PAX9 is a transcription factor of the PAX family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of PAX9 in squamous cell differentiation and carcinogenesis of the oro-oesophageal epithelium. However, its functional roles in differentiation and carcinogenesis remain unclear. In this study, Pax9 deficiency in mouse oesophagus promoted cell proliferation, delayed cell differentiation, and altered the global gene expression profile. Ethanol exposure downregulated PAX9 expression in human oesophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was downregulated in human oro-oesophageal squamous cell carcinoma (OESCC), and its downregulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with a liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted N-nitrosomethylbenzylamine-induced squamous cell carcinogenesis in mouse tongue, oesophagus, and forestomach. In conclusion, PAX9 regulates squamous cell differentiation in the oro-oesophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. PAX9 downregulation may contribute to alcohol-associated oro-oesophageal squamous cell carcinogenesis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Alcohol drinking has been established as a major risk factor for esophageal diseases. Our previous study showed that ethanol exposure inhibited PAX9 expression in human esophageal squamous epithelial cells in vitro and in vivo. In this study, we aimed to investigate the molecular pathways through which alcohol drinking suppresses PAX9 in esophageal squamous epithelial cells. We first demonstrated the inhibition of NOTCH by ethanol exposure in vitro. NOTCH regulated PAX9 expression in KYSE510 and KYSE410 cells in vitro and in vivo. RBPJ and NOTCH intracellular domain (NIC) D1 ChIP‐PCR confirmed Pax9 as a direct downstream target of NOTCH signaling in mouse esophagus. NOTCH inhibition by alcohol drinking was further validated in mouse esophagus and human tissue samples. In conclusion, ethanol exposure inhibited NOTCH signaling and thus suppressed PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. Our data support a novel mechanism of alcohol‐induced esophageal injury through the inhibition of NOTCH–PAX9 signaling. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pax9 is a transcription factor of the Pax family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of Pax9 in squamous cell differentiation and carcinogenesis of oro-esophageal epithelium. However, its functional role in differentiation and carcinogenesis remains unclear. In this study, Pax9 deficiency in mouse esophagus promoted cell proliferation, delayed cell differentiation and altered the global gene expression profile. Ethanol exposure down-regulated Pax9 expression in human esophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was down-regulated in human oro-esophageal squamous cell carcinoma (OESCC), and its down-regulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with an isocaloric Lieber-DeCarli liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted NMBA-induced squamous cell carcinogenesis in mouse tongue, esophagus, and forestomach. In conclusion, Pax9 regulates squamous cell differentiation in the oro-esophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. Pax9 down-regulation, at least in part, contributes to alcohol-associated oro-esophageal squamous cell carcinogenesis. Citation Format: Zhaohui Xiong, Shuang Ren, Hao Chen, Yao Liu, Caizhi Huang, Yawan Lyvia Zhang, Joab Otieno Odera, Tong Chen, Ralf Kist, Heiko Peters, Katherine Garman, Zheng Sun, Xiaoxin Luke Chen. Pax9 regulates squamous cell differentiation and alcohol-associated carcinogenesis in the oro-esophageal epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4471.