Summary Sox2 regulates the self-renewal of multiple types of stem cells. Recent studies suggest it also plays oncogenic roles in the formation of squamous carcinoma in several organs, including the esophagus where Sox2 is predominantly expressed in the basal progenitor cells of the stratified epithelium. Here, we use mouse genetic models to reveal a novel mechanism by which Sox2 cooperates with microenvironmental signals to malignantly transform epithelial progenitor cells. Conditional overexpression of Sox2 in basal cells expands the progenitor population in both the esophagus and forestomach. Significantly, carcinoma only develops in the forestomach where pathological progression correlates with inflammation and nuclear localization of Stat3 in progenitor cells. Importantly, co-overexpression of Sox2 and activated Stat3 (Stat3C) also transforms esophageal basal cells but not the differentiated suprabasal cells. These findings indicate basal stem/progenitor cells are the cells-of-origin of squamous carcinoma and that cooperation between Sox2 and microenvironment-activated Stat3 is required for Sox2-driven tumorigenesis.

The barrier function of the esophageal epithelium is a major defense against gastroesophageal reflux disease. Previous studies have shown that reflux damage is reflected in a decrease in transepithelial electrical resistance associated with tight junction alterations in the esophageal epithelium. To develop novel therapies, it is critical to understand the molecular mechanisms whereby contact with a refluxate impairs esophageal barrier function. In this study, surgical models of duodenal and mixed reflux were developed in mice. Mouse esophageal epithelium was analyzed by gene microarray. Gene set enrichment analysis showed upregulation of inflammation-related gene sets and the NF-κB pathway due to reflux. Significance analysis of microarrays revealed upregulation of NF-κB target genes. Overexpression of NF-κB subunits (p50 and p65) and NF-κB target genes (matrix metalloproteinases-3 and -9, IL-1β, IL-6, and IL-8) confirmed activation of the NF-κB pathway in the esophageal epithelium. In addition, real-time PCR, Western blotting, and immunohistochemical staining also showed downregulation and mislocalization of claudins-1 and -4. In a second animal experiment, treatment with an NF-κB inhibitor, BAY 11-7085 (20 mg·kg⁻¹·day⁻¹ ip for 10 days), counteracted the effects of duodenal and mixed reflux on epithelial resistance and NF-κB-regulated cytokines. We conclude that gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice and that targeting the NF-κB pathway may strengthen esophageal barrier function against reflux.

Objective As a major cellular defense mechanism, the Nrf2/Keap1 pathway regulates expression of genes involved in detoxification and stress response. Our previous study revealed activation of the Nrf2/Keap1 pathway at the maturation phase during mouse esophageal development, suggesting a potential function in epithelial defense. Here we hypothesize that Nrf2 is involved in the barrier function of esophageal epithelium, and plays a protective role against gastroesophageal reflux disease (GERD). Design Human esophageal biopsy samples, mouse surgical models and Nrf2-/- mice were used to assess the role of the Nrf2/Keap1 pathway in esophageal mucosal barrier function. Trans-epithelial electrical resistance (TEER) was measured with mini-Ussing chambers. Hematoxylin and eosin (HE) staining and transmission electron microscopy were used to examine cell morphology, while gene microarray, immunohistochemistry, Western blotting and ChIP analysis were used to assess the expression of pathway genes. Results Nrf2 was expressed in normal esophageal epithelium and activated in GERD of both humans and mice. Nrf2 deficiency and gastroesophageal reflux in mice, either alone or in combination, reduced TEER and increased intercellular space diameter in esophageal epithelium. Nrf2 target genes and gene sets associated with oxidoreductase activity, mitochondrial biogenesis and energy production were down-regulated in the esophageal epithelium of Nrf2-/- mice. Consistent with the antioxidative function of Nrf2, a DNA oxidative damage marker (8OHdG) dramatically increased in esophageal epithelial cells of Nrf2-/- mice compared with those of wild-type mice. Interestingly, ATP biogenesis, Cox IV (a mitochondrial protein) and Claudin-4 (Cldn4) expression were down-regulated in the esophageal epithelium of Nrf2-/- mice, suggesting that energy-dependent tight junction integrity was subject to Nrf2 regulation. ChIP analysis confirmed the binding of Nrf2 to Cldn4 promoter. Conclusion Nrf2 deficiency impairs esophageal barrier function through disrupting energy-dependent tight junction. Elucidating the role of this pathway in GERD has potential implications for the pathogenesis and therapy of the disease.

In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography–tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2–/– mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms.

Background and AimsMorphological changes during human and mouse esophageal development have been well characterized. However, changes at the molecular level in the course of esophageal morphogenesis remain unclear. This study aims to globally profile critical genes and signaling pathways during the development of mouse esophagus. By using microarray analysis this study also aims to determine how the Nrf2/Keap1 pathway regulates the morphogenesis of the esophageal epithelium.MethodsGene expression microarrays were used to survey gene expression in the esophagus at three critical phases: specification, metaplasia and maturation. The esophagi were isolated from wild-type, Nrf2−/−, Keap1−/−, or Nrf2−/−Keap1−/− embryos or young adult mice. Array data were statistically analyzed for differentially expressed genes and pathways. Histochemical and immunohistochemical staining were used to verify potential involvement of the Wnt pathway, Pparβ/δ and the PI3K/Akt pathway in the development of esophageal epithelium.ResultsDynamic gene expression patterns accompanied the morphological changes of the developing esophagus at critical phases. Particularly, the Nrf2/Keap1 pathway had a baseline activity in the metaplasia phase and was further activated in the maturation phase. The Wnt pathway was active early and became inactive later in the metaplasia phase. In addition, Keap1−/− mice showed increased expression of Nrf2 downstream targets and genes involved in keratinization. Microarray and immunostaining data also suggested that esophageal hyperkeratosis in the Keap1−/− mice was due to activation of Pparβ/δ and the PI3K/Akt pathway.ConclusionsMorphological changes of the esophageal epithelium are associated with dynamic changes in gene expression. Nrf2/Keap1 pathway activity is required for maturation of mouse esophageal epithelium.

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.

Barrett’s esophagus (BE) is defined as metaplastic conversion of esophageal squamous epithelium to intestinalized columnar epithelium. As a premalignant lesion of esophageal adenocarcinoma (EAC), it develops as a result of chronic gastroesophageal reflux disease (GERD). Many studies have been conducted to undertand the molecular mechanism of this disease. This review summarizes recent results of involving squamous transcription factors, intestinal transcription factors, signaling pathways, stromal factors, microRNAs, and other factors in the development of BE. A conceptual framework is proposed to guide future studies. We expect elucidation of the molecular mechanism of BE will help us develop proper management of GERD, BE, and EAC.

Alcohol drinking is a major etiological factor of oro-esophageal squamous cell carcinoma (OESCC). Both local and systemic effects of ethanol may promote carcinogenesis, especially among chronic alcoholics. However, molecular mechanisms of ethanol-associated OESCC are still not well understood. In this review, we summarize current understandings and propose three mechanisms of ethanol-associated OESCC: (1) Disturbance of systemic metabolism of nutrients: during ethanol metabolism in the liver, systemic metabolism of retinoids, zinc, iron and methyl groups is altered. These nutrients are known to be associated with the development of OESCC. (2) Disturbance of redox metabolism in squamous epithelial cells: when ethanol is metabolized in oro-esophageal squamous epithelial cells, reactive oxygen species are generated and produce oxidative damage. Meanwhile, ethanol may also disturb fatty-acid metabolism in these cells. (3) Disturbance of signaling pathways in squamous epithelial cells: due to its physico-chemical properties, ethanol changes cell membrane fluidity and shape, and may thus impact multiple signaling pathways. Advanced molecular techniques in genomics, epigenomics, metabolomics and microbiomics will help us elucidate how ethanol promotes OESCC.