Noxious Combination of Tobacco Smoke Nitrosamines with Bile, Deoxycholic Acid, Promotes Hypopharyngeal Squamous Cell Carcinoma, via NFκB, In Vivo

Abstract

Abstract Tobacco smoking is the most known risk factor for hypopharyngeal cancer. Bile reflux has recently been documented as an independent risk factor for NFκB-mediated hypopharyngeal squamous cell carcinoma. However, the carcinogenic effect of tobacco smoke on the hypopharynx and its combination with bile has not yet been proven by direct evidence. We investigated whether in vivo chronic exposure (12–14 weeks) of murine (C57Bl/6J) hypopharyngeal epithelium to tobacco smoke components (TSC) [N-nitrosamines; 4-(N-Methyl-N-Nitrosamino)-1-(3-pyridyl)-1-butanone (0.2 mmol/L), N-nitrosodiethylamine (0.004 mmol/L)], as the sole drinking fluid 5 days per week, along with topically applied (two times/day) bile [deoxycholic acid (0.28 mmol/L)], can accelerate a possible TSC-induced neoplastic process, by enhancing NFκB activation and the associated oncogenic profile, using histologic, IHC, and qPCR analyses. We provide direct evidence of TSC-induced premalignant lesions, which can be exacerbated by the presence of bile, causing invasive carcinoma. The combined chronic exposure of the hypopharynx to TSC with bile causes advanced NFκB activation and profound overexpression of Il6, Tnf, Stat3, Egfr, Wnt5a, composing an aggressive phenotype. We document for the first time the noxious combination of bile with a known risk factor, such as tobacco smoke nitrosamines, in the development and progression of hypopharyngeal cancer, via NFκB, in vivo. The data presented here encourage further investigation into the incidence of upper aerodigestive tract cancers in smokers with bile reflux and the early identification of high-risk individuals in clinical practice. This in vivo model is also suitable for large-scale studies to reveal the nature of inflammatory-associated aerodigestive tract carcinogenesis and its targeted therapy. Prevention Relevance: Early assessment of bile components in refluxate of tobacco users can prevent the chronic silent progression of upper aerodigestive tract carcinogenesis. This in vivo model indicates that bile reflux might have an additive effect on the tobacco-smoke N-nitrosamines effect and could be suitable for large-scale studies of diagnostic and therapeutic interventions.