Establishment Of Multi-Stage Chemical Carcinogenesis In Rat Esophagus And YAP Signaling Drives The Progression Of Esophagus Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is one of the most malignant squamous cell carcinomas and the sixth leading cause of cancer-related mortality worldwide.ESCC is highly prevalent in China,with an estimated 300,000 new cases per year,and the mortality rate ranks the fourth among all cancers.Due to the lack of targeted therapy and precision medicines,the prognosis of ESCC patients is poor.N-nitrosomethylbenzylamine(NMBzA)can specifically induce carcinogenesis of esophageal epithelium.The NMBzA-induced rat ESCC model is a commonly used animal model in the study of etiology and prevention of ESCC.The histopathology of tumor induced by NMBzA in the rat esophagus are similar to human ESCC origin,with stages from normal epithelium,hyperplasia,papilloma,dysplasia to squamous cell carcinoma.However,this method has long carcinogenesis cycle and low rate.So,in this study,we use NMBzA as the initial carcinogen and sorafenib as a promoting agent to establish a multiple-stage chemical carcinogenesis model which has short cycle and high success rate.It remains unclear whether NMBzA-induced esophageal carcinogenesis in rats is driven by the same spectrum of genomic alterations found in humans.In this study,whole-genome sequencing(WGS)was performed on samples from papilloma and squamous cell carcinoma in this model.We find that rat esophageal tumors present recurrent mutations in RAS,which is not common in human ESCC.The rat ESCC mutational landscape is similar to that of human ESCC,with C>T mutations dominate the spectra.Some significantly mutated genes in human ESCC,such as TP53?Notch?FAT?PIK3CA?CREBBP?PLEC,etc.,are also present in rat esophageal tumors.There are no significant differences in mutated genes and mutation burden between papilloma and squamous cell carcinoma,but squamous cell carcinoma present substantial copy number variation(CNV).These results provide a genomic background for future functional studies in rat ESCC model.Based on this model,immortalized cells from rat esophageal tumors,including papilloma and squamous cell carcinoma,were established in vitro.We also successfully establish a 3D organoids culture system with these cells.Through 2D and 3D culture system in vitro,we find that benign papilloma cells retain more characteristics of normal epithelial cells than malignant squamous cell carcinoma cells.For example,they could not form tumor in immunodeficient mice,the karyotype of cells is normal,and the organoids formed are similar to those formed by normal esophageal epithelial cells in structure and morphology.As one of the most significant mutant genes in rat and human ESCC,the loss of function(LOF)mutations of FAT family can activate the Hippo-YAP pathway.Using RNA sequencing,we uncover that both papilloma and squamous cell carcinoma showing activation of YAP1 signal compared with normal rat esophageal epithelium,and the progression from papilloma to carcinoma is accompanied by further activation of YAP 1.Strikingly,siRNA or pharmacological inhibition of YAP1 can inhibit the growth and organoid formation of rat ESCC cells in vitro and vivo.In conclusion,our study find that YAP 1 activation may be a potent driver of ESCC,and targeting YAP 1 can be a new strategy for the treatment of ESCC.