Abstract 2726: Pepsin promotes growth and proliferation of laryngopharyngeal squamous cell carcinoma

Abstract

Abstract Squamous cell carcinoma (SCC) of the head in neck is currently the 5th most common cancer in the United States, with over 35,000 cases diagnosed in 2009. In addition to tobacco and alcohol, laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngopharyngeal SCC, but definitive associations between LPR and carcinogenesis are lacking. The purpose of this study was to investigate a role for refluxed pepsin in the promotion of SCC of the laryngopharynx. We recently discovered that inactive but stable pepsin (at pH7, in non-acidic reflux) is actively taken up by laryngeal epithelial cells by endocytosis in a receptor mediated manner and leads to intracellular damage and pro-inflammatory cytokine gene expression changes. This reveals a novel mechanism by which pepsin can cause cell/tissue damage and thereby promote carcinogenesis in patients with LPR. These studies were therefore done to specifically study the effect of neutral pH pepsin on laryngopharyngeal SCC proliferation. Pepsin levels were measured in posterior laryngeal biopsy specimens from patients with clinically diagnosed LPR (n = 5) and normal control subjects (n = 5) by Western blot analysis. Human hypopharyngeal SCC FaDu cells and normal human primary laryngeal epithelial cells were exposed to affinity purified human pepsin (0.1mg/ml, pH7) and gene expression levels measured on a Human Cancer PathwayFinder SuperArray by real-time RT-PCR. Cell proliferation was measured by growth curve, propidium iodide staining, and Click-iT Edu assay. Pepsin was detected in 3/5 patient laryngeal cancer biopsies, and was absent in all normal control laryngeal biopsies (0/5). Pepsin (0.1mg/ml, pH7) significantly altered the expression level of 27 genes implicated in carcinogenesis on SuperArray analysis with a pattern promoting generalized increased cellular proliferation. Flow cytometry analysis with propidium iodide staining demonstrated an increase in S phase distribution following exposure to pepsin in a time and dose dependent manner. Growth curve and Click-iT EdU proliferation assays also demonstrated increased proliferation following pepsin exposure. Pepsin increases cell proliferation and alters the expression of multiple genes implicated in carcinogenesis. Pepsin also appears to be selectively present in human laryngeal tumors and not in control specimens. Thus, uncontrolled chronic reflux of pepsin as in LPR may increase risk for laryngopharyngeal SCC development. Studies are ongoing to investigate the effect of pepsin on cell transformation, clone-forming ability, resistance to anoikis, DNA damage and reactive oxygen species levels. New therapies to prevent pepsin uptake and damage, such as pepsin inhibitors and receptor antagonists, are also being tested for patients with LPR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2726. doi:10.1158/1538-7445.AM2011-2726