Inhibition of NF-κB prevents the acidic bile-induced oncogenic mRNA phenotype, in human hypopharyngeal cells

Abstract

Bile-containing gastro-duodenal reflux has been clinically considered an independent risk factor in hypopharyngeal carcinogenesis. We recently showed that the chronic effect of acidic bile, at pH 4.0, selectively induces NF-κB activation and accelerates the transcriptional levels of genes, linked to head and neck cancer, in normal hypopharyngeal epithelial cells. Here, we hypothesize that NF-κB inhibition is capable of preventing the acidic bile-induced and cancer-related mRNA phenotype, in treated normal human hypopharyngeal cells. In this setting we used BAY 11-7082, a specific and well documented pharmacologic inhibitor of NF-κB, and we observed that BAY 11-7082 effectively inhibits the acidic bile-induced gene expression profiling of the NF-κB signaling pathway (down-regulation of 72 out of 84 analyzed genes). NF-κB inhibition significantly prevents the acidic bile-induced transcriptional activation of NF-κB transcriptional factors, RELA (p65) and c-REL, as well as genes related to and commonly found in established HNSCC cell lines. These include anti-apoptotic bcl-2, oncogenic STAT3, EGFR, ∆Np63, TNF-α and WNT5A, as well as cytokines IL-1β and IL-6. Our findings are consistent with our hypothesis demonstrating that NF-κB inhibition effectively prevents the acidic bile-induced cancer-related mRNA phenotype in normal human hypopharyngeal epithelial cells supporting an understanding that NF-κB may be a critical link between acidic bile and early preneoplastic events in this setting.

NF‐κB inhibition reverses acidic bile‐induced miR‐21, miR‐155, miR‐192, miR‐34a, miR‐375 and miR‐451a deregulations in human hypopharyngeal cells

“…In line with our recent study,33 our current data suggest that NF‐κB inhibition may reverse acidic bile‐induced molecular events in normal human hypopharyngeal cells that are known to link inflammation to cancer, thereby in a sense shielding HHPC from the effects of bile‐induced oncogenic molecular events.…”

“…We used the same pool of total RNA to determine, by qPCR, the effect of BAY 11‐7082 on transcriptional levels of RELA(p65), TNF‐α, IL‐1β, IL‐6 and STAT3 in acidic bile‐treated and control HHPC with or without BAY 11‐7082, as previously described33 (Supplementary Methods & Table S2). These genes were selected because they demonstrated an increased transcriptional activity under acidic bile exposure of HHPC 8, 9 that was prevented by BAY 11‐7082 in our previous study 33…”

Biliary reflux as a causal factor in hypopharyngeal carcinoma: New clinical evidence and implications

“…Because PI3K activation may lead to downstream NF-κB activation, 35 its role in bile-induced NF-κB activation requires further exploration. 9,33,34,43,44 Bile(+) tumors also demonstrate an intense downregulation of tumor suppressors miR-489, miR-504, and miR-99a compared with their ANTs, a finding that is in keeping with previous studies suggesting their involvement in the initiation and progression of HSCC. 46,47 The data from the current study do not fully support a strong association between the NOTCH family and bile(+) HSCC.…”

“…7,32,33 Our analysis using qPCR demonstrated that bile(+) HSCC produced an upregulated NF-κB-related mRNA phenotype (Fig. 7,32,33 Our analysis using qPCR demonstrated that bile(+) HSCC produced an upregulated NF-κB-related mRNA phenotype (Fig.…”

“…It is apparent that activation of NF-κB is a common finding in HSCC from patients with documented biliary-esophageal reflux. [4][5][6][7][8][9]16,[32][33][34][43][44][45] In aggressive bile(+) HSCC, there appears to be a selective upregulation of NF-κB transcriptional factors RELA(p65) and c-REL; antiapoptotic bcl-2; and oncogenic EGFR, STAT3, and WNT5A, which is consistent 14,15,29 Clinical observations described herein further suggest that in bile(+) HSCC, constitutive activation of NF-κB also is capable of producing a cancer-related mRNA and miRNA phenotype similarly found in our preclinical in vitro and in vivo models and negatively affected by NF-κB inhibition ( Table 2).…”