Pepsin Promotes Activation of Epidermal Growth Factor Receptor and Downstream Oncogenic Pathways, at Slightly Acidic and Neutral pH, in Exposed Hypopharyngeal Cells

Abstract

Pepsin refluxate is considered a risk factor for laryngopharyngeal carcinogenesis. Non-acidic pepsin was previously linked to an inflammatory and tumorigenic effect on laryngopharyngeal cells in vitro. Yet there is no clear evidence of the pepsin-effect on a specific oncogenic pathway and the importance of pH in this process. We hypothesized that less acidic pepsin triggers the activation of a specific oncogenic factor and related-signalling pathway. To explore the pepsin-effect in vitro, we performed intermittent exposure of 15 min, once per day, for a 5-day period, of human hypopharyngeal primary cells (HCs) to pepsin (1 mg/mL), at a weakly acidic pH of 5.0, a slightly acidic pH of 6.0, and a neutral pH of 7.0. We have documented that the extracellular environment at pH 6.0, and particularly pH 7.0, vs. pH 5.0, promotes the pepsin-effect on HCs, causing increased internalized pepsin and cell viability, a pronounced activation of EGFR accompanied by NF-κB and STAT3 activation, and a significant upregulation of EGFR, AKT1, mTOR, IL1β, TNF-α, RELA(p65), BCL-2, IL6 and STAT3. We herein provide new evidence of the pepsin-effect on oncogenic EGFR activation and its related-signaling pathway at neutral and slightly acidic pH in HCs, opening a window to further explore the prevention and therapeutic approach of laryngopharyngeal reflux disease.