According to Nehra et al (35) and Kauer et al (39,40), the majority of bile acids in esophageal aspirates of patients with GERD are conjugated with taurine or glycine and may be sulfated. Specifically, glycine conjugates of cholic, deoxycholic and chenodeoxycholic acids are the predominant bile acids aspirated from the esophagus of patients with GERD (the ratio of glycine to taurine conjugates in normal human bile is 3:1). It has also been indicated that unconjugated secondary bile acids, such as deoxycholic acid (DCA), may be present in the esophageal refluxate, particularly in patients with erosive esophagitis and Barrett's esophagus (35,41,55). It is known that pH affects the solubility of each bile component in various manners. At acidic pH (≤4), the conjugated bile acids are more un-ionized and therefore capable of penetrating or interacting with the cell membrane (taurine conjugates: pKa=1.8–1.9; glycine conjugates: pKa=4.3–5.2) (56). At pH <3.0, bile salts tend to precipitate, whereas between pH 5.5 and 7.0, most conjugated primary bile acids are found to be ionized and therefore relatively inactive. However, unconjugated secondary bile acid DCA remains unionized (pKa 5.5–6.2) and may therefore exert its harmful effects, causing mucosal injury even at a less acidic pH (12,16,17,55). According to Stamp (57), duodenal fluid, at a less acidic pH, may also contribute to gastrointestinal tract tumorigenesis. Specifically, glycine conjugates may remain un-charged and therefore be harmful to the epithelial cells at a less acidic pH. Ireland et al (58) indicated that duodenal fluid significantly contributes to the carcinogenic potential of methyl-N-amyl nitrosamine, particularly at less acidic pH. In addition, as bile acids are natural detergents when in high concentration, they may interact with the cell membrane even at a neutral pH.

Although the bile composition is a crucial factor of bile reflux-related tumorigenesis, the concentration of bile may also potentiate its oncogenic effect. This view may be supported by previous in vivo studies indicating that the application of bile reflux components, chenodeoxycholic acid or DCA, at pharmacologic concentrations, both at a neutral pH of 7.0, to murine laryngopharyngeal mucosa was able to cause early premalignant changes, such as hyperplasia and dysplasia, as well as marked activation of NF-κB and its related oncogenic molecular phenotype (13,14).

It appears that acidity of LPR refluxate is a critical factor for bile to induce a harmful effect on laryngopharyngeal mucosa (39,59,60). In the clinic, intraesophageal pH monitoring has been used extensively to identify reflux episodes. Although pH varies during gastroesophageal reflux episodes, according to Ulualp et al (61), 24-h ambulatory pH monitoring in the pharynx of patients with reflux laryngitis confirmed that a drop below pH 4.0 is common and is considered diagnostic of a reflux event, suggesting that acid may contribute to duodenogastric-induced inflammatory and neoplastic events. Lillemoe et al (62) demonstrated the injurious effect of the various duodenal components on rabbit esophageal mucosa at strongly acidic pH, supporting a synergism between bile and HCl. A study by our group from 2019 (17) documented that the tumorigenic effect of bile on hypopharyngeal cells is pH-dependent. Other studies from our group (13–15) also demonstrated that a strongly acidic pH (≤4.0) serves a critical role in the bile-induced tumorigenic effect in murine laryngopharyngeal mucosa. It was indicated that chronic intermittent exposure of murine HM to a mixture of bile salts at a strongly acidic pH of 3.0 was able to progressively induce premalignant changes, microinvasion and invasive squamous cell carcinoma, causing increased DNA damage and oncogenic molecular alterations (15). Specifically, it was demonstrated that histopathologic changes caused by acidic bile were accompanied by underlying molecular alterations, such as increased levels of i) oxidative DNA/RNA damage and double-strand break (DSB) markers, ii) p53 and cell proliferation markers (Ki67, cytokeratin 14, and p63), as well as iii) alterations of the expression of cell adhesion molecules, like E-Cadherin and β-catenin, and iv) activation of NF-κB and other cancer-related transcription factors, such as signal transducer and activator of transcription 3 (STAT3) (13–15). However, hypopharyngeal cells or mucosa exposed to the same mixture of conjugated primary bile acids at neutral pH (7.0) produced hyperplastic or mild dysplastic changes and significantly less intense underlying molecular changes compared to acidic bile salts (12,13,17). In parallel, it was indicated that chronic exposure to acid alone or concentrated glucose was not able to produce any histological changes (13,15). The negative or reduced effect of acid alone and/or bile salts at neutral pH, compared to acidic bile salts, indicates that the latter is particularly injurious.

It is clear that the presence of conjugated primary bile acids in a highly acidic refluxate exerts a tumorigenic potency on the long-term exposed upper aerodigestive tract. This theory may explain findings from our group (13,15) indicating that chronic local exposure of murine HM to a mixture of conjugated primary bile acids, at concentrations previously measured in patients with GERD (35,40,43,35) at a strongly acidic pH (≤4.0), is able to progressively cause precancerous lesions and invasive cancer. Specifically, as taurine conjugates are active at low pH (≤4.0), it appears that taurine-conjugated bile acids may be responsible for the tumorigenic effect of bile at lower pH (15,17). There is also recent in vitro evidence supporting that acidity (pH ≤4.0) and bile composition may have a role in the progression of HSCC (63).

The above observations strongly support that controlling the pH during reflux episodes may have a protective effect by reducing the risk of bile-induced hypopharyngeal cancer. However, there is epidemiologic evidence that numerous patients with refractory GERD may also experience symptoms at a weakly acidic pH of 5.5–6.0 (60,64). Since unconjugated DCA and glycine-conjugated bile acids may be partially active at a weakly acidic pH, it appears that as the pH grows less acidic, approaching pH 5.5, the partially activated primary bile acids and the activated DCA may exert their influence (17). A recent study by our group (16) supported that DCA and glycine-conjugated bile acids are potent activators of DNA damage and oncogenic pathways in HM in a weakly acidic environment. Previous findings have demonstrated a similar association between DCA and its tumorigenic activity in the esophagus and colon (31,65,66). Regarding the hypopharynx, it has been documented that bile at a weakly acidic pH (5.0–5.5) with or without DCA, similarly to a strongly acidic pH 3.0, is able to increase the risk of bile-related hypopharyngeal neoplasia by promoting premalignant lesions, DNA damage and oncogenic molecular alterations, compared to controls (16). Of note, it was indicated that long-term exposure to a weakly acidic control (pH 5.5) was not able to induce any histological changes (16). This observation strongly supports that the oncogenic properties of biliary esophageal reflux on laryngopharyngeal mucosa may not be fully modified when antacid therapy is applied.

Although further exploration with clinical evidence is expected to strengthen these previous preclinical observations, investigation of the mechanism by which bile refluxate exerts its oncogenic properties is expected to contribute not only to a better understanding of the pathophysiology of hypopharyngeal cancer but also to alternative therapeutic strategies for patients with refractory GERD, using specific inhibitors of relevant molecular pathways or bile receptors.

There is evidence that numerous types of cancer arise from sites of chronic inflammation (95). Specifically, a wide array of chronic inflammatory conditions predisposes susceptible cells of epithelial origin to neoplastic transformation (carcinomas) as a multistep process (focal proliferation of dysplastic cells with potential progression to malignant carcinoma). An example includes reflux esophagitis that may lead to DNA damage, development of Barrett's esophagus and esophageal carcinoma (96). In certain cases, the progenitors of the inflammation are known, such as bacterial infections or gastric acids that have been associated with increased risk of adenocarcinoma of the stomach and esophagus, respectively (97,98). It may be assumed that bile acids interact directly with HM, as described in a paragraph below, but may also be the progenitors of an LPR-induced chronic inflammatory microenvironment associated with an increased risk of hypopharyngeal cancer. It has been indicated that a chronic inflammatory microenvironment or harmful stimuli are able to induce a constitutive activation of NF-κB, which may lead to a cascade of molecular alterations. Specifically, constitutive activation of NF-κB may lead to subsequent transcriptional activation of genes that are implicated in a variety of signaling pathways via aberrant overexpression of cytokines, transcription factors and growth factor receptors, such as TNF-α, TLR and EGFR (76,77,79–89,93,94,99–103).

Preclinical studies from our group (12–25) documented that acidic bile is able to induce activation of NF-κB and significant overexpression of several cancer-related genes. Specifically, acidic bile was reported to induce significant transcriptional activation of anti-apoptotic BCL2 and other genes previously linked to HNSCC, such as STAT3, EGFR, WNT5A, TNF-α, ΔNp63, cREL, IL6, IL1β, AKT1 and PTGS2 (82–94,102–111) (Fig. 1). Furthermore, a clinical pilot study revealed that bile-related HSCC had significantly higher levels of NF-κB and differential expression of the above genes compared to the adjacent non-pathologic tissue or bile-negative HSCC, providing further evidence of the central role of NF-κB (18).

Targeting NF-κB negatively affects the NF-κB signaling pathway and has been indicated to be an encouraging strategy to improve anticancer therapies (112). Several pharmacologic and dietary inhibitors of NF-κB are considered promising thera-peutic options, demonstrating chemo-preventive or chemo-sensitizing properties in head and neck cancer (99,112). BAY 11-7082 [(E)-3-(4-methylphenylsulphonyl)-1-propenenitrile] is a reliable inhibitor of the NF-κB pathway that has been widely used in numerous studies exploring the effect of NF-κB (112,113). It has been suggested that BAY 11-7082 offers the most rapid and potent anti-tumor effect among other NF-κB inhibitors (112) and it may be used as a sensitizer for anti-cancer treatment (114,115). Furthermore, curcumin is a turmeric natural supplement with known antioxidant, anti-inflammatory and anti-cancer properties, previously demonstrated to have potential chemo-preventive effects in head and neck malignancies (116), by blocking NF-κB activation and halting the proliferation of cancer cells (117) due to its pleiotropic properties (118).

Both BAY 11-7082 and dietary curcumin have been used in in vitro and in vivo experimental studies to investigate the underlying mechanism of bile reflux-induced carcinogenic effect into the hypopharynx. Studies by our group (19,25) suggested that application of BAY 11-7082 effectively suppressed cell proliferation rates, the activation of NF-κB and related oncogenic mRNA profiles induced by acidic bile exposure. This oncogenic phenotype included the significant overexpression of anti-apoptotic BCL2 and other genes implicated in the initiation and progression of HNSCC, including TNF-α, EGFR, STAT3, ΔNp63, cREL, IL6, IL1β, AKT1, PTGS2 and WNT5A (82–85,87,89–94,102–111) (Fig. 1). Parallel evidence that acidic bile stimulus is able to activate NF-κB and its related pathways in HHCs arose from the in vitro treatment with curcumin, which successfully blocked the transcriptional activity of NF-κB (20), similar to BAY 11-7082 (21,23). A study by our group from 2020 (24) documented the preventive and therapeutic properties of curcumin on murine HM against the acidic bile effect, thus shaping the future translational development of effective targeted therapies using topical non-pharmacologic inhibitors of NF-κB.

Strong evidence that NF-κB activation is able to influence the acidic bile-induced oncogenic mRNA profile inspired a further study on whether synchronizing its inhibition with acidic bile exposure is significant. Thus, a study by our group from 2019 (23) reported the temporal characteristics of NF-κB inhibition in blocking the acidic bile-induced oncogenic molecular events in HHCs. A series of studies also documented that topical application of BAY 11-7082 or curcumin to HM, either before, after or simultaneous to acidic bile exposure, successfully prevented or suppressed cell proliferation and NF-κB-related molecular events (19,24,25).

These results revealed that the upregulation of RELA, BCL2, STAT3, EGFR, WNT5A, TNF-α, IL6 and PTGS2 is directly promoted by acidic bile through NF-κB, shortly after its exposure (19,24,25) (Fig. 1), and strongly suggested that it may be clinically feasible to topically apply NF-κB inhibitors, without any precise synchronization with acidic bile exposure, to prevent acidic bile-induced oncogenic molecular changes.

The application of NF-κB inhibitors also revealed important information about possible interactions between acidic bile-induced NF-κB activation and other central molecules in head and neck cancer (Fig. 1).

The NF-κB/STAT3 crosstalk has been indicated to be fundamental in inflammation-associated carcinogenesis in head and neck cancer (108,119,120). Application of NF-κB inhibitors successfully prevented the acidic bile-induced activation and nuclear translocation of STAT3, which is an important regulator of cell proliferation, and reduced the transcriptional levels of IL6 and STAT3, in treated hypopharyngeal cells (19–25) (Fig. 1). These data strongly support the theory that the acidic bile-induced activation of IL-6/STAT3 is NF-κB-dependent (108) (in a paragraph below, the role of STAT3 in bile-induced carcinogenesis is discussed). In addition, prior findings implied strong interactions between NF-κB and STAT3 in acidic bile-exposed premalignant HM (13,14), further supporting the theory that the inflammatory response induced by acidic bile may increase the risk of laryngopharyngeal cancer.

EGFR is frequently overexpressed in HNSCC (121,122). Crosstalk between NF-κB and downstream pathways of EGFR has been observed (123,124). Although the exact role of EGFR in bile-related hypopharyngeal carcinogenesis has remained to be elucidated, the application of NF-κB inhibitors resulted in the successful suppression of acidic bile-induced overexpression of EGFR, supporting the interactions between NF-κB and EGFR pathways during this process (Fig. 1). In addition, as both STAT3 and EGFR are important contributors to HNSCC pathogenesis (80,121), the above observations further emphasized the requirement to develop a therapeutic strategy for targeting NF-κB in head and neck malignancies and particularly in bile reflux-related HSCC.

Furthermore, NF-κB inhibition had a strong effect in suppressing the acidic bile-induced overexpression of WNT5A (Fig. 1), a factor related to cancer-associated inflammation and epithelial-to-mesenchymal transition (111), indicating that NF-κB is able to mediate acidic bile-induced changes in hypopharyngeal cell-cell interactions.

In addition, COX-2 is regularly highly overexpressed during inflammatory and neoplastic processes (125) and is significantly overexpressed in acidic bile-exposed HM (24,25). In vivo application of NF-κB inhibitor significantly abrogated the acidic bile-induced overexpression of PTGS2 (24,25) (Fig. 1), further supporting the regulatory role of NF-κB in early inflammatory and cancer-related pathways, such as COX-2 (126).

In addition to the above, the PI3K/Akt pathway (127) is one of the most frequently activated pathways in head and neck cancer (128). The successful suppression of acidic bile-induced AKT1 overexpression using topical application of NF-κB inhibitor on murine HM suggested that NF-κB may mediate acidic bile-induced deregulations of PI3K/Akt downstream pathways (127) (Fig. 1).

In summary, the NF-κB pathway is a core central pathway that interacts with multiple upstream and downstream signaling pathways linked to the carcinogenic process. Using both a specific NF-κB inhibitor, such as BAY 11-7082, and a more pleiotropic NF-κB inhibitor, such as curcumin, it was documented that the acidic bile-induced deregulations of cancer-related genes or inflammatory factors are mediated by the NF-κB (Fig. 1). Furthermore, as curcumin is able to prevent the bile-related anti-apoptotic effect independently of the pH status, it may have an advantage over other NF-κB inhibitors. Of note, curcumin specifically reduced a lower percentage of analyzed NF-κB signaling genes compared to BAY 11-7082 (25 vs. 85%) (20,21). Thus, curcumin may confer a clinical advantage by preventing generalized suppression of NF-κB signaling, which is essential to the basic metabolic function of healthy mucosa and thereby reducing global toxicity (24,129).

miRNA molecules have also been important in both inflammation and cancer (130), modulating the expression of genes by causing target mRNA degradation or inhibiting their translation (131). The expression levels of certain miRNAs, such as ‘oncomiRs’ and ‘tumor suppressor’ miRNAs, have been indicated to be altered in tumor cells compared to normal cells (upregulated or downregulated), and capable of contributing to carcinogenesis, thereby demonstrating a significant regulatory role in the multistep process of cancer initiation and progression (132).

There is further evidence that miRNA markers, such as ‘oncomiR’ miR-21 and ‘tumor suppressor’ miR-375, have a crucial role in the initiation and progression of HNSCC (14,22,133,134). Arantes et al (135) reported the fundamental role of miR-21 as a biomarker in head and neck carcinogenesis, while miR-375 has been proposed as a predictive biomarker for early diagnosis in laryngeal cancer (136). In addition, interactions between NF-κB and miRNA markers, such as miR-21, miR-34a and miR-451a, have been importantly described by others in human cancer cells, including HNSCC (104,137,138). Specifically, a cluster of miRNA markers was reported to be associated with NF-κB that may be associated with the aggressive biological behavior of HNSCC (104).

Explorations by our group (14,15,19,14) revealed that acidic bile caused deregulations of the expression of oncogenic miRNA markers, previously associated with laryngopharyngeal cancer (133–141). Specifically, the ‘oncomiRs’ miR-21, miR-192 and miR-155, and the ‘tumor suppressors’ miR-34a, miR-375, miR-451a, miR-99a and miR-504 were indicated to be significantly altered in exposed HHCs and murine laryngopharyngeal mucosa (14,15,19,14) (Fig. 2). Of note, results from our group (14,15) highlighted the role of miR-21 and miR-375 deregulations in acidic bile-related neoplasia.

In addition, findings from HSCC tumor specimens from patients with documented bile reflux supported its strong association with upregulation of ‘oncomiR’ miR-21 and downregulation of ‘tumor suppressors’ miR-34a and particularly of miR-375, along with strong positivity for NF-κB (18) (Fig. 2). Bile reflux-associated HSCC was also associated with a marked reduction of ‘tumor suppressors’ miR-489, miR-504 and miR-99a compared with their adjacent non-pathologic tissue (18), suggesting their involvement in the onset and progression of HSCC (142,143). Finally, bile exposure-associated HSCC exhibited differential expression of miR-489 and miR-504, and particularly of miR-375, compared to bile-negative HSCC, which had significantly lower NF-κB levels. A previous view by our group (18) suggested that these miRNA markers may have a distinct role in biliary reflux-associated hypopharyngeal cancer.

Application of BAY 11-7082 in HHCs or murine HM was proven to prevent miRNA deregulations caused by acidic bile, providing insight into the interactions of transcriptionally active NF-κB with cancer-related miRNA markers (19,22,23,25). Specifically, it was demonstrated that BAY 11-7082 is able to effectively reverse the acidic bile-induced downregulation of ‘tumor suppressor’ miR-451a and miR-99a, and upregulation of ‘oncomiRs’ miR-21, miR-155 and miR-192 (19,22,23,25) (Fig. 2), which are considered important markers for poor prognosis in head and neck cancer or linked to gastroesophageal reflux (22,135,144–150). Other studies have also indicated that NF-κB has a direct regulatory effect on the expression of miR-21 and miR-155 through their binding promoters (151,152). Through in vitro and in vivo applications of BAY 11-7082 on hypopharyngeal cells and mucosa, respectively, a direct effect of acidic bile on the above miRNAs was demonstrated shortly after exposure (22,23,25). This observation proposed the use of these miRNAs as biomarkers of early neoplastic events in acidic bile-exposed HM, strongly supporting the role of NF-κB as a mediator in this process. In addition, topical in vivo application of BAY 11-7082 either before, after or simultaneous to acidic bile significantly inhibited the acidic bile-induced upregulation of ‘oncomiR’ miR-192 (19,25), previously associated with GERD (148), and downregulation of ‘tumor suppressor’ miR-504, a promising target for HSCC (142) (Fig. 2). This observation also suggested the utility of these miRNAs as biomarkers of early neoplastic events in acidic bile-exposed HM.

In summary, the above preclinical data provided evidence of the role of NF-κB as a regulator of miR-192, miR-21, miR-155, miR-451a, miR-375, miR-99a and miR-504 (Fig. 2), and proposed these miRNAs as potential therapeutic targets of bile-related mutagenic evolution in the HM.

Previous studies suggested that interactions between miRNAs and mRNA molecules may be NF-κB-dependent during carcinogenesis (141,153,154). Rokavec et al (153) proposed that miRNA molecules, such as miR-34a, interact with STAT3 in an NF-κB-dependent manner. According to Tili et al (154), permanent upregulation of miR-155 may mediate a prolonged inflammatory reaction leading to cancer.

It has been proposed that acidic bile-induced NF-κB activation, BCL2 overexpression and significant alterations of oncogenic EGFR, STAT3, TNF-α, IL6, IL1β and WNT5A may directly or indirectly interact with cancer-related miRNA markers, such as ‘oncomiRs’ miR-21 and miR-155, as well as ‘tumor suppressors’ miR-34a, miR-375 and miR-451a (14–16,141) (Fig. 3). These observations suggest that inflammatory episodes caused by acidic bile may be associated with downstream oncogenic pathways and may be effectively prevented by NF-κB inhibition.

Recent findings documenting the crucial role of NF-κB in bile reflux-related hypopharyngeal carcinogenesis, pose a challenge for researchers and clinicians on how to identify patients who are more likely to benefit from NF-κB inhibition treatment. An NF-κB-related gene expression signature associated with bile reflux in patients with HSCC (Figs. 1 and 2) may provide a better prediction for inhibition selection and also allow the development of diagnostic and prognostic biomarkers of NF-κB inhibition response.

Although NF-κB targeted therapy has been already applied in clinical practice with promising results in anti-cancer therapy (129,155,156), including HNSCC treatment (157,158), there is an increasing effort in the pharmaceutical industry to develop advanced NF-κB inhibitors (129,159,160). In particular, research focuses on the identification of IKK/NF-κB inhibitors for targeted therapy that would prevent NF-κB activation without affecting other signaling pathways and selectively affect malignant cells rather than normal cells. As one of the major adverse effects of using NF-κB inhibitors as anticancer drugs is their ability to impair innate immunity when applied in excessive and prolonged periods (129,159–161), research should also focus on both minimizing systemic toxicity and prevention of long-term immunosuppression. Thus, an ideal inhibition of NF-κB should be transient and reversible, as well as effective when combined with other anti-cancer treatments. Prior in vivo explorations strongly supported the effectiveness of models using intermittent and topical treatment as opposed to prolonged and systemic treatment. Specifically, the marked efficacy of short-term topical treatment with NF-κB inhibitors, such as curcumin (2 mg/kg/day) and BAY 11-7082 (6.25 mg/kg/day), in suppressing bile reflux-induced early preneoplastic changes in the hypopharynx (19,24,25), strongly supports the view that non-systemic and transient NF-κB inhibition may be clinically feasible in preventing bile-reflux-related oncogenic effects. This area of research is progressing rapidly; however, another adverse effect is of high importance prior to the targeting of IKK or NF-κB in the clinic. This refers to the enhanced production of IL-1β and related cytokines by inhibitors of NF-κB activation during bacterial infections (161), suggesting the short-term use of NF-κB inhibitors in combination with antibiotics.