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.

Tobacco smoking has long been considered a causative risk factor for laryngopharyngeal or hypopharyngeal cancer (1). Components of tobacco smoke, such as N-nitrosamines [4-(N-Methyl-N-Nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosodiethylamine (NDEA)] are potential carcinogens (2, 3). Previous in vivo studies in rodents have specifically linked these components to esophageal and upper aerodigestive tract cancer (3–5). In particular, an in vivo study with Syrian hamsters linked NNK to laryngeal tumorigenesis (5). Although their carcinogenicity is mainly related to the induction of TP53 mutations (2), N-nitrosamines can also activate NFκB, promoting cell proliferation (6, 7).

Bile reflux disease, a variant of gastroesophageal reflux disease (GERD), has also been considered a risk factor that exerts an independent carcinogenic effect related to the chronic effect of bile refluxate on hypopharyngeal mucosa (8, 9). GERD prevalence has significantly increased in the United States over the last decade (10). In addition, more than half (50%–86%) of patients with GERD present with bile acids in their refluxate (11–14). The carcinogenic potential of bile acids has been previously discussed in gastrointestinal tract, supporting the clinical association of bile reflux with Barrett esophagus (9, 12, 13), a known precursor of esophageal adenocarcinoma. It is also well known that the components of bile refluxate may include conjugated bile acids and unconjugated secondary bile acid, deoxycholic acid (DCA), which is one of the most noxious of these components (15). DCA can be identified in esophageal aspirates (11, 13, 16) and is considered an important contributor to the development of Barrett esophagus and esophageal adenocarcinoma, by constitutive activation of NFκB (17, 18). The previously established in vivo murine model from our team recently documented bile reflux as an independent causative risk factor of hypopharyngeal cancer (9, 19–22), characterized by significant activation of NFκB and its related oncogenic signaling, particularly in the presence of DCA (22). In addition, clinical findings from our pilot study showed that human papilloma virus (HPV)-negative hypopharyngeal squamous cell carcinoma (HSCC) from patients with a history of tobacco smoking and bile reflux presented a characteristic NFκB-related molecular profile, which was distinguished by patients with smoking history without bile reflux disease (23).

In a previous study of others, it was also shown that the combined effect of duodenal fluid with NDEA can develop epidermoid carcinomas in rodent esophagus (24). Yet, there is no direct evidence of the carcinogenic effect of tobacco smoke N-nitrosamines, such as NNK and NDEA, and their combination with bile acids in the hypopharynx, as well as the role of NFκB in this process. Here we hypothesize that the chronic exposure to a mixture of tobacco smoke components (TSC), such as NNK and NDEA, would provide direct evidence of its neoplastic effect in the hypopharynx in vivo, while the combined in vivo effect of TSC along with bile acid, DCA, would accelerate the neoplastic process, by enhancing the activation of NFκB and related oncogenic phenotype, and thus increasing the risk of malignancy.

Constitutive activation of NFκB has been observed in head and neck squamous cell carcinomas (HNSCC; refs. 25, 26). Specifically, HNSCC exhibits abundant NFκB activation, and several studies indicate that NFκB is upregulated from premalignant lesions to invasive cancer (25, 26) with subsequent transcriptional activation of genes that are implicated in a variety of antiapoptotic or cell proliferation signaling pathways, such as BCL2 (23, 27), STAT3 (27, 28), TNFα (28), various cancer-related cytokines, like IL6 (29), and epithelial-to-mesenchymal transition mediator WNT5A (30). NFκB activation has also been associated with the oncogenic pathway PI3K/mTOR, while cross-talk between NFκB and downstream pathways of oncogenic EGFR, such as STAT3, has been identified in head and neck cancer (29). In addition, it has been previously demonstrated by our team that inhibition of NFκB, using pharmacologic drugs or dietary NFκB inhibitors, can prevent early molecular neoplastic events caused by bile acids in the hypopharynx (31–37), such as transcriptional activation of BCL2, IL6, STAT3, TNFα, in this process.

The assessment of direct in vivo evidence of how chronic diseases, such as GERD with bile acid components, may contribute to carcinogenesis of the upper aerodigestive tract, when present with other risk factors, such as tobacco smoke N-nitrosamines, could be considered novel and promote further clinical investigation in this process. In addition, by understanding that a complex network of molecular events may be involved in this process, data from this study may further help to define the precise role of NFκB and its related gene expression profile in tobacco smoking with bile reflux–induced neoplasia, as well as their potential use as promising biomarkers in high-risk groups for developing hypopharyngeal cancer.

An in vivo model of wild mice C57Bl/6J mice (Mus Musculus) was performed to identify the chronic exposure to TSC and its combination with the local application of bile acid, DCA, on hypopharyngeal mucosa, to induce (i) histopathologic changes, (ii) strong nuclear positivity of NFκB, and (iii) transcriptional activation of its related oncogenic phenotype, compared with controls. In our previous study, we showed that DCA alone could produce premalignant lesions of murine hypopharyngeal mucosa as early as 6–7 weeks of treatment (19). In this study, we investigated in double time (12–14 weeks) the effect of DCA alone or in combination with TSC.

Animal model

A previously established in vivo model of wild mice C57Bl6J mice (Mus Musculus) was performed. Animals were treated for a period of 12–14 weeks. Forty mice (20 females and 20 males) 4-week-old age were randomly divided into three experimental groups and two controls (n = 8 animals in each group; see Table 1). After acclimatization of mice (2–3 days), all treatment exposures were performed in parallel, following procedures (Table 1) and intraprocedural or postprocedural monitoring plan according to the approved protocol [11039, by Institutional Animal Care and Use Committee (IACUC); Yale University, New Haven, CT]. The mice were obtained from Jackson Laboratory (Jax mice).

Table 1.

Experimental and control treated groups of wt-C57BL/6J.

Experimentaland control treated groupsN-NitrosaminesDCA
 (NNK+NDEA)  
Saline — — 
TSC — 
DCA — 
TSC+DCA 
Untreated control — — 
Experimentaland control treated groupsN-NitrosaminesDCA
 (NNK+NDEA)  
Saline — — 
TSC — 
DCA — 
TSC+DCA 
Untreated control — — 

*, component of the experimental or control fluids.

Intraprocedural monitoring plan

Animals were monitored for signs of distress whether fluid would come out of the mouth/nose (e.g., animal coughing, choking, vomiting). The procedure was discontinued immediately if any sign of distress, as described above, and following the animal was put in prone position and monitor for respiratory distress for up to 15 minutes. If necessary, veterinarians (VCS) were contacted to advise and/or we will euthanize the animal.

Postprocedural monitoring plan

The animal was kept upright, after treatment, for 1–2 minutes, to prevent possible aspiration into the pulmonary tree. The animal was returned to the cage and monitored for 15 minutes and at least once for 12–24 hours, after treatment, for signs of fluid coming into the lungs. If signs of fluid coming into the lungs (the animal coughs, chokes or fluid is seeing coming through the mouth or the nose) or respiratory distress (labored breathing that is indicated by increased respiratory rate and/or effort, that could be accompanied by a strong abdominal component to breathing), the animal was removed from the procedure immediately and monitored for up to 6 hours. If an animal was found to be in pain and/or distress, VCS was consulted and/or the animal was euthanized.

Chronic exposure to bile acid (DCA)

Murine hypopharyngeal mucosa was topically treated two times per day, 5 days per week for 12–14 weeks, using a plastic feeding tube (20–25 g), by unconjugated secondary bile acid DCA (0.28 mmol/L; Alfa Aesar) at concentrations previously described in patients with GERD (11, 13, 38), in 0.15 mL buffered saline, as described previously (22).

Chronic exposure to TSCs

Previous studies have shown concentrations of N-nitrosamines, 4-(N-Methyl-N-Nitrosamino)-1-(3-pyridyl)-1-butanone NNK) up to 2,950 ng/cig and NDEA up to 28 ng/cig in tobacco smoke mainstream (39–41). Therefore, a 20 pack-year smoker can be exposed approximately to 5 mg/kg NNK and 0.05 mg/kg NDEA in total. On the basis of these data and previous in vivo models (3, 4, 42), we used a mixture of N-nitrosamines, NNK (0.2 mmol/L; Santa Cruz Biotechnology) and NDEA (0.004 mmol/L) that was added and administered in drinking bottles as the sole drinking fluid 5 days per week for 12–14 weeks.

Combined chronic exposure to TSCs and bile acid (TSC+DCA)

Topical application of bile acid, DCA, on hypopharyngeal mucosa using a plastic feeding tube was performed as described above twice per day for 12–14 weeks in animals exposed to TSC (Table 1).

Controls

(i) Saline-treated control: Hypopharyngeal mucosa was repetitively treated twice per day, for 12–14 weeks, with 0.15 mL of buffered saline (ii) Untreated control: a group of untreated animals (15 weeks old) was used as a negative control.

At the end of experimental procedures, animals were euthanized, using carbon dioxide (new IACUC policy), and kept on ice. Hypopharyngeal tissue fragments (HTF) from 4 animals (2 females and 2 males) of each group were placed immediately into 10% neutral buffered formalin (Thermo Fisher Scientific), and submitted for embedding in paraffin blocks (Yale Pathology Facilities), for subsequent histopathologic and IHC analyses. The remaining four HTF from each experimental and control group (2 females and 2 males) were immersed in RNA stabilization solution (RNAlater, Life Technologies) and kept at −80°C, for RNA isolation.

Tissue examination histology

Normal hypopharyngeal mucosa was characterized by stratified keratinizing squamous epithelium: Histologic staining was performed using hematoxylin and eosin (H&E) staining to reveal histologic changes in hypopharyngeal mucosa exposed to TSC and its combination with bile acid compared with controls. A total of 3 µmol/L tissue sections of formalin-fixed and paraffin-embedded HTF, of experimental and control groups (2 females and 2 males), were stained for H&E, following standard protocols (19–22). At least two H&E-stained tissue sections were examined from each specimen (eight tissue sections per group; four sections from each gender per group) and examined by light microscopy. Images were captured and analyzed by Aperio CS2, Image Scope software (Leica microsystems). Histopathologic alterations were assessed according to established criteria (43, 44) and laboratory mouse histology (45), as described previously (19–22).

IHC analysis

IHC analysis was performed for phospho-NFκB (p-NFκB; p65 S536) protein to observe NFκB activation and its nuclear localization in hypopharyngeal tissue sections from all experimental and control specimens (21, 22). Four to 5 µmol/L serial sections of formalin-fixed and paraffin-embedded tissues, from each experimental and control group, were stained for p-NFκB (p65 S536), following standard protocols (19–22). At least two tissue sections from each specimen (eight tissue sections per group; four tissue sections per gender per group) including those presented histopathologic lesions were stained using dilutions of 1:100 of anti-p-NFκB p65 antibody (27.Ser 536; mouse monoclonal, Santa Cruz Biotechnology, Inc.), 1:100 of anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP; m-IgG1 BP-HRP; Santa Cruz Biotechnology, Inc.); and peroxidase substrate (3–3′ diaminobenzidine tetrachloride; Santa Cruz Biotechnology, Inc.). Positive controls, such as human tonsil, which showed nuclear p-NFκB staining of cells in germinal and nongerminal centers, and nontemplate negative control (sections treated with mouse secondary antibody omitting the primary antibody) were used according to the manufacturer's instructions. Slides were examined using a light Leica microscope and images were captured using Aperio CS2. The images were analyzed by Image Scope software (Leica microsystems), which generates an algorithm(s) illustrating the p-NFκB staining in mucosal and cellular compartments. A multiple comparison t test (means ± SD; by multiple t tests; by GraphPad Prism 7.0) was used to compare strong positivity of p-NFκB in basal/parabasal/suprabasal cells of murine hypopharyngeal mucosa and in cancerous cells invading the submucosa, among experimental groups and controls, as well as between malignant and nonaffected mucosal sites of specimens with histopathologic alterations. Strong positivity (Nsr) is assigned as the ratio of strong positive (Nsp) relative to the number of weakly positive (Nwp) plus the number of positive (Np) plus the number of strongly positive (Nsp) (Nsr = Nsp/(Nwp+Np+Nsr; positivity defined as the number of positive nuclei relative to the total number of nuclei). IHC analysis scores were derived from at least two independent images per tissue section (sixteen images in total per group; eight images per gender per group).

qPCR analysis

qPCR analysis was performed to determine the mRNA expression levels of Il6, Tnf, Egfr, Rela, Wnt5a, Bcl2, Stat3, and Mtor genes, in hypopharyngeal mucosa under its chronic exposure to TSC and its combination with bile acid, compared with controls. We selected these genes because they previously appeared to be overexpressed in bile reflux–related hypopharyngeal cancer and linked to NFκB (19, 21–23, 31–37). Total RNA was isolated from murine HTF of experimental and control groups (four tissue specimens per group of 2 males and 2 females), using an RNeasy mini kit (Qiagen). RNA quality was determined by absorption ratios at 260/280 nm (≥2.0) and concentration ratios by absorption at 260 nm, using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription to cDNA was performed using a Whole Transcriptome kit (Qiagen), following the manufacturer's instructions. The qPCR analysis (Bio-Rad real-time thermal cycler CFX96TM) was performed using specific primers for mouse genome (QuantiTect primers assay, Qiagen), as presented in Supplementary Table S1, and iQ SYBR Green Supermix (Bio-Rad). Gapdh gene was used as a reference control gene. qPCR assays were performed in 96-well plates and each sample was assayed in triplicate. The PCR conditions included an initial denaturation and enzyme activation step at 95°C for 3 minutes, followed by two-step optimizing cycling of denaturation at 95°C for 10 seconds and annealing and extension at 55°C for 40 seconds, for 40 cycles, according to the manufacturer's instructions (19, 21). Melting curve analysis was performed after cycling steps (55–95°C; 0.5°C increment). Relative expression ratios for each specific gene (target gene/Gapdh) were estimated by CFX96 (Bio-Rad) software, for all experimental and control mRNAs.

Statistical analysis

Statistic analysis was performed using GraphPad Prism 7.0 software and t-test analysis (multiple comparisons by Holm-Sidak) to reveal statistically significant changes of NFκB positivity levels or mRNA levels of the Il6, Tnf, Egfr, Rela, Wnt5a, Bcl2, Stat3, and Mtor genes, among different exposures. χ2 test was used to reveal significant difference of the developed histopathologic changes among different exposures (P < 0.05). Spearman correlation was performed to estimate the correlation coefficient (i) between p-NFκB positivity and mRNA levels of each specific gene or (ii) among the mRNA levels of the analyzed genes, in the studied groups (P < 0.05).

Data availability statement

Data are contained within the article or Supplementary Data.

In vivo effect of TSCs and their combination with bile acid exposure in developing HSCC

Microscopic examination of hypopharyngeal mucosa long-term exposure to TSCs, N-nitrosamines (NNK-NDEA), revealed premalignant changes, such as severe dysplasia or in situ carcinoma, compared with saline-treated hypopharyngeal mucosa presented normal histologic figure (Fig. 1). Examination of hypopharyngeal mucosa exposed to bile acid, DCA, alone, induced premalignant changes, such as dysplasia (Fig. 1), while hypopharyngeal mucosa exposed to TSC along with bile acid treatment demonstrated a progression to malignancy that was documented by figures of invasive cancer (Fig. 1; Supplementary Fig. S1). Saline-treated hypopharyngeal mucosa revealed normal histologic structure (Fig. 1), similarly to untreated hypopharyngeal mucosa (Supplementary Fig. S2).

Figure 1.

The in vivo effect of TSC and their combination with bile acid exposure in developing HSCC. Histologic staining (H&E) of murine hypopharyngeal mucosa (HM) of C57Bl/6J mice after 12–14 weeks of exposure to TSC, N-nitrosamines, NNK-NDEA), bile acid (DCA) or their combination (TSC+DCA) and saline-treated control. A, Normal HM: keratinized stratified squamous epithelium/single layer of basal cells. B, Mild/moderate dysplastic HM: thickness/full thickness of stratified epithelium, nuclear hyperchromatic or pleiomorphic basal cells expanding in the stratum spinosum, and/or loss of large cells polarity. C, Severe dysplasia or in situ carcinoma: Architectural changes extend into the upper levels of the mucosa with mitotic features evident throughout the depth of the mucosa, while cellular atypia and submucosal invasion by basal cells maintaining full-thickness nuclear hyperchromatism without surface maturation are marked. D, Invasive carcinoma: Malignant phenotype characterized by architectural changes, nests of atypical/immature cells with nuclear pleomorphism, and mitotic figures without apparent stratification or keratinization in the submucosa moderate differentiated squamous cell carcinoma.

Figure 1.

The in vivo effect of TSC and their combination with bile acid exposure in developing HSCC. Histologic staining (H&E) of murine hypopharyngeal mucosa (HM) of C57Bl/6J mice after 12–14 weeks of exposure to TSC, N-nitrosamines, NNK-NDEA), bile acid (DCA) or their combination (TSC+DCA) and saline-treated control. A, Normal HM: keratinized stratified squamous epithelium/single layer of basal cells. B, Mild/moderate dysplastic HM: thickness/full thickness of stratified epithelium, nuclear hyperchromatic or pleiomorphic basal cells expanding in the stratum spinosum, and/or loss of large cells polarity. C, Severe dysplasia or in situ carcinoma: Architectural changes extend into the upper levels of the mucosa with mitotic features evident throughout the depth of the mucosa, while cellular atypia and submucosal invasion by basal cells maintaining full-thickness nuclear hyperchromatism without surface maturation are marked. D, Invasive carcinoma: Malignant phenotype characterized by architectural changes, nests of atypical/immature cells with nuclear pleomorphism, and mitotic figures without apparent stratification or keratinization in the submucosa moderate differentiated squamous cell carcinoma.

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Summarizing the above observations, histologic examination of hypopharyngeal mucosa exposed to TSC and their combination with bile acid treatment documented that bile promotes TSC-induced tumorigenic effect. Table 2 presents the survival data, the total number of mice analyzed and the prevalence of those with premalignant/malignant histopathologic alterations in hypopharyngeal mucosa among different exposures. As shown in Table 2, of the 24 animals treated in the different control and experimental groups, only 1 animal demonstrated signs of distress and died during the third week of treatment, possible due to aspiration. No signs of behavioral change or distress were noted in the rest of the animals of this study. Overall, the number of mice developed malignant lesions were significantly higher in TSC plus DCA treated group compared with TSC alone, DCA alone, or saline-treated control (χ2 test, P = 0.0023, by GraphPad Prism 7.0). Finally, no difference was noticed between males and females, in line to our previous in vivo studies (21, 22, 32, 36, 37).

Table 2.

Survival data, total C57Bl/6J mice analyzed, and prevalence of those with premalignant/malignant lesions of the hypopharyngeal epithelium under its chronic exposure to TSC, N-nitrosamines (NNK-NDEA), along with exposure to bile acid (DCA).

TSCBile acidTSC plus bile acid
Saline(NNK-NDEA)(DCA)(NNK-NDEA+DCA)
C57Bl/6J Mice 100% 100% 100% 87.5% 
% (surv./total) (8/8) (8/8) (8/8) (7/8) 
Total analyzed 100% 100% 100% 100% 
% (surv./total) (4/4) (4/4) (4/4) (4/4) 
Hyperplasia/Dysplasia 0% 100% 100% 100% 
% (observ./surv.) (0/4) (4/4) (4/4) (4/4) 
Malignant lesion 0% 0% 0% 100% 
% (observ./surv.) (0/4) (0/4) (0/4) (4/4) 
TSCBile acidTSC plus bile acid
Saline(NNK-NDEA)(DCA)(NNK-NDEA+DCA)
C57Bl/6J Mice 100% 100% 100% 87.5% 
% (surv./total) (8/8) (8/8) (8/8) (7/8) 
Total analyzed 100% 100% 100% 100% 
% (surv./total) (4/4) (4/4) (4/4) (4/4) 
Hyperplasia/Dysplasia 0% 100% 100% 100% 
% (observ./surv.) (0/4) (4/4) (4/4) (4/4) 
Malignant lesion 0% 0% 0% 100% 
% (observ./surv.) (0/4) (0/4) (0/4) (4/4) 

In vivo effect of TSCs and their combination with bile acid exposure in inducing strong activation of NFκB in HSCC

IHC analysis demonstrated strong p-NFκB nuclear staining of tumor specimens from hypopharyngeal mucosa exposed to TSC in combination with bile acid treatment supporting a constitutive activation of NFκB. Specifically, IHC, documented that exposure to TSC, N-nitrosamines (NNK-NDEA), caused NFκB activation, while their combination with bile acid, DCA, treatment accelerates the effect of TSC by producing a strong activation of NFκB, particularly at sites of precancerous lesions or invasive cancer (Fig. 2A; Supplementary Fig. S3). On a contrary, it is shown that saline-treated control (Fig. 2A) and untreated normal mucosa (Supplementary Fig. S2) did not show nuclear staining for NFκB and thus considered negative for NFκB activation.

Figure 2.

The in vivo effect of TSC and their combination with bile acid treatment in inducing strong activation of NFκB in hypopharyngeal SCC. A, IHC analysis (brown staining) and image analysis algorithm(s) for p-NFκB (p65 S536) of murine hypopharyngeal mucosa (HM) of C57Bl6J mice after 12 weeks of exposure to tobacco smoke components (TSC; NNK-NDEA), bile acid (DCA), or their combination (TSC+DCA) and corresponding saline-treated control. In the image analysis algorithm(s), red indicates positive nuclear staining of p-NFκB, orange indicates intense positive cytoplasmic staining of p-NFκB, yellow indicates weak cytoplasmic staining of p-NFκB, and blue indicates negative p-NFκB staining. Images were captured using Aperio CS2 and analyzed using Image Scope software (Leica Microsystems) to generate the algorithm(s) illustrating mucosal and cellular compartments demonstrating p-NFκB staining. Saline-treated control (normal HM): sporadic and weak cytoplasmic staining in a few basal or parabasal cells; DCA-treated HM (Dysplastic HM): strong nuclear and intense cytoplasmic staining in cells of basal and suprabasal layers; TSC-treated-HM (Severe dysplastic/in situ carcinoma): strong nuclear and intense cytoplasmic staining in cells of basal and suprabasal layers or projections of the epithelium; TSC+DCA treated-HM (invasive HSCC): Strong nuclear and intense cytoplasmic staining through the mucosal thickness and the invasive carcinoma sites. B, Quantification of p-NFκB nuclear expression (strong positivity) in murine HM exposed TSC and their combination with DCA, compared with saline-treated control, TSC or DCA alone. Positive nuclear p-NFκB (p65 S536) levels are assigned as the ratio of strong nuclear positivity (Nsp/Nwp+Np+Nsp, Np: strong positivity; Nwp: weakly positive; Np: nuclear positive; positivity indicates the number of positive nuclei/number of total nuclei). Scores were derived from five independent images per tissue section (at least two tissue sections per group; t test with multiple comparisons using the Holm-Sidak method: *, P < 0.05; **, P < 0.005; and ***, P < 0.0005; statistical analysis was performed using GraphPad Prism software version 7.0).

Figure 2.

The in vivo effect of TSC and their combination with bile acid treatment in inducing strong activation of NFκB in hypopharyngeal SCC. A, IHC analysis (brown staining) and image analysis algorithm(s) for p-NFκB (p65 S536) of murine hypopharyngeal mucosa (HM) of C57Bl6J mice after 12 weeks of exposure to tobacco smoke components (TSC; NNK-NDEA), bile acid (DCA), or their combination (TSC+DCA) and corresponding saline-treated control. In the image analysis algorithm(s), red indicates positive nuclear staining of p-NFκB, orange indicates intense positive cytoplasmic staining of p-NFκB, yellow indicates weak cytoplasmic staining of p-NFκB, and blue indicates negative p-NFκB staining. Images were captured using Aperio CS2 and analyzed using Image Scope software (Leica Microsystems) to generate the algorithm(s) illustrating mucosal and cellular compartments demonstrating p-NFκB staining. Saline-treated control (normal HM): sporadic and weak cytoplasmic staining in a few basal or parabasal cells; DCA-treated HM (Dysplastic HM): strong nuclear and intense cytoplasmic staining in cells of basal and suprabasal layers; TSC-treated-HM (Severe dysplastic/in situ carcinoma): strong nuclear and intense cytoplasmic staining in cells of basal and suprabasal layers or projections of the epithelium; TSC+DCA treated-HM (invasive HSCC): Strong nuclear and intense cytoplasmic staining through the mucosal thickness and the invasive carcinoma sites. B, Quantification of p-NFκB nuclear expression (strong positivity) in murine HM exposed TSC and their combination with DCA, compared with saline-treated control, TSC or DCA alone. Positive nuclear p-NFκB (p65 S536) levels are assigned as the ratio of strong nuclear positivity (Nsp/Nwp+Np+Nsp, Np: strong positivity; Nwp: weakly positive; Np: nuclear positive; positivity indicates the number of positive nuclei/number of total nuclei). Scores were derived from five independent images per tissue section (at least two tissue sections per group; t test with multiple comparisons using the Holm-Sidak method: *, P < 0.05; **, P < 0.005; and ***, P < 0.0005; statistical analysis was performed using GraphPad Prism software version 7.0).

Close modal

Scoring of nuclear positivity for p-NFκB (p65 S536), by ImageScope software, revealed that long-term exposure of hypopharyngeal mucosa to TSC, bile acid, or their combined treatment produced significantly higher levels of strong nuclear positivity of p-NFκB compared with saline-treated control (Fig. 2B). However, our analysis revealed that exposure of hypopharyngeal mucosa to TSC in combination with DCA treatment induced statistically significant higher levels of activated NFκB (strong nuclear positivity) compared with TSC alone (Fig. 2B; P< 0.05, t-test; means ± SD; multiple comparisons by Holm-Sidak). We also observed that the adjacent non-pathologic epithelium showed a notably weak nuclear and cytoplasmic staining for p-NFκB compared with cancerous lesions caused by the TSC with DCA effect (Supplementary Fig. S3). This finding may conclude that NFκB is primarily activated in cancerous lesion and is relevant to TSC with DCA-induced progression to malignancy.

In vivo effect of TSC and their combination with bile acid exposure in inducing transcriptional activation of NFκB-related genes with oncogenic profile

Gene expression analysis by qPCR revealed that the long-term exposure of hypopharyngeal mucosa to TSC (NNK-NDEA) alone produced significant transcriptional activation of mRNA oncogenic phenotype, compared with saline-treated controls (Fig. 3A). However, TSC exposure, in combination with bile acid, DCA, treatment, accelerated the TSC effect, causing the highest transcriptional levels of the analyzed genes (Fig. 3A).

Figure 3.

TSC exposure in combination with bile acid treatment induced a progressive transcriptional activation of the NFκB-related oncogenic phenotype. A, Transcriptional levels of each analyzed NFκB-related gene (mRNA levels of each target gene were normalized to Gapdh by qPCR analysis) after chronic exposure to TSC (NNK-NDEA), DCA, and their combined exposure versus saline-treated control. The mRNA levels caused by TSC exposure in combination with bile acid (DCA) treatment were compared with saline-treated control, TSC or DCA alone (−/−/−). (*, P < 0.01; **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001; t test; multiple comparisons using Holm–Sidak; GraphPad Prism 7.0; data obtained from four analyzed samples; Graph Pad Prism software 7.0). B, mRNA oncogenic phenotype (fold change of mRNAs) in TSC or DCA induced premalignant HM versus saline-treated control. C, mRNA oncogenic phenotype (fold change of mRNAs) in TSC with DCA induced HSCC versus saline-treated control. D, Progressive mRNA changes during the neoplastic process in HM exposed to TSC along with DCA relative to TSC alone.

Figure 3.

TSC exposure in combination with bile acid treatment induced a progressive transcriptional activation of the NFκB-related oncogenic phenotype. A, Transcriptional levels of each analyzed NFκB-related gene (mRNA levels of each target gene were normalized to Gapdh by qPCR analysis) after chronic exposure to TSC (NNK-NDEA), DCA, and their combined exposure versus saline-treated control. The mRNA levels caused by TSC exposure in combination with bile acid (DCA) treatment were compared with saline-treated control, TSC or DCA alone (−/−/−). (*, P < 0.01; **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001; t test; multiple comparisons using Holm–Sidak; GraphPad Prism 7.0; data obtained from four analyzed samples; Graph Pad Prism software 7.0). B, mRNA oncogenic phenotype (fold change of mRNAs) in TSC or DCA induced premalignant HM versus saline-treated control. C, mRNA oncogenic phenotype (fold change of mRNAs) in TSC with DCA induced HSCC versus saline-treated control. D, Progressive mRNA changes during the neoplastic process in HM exposed to TSC along with DCA relative to TSC alone.

Close modal

Specifically, hypopharyngeal mucosa exposed to TSC alone produced significantly higher mRNA levels of Il6, Tnf, Stat3, Egfr, Wnt5a, Rela, Mtor, and Bcl2, compared with saline-treated control. However, hypopharyngeal mucosa exposed to TSC along with DCA treatment produced a profound activation of Il6, TNf, and Stat3, followed by Egfr, Wnt5a, Rela, Mtor, and Bcl2, with significantly higher mRNA levels, compared with saline-treated control or TSC alone (Fig. 3A; Supplementary Table S2).

In addition, although, DCA alone–treated hypopharyngeal mucosa induced a significant overexpression of all the analyzed genes (Fig. 3A), the combination of TSC with DCA treatments, induced significantly higher mRNA levels of Il6, Tnf, Stat3, Rela, Mtor, and Bcl2, compared with DCA alone (Fig. 3A).

Panels B and C in Fig. 3, present the mRNA oncogenic phenotypes caused by exposure to TSC or DCA alone, and TSC along with DCA, compared with saline-treated controls, respectively. The panel D in Fig. 3 also shows the relative overexpression of the analyzed genes between combined exposure to TSC along with bile acid, relative to TSC alone, which documented high relative mRNA expression ratios.

Correlation between preclinical and clinical mRNA oncogenic phenotypes detected in HSCC associated with the combined chronic effect of TSC with bile reflux

Table 3 presents the molecular phenotypes from current in vivo and previous clinical findings from hypopharyngeal carcinoma associated with exposure to TSC in combination with bile acid. Clinical data were obtained from previous analysis of HSCC tumors relative to their adjacent non-pathologic hypopharyngeal mucosa in patients who were tobacco smokers and were diagnosed with bile reflux (23). Similar to our current in vivo findings, bile reflux–related HSCC demonstrated an extensive NFκB positivity and gene expression phenotype when compared with their normal controls (23). As shown in Table 3, IL6, TNFα, STAT3, WNT5α, EGFR, RELA, and BCL2 were found to be similarly overexpressed in both murine and human specimens exposed to TSC in combination with bile, compared with their normal controls. IL6 was commonly found in high overexpression (>more than 100 times) in both in vivo and clinical specimens, followed by TNFα (>50 times), STAT3, WNT5α, EGFR (>10 times) and RELA and BCL2 (>2 times).

Table 3.

Common mRNA aphenotypes identified in tobacco smoke and bile reflux–associated HSCC by preclinical and clinical studies.

Common mRNA phenotypes of tobacco smoke and bile associated HSCCa
Preclinical databClinical datac
IL6 +++++ ++++ 
TNFα +++ +++ 
STAT3 ++ +++ 
EGFR ++ ++ 
WNT5A ++ ++ 
RELA + ++++ 
MTOR + NP 
BCL2 + ++ 
Common mRNA phenotypes of tobacco smoke and bile associated HSCCa
Preclinical databClinical datac
IL6 +++++ ++++ 
TNFα +++ +++ 
STAT3 ++ +++ 
EGFR ++ ++ 
WNT5A ++ ++ 
RELA + ++++ 
MTOR + NP 
BCL2 + ++ 

Note: +, 2–10; ++, 10–50; +++, 50–100; ++++, 100–500; +++++, >500.

Abbreviation: NP, not performed.

amRNA normalized expression ratios between tobacco smoke and bile-associated HSCC versus normal hypopharyngeal mucosa.

bmRNA oncogenic phenotype induced by TSC along with bile acid exposure in murine HSCC.

cmRNA oncogenic phenotype identified in HSCC from tobacco smoker patients with bile in their refluxate.

Correlation between NFκB and mRNA alterations caused by TSC in combination with bile acid exposures

Strong linear correlations were found between p-NFκB positivity and mRNA levels of NFκB-related genes, in treated hypopharyngeal mucosa. Specifically, a Spearman nonparametric test revealed statistically significant linear correlations between TSC with bile-induced p-NFκB strong positivity and Il6, Tnf, Stat3, Egfr, Wnt5a, Mtor, or Bcl2 mRNAs (r = 1, P = 0.0417; by Spearman one-tailed test). In addition, the Spearman test revealed statistically significant linear correlations among Il6, Tnf, Stat3, Egfr, Wnt5a, Mtor, and Bcl2 mRNAs (r = 1, P = 0.0417; by Spearman two-tailed test), supporting interactions among TSC plus bile-induced NFκB-related gene expression profiles and again supporting NFκB as a key mediator in laryngopharyngeal carcinogenesis.

Tobacco smoking is historically associated with HPV-negative hypopharyngeal cancer (46). TSCs, such as N-nitrosamines, are potential carcinogens (2, 3) and have been shown to induce activation of NFκB (6, 7) and are therefore linked to this process. However, the direct carcinogenic effect of TSC in the hypopharynx and its combined effect with bile reflux remained unexplored. We recently documented the role of bile reflux as an independent risk factor for hypopharyngeal cancer (9), as well as NFκB as a mediator of bile reflux-induced oncogenic molecular events (9, 31–37). Although physical and chemical stress may be associated with NFκB activation, multiple previous studies in our laboratory showed that bile specifically and selectively can promote NFκB activation and cause premalignant and malignant lesions compared with acid at pH 4.0 or 5.0 or saline at 7.0 or other stress factors, such as highly concentrated glucose, which could not cause any histologic change when applied topically with the same technique (9, 19, 21, 22). Moreover, none of physical and chemical stress factors or acidic pepsin were found capable of inducing activation of NFκB-related genes with inflammatory or oncogenic function previously linked to HNSCC (9, 19). We have also recently shown that DCA alone or in a mixture of bile acids can produce a significant activation of NFκB and promote premalignant lesions in the hypopharyngeal mucosa of mice, as early as 6–7 weeks of treatment (19–22). Here, we performed an experimental approach involving double time (12–14 weeks) of chronic exposure of the hypopharynx to tobacco smoke N-nitrosamines, NNK and NDEA, in combination with bile acid, DCA, to investigate whether bile refluxate can affect the oncogenic process in TSC-exposed hypopharyngeal mucosa. We used DCA at a concentration of 0.28 mmol/L, based on values previously described in patients with GERD (13, 38) and a mixture of NNK and NDEA at concentrations of 0.2 and 0.004 mmol/L, based on values previously measured in the mainstream of tobacco smoke (39–41) and in vivo models (3, 4, 42). Because the anatomic site of focus was the hypopharynx, we administrated TSC orally to maximize its effect on the hypopharyngeal epithelium. In parallel, to mimic the episodes of reflux into the hypopharynx, we performed topical intermittent hypopharyngeal mucosa treatment with bile acid, using a previously established in vivo model (19–22). Because a variety of risk factors can promote reflux events, bile refluxate can be spontaneously detected in patients with laryngopharyngeal reflux (LPR) throughout the day, two or three times a day. Thus, we used a model, which included a random exposure of murine hypopharyngeal mucosa twice daily, to be biologically similar to what the patient experiences (8, 35). Because the current study revealed significant pathologic changes after 14 weeks of treatment especially in the groups treated with TSC plus DCA, our data encourage large-scale studies including a large animal sample and multiple timepoints of this process to reveal the natural history of carcinogenesis as well as occult gender-related differences.

Our novel in vivo data provide direct evidence that chronic exposure of the hypopharyngeal epithelium to tobacco smoke in combination with bile refluxate carries a high risk of developing invasive cancer, possibly due to enhanced NFκB activation and oncogenic signaling. Although we recognize that other factors may contribute to this process, we focused our study on the oncogenic profile associated with NFκB, which was previously associated with biliary reflux-induced hypopharyngeal carcinogenesis (9).

Our findings document that chronic exposure of hypopharyngeal mucosa to TSC can induce premalignant lesions, activation of NFκB, and significant overexpression of its related oncogenic mRNA phenotype compared with controls, supporting the tumorigenic effect of TSC. When intermittent exposure to bile was combined with TSC exposure, it caused a notable progression to invasive squamous cell carcinoma, as well as enhanced activation of NFκB and profound transcriptional activation of its related oncogenic profile (Fig. 3B). The fact that the treated groups (TSC, DCA, or TSC plus DCA) compared with controls not only demonstrated a strong activation of NFκB but also histologic and molecular changes indicating progression to malignancy further support the possible central role of NFκB in this process. Specifically, the abundant activation of NFκB, particularly at malignant sites, and the profound overexpression of Il6, Tnf, Stat3, followed by Egfr, Wnt5a, Rela, Mtor, and Bcl2, support the theory that bile refluxate can accelerate the malignant transformation of the hypopharyngeal epithelium when chronically exposed to TSC, promoting a constitutive strong activation of NFκB and related oncogenic pathways.

Interestingly, data from previous analysis of HSCC tumors extracted from tobacco smokers previously diagnosed with bile reflux (23) showed a similar molecular phenotype to our current in vivo data (Table 3). The most substantial similarity was associated with the extensive NFκB positivity shown in bile reflux–related HSCC compared with bile-negative tumors, especially in the core of these tumors. This observation further supports the role of NFκB in promoting cancer of the hypopharyngeal mucosa exposed to tobacco smoke in combination with bile refluxate. In addition, the gene expression panel characterized the TSC plus bile acid effect, in vivo, as shown in Fig. 3C, is consistent with previous findings from human HSCC specimens associated with bile reflux (ref. 23; Table 3). Genes, such as Il6, Tnf, Egfr, Rela, Wnt5a, Bcl2, and Stat3, which have been linked to NFκB oncogenic activity, usually appear to be overexpressed in bile reflux–related hypopharyngeal cancer, as evidenced by clinical analyses or preclinical in vivo explorations, like the one presented here.

Current research focuses on identifying biomarkers for early detection and prognosis of head and neck cancer (47, 48). Therefore, new evidence of molecular profile characterizing a group of individuals at high risk for HSCC may contribute to preventive and potential therapeutic applications. On the basis of the data here, NFκB and its related factors IL6, STAT3, TNFα, EGFR, BCL2, and WNT5A remain the main factors in hypopharyngeal carcinogenesis, particularly characterizing the bile acid contribution to this process. Strong activation of NFκB, accompanied by increased expression levels of these genes, could be assigned to a high-risk phenotype associated with tobacco smoking in combination with bile reflux, which may substantially differ from those associated with tobacco smoking or bile reflux alone. On the basis of our findings, documentation of the high-risk phenotype in tobacco smokers with bile acid in their refluxate may prove useful biomarkers in upper aerodigestive tract neoplasia by broad clinical studies.

The strong linear correlation found between nuclear NFκB positivity and Il6, Stat3, Tnf, Egfr, Mtor, and Wnt5a mRNA levels may support possible interactions between NFκB and downstream oncogenic signaling pathways of these factors in hypopharyngeal carcinogenesis caused by the combined effect of TSC along with bile acid, and this is consistent with previous clinical data (ref. 23; Table 3). In particular, the observed significant linear correlation between nuclear NFκB and Mtor mRNA levels may indicate crosstalk between the NFκB and AKT1/mTOR oncogenic pathway, possibly mediated by other oncogenic factors, such as EGFR and its related downstream signaling, playing a central role in head and neck cancer (29). In addition, previous studies from our team documented NFκB-mediated STAT3 activation (31–37), while recent data supported the important role of STAT3 in bile reflux–associated hypopharyngeal carcinogenesis in maintaining the continuous production of inflammatory and cancer-related molecules, such as Il6 and Tnf (49). Here, a strong linear correlation was observed between nuclear positivity of NFκB and Stat3, Il6, and Tnf mRNAs, which are the most overexpressed in hypopharyngeal mucosa exposed to TSC in combination with bile acid, highlighting the contributory role of STAT3 signaling in this carcinogenic process. Overall, our data recognize the importance of NFκB in the onset and progression of head and neck malignancies by interacting with a complex network of other cancer-related transcriptional factors, cytokines, and growth factors, including EGFR/Ras/RAF/ MAPK, Akt/PI3K/mTOR, ΙΚΚ/NFκB, STAT3, and Wnt/β-catenin (25–30). Therefore, our data encourage the extension of this study using wide-analysis techniques, such as whole transcriptome and/or proteomics assays that along with histologic data may reveal all NFκB-related genes and multiple pathways in HSCC and other upper aerodigestive tract cancers associated with the combined effect of TSC with bile reflux or multiple hazardous cancer-related factors.

It is worth mentioning that this study presents in vivo evidence of the carcinogenic effect of N-nitrosamines, which are present in the mainstream of tobacco smoke, in the hypopharynx. However, it is of equal importance to document for the first time the tremendous carcinogenic combination of tobacco components, NNK and NDEA, which are also found in the environment and dietary products, with bile-refluxate in the upper aerodigestive tract. Although there are several studies alarming the poisoning and carcinogenic potential of N-nitrosamines in nontobacco substances (50), here we can recognize that chronic epithelial exposure to N-nitrosamines, regardless of the relationship of patients with bile reflux, carries a considerable risk of developing cancer.

It is widely accepted that prevention and prognosis of aggressive cancer such as hypopharyngeal remain of high priority. Our investigation elucidates how chronic diseases, such as mixed gastroesophageal reflux with bile components that may remain silent and undetectable, can greatly contribute to head and neck carcinogenesis when associated with other risk factors such as tobacco smoke–related nitrosamines. Until the present day, the percentage of patients with LPR who simultaneously experience bile acid reflux remains to be fully determined. However, it has been recently shown that the percentage of patients with bile contents in their refluxate is higher than previously considered (11, 12, 14). Furthermore, it has been suggested that refractory GERD in most patients may be due to bile content in their refluxate (9). We believe that this new evidence will advance further investigation into the incidence of hypopharyngeal cancer and other upper aerodigestive tract cancers or precancerous conditions, including Barrett esophagus, in tobacco users with reflux-related disorders in clinical practice, possibly including the assessment of bile acid components in their refluxate. In addition, the data of our in vivo model encourage its application in large-scale studies for wide molecular analyses to reveal the nature of carcinogenesis in the hypopharynx and other sites of the upper aerodigestive tract associated with inflammation, as well as for preclinical studies and targeted therapy.

We conclude that tobacco smoke nitrosamines when combined with bile acid significantly increase the risk of hypopharyngeal cancer. Our data suggest that the noxious combination of tobacco smoke nitrosamines with bile may also contribute to the development of other known premalignant or malignant changes in the upper aerodigestive tract, such as Barrett esophagus and esophageal cancer. Our findings also support that the bile-induced activation of NFκB is a critical event that promotes carcinogenesis in hypopharyngeal mucosa exposed to TSCs. Finally, our data suggest NFκB, as well as IL6, TNFα, STAT3, EGFR, and WNT5A as promising biomarkers and potential therapeutic targets in this process.

No disclosures were reported.

D.P. Vageli: Conceptualization, resources, data curation, software, formal analysis, supervision, validation, investigation, visualization, methodology, writing–original draft, funding acquisition, project administration, writing–review and editing. P.G. Doukas: Conceptualization, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. S.G. Doukas: Conceptualization, data curation, software, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing, this author and the first author contributed equally. A. Tsatsakis: Data curation, validation, writing–review and editing. B.L. Judson: Resources, data curation, funding acquisition, project administration, writing–review and editing.

Research reported in this publication was partially supported by the Virginia Alden Wright Fund to D.P. Vageli.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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