Gastro-duodenal fluid induced Nuclear Factor-KappaB activation and early pre-malignant alterations in murine hypopharyngeal mucosa

We recently described the role of gastro-duodenal fluids (GDFs) in generating changes consistent with hypopharyngeal neoplasia through activation of NF-kappa B pathway, using an in vitro model of human hypopharyngeal normal keratinocytes. Here, we further provide evidence that gastro-duodenal reflux is a risk factor for early pre-malignant alterations in hypopharyngeal mucosa (HM) related to an activated NF-kappa B oncogenic pathway, using both an in vitro and a novel in vivo model of C57BI/6J mice. Histological, immunohistochemical and automated quantitative analysis documents significant NF-kappa B activation and early pre-malignant alterations in HM topically exposed to GDFs, compared to acid alone and other controls. Early pre-malignant histologic lesions exhibited increased Ki67, CK14 and Delta Np63, cell proliferation markers, changes of cell adhesion molecules, E-Cadherin and beta-catenin, and STAT3 activation. The in vivo effect of NF-kappa B activation is positively correlated with p-STAT3, Ki67, CK14 or beta-catenin expression, while GDFs induce significant transcriptional activation of RELA(p65), bcl-2, TNF-alpha, STAT3, EGFR and wnt5A, in vivo. Our in vivo model demonstrates selectively activated NF-kappa B in response to topically administrated GDFs, leading to early pre-malignant events in HM.