可抑制口腔鳞癌细胞的增殖及裸鼠体内成瘤，病理分化程度变好。通过检测口腔鳞癌细胞中EGFR磷酸化及EGFR相关信号通路EGFR/AKT、EGFR/MAPK通路中相关分子的表达及磷酸化情况，发现在口腔鳞癌中Annexin A1表达下调后两条信号通路均被激活，Annexin A1的表达下调后EGFR蛋白磷酸化水平提高，同时AKT和ERK1/2的磷酸化水平也有相应的提高。在AKT信号通路中，Annexin A1表达下调后AKT上游的PDK1磷酸化水平和下游的c-RAF的磷酸化也有明显的提高，但是细胞内cyclin D1的表达未受到Annexin A1水平的影响。在MAPK信号通路中，当Annexin A1的表达水平下调后ERK上游c-RAF的磷酸化升高，其下游的MSK1的表达上调。通过干预口腔鳞癌细胞系中的Annexin A1表达联合多西他赛、顺铂、5氟尿嘧啶药物处理，发现Annexin A1表达下调后口腔鳞癌细胞对化疗药物更加敏感，细胞中凋亡直接相关蛋白PARP和caspase3活性片段的表达升高，这可能是Annexin A1表达下调后对TPF抗肿瘤药物敏感的原因，为Annexin A1作为头颈鳞癌预测性标志物选择个体化治疗方案提供了研究基础。