An effective two-stage NMBzA-induced rat esophageal tumor model revealing that the FAT-Hippo-YAP1 axis drives the progression of ESCC

Rat model of N-nitrosomethylbenzylamine (NMBzA)-induced esophageal squamous cell carcinoma (ESCC) is routinely used to study ESCC initiation, progression and new therapeutic strategies. However, the model is time-consuming and malignant tumor incidences are low. Here, we report the usage of multi-kinase inhibitor sorafenib as a tumor promoter to establish an efficient two-stage NMBzA-induced rat ESCC carcinogenesis model, resulting in increments of tumor incidences and shortened tumor formation times. By establishing the model and applying whole-genome sequencing, we discover that benign papillomas and malignant ESCCs harbor most of the "driver" events found in rat ESCCs (e.g. recurrent mutations in Ras family, the Hippo and Notch pathways and histone modifier genes) and the mutational landscapes of rat and human ESCCs overlap extensively. We generate tumor cell lines derived from NMBzA-induced papillomas and ESCCs, showing that papilloma cells retain more characteristics of normal epithelial cells than carcinoma cells, especially their exhibitions of normal rat cell karyotypes and inabilities of forming tumors in immunodeficient mice. Three-dimensional (3-D) organoid cultures and single cell RNA sequencing (scRNA-seq) indicate that, when compared to control- and papilloma-organoids, ESCC-organoids display salient abnormalities at tissue and single-cell levels. Multi-omic analyses indicate that NMBzA-induced rat ESCCs are accompanied by progressive hyperactivations of the FAT-Hippo-YAP1 axis and siRNA or inhibitors of YAP1 block the growth of rat ESCCs. Taken together, these studies provide a framework of using an effective rat ESCC model to investigate multilevel functional genomics of ESCC carcinogenesis, which justify targeting YAP1 as a therapeutic strategy for ESCC.