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

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, IL1ß, 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.

The in vivo preventive and therapeutic properties of curcumin in bile reflux-related oncogenesis of the hypopharynx.

Bile at strongly acidic pH exerts a carcinogenic effect on the hypopharynx, based upon recent pre-clinical studies that support its role as an independent risk factor. We recently demonstrated in vitro that curcumin can prevent oncogenic profile of bile in human hypopharyngeal cells, by inhibiting NF-κB. We hypothesize that topically applied curcumin to the hypopharynx can similarly block early oncogenic molecular events of bile, by inhibiting NF-κB and consequently altering the expression of genes with oncogenic function. Using Mus musculus (C57Bl/6J), we topically applied curcumin (250 µmol/L; three times per day; 10 days) to the hypopharynx, 15 minutes before, 15 minutes after or in combination with bile acids (pH 3.0). Immunohistochemical analysis and qPCR revealed that topically applied curcumin either before, after or in combination with acidic bile exposure significantly suppressed its induced NF-κB activation in regenerating epithelial cells, and overexpression of Rela, Bcl2, Egfr, Stat3, Wnt5a, Tnf, Il6, Ptgs2. Akt1 was particularly inhibited by curcumin when applied simultaneously with bile. We provide novel evidence into the preventive and therapeutic properties of topically applied curcumin in acidic bile-induced early oncogenic molecular events in hypopharyngeal mucosa, by inhibiting NF-κB, and shaping future translational development of effective targeted therapies using topical non-pharmacologic inhibitors of NF-κB.

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

BACKGROUND: Recent preclinical explorations strongly support the tumorigenic potential of bile on laryngopharyngeal mucosa. Herein, the authors describe, in bile-related human hypopharyngeal squamous cell carcinoma (HSCC), NF-κB-related messenger RNA (mRNA) and microRNA (miRNA) oncogenic phenotypes similar to those previously identified in acidic bile-exposed premalignant murine hypopharyngeal mucosa. METHODS: In this pilot study, the authors included human HSCC specimens paired with their adjacent normal tissue (ANT) derived from 3 representative patients with documented biliary laryngopharyngeal reflux (bile[+]) compared with 5 control patients without signs of bile reflux disease (bile[-]). Immunohistochemical, quantitative polymerase chain reaction, and miRNA analyses were used to detect the levels of activated NF-κB and expression levels of STAT3, EGFR, BCL2, WNT5A, IL-6, IL-1B, ΔNp63, cREL, TNF-α, TP53, NOTCH1, NOTCH2, NOTCH3, miR-21, miR-155, miR-192, miR-34a, miR-375, miR-451a, miR-489, miR-504, and miR-99a. RESULTS: Bile(+) HSCC demonstrated an intense NF-κB activation accompanied by significant overexpression of RELA(p65), EGFR, STAT3, BCL-2, cREL, ΔNp63, WNT5A, IL-6, and IL1B; upregulation of oncomir miR-21; and downregulation of tumor suppressor miR-375 compared with their respective ANTs. Bile(+) HSCC demonstrated significantly higher mRNA levels of all the analyzed genes, particularly RELA(p65), IL-6, EGFR, and TNF-α compared with bile(-) tumors. The miR-21/miR-375 ratio, which previously has been linked to tumor aggressiveness, was found to be >260-fold and >30-fold higher, respectively, in bile(+) HSCCs compared with their ANTs and bile(-) tumors. CONCLUSIONS: Although limitations apply to this pilot study due to the small number of patients with HSCC, the novel findings suggest that a history of bile as a component of esophageal reflux disease may represent an independent risk factor for hypopharyngeal carcinogenesis.

Hypopharyngeal Diverticulum: Toward a Unified Understanding of Its Etiopathogenesis.

The etiopathogenesis of Zenker's diverticulum (ZD) remains uncertain. Increased hypopharyngeal pressure due to a hypertonic upper esophageal sphincter results in herniation proximal to the sphincter producing a pulsion diverticulum. Gastroesophageal reflux, which is known to induce shortening of the injured esophagus, likely plays a prominent role in ZD formation by pulling the cricopharyngeus muscle (CPM) away from the anchored inferior constrictor muscle. This creates a "weak zone" encouraging herniation. A bilobed diverticulum may originate from continuation of the fibrous midline raphe inferiorly to developmentally include part of the CPM. We report using laser endoscopy to divide the inter-diverticular septum followed by transmucosal cricopharyngeus myotomy. Presentation of a rare, bilobed diverticulum emphasizes the importance of the midline prevertebral raphe in anchoring the pharyngeal constrictor muscles with respect to the CPM. This lends support to the hypothesis that the etiopathogenesis of ZD is multifactorial while guiding us to a unified understanding of ZD.

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.

We previously demonstrated that acidic bile activates NF-κB, deregulating the expression of oncogenic miRNA markers, in pre-malignant murine laryngopharyngeal mucosa. Here, we hypothesize that the in vitro exposure of human hypopharyngeal cells to acidic bile deregulates cancer-related miRNA markers that can be reversed by BAY 11-7082, a pharmacologic NF-κB inhibitor. We repetitively exposed normal human hypopharyngeal primary cells and human hypopharyngeal keratinocytes to bile fluid (400 µmol/L), at pH 4.0 and 7.0, with/without BAY 11-7082 (20 µmol/L). We centred our study on the transcriptional activation of oncogenic miR-21, miR-155, miR-192, miR-34a, miR-375, miR-451a and NF-κB-related genes, previously linked to acidic bile-induced pre-neoplastic events. Our novel findings in vitro are consistent with our hypothesis demonstrating that BAY 11-7082 significantly reverses the acidic bile-induced oncogenic miRNA phenotype, in normal hypopharyngeal cells. BAY 11-7082 strongly inhibits the acidic bile-induced up-regulation of miR-192 and down-regulation of miR-451a and significantly decreases the miR-21/375 ratios, previously related to poor prognosis in hypopharyngeal cancer. This is the first in vitro report that NF-κB inhibition reverses acidic bile-induced miR-21, miR-155, miR-192, miR-34a, miR-375 and miR-451a deregulations in normal human hypopharyngeal cells, suggesting that acidic bile-induced events are directly or indirectly dependent on NF-κB signalling.

Curcumin prevents the bile reflux-induced NF-κB-related mRNA oncogenic phenotype, in human hypopharyngeal cells.

The presence of bile is not an uncommon finding in acidic oesophageal and extra-oesophageal refluxate, possibly affecting the hypopharyngeal mucosa and leading to neoplastic events. We recently demonstrated that acidic bile (pH ≤ 4.0) can induce NF-κB activation and oncogenic mRNA phenotype in normal hypopharyngeal cells and generate premalignant changes in treated hypopharyngeal mucosa. We hypothesize that curcumin, a dietary inhibitor of NF-κB, may effectively inhibit the acidic bile-induced cancer-related mRNA phenotype, in treated human hypopharyngeal primary cells (HHPC), supporting its potential preventive use in vivo. Luciferase assay, immunofluorescence, Western blot, qPCR and PCR microarray analysis were used to explore the effect of curcumin in HHPC exposed to bile (400 µmol/L) at acidic and neutral pH. Curcumin successfully inhibited the acidic bile-induced NF-κB signalling pathway (25% of analysed genes), and overexpression of NF-κB transcriptional factors, c-REL, RELA(p65), anti-apoptotic bcl-2, oncogenic TNF-α, EGFR, STAT3, WNT5A, ΔNp63 and cancer-related IL-6. Curcumin effectively reduced bile-induced bcl-2 overexpression at both acidic and neutral pH. Our novel findings suggest that, similar to pharmacologic NF-κB inhibitor, BAY 11-7082, curcumin can suppress acidic bile-induced oncogenic mRNA phenotype in hypopharyngeal cells, encouraging its future in vivo pre-clinical and clinical explorations in prevention of bile reflux-related pre-neoplastic events mediated by NF-κB.

Is partial laryngectomy safe forever?

OBJECTIVES: Over past decades, function-preserving surgery has been oncologically effective for specific types of laryngeal cancer. Although safe short-term swallow function has been reported, swallow safety during long-term survival has received less attention. The purpose of this report is to highlight potential consequences of late dysphagia and chronic aspiration after partial laryngectomy. METHODS: A retrospective case series was performed. The head and neck cancer database from Yale-New Haven Hospital identified 3 patients requiring completion laryngectomy due to chronic aspiration 11-15 years after oncologically successful partial laryngectomy. Demographics, presentation, treatment, and course are included. RESULTS: Primary treatment was open supraglottic laryngectomy with adjuvant radiation therapy (n=2) and vertical hemilaryngectomy (n=1). All patients demonstrated locoregional control and preservation of swallow function for >10 years postoperatively. Due to late dysphagia and chronic aspiration, two patients required completion laryngectomy 11 and 15 years postoperatively and the third patient will require this 14 years postoperatively. CONCLUSIONS: Successful swallowing after function-preserving laryngeal surgery may not last forever despite adequate control of cancer. Three patients presented with aspiration 11-15 years after partial laryngectomy and required definitive completion laryngectomy. This observation may affect preoperative counseling and consideration for longer post-operative follow-up. The data encourage a larger sample size.

Delayed platysma myocutaneous turnover flap for repair of pharyngocutaneous fistula.

INTRODUCTION: Pharyngocutaneous fistula (PCF) is a common and serious complication after total laryngectomy. Numerous surgical and non-surgical treatment approaches have been described. Here we describe a platysma myocutaneous turnover flap for repair of PCF. MATERIALS AND METHODS: Platysma myocutaneous turnover flap is described and two patients are used as examples. RESULTS: Repair was initially successful in both patients; however, one patient had recurrence of fistula after her cancer recurred at the stoma. DISCUSSION: Numerous surgical techniques have been described for repair of PCF. Here a turnover flap was used, a technique not previously described for this problem. The delay technique enhances the viability of the flap thought to be through numerous mechanisms. CONCLUSION: The platysma myocutaneous turnover flap is useful for closure of pharyngocutaneous fistula when non-operative measures have failed.

An Unusual Morphology of a Pleomorphic Adenoma.

We present a case of a deep lobe parotid pleomorphic adenoma extending into the parapharyngeal space via an unusual morphology. This is a single patient case report of an unusual morphology with an associated review of the literature. The patient successfully underwent right parotidectomy during which it was seen that the tumor was extending above rather than through the stylomandibular tunnel, an unusual morphology for a pleomorphic adenoma. Despite the classic teaching that deep lobe parotid tumors reach the parapharyngeal space by traveling through, or below, the stylomandibular tunnel, it is possible for tumors to occur outside this tunnel. Recognition that this unusual growth path, although rare, can occur is important when designing an appropriate surgical resection.

The impact of dairy consumption on salivary inoculum.

Quantitative levels of harmful oral microbes present following complex surgical excisions of head and neck cancer are important since wounds are often contaminated through direct connection to the oral cavity and its flora. This possibility is especially important in irradiated patients who have decreased protective salivary function. In addition, high oral microbial levels increase and intensify oral mucositis leading to significant morbidity in patients treated with radiation therapy. One previously untested surgical teaching to decrease the bacterial inoculum present in the oral cavity is to counsel patients against consuming otherwise nutritious dairy products, as they are thought to coat the oral cavity with rate-limiting nutrients vital for bacterial growth. This risk may extend to individuals with chronic laryngeal penetration or aspiration, since salivary bacterial load might represent a lethal threat in the presence of marginal pulmonary reserve. A crossover study using six healthy adult volunteers and six patients who had previously undergone radiation therapy to an oropharyngeal primary site was performed. Saliva samples were quantitatively cultured in both groups with and without the consumption of dairy products at 1-h and 5-h intervals. Analysis of quantitative cultures demonstrated that the consumption of dairy products had no influence on bacterial levels present in previously radiated subjects and nonirradiated controls. Additionally, the consumption of dairy did not affect the composition of microbes present. Due to the lack of changes in both quantity and composition of oral bacteria seen in this study, patients would not benefit from the avoidance of dairy products.

Revision Zenker diverticulum: laser versus stapler outcomes following initial endoscopic failure.

OBJECTIVES: We used a retrospective chart review to analyze revision endoscopic carbon dioxide (CO2) laser and staple repairs of recurrent Zenker diverticulum (ZD). METHODS: The medical records of patients with recurrent ZD after primary endoscopic repair were selected. The chart data included method of repair (CO2 laser or stapler), demographics (age and sex), defect size (in centimeters), preoperative and postoperative symptoms, and complications. Patients' dysphagia was graded on a modified Functional Oral Intake Scale from 1 to 4 (1 being normal intake and 4 being severely limited intake or gastrostomy tube dependence). Regurgitation was also graded on a 1-to-4 scale (1 being no regurgitation and 4 being aspiration). RESULTS: A total of 148 consecutive patients with ZD were treated with endoscopic repair between 2000 and 2010. Twelve of these patients had revisions after failed primary endoscopic management procedures, all done with the stapler. Eight revision surgeries were performed by CO2 laser, and 4 by stapler repair. No difference was noted in patient age or defect size (laser, 3.06-cm defects; stapler, 2.75-cm defects). The length of hospital stay and the time to oral intake for the patients who had a revision stapler procedure were significantly greater (p values of 0.029 and 0.009) than those for the patients in the primary stapler procedure group. Better postoperative regurgitation scores were noted for patients who had a CO2 laser procedure. CONCLUSIONS: Secondary endoscopic repair for ZD recurrence is an effective treatment method. Better symptom outcomes were observed with secondary CO2 laser repair than with stapler revision. Patients with revision stapling had longer hospital stays and a longer time to oral intake than did patients with primary staple repairs.

Neuromuscular basis for ventricular fold function.

OBJECTIVES: We sought to examine the neuromuscular basis for ventricular fold function. The primary function of the ventricular folds is to assist in the regulation of intra-abdominal and intrathoracic pressure. They also influence phonation in the setting of vocal fold paralysis or ventricular dysphonia, or after partial laryngectomy. The neuromuscular basis of true vocal fold function has been well studied; however, its neuromuscular correlates in the ventricular folds are ambiguous. The literature is unclear as to whether ventricular fold contraction is passive or active. The musculature and innervation responsible for this action also have not been well defined. The aim of this study was to provide clarity in regard to these mechanisms. METHODS: We examined a whole-organ section of a human larynx from a patient with unilateral vocal fold paralysis. The region of the ventricular folds was compared on both the paralyzed and normal sides. Electrophysiological examination was performed in a porcine model. The superior laryngeal nerve was stimulated, and recording electrodes in both ventricular folds measured the electrical activity. The recurrent laryngeal nerve was then severed, and the experiment was repeated. RESULTS: The histologic slides from the patient with unilateral vocal fold paralysis demonstrated atrophied ventricularis and thyroarytenoid muscles on the paralyzed side. On the unaffected side, these muscles were of normal size. The electrophysiological examination in the porcine model demonstrated findings consistent with innervation of the ventricularis muscle by the recurrent laryngeal nerve. An association of ventricularis muscle activity with ventricular fold contraction also was demonstrated. CONCLUSIONS: Ventricular fold adduction appears to be a result of ventricularis muscle contraction that is mediated by the recurrent laryngeal nerve.

Polymer nanoparticles containing tumor lysates as antigen delivery vehicles for dendritic cell-based antitumor immunotherapy.

Encapsulation of tumor-associated antigens in polymer nanoparticles (NP) is a promising approach to enhance efficiency of antigen delivery for anti-tumor vaccines. Head and neck squamous carcinoma (HNSCC) cell lines were initially used to generate tumor-associated antigens (TAA)-containing poly (lactic-co-glycolic acid) (PLGA) NP; encapsulation efficiency and release kinetics were profiled. Findings were adopted to entrap fresh tumor lysate from five patients with advanced HNSCC. To test the hypothesis that NP enhance antigen presentation, dendritic cell (DC) produced from patient blood monocyte precursors were loaded with either the un-encapsulated or NP-encapsulated versions of tumor lysates. These were used to stimulate freshly-isolated autologous CD8+ T cells. In four of five patients, anti-tumor CD8+ T cells showed significantly increased immunostimulatory IFN-γ (p=0.071) or decreased immmunoinhibitory IL-10 production (p=0.0004) associated with NP-mediated antigen delivery. The observations represent an enabling step in the production of clinically-translatable, inexpensive, highly-efficient, and personalized polymer-based immunotherapy for solid organ malignancies. FROM THE CLINICAL EDITOR: Enhancing the antigen presentation may be a viable approach to increase the efficiency of tumor cell directed cytotoxicity via immune mechanisms. This study presents an example for this using head and neck cancer cell lines and nanotechnology-based encapsulated antigen presentation to dendritic cells. The observed CD8+ T-cell response was significantly enhanced. This method may pave the way to a highly efficient cancer cell elimination method with minimal to no toxicity.

Effect of altered core body temperature on glottal closing force.

OBJECTIVES: A basic function of the larynx is to provide sphincteric protection of the lower airway, initiated by a brain stem-mediated glottal closure reflex. Glottal closing force is defined as the measured pressure generated between the vocal folds during glottal closure. One of the factors thought to affect the glottal closure reflex is a variation in core body temperature. METHODS: Four adult male Yorkshire pigs were used in this study. The subjects were studied under control conditions (37 degreesC), hyperthermic conditions (38 degrees C to 41 degrees C), and hypothermic conditions (36 degrees C to 34 degrees C). RESULTS: We demonstrated that the glottal closing force increased significantly with an increase in core body temperature and also decreased significantly with decreased core body temperature. These results are supported by neurophysiological changes demonstrated by other studies in pups and adult dogs in response to altered core body temperatures. The mechanism for these responses is thought to reside centrally, rather than in the peripheral nervous system. CONCLUSIONS: We hope that a better understanding of these aspects of glottal closure will alter the care of many patients with postanesthesia hypothermia and many sedated inmates and will also further enhance preventive measures needed to decrease the incidence of sudden infant death syndrome in overheated or febrile infants.

Response of cricopharyngeus muscle to esophageal stimulation by mechanical distension and acid and bile perfusion.

OBJECTIVES: The aim of this study was to identify the response of the cricopharyngeus muscle (CPM) to esophageal stimulation by intraluminal mechanical distension and intraluminal acid and bile perfusion. METHODS: In 3 adult pigs, electromyographic (EMG) activity of the CPM was recorded at baseline and after esophageal stimulation at 3 levels: proximal, middle, and distal. The esophagus was stimulated with 20-mL balloon distension and intraluminal perfusion of 40 mL 0.1N hydrochloric acid, taurocholic acid (pH 1.5), and chenodeoxycholic acid (pH 7.4) at the rate of 40 mL/min. The EMG spike density was defined as peak-to-peak spikes greater than 10 microV averaged over 10-ms intervals. RESULTS: In all 3 animals, the spike density at baseline was 0. The spike densities increased after proximal and middle distensions to 15.2 +/- 1.5 and 5.1 +/- 1.2 spikes per 10 ms, respectively. No change in CPM EMG activity occurred after distal distension. The spike density following intraluminal perfusion with hydrochloric acid at the distal level was 10.1 +/- 1.1 spikes per 10 ms. No significant change in CPM EMG activity occurred after acid perfusion at the middle and proximal levels. No change in CPM EMG activity occurred after intraluminal esophageal perfusion with either taurocholic acid or chenodeoxycholic acid. CONCLUSIONS: Proximal esophageal distension, as well as distal intraluminal acid perfusion, appeared to be important mechanisms in generation of CPM activity. Bile acids, on the other hand, failed to evoke such CPM activity. The data suggest that transpyloric refluxate may not be significant enough to evoke the CPM protective sphincteric function, thereby placing supraesophageal structures at risk of bile injury.

Weakly Acidic Bile Is a Risk Factor for Hypopharyngeal Carcinogenesis Evidenced by DNA Damage, Antiapoptotic Function, and Premalignant Dysplastic Lesions In Vivo.

BACKGROUND: There is recent in vivo discovery documenting the carcinogenic effect of bile at strongly acidic pH 3.0 in hypopharynx, while in vitro data demonstrate that weakly acidic bile (pH 5.5) has a similar oncogenic effect. Because esophageal refluxate often occurs at pH > 4.0, here we aim to determine whether weakly acidic bile is also carcinogenic in vivo. METHODS: Using 32 wild-type mice C57B16J, we performed topical application of conjugated primary bile acids with or without unconjugated secondary bile acid, deoxycholic acid (DCA), at pH 5.5 and controls, to hypopharyngeal mucosa (HM) twice per day, for 15 weeks. RESULTS: Chronic exposure of HM to weakly acidic bile, promotes premalignant lesions with microinvasion, preceded by significant DNA/RNA oxidative damage, γH2AX (double strand breaks), NF-κB and p53 expression, overexpression of Bcl-2, and elevated Tnf and Il6 mRNAs, compared to controls. Weakly acidic bile, without DCA, upregulates the "oncomirs", miR-21 and miR-155. The presence of DCA promotes Egfr, Wnt5a, and Rela overexpression, and a significant downregulation of "tumor suppressor" miR-451a. CONCLUSION: Weakly acidic pH increases the risk of bile-related hypopharyngeal neoplasia. The oncogenic properties of biliary esophageal reflux on the epithelium of the upper aerodigestive tract may not be fully modified when antacid therapy is applied. We believe that due to bile content, alternative therapeutic strategies using specific inhibitors of relevant molecular pathways or receptors may be considered in patients with refractory GERD.

The temporal effects of topical NF-κB inhibition, in the in vivo prevention of bile-related oncogenic mRNA and miRNA phenotypes in murine hypopharyngeal mucosa: a preclinical model.

Supraesophageal bile reflux at strongly acidic pH can cause hypopharyngeal squamous cell cancer, through activation of the oncogenic NF-κB-related pathway. We hypothesize that topical pre- or post-application of pharmacologic NF-κB inhibitor, BAY 11-7082 (0.25 µmol), on murine (C57BL/6J) HM (twice a day for 10 days) can effectively inhibit acidic bile (10 mmol/l; pH 3.0) induced oncogenic molecular events, similar to prior in vitro findings. We demonstrate that the administration of BAY 11-7082, either before or after acidic bile, eliminates NF-κB activation, prevents overexpression of Bcl2, Rela, Stat3, Egfr, Tnf, Wnt5a, and deregulations of miR-192, miR-504, linked to bile reflux-related hypopharyngeal cancer. Pre- but not post-application of NF-κB inhibitor, significantly blocks overexpression of Il6 and prostaglandin H synthases 2 (Ptgs2), and reverses miR-21, miR-155, miR-99a phenotypes, supporting its early bile-induced pro-inflammatory effect. We thus provide novel evidence that topical administration of a pharmacological NF-κB inhibitor, either before or after acidic bile exposure can successfully prevent its oncogenic mRNA and miRNA phenotypes in HM, supporting the observation that co-administration of NF-κB inhibitor may not be essential in preventing early bile-related oncogenic events and encouraging a capacity for further translational exploration.

The Progressive Mutagenic Effects of Acidic Bile Refluxate in Hypopharyngeal Squamous Cell Carcinogenesis: New Insights.

Cancers of the laryngopharynx represent the most devastating of the head and neck malignancies and additional risk factors are now epidemiologically linked to this disease. Using an in vivo model (Mus musculus C57Bl/6J), we provide novel evidence that acidic bile (pH 3.0) progressively promotes invasive cancer in the hypopharynx. Malignant lesions are characterized by increasing: i) oxidative DNA-damage, ii) γH2AX expression, iii) NF-κB activation, and iv) p53 expression. Histopathological changes observed in murine hypopharyngeal mucosa exposed to acidic bile were preceded by the overexpression of Tnf, Il6, Bcl2, Egfr, Rela, Stat3, and the deregulation of miR-21, miR-155, miR-192, miR-34a, miR-375, and miR-451a. This is the first study to document that acidic bile is carcinogenic in the upper aerodigestive tract. We showed that oxidative DNA-damage produced by acidic bile in combination with NF-κB-related anti-apoptotic deregulation further supports the underlying two-hit hypothesized mechanism. Just as importantly, we reproduced the role of several biomarkers of progression that served as valuable indicators of early neoplasia in our experimental model. These findings provide a sound basis for proposing translational studies in humans by exposing new opportunities for early detection and prevention.

The role of bile reflux and its related NF-κB activated pathway in progression of hypopharyngeal squamous cell cancer.

BACKGROUND: Prognosis for hypopharyngeal cancer is usually poor, and recurrence is common. Identifying new factors or related mechanisms that promote its progression may have clinical implications. Although, recent studies support bile reflux in hypopharyngeal carcinogenesis, it remains to be explored how bile and its related NF-κB activated pathway may further affects its progression in already established hypopharyngeal cancer. METHODS: Hypopharyngeal squamous cell carcinoma (HSCC) cell lines, FaDu and UMSCC11A, both negative for HPV, were repetitively exposed to bile acids (400 µM) at variable pH points (4.0, 5.5 and 7.0). Immunofluorescence, western blotting, luciferase assay, and qPCR were used to detect NF-κB activation, bcl-2 overexpression and gene expression. RESULTS: Bile at strongly acidic pH (4.0) potentiated the activation of NF-κB and its related mRNA phenotype in HSCC cells. IL-6, TNF-α, and BCL2 were found among the highest overexpressed genes as was previously found in HSCCs excised from patients with documented biliary reflux. An enhanced transcriptional activity of EGFR, RELA, STAT3, and WNT5Α and higher survival rates were observed in HSCC cells exposed to acidic bile compared to those exposed to bile at weakly acidic or neutral pH. CONCLUSION: Our novel findings support the observation that bile reflux has the potential for actively influencing the progression of hypopharyngeal cancer, mediated by NF-κB. In patients with hypopharyngeal cancer and known gastroesophageal reflux disease, antacid therapy may exert a role in furthering control of disease recurrence and progression.

The Effect of NNK, A Tobacco Smoke Carcinogen, on the miRNA and Mismatch DNA Repair Expression Profiles in Lung and Head and Neck Squamous Cancer Cells.

Tobacco smoking is a common risk factor for lung cancer and head and neck cancer. Molecular changes such as deregulation of miRNA expression have been linked to tobacco smoking in both types of cancer. Dysfunction of the Mismatch DNA repair (MMR) mechanism has also been associated with a poor prognosis of these cancers, while a cross-talk between specific miRNAs and MMR genes has been previously proposed. We hypothesized that exposure of lung and head and neck squamous cancer cells (NCI and FaDu, respectively) to tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is capable of altering the expression of MSH2 and MLH1, key MMR components, by promoting specific miRNA deregulation. We found that either a low (1 µM) or high (2 µM) dose of NNK induced significant upregulation of "oncomirs" miR-21 and miR-155 and downregulation of "tumor suppressor" miR-422a, as well as the reduction of MMR protein and mRNA expression, in NCI and FaDu, compared to controls. Inhibition of miR-21 restored the NNK-induced reduced MSH2 phenotype in both NCI and FaDu, indicating that miR-21 might contribute to MSH2 regulation. Finally, NNK exposure increased NCI and FaDu survival, promoting cancer cell progression. We provide novel findings that deregulated miR-21, miR-155, and miR-422a and MMR gene expression patterns may be valuable biomarkers for lung and head and neck squamous cell cancer progression in smokers.