自噬通过诱导休眠多倍体巨大肿瘤细胞形成促进鼻咽癌复发的研究

Autophagy promotes recurrence of nasopharyngeal carcinoma via inducing the formation of dormant polyploid giant cancer cells

目的探究休眠的多倍体巨大肿瘤细胞(polyploid giant cancer cells,PGCC)对鼻咽癌(nasopharyngeal carcinoma,NPC)复发的影响,明确抑制自噬在阻止NPC复发中的作用。方法利用紫杉醇(paclitaxel,PTX)诱导NPC细胞来源的PGCC(NPC-PGCC)形成,并利用光学显微镜、细胞免疫荧光、活/死细胞双染色实验对PGCC形态、多倍体特性、细胞活性等进行鉴定。采用转录组测序(RNA-seq)检测NPC-PGCC和二倍体NPC细胞CNE2的差异表达基因。采用基因本体论(gene ontology,GO)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)对差异基因进行功能富集和通路注释分析。利用免疫蛋白印迹与细胞透射电镜实验评估NPC-PGCC细胞中的自噬水平。利用临床高度相关的裸鼠NPC复发模型研究自噬在NPC-PGCC形成中的作用及NPC-PGCC对NPC复发的影响。所有数据采用GraphPad Prism 6进行统计学分析,以P<0.05为差异具有统计学意义。结果紫杉醇诱导形成的NPC-PGCC具有休眠后爆炸性分裂的特征。NPC-PGCC和二倍体NPC细胞CNE2的差异基因GO富集、KEGG通路注释主要集中在自噬及其相关通路。NPC-PGCC细胞中的自噬水平显著增强。临床高度相关裸鼠NPC复发模型中,进行顺铂治疗的裸鼠原发肿瘤中PGCC数量高于其余各组;自噬抑制剂预处理后与顺铂联合治疗的裸鼠原发肿瘤中PGCC数量少,同时复发率显著低于其余各组。结论休眠多倍体巨大肿瘤细胞的形成机制与自噬有关,抑制自噬可通过抑制PGCC形成进而抑制NPC复发。

Objective To explore the effect of dormant polyploid giant cancer cells(PGCC)on nasopharyngeal carcinoma(NPC)recurrence and to clarify the role of inhibition of autophagy in inhibiting NPC-PGCC formation and preventing NPC recurrence.Methods NPC cells-derived PGCC(NPC-PGCC)were induced by paclitaxel(PTX),and the morphology,polyploid characteristics and cell activity of PGCC were identified by light microscopy,immunofluorescence and Live/Dead cell double staining assays.RNA-seq was used to analyze the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2.Functional enrichment and pathway annotation analysis of differentially expressed genes were performed using Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG).The level of autophagy in NPC-PGCC cells was assessed by Western Blot and transmission electron microscopy analysis.The role of autophagy in the formation of NPC-PGCC and the effect of NPC-PGCC on the recurrence of nasopharyngeal carcinoma were studied using a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model.Statistical analysis was performed using GraphPad Prism 6 and P-values<0.05 were considered statistically significant.Results NPC-PGCC induced by paclitaxel had the characteristics of burst-like division after dormancy.GO enrichment and KEGG pathway analyses identified the significant biological processes and pathways mainly concentrated in autophagy and related pathways involving the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2.The autophagy level was significantly enhanced in NPC-PGCC cells.In a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model,the number of PGCC in the primary tumor of the nude mice treated with cisplatin were higher than those of the other groups.In nude mice pretreated with autophagy inhibitor and then co-treatment with autophagy inhibitor and cisplatin,the number of PGCC in primary tumors was less and the recurrence rate was significantly lower than in other groups.Conclusions The mechanism of dormant polyploid giant cancer cells formation is related to autophagy.Inhibition of autophagy can inhibit the formation of PGCC and thus prevent the recurrence of nasopharyngeal carcinoma.