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Hypopharyngeal cancer is a distinct type of malignant head and neck tumor, which exhibits low sensitivity to anti-cancer drugs. The importance of developing methods for reducing chemotherapy resistance, and improving and enhancing prognosis has previously been emphasized and is considered a challenge for effective clinical treatment of hypopharyngeal cancer. The current study investigated the effects of co‑expression of inhibitor of growth protein 4 (ING4) and P53, a tumor suppressor gene, on chemosensitivity to cisplatin in human hypopharyngeal cancer xenografts in vivo, and the potential molecular mechanisms involved. A tumor model was established by injecting athymic nude mice with FADU human hypopharyngeal cancer cells. Five days after intratumoral and peritumoral injections of an empty adenoviral vector (Ad), Ad‑ING4‑P53, cisplatin, or a combination of Ad‑ING4‑P53 and cisplatin (Ad‑ING4‑P53 + cisplatin) every other day for 5 days, the mice were euthanized and their tumors, livers, and kidneys were removed. The tumor weights were used to calculate the inhibition rate, and the expression levels of ING4 and P53 were detected by reverse transcription‑polymerase chain reaction. Additionally, apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry determined the levels ING4, P53, B‑cell lymphoma‑2 (Bcl‑2) and Bcl‑2 associated X protein (Bax) protein expression. The results demonstrated increased expression of ING4 and P53 in the Ad‑ING4‑P53 groups compared with PBS and Ad groups, indicating successful introduction of the genes into the tumor cells. Notably, the Ad‑ING4‑P53 + cisplatin group exhibited a higher inhibition rate compared with the four other groups. The results of immunohistochemistry analysis demonstrated that Bax expression was increased and Bcl‑2 was decreased in the Ad‑ING4‑P53 + cisplatin group. This suggested that the enhanced cisplatin chemosensitivity with Ad-ING4-P53 gene therapy in hypopharyngeal cancer xenografts may be associated with apoptosis induction through upregulation of Bax expression and downregulation of Bcl‑2. The results of the present study indicated that gene therapy combined with cisplatin treatment may be a promising treatment for human hypopharyngeal cancer.

Background: Selenium-binding protein 1 (SELENBP1) expression is reduced markedly in many types of cancers and low SELENBP1 expression levels are associated with poor patient prognosis., Methods: SELENBP1 gene expression in head and neck squamous cell carcinoma (HNSCC) was analyzed with GEO dataset and characteristics of SELENBP1 expression in paraffin embedded tissue were summarized. Expression of SELENBP1 in nasopharyngeal carcinoma (NPC), laryngeal cancer, oral cancer, tonsil cancer, hypopharyngeal cancer and normal tissues were detected using immunohistochemistry, at last, 99 NPC patients were followed up more than 5 years and were analyzed the prognostic significance of SELENBP1., Results: Analysis of GEO dataset concluded that SELENBP1 gene expression in HNSCC was lower than that in normal tissue (P < 0.01), but there was no significant difference of SELENBP1 gene expression in different T-stage and N-stage (P > 0.05). Analysis of pathological section concluded that SELENBP1 in the majority of HNSCC is low expression and in cancer nests is lower expression than surrounding normal tissue, even associated with the malignant degree of tumor. Further study indicated the low SELENBP1 expression group of patients with NPC accompanied by poor overall survival and has significantly different comparing with the high expression group., Conclusion: SELENBP1 expression was down-regulated in HNSCC, but has no associated with T-stage and N-stage of tumor. Low expression of SELENBP1 in patients with NPC has poor over survival, so SELENBP1 could be a novel biomarker for predicting prognosis., Competing Interests: The authors report no conflicts of interest.

Livin, a member of the human inhibitor of apoptosis protein (IAP) family, is expressed at high levels in various human cancer tissues and may have prognostic significance. The aim of the present study was to evaluate the effect of Livin on tumor cell behavior and oncogenic signaling pathways in human hypopharyngeal squamous cell carcinoma (HSCC). Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to determine the mRNA and protein expression levels, respectively. A cell proliferation assay and cell cycle analysis were used to assess the functional effects of small interfering RNA‑mediated Livin knockdown. Livin was overexpressed in fresh HSCC tissues, compared with the adjacent normal mucosa. Livin knockdown led to significantly reduced cell proliferation and cell cycle arrest in the G1 phase of the human HSCC cells. The expression levels of c‑myc, cyclin D1, cyclin D3, cyclin‑dependent kinase (CDK)4 and CDK6 were decreased. The phosphorylation levels of extracellular signal‑regulated kinase 1/2, p38, c‑Jun N-terminal kinase and Akt were also decreased by Livin knockdown in the HSCC cells. Taken together, the results of the present study suggested that Livin may enhance tumorigenesis by modulating the mitogen‑activated/Akt signaling pathways in human HSCC.

Long noncoding RNAs (lncRNAs) are associated with the development, progression, and prognosis of human cancers. However, the clinical significance and biological function of lncRNAs in hypopharyngeal squamous cell carcinoma (HSCC) remain largely unknown. We characterized the novel lncRNA AB209630 in vivo and in vitro. First, using qRT-PCR, we evaluated whether AB209630 levels differ between HSCC tissues/cell lines and adjacent normal tissues/cell lines. We then assessed whether AB209630 expression levels stimulate or inhibit proliferation, invasion, apoptosis, and metastasis in vitro. Finally, we investigated whether AB209630 levels in tumor tissues were associated with survival outcomes. Our results demonstrated that AB209630 levels were markedly lower in HSCC tissues and cells than in normal tissues and cells, and increased expression of AB209630 level significantly inhibited growth, metastasis, and invasion and stimulated apoptosis in vitro. In addition, patients with decreased expression of AB209630 had a significantly poorer prognosis than those with high AB209630 expression. These data suggest that increased expression of AB209630 might either stimulate or inhibit biological activities involved in HSCC development, indicating a potential application of AB209630 in future treatment for this disease. This study suggest that AB209630 functions as a tumor suppressor in HSCC, and its decreased expression may help predict a poor prognostic outcome of HSCC. Our future work will focus on the mechanisms of whether and how AB209630 as a tumor suppressor gene is involved in HSCC development.

Th0 cells differentiate into Th1 or Th2 depending on multiple transcription factors acting on specific time points to regulate gene expression. Th17 cells, a subset of IL-17-producing T cells distinct from Th1 or Th2 cells, have been described as key players in inflammation and autoimmune diseases as well as cancer development. In the present study, 53 patients with hypopharyngeal cancer were included. The expression levels of Th1-, Th2- and Th17-associated cytokines in hypopharyngeal cancer tissues and pericarcinoma tissues were detected. The relationship between Th1, Th2, or Th17 infiltration and metastasis was studied. Our results showed that the mRNA and protein expressions of Th1 cytokines were lower, while the expressions of Th2 and Th17 cytokines were higher in tumor tissues, and the intensity of expression was strengthened with clinical stage increasing. Cancer tissues had higher level expressions of Th2 and Th17 cytokines than that of pericarcinoma tissues. From the above data, we speculated that high expressions of Th2- and Th17-associated cytokines in hypopharyngeal carcinoma may contribute to cancer development and metastasis.

Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common head and neck cancers with high invasiveness and poor prognosis. To identify targeted therapeutics against metastasis, a better understanding of the regulation of HSCC cell invasion is needed. In recent years, G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through phosphorylation of the activated form of G protein coupled receptors (GPCRs). However, there is little information regarding GRKs expression in HSCC. In the present study, we examined GRK6 expression in HSCC and also assessed the possible cause of its aberrant expression, as well as its clinical significance. Real-time quantitative PCR (qPCR) and western blotting were performed to analyze the expression of GRK6 in HSCC tissues and corresponding non-malignant tissues. Subsequently, paired HSCC and corresponding non-malignant tissues were evaluated for the methylation status of GRK6 gene promoter using methylation-specific PCR (MSP). Furthermore, we investigated the methylation status and the clinicopathological significance of GRK6 in 45 paired HSCC and corresponding non-malignant tissues. The suppression of GRK6 in hypopharyngeal cell line FaDu by GRK6-shRNA lentivirus transfection was utilized to detect the role of GRK6 in hypopharyngeal cancer progression. Our results showed that the expression of GRK6 mRNA and protein was significantly lower in HSCC than in corresponding adjacent non-tumor tissues, and this downregulation was found to be in accordance with aberrant methylation of the gene. Hypermethylation of the gene was observed in 77.8% (35/45) of the HSCC tissues, while it was found in only 42.2% (19/45) of the corresponding non-malignant tissues. GRK6 methylation was related to depth of tumor invasion and TNM stage. Upon treatment with 5-aza-2'-deoxycytidine, GRK6 expression was upregulated in FaDu cells, and cell invasion was signinficantly inhibited. Furthermore, the suppression of GRK6 by shRNA transfection enhanced FaDu cells invasion. Our results indicate that the aberrant methylation of GRK6 gene promoter may underlie its downregulation in HSCC, and may play an important role in the metastasis of HSCC.

Hypopharyngeal squamous cell carcinoma is a common type of malignant tumor among head and neck squamous cell carcinomas (HNSCCs). Heavy smoking and/or drinking is associated with the development of HNSCC. However, HNSCC also occurs in individuals that do not drink or smoke, possibly due to infection with the human papilloma virus (HPV). HPV-16 has been shown to be closely associated with the occurrence of several types of cancers. However, its role in hypopharyngeal squamous cell carcinoma remains unclear. In the present study, we investigated the effects of HPV-16 on hypopharyngeal squamous cell carcinoma and FaDu cells. Lentiviral vectors were used to establish FaDu cells that expressed the E6 and E7 proteins of HPV-16. We used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays and western blotting to detect and determine the levels of expression for E6-E7 mRNAs and proteins. Cell Counting Kit-8 (CCK-8) assays, enzyme-linked immunosorbent assays (ELISA), Transwell assays, and flow cytometry were used to assess the effects of HPV-16 E6-E7 on the proliferation, invasion, metastasis and apoptosis of FaDu cells. Expression of microRNAs was analyzed by qRT-PCR. We found that the expression levels of HPV-16 E6-E7 were increased in FaDu cells transfected with the lentiviral vector compared with that observed in the control cells. In addition, the rates of apoptosis were decreased in the transfected cells, while proliferation was increased. The average numbers of cells penetrating the Matrigel were significantly higher than those for the controls. We detected miR-363 and miR-15a, and their expression levels were significantly increased in the HPV-16-positive patients and in FaDu cells expressing HPV-16 E6-E7. We found that HPV-16 E6-E7 appeared to inhibit apoptosis, and to increase cell proliferation, invasion and metastasis. Furthermore, miR-363 and miR-15a were overexpressed in the hypopharyngeal squamous cell carcinoma samples infected with HPV-16, and in FaDu cells stably expressing HPV-16 E6-E7. These findings may provide a new clue of the mechanisms involved in the pathogenesis of HPV-16-positive hypopharyngeal squamous cell carcinoma.

Although the expression of tumor necrosis factor receptors (TNFRs) has been associated with clinicopathologic features of some other cancers, their roles in hypopharyngeal squamous cell carcinoma (HPSCC) have not been documented. Forty-five HPSCC specimens were analyzed for the expression of TNFR1 and TNFR2 and its relationship with clinicopathologic factors. Interaction between the two receptors and its effects on TNF-α was investigated by neutralizing TNFR1 and upregulation of TNFR2. The results indicated that, in HPSCC specimens, the expression of TNFR1 but not TNFR2 is associated with clinical staging, T stage, cervical lymph node metastasis, and histologic grade in HPSCC. In Fadu cells, when conjugating with its receptors, TNF-α mediates proliferation effects, and neutralizing TNFR1 and/or upregulating TNFR2 evokes proliferation-inhibiting and apoptosis-inducing effects and potentiates cisplatin (DDP)-induced growth inhibition and apoptosis induction. In conclusion, interaction of TNFR1 with TNFR2 determines the biological characters of HPSCC, and TNFR1 may dominate this process. Moreover, interaction between the two receptors plays important roles in determining the fates of HPSCC cells and thus may serve as a therapeutic target for developing new therapeutic strategies for HPSCC.

We investigated the relationship between claudin-1 and micro-lymphatic vessel density (MLVD) by detecting claudin-1 and protein D2-40 expression in cancer tissue specimens obtained from 97 patients with hypopharyngeal squamous cell carcinoma (HSCC). We also explored the correlation between the expression of these proteins and clinical tumor stage, pathological grading, and clinical prognosis in the patients. Moreover, we studied the mechanism of lymph node metastasis in HSCC, thereby providing information for treating HSCC and inhibiting lymph node metastasis. We detected levels of claudin-1 and protein D2-40 expression in cancer tissue from 97 patients with HSCC and para-tumor tissue from 90 patients by immunohistochemistry; we analyzed the correlation between markers and clinicopathological features by using the Pearson chi-square test and conducted survival analysis by the log-rank test. Claudin-1 expression was high in HSCC and was related to tumor differentiation and lymph node metastasis; Kaplan-Meier analysis showed that claudin-1 expression was related to patient survival rate (P = 0.012). There was a significant relationship between MLVD in the tissues adjacent to the carcinoma and the indices of histopathological grade, clinical stage, and lymph node metastasis. There was also a positive correlation between claudin-1 expression and MLVD. High expression of claudin-1 might induce the generation of tumor lymphatic vessels, which increases metastasis in the lymph node. Because claudin-1 is related to patient survival rate, it may be useful as a monitoring index for postoperative HSCC and might be a new target for treating the disease.

MicroRNAs (MiRNAs) have been recognized to regulate cancer initiation and progression in carcinogenesis as either oncogenes or tumor suppressor genes, but their role in hypopharyngeal cancer development is not clearly defined. To determine whether miRNA-203 can promote tumor growth in human hypopharyngeal squamous cell carcinoma, we conducted experiments on the functional study of miRNA-203 and identification of miRNA-203 regulated target genes in hypopharyngeal cancer cells. We found that cell proliferation and cell colony-forming increased more in the miRNA-203 up-regulated cancer cells than in the negative control cancer cells. Up-regulation of miRNA-203 accelerated cell cycle progression in hypopharyngeal cancer cells. TP63 and B3GNT5 mRNAs were identified and validated as targets of miRNA-203. However, transwell assay and wound scratch assay showed that miRNA-203 did not involve in invasion and metastasis in hypopharyngeal cancer cells. According to the results, we conclude that miRNA-203 can promote tumor growth in human hypopharyngeal squamous cell carcinoma. These results provide the convincing evidence for the first time that up-regulation of miRNA-203 contributes to the malignancy of hypopharyngeal squamous cell carcinoma, possibly through down-regulating TP63 and B3GNT5.

Epidermal growth factor receptor (EGFR) signaling plays an important role in tumor cell resistance to therapy. In addition to ligand binding, mutual and cooperative interactions of EGFR with integrin cell adhesion receptors critically influence proper downstream signaling through a number of bridging adapter proteins. In the present study, we analyzed the role of two of these adapter proteins, called PINCH1 and Nck2, for cellular radioresistance in combination with EGFR-targeting using the monoclonal antibody cetuximab. siRNA-mediated knockdown of PINCH1 or Nck2 resulted in enhanced radiosensitivity of 3D grown human squamous cell carcinoma cell lines FaDu (head and neck) and A431 (epidermis) comparable with effects seen after cetuximab treatment. Combination of knockdown and cetuximab did not result in additive nor synergistic effects regarding clonogenic radiation survival. Modifications in MAPK, Akt and FAK phosphorylation occurred upon cetuximab treatment as well as PINCH1 or Nck2 depletion. We further found this tumor cell radiosensitization to be due to attenuated repair of DNA double strand breaks and altered Rad50 and Nbs1 expression but without changes in other DNA repair proteins such as ATM, DNA-PK and Mre11. Our data suggest that the adaptor proteins PINCH1 and Nck2 critically contribute to cellular radioresistance and proper EGFR signaling in 3D lrECM grown human squamous cell carcinoma cells. Further investigations are warranted to identify the intracellular signaling network controlled by EGFR, PINCH1 and Nck2.

Objective: This study aimed to link expression patterns of AQP1, AQP5, Bcl-2 and p16 to clinicopathological characteristics of oro-hypopharyngeal squamous cell carcinomas., Methods: Immunohistochemical expression of AQP1, AQP5, Bcl-2 and p16 was investigated in 107 consecutive oro-hypopharyngeal squamous cell carcinoma cases. Molecular interrelationship and correlations with clinicopathological parameters and survival were computed., Results: AQP1 was expressed exclusively by a subgroup of basaloid-like squamous cell carcinomas. AQP5 was detected in 25.2 per cent of the samples, showing significant association with the absence of p16 and Bcl-2 (p = 0.018; p = 0.010). In multivariate analysis, overexpression of p16 was significantly correlated with favourable overall survival (p = 0.014)., Conclusion: AQP5 defined a subset of patients with Bcl-2-negative and p16-negative tumours with a poor clinical outcome. AQP1 was found to be a marker of a subgroup of aggressive basaloid-like squamous cell carcinomas. These findings suggest that AQP1 and AQP5 are interesting candidates for further studies on risk group classification and personalised treatment of oro-hypopharyngeal squamous cell carcinomas.

As an important pathway maintaining the balance of intracellular calcium (Ca(2+)), store-operated Ca(2+) entry (SOCE) is critical for cellular functions. Stromal interaction molecule 1 (STIM1), a key component of SOCE, plays a dual role as an endoplasmic reticulum Ca(2+) receptor and an SOCE exciter. Aberrant expression of STIM1 could be discovered in several human cancer cells. However, the role of STIM1 in regulating human hypopharyngeal carcinoma still remains unclear. Real-time polymerase chain reaction (PCR) was used to detect expression of STIM1 in human hypopharyngeal carcinoma cell line FaDu. STIM1 on FaDu cells was knocked down by lentiviral transduction method. The biological impacts after knocking down of STIM1 on FaDu cells were investigated in vitro and in vivo. The result of real-time PCR showed that STIM1 was expressed in FaDu cells. Lentiviral transduction efficiently downregulated the expression of STIM1 in FaDu cells at both mRNA and protein levels. Significant downregulation of STIM1 on FaDu cells inhibited cell proliferation, induced cell cycle arrest in G0/G1 phase, promoted cell apoptosis, and restrained cell growth rate. The antigrowth effect of STIM1 silencing was also discovered in FaDu hypopharyngeal tumor model. Our findings indicate that STIM1 is likely to become a new therapeutic target for hypopharyngeal carcinoma treatment.

Hypopharyngeal squamous cell carcinoma (HSCC) has the worst prognosis among head and neck cancers. Cisplatin (DDP)-based chemotherapy is an important part of multimodal treatments. However, resistance to DDP severely impairs the effectiveness of chemotherapy for HSCC. Chloroquine (CQ) has been reported to enhance the effectiveness of chemotherapy and radiotherapy in liver, pancreas, breast, prostate and colon tumors, but it is unclear whether CQ could increase the efficacy of DDP for treating HSCC. We inoculated BALB/c nude mice with a subcutaneous injection of human hypopharyngeal FaDu cells to generate our animal model. Mice were randomly divided into 4 groups and treated with vehicle control, CQ (60 mg/kg/day), DDP (5 mg/kg/6 days), or a combination of DDP and CQ. Tumor growth and survival of the mice were monitored. We found that CQ inhibited autophagy and increased DDP-induced apoptosis in the xenograft mouse model. CQ enhanced the efficacy of DDP, resulting in decreased tumor growth and prolonged survival of the mice. To test whether blocking autophagy enhanced the efficacy of DDP, FaDu cells were infected with lentiviral shRNA to Beclin-1 and inoculated into the flanks of nude mice. Inhibition of autophagy markedly enhanced the DDP-induced antitumor effect. Our study suggests that the addition of CQ to DDP-based chemotherapy could be a potential therapeutic strategy for treating HSCC, and the inhibition of autophagy may contribute to chemotherapy sensitization in HSCC.

The PHLPP (pleckstrin homology [PH] domain leucine rich repeat protein phosphatase) family, which represents a family of novel Ser/Thr protein phosphatases, is composed of 2 members: PHLPP1 and PHLPP2. PHLPPs partake in diverse cellular activities to exhibit their antitumor and metastasis suppressor functions. It is necessary to investigate the expression patterns of PHLPP1 and PHLPP2 in hypopharyngeal squamous cell carcinomas (HSCCs) and clarify their clinical significance. A total of 138 patients with primary HSCC who underwent curative surgical treatment as an initial treatment were enrolled in this study. A total of 138 HSCC specimens and 64 adjacent noncancerous mucosal epithelial tissues were collected. The expression levels of PHLPP1 and PHLPP2 were examined by quantitative reverse transcription polymerase chain reaction and immunohistochemistry assays. Correlations between clinicopathological parameters of the patients were further evaluated. PHLPP1 and PHLPP2 mRNA transcript levels were significantly lower in tumor samples than in paired adjacent nontumor mucosae (P<0.0001, both). Positive correlations were observed between the mRNA levels of PHLPP1 and PHLPP2 in HSCC tissues (correlation coefficient r = 0.678, P<0.001) and in adjacent nontumor mucosae (r = 0.460, P<0.001). The majority of the noncancerous tissues showed high expression levels of PHLPP1 (87.5%, 56/64) and PHLPP2 (85.9%, 55/64). However, the expressions of PHLPP1 and PHLPP2 were significantly decreased in 83.3% (115/138) and 82.6% (114/138) of tumor tissues, respectively (P<0.0001, both). The expressions of both PHLPP isoforms were significantly related to the tumor clinical stage, differentiation, and cervical lymph node metastasis (P<0.05, all). It was PHLPP1 but not PHLPP2 that was significantly related to the tumor T stage. Low PHLPP1 and PHLPP2 expressions were associated with poor overall survival (OS) in HSCC patients (P = 0.004, P = 0.008, respectively). Multivariate analysis revealed that PHLPP1 was an independent prognostic factor for OS. This study indicates that, in HSCC, aberrant expressions of PHLPP1 and PHLPP2 are common events, and loss of PHLPPs might identify patients with poor prognostic outcomes.

Background: Development of squamous cell cancer of head and neck (SCCHN) is associated with human papillomavirus (HPV) infection, which in turn is closely related with expression of p16 INK4A. Loss of p16 INK4A expression by deletion, mutation, or hypermethylation is common in SCCHN. We here evaluated p16 INK4A as a prognostic marker of treatment response and survival in our SCCHN patients with laryngeal, hypopharyngeal or nasopharyngeal cancers., Materials and Methods: 131 patients diagnosed with SCCHN between January 2,2006 and July 17, 2010 were examined for p16 INK4A. The median age was 60 years (15-82 years). Fifty one patients were stage I-II and 80 were stage III-IV. Immunohistochemical expression of p16 INK4A was analyzed in pretreatment paraffin-embedded tumor blocks. The influence of p16 INK4A status on disease-free survival, and overall survival after treatment was evaluated., Results: P16 INK4A positivity was found in 58 patients (44%). Tumor-positivity for p16INK4A was correlated with improved disease free survival (70.1 months vs 59 months) and improved overall survival (2, 3 and 5-year values; 77% vs 72%, 70% vs 63% and, 63% vs 55%; respectively). On multivariate analysis, stage was determined as independent prognostic factor for disease-free survival., Conclusions: Stage was the major prognostic factor on treatment response and survival in our patients. P16 INK4A status predicts better outcome in laryngeal, hypopharyngeal or nasopharyngeal cancer cases treated with surgery plus adjuvant radiochemotherapy as well as with definitive radiation therapy and/or chemotherapy.

Background: Emerging evidence has indicated the significance of RbAp48 in tumorigenesis. Although many genetic and epigenetic factors have been found to be involved in the pathogenesis of hypopharyngeal carcinoma, the effect of RbAp48 in hypopharyngeal carcinoma is still unclear., Methods: A stable cell line overexpressing RbAp48 was generated in FaDu cells. Cell proliferation and colony formation were detected using FaDu-RbAp48 cells. Next we utilized nude mouse xenografts to determine the role of RbAp48. Flow cytometry was employed to investigate the effect of RbAp48 in cell cycle distribution and apoptosis. Real-time PCR was used to detect the expression of tumor suppressors and apoptosis-related factors., Results: The overexpression of RbAp48 inhibited cell proliferation, colony formation, and tumor formation in nude mice. The overexpression of RbAp48 affected cell cycle distribution and induced apoptosis. The expression of p53, Rb, Bax, caspase 3, caspase 8, and caspase 9 was upregulated, whereas the expression of Bcl-2 was downregulated resulting from the overexpression of RbAp48., Conclusion: RbAp48 was identified as critical in the proliferation of hypopharyngeal carcinoma in both in vitro and in vivo experiments. It is conceivable that the regulation of tumor suppressors (Bcl-2 family and caspase enzymes) by RbAp48 contributes, at least in part, to the RbAp48-mediated proliferation in hypopharyngeal carcinoma., (© 2015 S. Karger AG, Basel.)

Conclusion: These findings indicate that in hypopharyngeal squamous cell carcinoma (HSCC), hypermethylation and down-regulation of death-associated protein kinase-1 (DAPk1) are common events, which are associated with a poor prognosis., Objectives: This study aimed to investigate the methylation and expression of DAPk1, a tumor suppressor gene, in HSCC, and explore its clinical significance., Methods: The tumor and adjacent non-tumor tissues were collected from 53 patients with HSCC. The methylation status of DAPk1 was detected by methylation-specific polymerase chain reaction (MSP), and expression of DAPk1 was determined with real-time reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot at mRNA or protein levels. Correlations between the findings and patients' clinicopathological parameters were further evaluated., Results: The methylation ratio of DAPk1 in tumor tissues (60.38%) was significantly higher than that in the adjacent non-tumor tissues (26.42%) (p = 0.001), while DAPk1 expression in the tumors was down-regulated markedly (real-time RT-PCR, p = 0.002; immunohistochemistry, p = 0.006; Western blot, p < 0.001). DAPk1 methylation was negatively correlated with its mRNA expression (p = 0.002, r = -0.521). Both hypermethylation and down-regulation of DAPk1 were closely related to lymph node metastasis (p = 0.001 and 0.001, respectively), advanced TNM stage (p = 0.009 and 0.019, respectively), and low survival rates (p = 0.031 and 0.045, respectively).

Objective: To investigate the expression of microRNA-214(miR-214) in advanced hypopharyngeal carcinoma tissues and its effects on the invasion, migration and colone formation of FaDu cells., Methods: miR-214 expression in 30 cases of advanced hypopharyngeal carcinoma tissues and normal hypopharyngeal mucosa tissues was detected by real-time quantitative polymerase chain reaction (RT-qPCR). miR-214 was upregulated through transfecting the overexpression vector hsa-mir-214 into FaDu cells. The influences of miR-214 upregulation on the invasion, migration, clone formation and Twist expression were measured by Transwell invasion, Transwell migration, plate clone formation and Western blot assays, respectively., Results: The expression of miR-214 in advanced hypopharyngeal carcinoma tissues (0.311 ± 0.206) was significantly less than normal hypopharyngeal mucosa tissues (1.620 ± 1.394; t = 5.09, P < 0.05) . The expression of miR-214 was notably upregulated after tranfected with hsa-mir-214 compared with the negative control group (t = 6.347, P < 0.05). The migration and invasion ability of FaDu cells transfeced with hsa-mir-214 was decreased by comparison with negative control cells (t = 11.6, P < 0.01; t = 6.499, P < 0.05). There was no significant difference of the average clony number and the cloning efficiency between the experimental and negative control groups (t = 0.592, P > 0.05)., Results: of Western blot assay showed that, Twist expression in the miR-214-overexpressed group was apparently decreased compared with that in the control group (t = 6.545, P < 0.05)., Conclusions: miR-214 is expressed at a low level in advanced hypopharyngeal carcinoma tissues, and can obviously inhibit the invasion and migration abilities of FaDu cells, possibly because of its inhibiting effect on Twist expression. Additionally, miR-214 plays no significant role in the proliferation of FaDu cells.

Aim: To evaluate the expression of Livin in human laryngohypopharyngeal squamous cell carcinoma (LHSCC) and investigate whether Livin-knockdown using small interfering-RNA (siRNA) affects tumor aggressiveness in LHSCC cells., Materials and Methods: Immunohistochemistry, reverse transcription-polymerase chain reaction, western blotting, cell invasion assay, cell migration assay, and cell apoptosis assays were performed to assess the impact of Livin on cancer cell behavior in human LHSCC., Results: High immunoreactivity of Livin was observed in 22 (36.7%) of the 60 LHSCC tissues relative to adjacent normal mucosa. In the positive-Livin expression group, distant metastasis tended to occur frequently, but the difference was not statistically significant (p=0.06). Livin-knockdown by siRNA induced cell apoptosis through activation of caspase 3, caspase 7, and poly ADP ribose polymerase in LHSCC cells. Livin-knockdown also resulted in significantly reduced cell invasion and migration in LHSCC cells., Conclusion: siRNA-mediated silencing of Livin may be associated with the reversal of invasive capacity in LHSCC., (Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

This study aimed to determine the expression of integrin β1 and vascular endothelial growth factor (VEGF) and microvascular density (MVD) by CD105 staining in hypopharyngeal squamous cell carcinoma to determine their association with clinicopathologic characteristics, and to determine their role and the effects of their interactions in the development and progression of hypopharyngeal squamous cell carcinomas. The expression of integrin β1 and VEGF and MVD in hypopharyngeal squamous cell carcinomas and normal hypopharyngeal tissues were evaluated using immunohistochemistry. The Image-Pro Plus software was used to determine the mean optical density of the immunohistochemical images. Integrin β1 expression was significantly higher in hypopharyngeal squamous cell carcinoma tissues (78.00%) than in normal hypopharyngeal tissues (35.00%; P = 0.001) and significantly differed across pathologic grades and different T stages, and regarding the presence of cervical lymph node metastasis (P < 0.05). VEGF expression was significantly higher in hypopharyngeal squamous cell carcinoma tissues (74.00%) than in normal hypopharyngeal tissues (30.00%; P = 0.002), VEGF overexpression differed significantly across different pathologic grades and different T stages, and regarding the presence of cervical lymph node metastasis (P < 0.05). The MVD count was significantly higher in hypopharyngeal squamous cell carcinoma tissues (37.10 ± 5.95) than in normal hypopharyngeal tissues (8.70 ± 3.34; P = 0.000). MVD differed significantly across different pathologic grades and different T stages, and regarding the presence of cervical lymph node metastasis (P < 0.05). The expression of integrin β1 and VEGF and the MVD count exhibited no significant differences in terms of age, gender, history of smoking, and clinical stages (P > 0.05). VEGF expression was positively associated with the MVD count of hypopharyngeal squamous cell carcinomas (r = 0.582, P = 0.000); however, integrin β1 was not associated with VEGF or MVD (P > 0.05). Integrin β1 and VEGF are overexpressed and MVD increased in hypopharyngeal squamous cell carcinomas. VEGF is positively correlated with MVD.

Background: Hypopharyngeal squamous cell carcinoma (HSCC) has a very poor prognosis because of its high rates of regional and distant metastasis. Identification of differentially expressed miRNAs and their regulated molecular targets in tumour cells might enhance our understanding of the molecular mechanisms of metastasis in human cancers., Methods: A HSCC miRNA signature was constructed by array-based methods. Functional studies of microRNA-451a (miR-451a) and target genes were performed to investigate cell proliferation, migration and invasion by cancer cell lines. To identify miR-451a-regulated molecular targets, we adopted gene expression analysis and in silico database analysis., Results: Our miRNA signature revealed that miR-451a was significantly downregulated in HSCC. Restoration of miR-451a in cancer cell lines revealed that this miRNA significantly inhibited cancer cell migration and invasion. Our data demonstrated that the gene coding for endothelial and smooth muscle cell-derived neuropilin-like molecule (ESDN/DCBLD2) was a direct target of miR-451a regulation. Silencing of ESDN inhibited cell migration and invasion by cancer cells., Conclusions: Loss of tumour suppressive miR-451a enhanced cancer cell migration and invasion in HSCC through direct regulation of ESDN. Our miRNA signature and functional analysis of targets regulated by tumour suppressive miR-451a provide new insights into the potential mechanisms of HSCC oncogenesis and metastasis.

Background: The antitumor activity of lymphokine activated killer (LAK) cells immunotherapy is not always effective in all patients, especially when used alone. In this study, we investigated the in vitro antitumor activities of a combination of LAK immunotherapy and gene therapy employing an adenovirus carrying the p53 gene (Ad-p53) in human head and neck squamous cell carcinoma., Materials and Methods: The in vitro cytotoxicity of LAK cells was tested in H891 cells infected with or without Ad-p53, and the mRNA expression levels of natural killer group 2D ligands (UL16 binding protein (ULBP) 1 to 5) and tumor necrosis factor (TNF-α) in these cells were measured by real-time reverse transcription polymerase chain reaction., Results: Ad-p53 infection increased the cytotoxicity of LAK cells against H891 cells, and also increased the mRNA expression levels of the ULBPs in H891 cells and TNF-α in the LAK cells., Conclusion: The antitumor activities of LAK cells in H891 cells were enhanced by Ad-p53., Conclusion: The combinational therapy of LAK immunotherapy and Ad-p53 gene therapy may represent a new paradigm for the treatment of head and neck cancer., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Our recent study of the microRNA (miRNA) expression signature of hypopharyngeal squamous cell carcinoma (HSCC) revealed that microRNA-504 (miR-504) is significantly downregulated in HSCC tissues, suggesting that this miRNA is a candidate tumor suppressor. However, several previous reports indicated that miR-504 has an oncogenic function through targeting TP53. The aim of this study was to investigate the functional significance of miR-504 in cancer cells and to identify novel targets regulated by this miRNA in HSCC cells. First, we confirmed the downregulation of miR-504 in HSCC clinical specimens (P<0.0001) by qPCR. Using two sources of miR-504 to restore function, we observed significant inhibition of cancer cell proliferation in head and neck SCC (HNSCC) cell lines (FaDu, SAS and HSC3) and HCT116 colon carcinoma cells (p53+/+ and p53-/-). In HNSCC cells, induction of cell cycle arrest was observed by miR-504 transfection. To identify the molecular targets of miR-504, we performed gene expression analysis of miR-504 transfectants and in silico database analyses. Our data showed that cell cycle-related genes (RB1, CDK6, CDC23 and CCND1) were candidate target genes of miR-504. In HSCC clinical specimens, the expression of cyclin-dependent kinase 6 (CDK6) was significantly higher in cancer tissues compared to non-cancer tissues (P=0.0004). A significant inverse correlation between CDK6 and miR-504 expression was found (r=-0.43, P=0.0039). Expression of miR-504 inhibited CDK6 expression in HNSCC cells. Loss of tumor-suppressive miR-504 enhanced HSCC cell proliferation through targeting CDK6. The identification of novel tumor-suppressive miR-504-mediated molecular pathways and targets provide new insights into HSCC oncogenesis.

In order to reverse the malignant characteristics of hypopharyngeal cancer, the proteasome inhibitor MG132 was introduced into FaDu/T cells and the mechanisms underlying its effects were investigated. The multi-drug resistance (MDR) sensitivities of FaDu/T and FaDu/T-MG132 cancer cells to several chemotherapeutics were investigated by a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured by staining cells with Annexin V and propidium iodide (PI) double staining. Reverse transcription-polymerase chain reaction and western blot analysis were conducted to detect mRNA and corresponding protein levels of the MDR- and apoptosis-related genes P-glycoprotein (P-gp), caspase-3, Bcl-2 and Bax. The nuclear protein of nuclear factor κ-light-chain-enhancer of activated B cells. (NF-κB) and p53 were also investigated via western blot analysis. Compared with FaDu/T cells, the drug resistance of FaDu/T + MG132 cells to cisplatin (DDP), 5-fluorouracil (5-FU), doxorubicin (Dox) and vincristine (VCR) decreased. With increased expression of caspase-3 and Bax and decreased expression of Bcl-2, the anti-apoptotic ability markedly decreased in FaDu/T + MG132 cells. P-gp and NF-κB significantly decreased; however, p53 increased in FaDu/T + MG132 cells. These results suggested that the proteasome inhibitor MG132 reversed the malignant characteristics of FaDu/T by enhancing apoptosis and inhibiting P-gp. MG132 was also able to inhibit the nuclear translocation of NF-κB and increase the expression of p53.

Metastasis remains a major cause of mortality in head and neck squamous cell carcinoma (HNSCC). Current clinicopathological features have shown limited predictability for the risk of distant metastasis in individual patients, and therefore more accurate and reliable markers are needed. The aim of this study was to investigate the ability of various molecular markers present in primary tumors to predict the risk of developing distant metastasis. Restrictive clinical criteria were applied for patient selection in order to carry out a case-control study with comparable clinical features on a group-wide basis and a similar risk of metastasis. All patients were surgically treated (with postoperative radiotherapy when appropriate) and classified as stage IV disease. Immunohistochemical analysis was performed for a panel of proteins known to participate in cellular processes relevant to metastatic dissemination (E-cadherin, annexin A2, cortactin, FAK, EGFR, p53, and p-AKT). Results showed that the loss of E-cadherin expression was significantly correlated with the risk of distant metastasis (P = 0.002; log-rank test), while the loss of annexin A2 expression was nearly statistically significant (P = 0.06). None of the other protein markers assessed were associated with the development of distant metastasis. Therefore, according to our data the loss of epithelial adhesion seems to play a central role in the development of metastasis in HNSCC, and more importantly, immunohistochemical assessment of key proteins involved in cell adhesion regulation, such as E-cadherin could represent a useful tool to evaluate easily and routinely the metastatic potential of these carcinomas.

Background: Patients with hypopharyngeal cancer have a 5-year survival of only 15% to 30%. Human papillomavirus (HPV) is a risk factor and a favorable prognostic factor for oropharyngeal carcinoma and p16 has been suggested as a surrogate marker for HPV-induced cancer. However, few studies have been performed on HPV and p16 in hypopharyngeal cancer., Methods: One hundred nine pretreatment hypopharyngeal cancer biopsies were analyzed for presence of HPV and p16 overexpression, and the results were correlated to patient survival., Results: Of 109 tumors, 7 were HPV-positive (4 HPV16) and 18 overexpressed p16. There was some correlation between survival and HPV status, but not with regard to p16 expression. Notably, all patients with HPV16-positive tumors, also overexpressing p16, lived tumor free for more than 3 years., Conclusion: Our results indicate that HPV-induced hypopharyngeal cancer is rare and that p16 is not a suitable biomarker for presence of HPV in this tumor type., (Copyright © 2013 Wiley Periodicals, Inc.)

Paclitaxel has been proved to be active in treatment and larynx preservation of HNSCC, however, the fact that about 20-40% patients do not respond to paclitaxel makes it urgent to figure out the biomarkers for paclitaxel-based treatment in Hypopharynx cancer (HPC) patients to improve the therapy effect. In this work, Fadu cells, treated or untreated with low dose of paclitaxel for 24 h, were applied to DNA microarray chips. The differential expression in mRNAs and miRs was analyzed and the network between expression-altered mRNAs and miRs was constructed. Differentially expressed genes were mainly enriched in superpathway of cholesterol biosynthesis (ACAT2, MSMO1, LSS, FDFT1 and FDPS etc.), complement system (C3, C1R, C1S, CFR and CFB etc.), interferon signaling (IFIT1, IFIT3, IFITM1 and MX1 etc.), mTOR signaling (MRAS, PRKAA2, PLD1, RND3 and EIF4A1 etc.) and IGF1 signaling (MRAS, IGFBP7, JUN and FOS etc.), most of these pathways are implicated in tumorigenesis or chemotherapy resistance. The first three pathways were predicted to be suppressed, while the last two pathways were predicted to be induced by paclitaxel, suggesting the combination therapy with mTOR inhibition and paclitaxel might be better than single one. The dramatically expression-altered miRs were miR-112, miR-7, miR-1304, miR-222*, miR-29b-1* (these five miRs were upregulated) and miR-210 (downregulated). The 26 putative target genes mediated by the 6 miRs were figured out and the miR-gene network was constructed. Furthermore, immunoblotting assay showed that ERK signaling in Fadu cells was active by low dose of paclitaxel but repressed by high dose of paclitaxel. Collectively, our data would provide potential biomarkers and therapeutic targets for paclitaxel-based therapy in HPC patients.

Identification of a reliable biomarker for predicting prognosis in head and neck cancers is highly desirable and has long been sought. There have been several reports that members of the regenerating gene (REG) family are highly expressed in chronic inflammation and in tumors of the digestive organs. In addition, it has been described in several reports that REG expression is associated with the progression of digestive cancers. In the present study, we evaluated the effect of REG expression on the prognosis of hypopharyngeal cancer. We investigated 37 cases with hypopharyngeal squamous cell carcinoma, determined REG mRNA expression, which is easily detected in formalin-fixed paraffin-embedded tissues using the real-time reverse transcriptase-polymerase chain reaction method, and evaluated the survival rate using the Kaplan-Meier method. According to these results, REG III mRNA expression was significantly associated with prolonged survival. Therefore, we constructed hypopharyngeal cancer cell lines transfected with REG III and assessed the cell proliferation and chemosensitivity and/or radiosensitivity in vitro. Cells transfected with REG III exhibited significantly lower cell proliferation and higher chemosensitivity and/or radiosensitivity compared with the control cells. These data suggest that REG III may be a reliable biomarker of prognosis in hypopharyngeal cancer. This is the first report concerning the association of REG III expression and the prognosis of head and neck squamous cell carcinoma including hypopharyngeal cancer.

Background: Beclin-1, a key regulator of autophagy. Microtubule-associated protein 1 light chain 3 (LC3), is involved in autophagsome formation during autophagy. The autophagic genes beclin-1 and LC3 paly an important role in the development and progression of tumor. This study was designed to investigate the expression of beclin-1 and LC3 and to clarify their clinical significance in hypopharyngeal squamous cell carcinoma (HSCC)., Methods: Eighty-two surgical hypopharyngeal squamous cell carcinoma specimens and fifty-four adjacent non-cancerous mucosal epithelial tissues were obtained. Beclin-1 and LC3-II expression was examined by immunohistochemistry, real-time RT-PCR and Western blotting assays. Correlations with patient clinical characteristics and overall survival were evaluated., Results: Beclin-1 was positively expressed in 42.7% (35/82) of HSCC specimens (adjacent non-cancerous tissues, 79.6%, 43/54; P<0.0001). Furthermore, 41.5% (34/82) of HSCC specimens exhibited high LC3 immunoreactivity (adjacent non-cancerous tissues, 74.1%, 40/54; P=0.0002). Beclin-1 and LC3-II mRNA transcript levels were significantly lower in HSCCs than in paired non-cancerous tissues (P<0.0001, P=0.0001, respectively). Similarly, western blotting assays showed that beclin-1 and LC3-II were markedly decreased in HSCCs (P=0.02, P=0.004, respectively). A positive correlation was observed between the mRNA transcript levels of beclin-1 and LC3-II in HSCCs (r=0.51, P<0.0001; 95%CI: 0.273 to 0.689). Beclin-1 and LC3 expression were significantly correlated with T categories, differentiation and lymph node metastasis. Negative beclin-1 immunoreactivity and low LC3 expression were associated with poorer overall survival in HSCC patients (P<0.0001, P=0.0145, respectively). Multivariate analysis revealed that beclin-1 was an independent prognositic factor for overall survival., Conclusion: Beclin-1 and LC3-II are downregulated in HSCCs and their aberrant expression correlates with poor prognosis of HSCCs.

Accumulating evidences indicate that the functional FAS-1377G>A, -670A>G and FASL-844T>C polymorphisms affect the risk of several kinds of cancers. However, their roles in the development of larynx and hypopharynx squamous cell carcinoma (SCC) were still unknown in the Chinese. In the current study, we examined whether these functional genetic variants were associated with the risk of larynx and hypopharynx squamous SCC in a Han Chinese population. The FAS and FASL polymorphisms were genotyped in 300 patients with laryngeal and hypopharyngeal SCC and 300 control subjects by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Logistic regression analysis revealed that subjects carrying the FASL-844CT or TT genotype had a significantly decreased risk of developing laryngeal and hypopharyngeal SCC [odds ratio (OR)=0.69; 95% confidence interval (CI)=0.51-0.93; P=0.016; or, OR=0.41; 95% CI=0.20-0.86; P=0.009] compared with those carrying the CC genotype. Joint gene-smoking and gene-drinking effects were also observed, with the OR of CC genotype for smokers or drinkers were 5.15 (95%CI=3.24-8.97) or 12.52 (95%CI=7.31-22.47), respectively. Therefore, the FASL-844T>C polymorphism is associated with genetic susceptibility of developing laryngeal and hypopharyngeal SCC in a Han Chinese population., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cancer stem cells contribute to the malignant phenotypes of a variety of cancers, but markers to identify human hypopharyngeal cancer (HPC) stem cells remain poorly understood. Here, we report that the CD271(+) population sorted from xenotransplanted HPCs possesses an enhanced tumor-initiating capability in immunodeficient mice. Tumors generated from the CD271(+) cells contained both CD271(+) and CD271(-) cells, indicating that the population could undergo differentiation. Immunohistological analyses of the tumors revealed that the CD271(+) cells localized to a perivascular niche near CD34(+) vasculature, to invasive fronts, and to the basal layer. In accordance with these characteristics, a stemness marker, Nanog, and matrix metalloproteinases (MMPs), which are implicated in cancer invasion, were significantly up-regulated in the CD271(+) compared to the CD271 (-) cell population. Furthermore, using primary HPC specimens, we demonstrated that high CD271 expression was correlated with a poor prognosis for patients. Taken together, our findings indicate that CD271 is a novel marker for HPC stem-like cells and for HPC prognosis.

Brachytherapy is a common clinical technique involving implantation of sealed radioactive "seeds" within a tumor to selectively irradiate the tumor mass while minimizing systemic toxicity. To mitigate the disadvantages associated with complex surgical implantation and subsequent device removal procedures, we have developed an alternative approach using a genetically encoded peptide polymer solution composed of a thermally responsive elastin-like polypeptide (ELP) radiolabeled with (131)I that self-assembles into radionuclide seeds upon intratumoral injection. The formation of these nontoxic and biodegradable polymer seeds led to prolonged intratumoral retention (~85% ID/tumor 7 days postinjection) of the radionuclide, elicited a tumor growth delay in 100% of the tumors in two human xenografts (FaDu and PC-3), and cured more than 67% of tumor-bearing animals after a single administration of labeled ELP. These results suggest that in situ self-assembly of biodegradable and injectable radionuclide-containing polypeptide seeds could be a promising therapeutic alternative to conventional brachytherapy., (©2012 AACR.)

Evidence indicates that human ether à-go-go-related gene 1 (HERG1) voltage-gated potassium channels could represent new valuable membrane therapeutic targets and diagnostic/prognostic biomarkers in various cancers. This study is the first to investigate the expression pattern of HERG1 potassium channel subunit in both primary tumors and precancerous lesions to establish its clinical and biological role during the development and progression of head and neck squamous cell carcinomas. HERG1 protein expression was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 133 patients with laryngeal/hypopharyngeal squamous cell carcinomas and 75 patients with laryngeal dysplasia, and correlated with clinical data. Our findings demonstrate that HERG1 is frequently aberrantly expressed in a high percentage of primary tumors (87%), whereas expression was negligible in both stromal cells and normal-adjacent epithelia. HERG1 expression increased during head and neck squamous cell carcinoma progression and was significantly associated with lymph node metastasis (P=0.04), advanced disease stages (P<0.001), regional tumor recurrence (P=0.004), distant metastasis (P=0.03) and reduced disease-specific survival (P=0.012, log-rank test). HERG1-positive expression was also detected in 31 (41%) of 75 laryngeal dysplasias. Interestingly, HERG1 expression increased with the grade of dysplasia; however, HERG1 expression but not histology correlated significantly with increased laryngeal cancer risk (P=0.007). In addition, functional studies in head and neck squamous cell carcinoma-derived cell lines further revealed that HERG1 expression promotes anchorage-dependent and -independent cell growth and invasive capability, although independently of its ion-conducting function. Our data demonstrate that HERG1 expression is a biologically and clinically relevant feature in head and neck squamous cell carcinoma progression and also during malignant transformation, and a promising candidate as cancer risk marker and therapeutic target for head and neck squamous cell carcinoma prevention and treatment.

Objective: To investigate the expression of multidrug resistance gene ABCB1 and ABCG2 in FaDu cells (human hypopharyngeal carcinoma cell line) and the multidrug resistance (MDR) cell lines FaDu/T transformed from FaDu cells by taxol and underlying mechanisms of MDR., Methods: The multidrug resistance sensitivities of FaDu and FaDu/T to cisplatin (DDP), 5-fluorouracil (5-FU), doxorubicin (Dox), and vincristine (VCR) were examined by methyl-thiazolyl-tetrazolium (MTT) assay. The mRNA and protein expressions of multidrug resistance genes ABCB1 and ABCG2 were analysed with RT-PCR, Western blot and laser confocal microscopy. JNK signal proteins were detected through Western blot., Results: The multidrug resistance of FaDu/T cells to Taxol, DDP, 5-FU, ADM and VCR was more than that of FaDu cells. The expression of ABCB1 in FaDu/T cells was significantly higher than that in FaDu cells (t = 22.42, P < 0.05), but the expression of ABCG2 in FaDu/T cells was significantly lower than that in FaDu cells (t = 10.06, P < 0.05). JNK signal was inhibited in FaDu or FaDu/T cells and the inhibited JNK was reactivated by taxol or anisomycin (an activator for MAPK signal transduction pathways). Anisomycin down-regulated the expression of ABCB1 (F = 33.72, P < 0.05) and up-regulated the expression of ABCG2 (F = 220.16, P < 0.05) in FaDu/T cells, but not in FaDu/T cells pretreated by JNK inhibitor SP600125 (P > 0.05)., Conclusion: The overexpression of ABCB1 and the down-regulation of ABCG2 in FaDu/T cells were the main features of MDR in hypopharyngeal carcinomas, in which JNK signal transduction pathways could play an important role.

Background: The objective of this study was to evaluate the efficacy of Nimesulide, a selective cyclooxygenase-2 (COX-2) inhibitor, on the growth of hypopharyngeal carcinoma cells (FaDu) in vitro, and investigate its potential mechanism., Methods: After FaDu cells were treated with graded concentrations of Nimesulide for divergent time, sensitivity of cells to drug treatment was analyzed by MTT assay. Morphological changes of FaDu cells in the presence of Nimesulide were observed by acridine orange cytochemistry staining. Proliferating cells were detected using the 5-Bromo-2'-deoxy-uridine (BrdU) incorporation assay. Following cells were subjected to Nimesulide (500 μmol/l) for 6 h, 12 h and 24 h, the percentage of apoptosis was examined by flow cytometry. We detected COX-2 and Survivin expression change by RT-PCR and Western blot, and analyzed the correlation of them with the growth of FaDu cells. Additionally, we also analyzed Caspase-3, Bcl-2 and Bax expressions as markers to investigate the related pathway of Nimesulide-indued apoptosis., Results: Compared with the control group, the viabilities rates were decreased by Nimesulide in time- and dose-dependent manners, typical morphological changes of apoptotic cells were observed in the Nimesulide-treatment groups, Nimesulide could suppress the proliferation of FaDu cells significantly. The percentage of apoptosis in FaDu cells were markedly increased after Nimesulide-treatment for 6 h, 12 h and 24 h. Nimesulide down-regulated the Survivin and COX-2 expressions at mRNA and protein levels in FaDu cells. Additional analyses indicated that Bcl-2 expression was significantly decreased and the expressions of Caspase-3 as well as Bax were increased at both mRNA and protein levels., Conclusions: Based on the induction of apoptosis and suppression of proliferation, Nimesulide could inhibit the growth of FaDu cells. Furthermore, the suppression of Survivin expression may play an important role in Nimesulide-induced growth inhibition. Nimesulide could act as an effective therapeutic agent for hypopharyngeal carcinoma therapy.

The transcription factor TWIST is an important factor in regulating epithelial-mesenchymal transition (EMT) and tumor metastasis. To explore the functions of TWIST in hypopharyngeal cancer, we investigated if overexpression of TWIST has an effect on FaDu cell morphology, and if alteration of TWIST has an effect on E-cadherin, N-cadherin, c-fos, MMP-9, as well as in cell migration, and the invasion ability of FaDu cells. Moreover, we also studied the relationship between TWIST overexpression and clinicopathological characteristics in hypopharyngeal cancer tissue samples by immunohistochemical assays. The results showed that overexpression of TWIST-induced morphological changes, such as occurrence of EMT. TWIST overexpression also increased cell migration and invasion ability, accompanied by an alteration of E-cadherin, N-cadherin, c-fos and MMP-9 expression. Furthermore, immunohistochemical assays showed that TWIST overexpression was related with tumor differentiation (P=0.038), tumor size (P=0.048) and lymph node metastasis (P=0.044). The data presented reveal that overexpression of TWIST plays a significant role in the metastasis of hypopharyngeal tumors, and alteration of TWIST has an effect on the EMT, c-fos and MMP-9 expression in FaDu cells. We conclude that TWIST promotes hypopharyngeal carcinoma metastasis, and the TWIST/c-fos/MMP-9 signaling pathway may play an important role in the metastasis of FaDu cells.

The major obstacle to tumor chemotherapy is drug resistance. In the present study, we investigated the characteristics of FaDu cells (a hypopharyngeal carcinoma cell line) with multidrug resistance (MDR) induced by Taxol. The resistant cell line, FaDu/T, was grown by exposing normal FaDu cells to escalating concentrations of Taxol stepwise for over 12 months. The multidrug resistant sensitivities of the FaDu/T cells to cisplatin (DDP), 5-fluorouracil (5-FU), doxorubicin (Dox) and vincristine (VCR) were investigated by methyl-thiazolyl-tetrazolium (MTT) assay. Cell apoptosis was measured by acridine orange and Hoechst 33342/propidium iodide double staining. Cell cycle distribution and the cell apoptosis index were quantified using flow cytometry. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted to determine the mRNA levels of the MDR-related genes MDR1/ABCB1 and BCRP/ABCG2. Western blotting was used to assay the expression of MDR1/ABCB1 and BCRP/ABCG2 and the apoptosis-related proteins caspase-3, Bcl-2 and Bax. Compared with the FaDu cells, the drug resistance of FaDu/T cells to DDP, 5-FU, Dox and VCR was increased 8.99-, 21.96-, 31.42- and 10.00-fold, respectively. The percentages of FaDu/T cells in the G0/G1 and G2/M phases were increased while the cell percentage in the S phase decreased as compared with the percentages of FaDu cells. The anti-apoptotic ability increased prominently, as the index of apoptosis decreased. Furthermore, caspase-3, Bcl-2 and Bax expression was altered accordingly to resist apoptosis in the FaDu/T cells. MDR1/ABCB1 expression increased significantly at both the mRNA and protein levels, while BCRP/ABCG2 expression appeared to inversely affected, i.e. decreased, in a concentration-dependent manner. These -findings may provide theoretical support for the prevention of MDR in clinical cancer chemotherapy.

Purpose: Micromilieu-dependent quantification of γH2AX after irradiation in vivo and correlation with local tumour control., Materials and Methods: Local tumour control was evaluated after irradiation of FaDu and SKX xenografts with ambient single doses. γH2AX foci were quantified in perfused and unperfused regions after different irradiation doses and at different time points., Results: The TCD(50) of FaDu was 2-times higher compared to SKX (28.0Gy [95% C.I. 24.6; 31.3Gy] for FaDu; 14.9Gy [10.9; 18.9] for SKX, p<0.001). The induction of foci did not differ between the tumour models. Residual foci were twice higher in perfused SKX regions compared to FaDu, no difference was observed in the non-perfused region between both tumour models. The number of residual foci increased with a 2-times higher slope in perfused SKX-regions compared to FaDu, while no difference was detected in unperfused regions. Already within the perfused regions, this slope decreased with distance from perfused vessels., Conclusion: The dose-response of residual γH2AX foci is highly dependent on tumour cell oxygenation in well perfused areas. This dependence decreases further away from tumour vessels. Only γH2AX evaluation in perfused tumour areas can distinguish between the different radiocurability of the two tumour models., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

RECQL1 and WRN proteins are RecQ DNA helicases that participate in suppression of DNA hyper-recombination and repair. In this study, we report evidence supporting their candidacy as cancer therapeutic targets. In hypopharyngeal carcinomas, which have the worst prognosis among head and neck squamous cell carcinomas (HNSCC) that are rapidly rising in incidence, we found that RECQL1 and WRN proteins are highly expressed and that siRNA-mediated silencing of either gene suppressed carcinoma cell growth in vitro. Similarly, siRNA administration in a murine xenograft model of hypopharyngeal carcinoma markedly inhibited tumor growth. Moreover, combining either siRNA with cis-platinum (II) diammine dichloride significantly augmented the in vivo anticancer effects of this drug that is used commonly in HNSCC treatment. Notably, we observed no recurrence of some tumors following siRNA treatment in this model. Our findings offer a preclinical proof of concept for RECQL1 and WRN proteins as novel therapeutic targets to treat aggressive HNSCC and perhaps other cancers., (©2011 AACR.)

Objective: Microsatellite alterations, especially those that cause loss of heterozygosity (LOH), have recently been postulated as a novel mechanism of carcinogenesis and a useful prognostic factor in many kinds of malignant tumors. However, few studies have focused on a specific site, hypopharynx. The aim of this study was to evaluate the relationship between LOH and hypopharyngeal squamous cell carcinoma (HPSCC)., Study Design: Laboratory-based study., Setting: Integrated health care system., Subjects and Methods: Matched normal and cancerous tissues from 30 patients with HPSCC were examined for LOH in 4 tumor suppressor genes (TSGs) (p16, Rb, E-cadherin, and p53) at loci 9p21, 13q21, 6q22, and 17p13, respectively, using microsatellite markers amplified by polymerase chain reaction. The results for each loci were compared with clinicopathological features., Results: Among the 30 cases, 26 (86.7%) exhibited LOH, with the most common alteration being LOH at p53 (52.6%). Significantly higher rates of LOH detection were seen in Rb, p53, and the LOH-high group (cases where 2 or more loci with LOH were found) in cases of lymph node metastasis. Compared with stage I and II carcinoma, tumors of stages III and IV had significantly higher frequencies of LOH in Rb, p53, and the LOH-high group. However, the presence of LOH was not significantly correlated with survival., Conclusion: These results suggest that LOH in TSGs such as Rb and p53 may contribute to the development and progression of HPSCC. The presence of LOH in the primary tumor may also be predictive of lymph node metastasis.

Purpose: The purpose of this study was to examine the expression of mir-21 and phosphatase and tensin homologue (PTEN) in laryngeal squamous cell carcinomas (LSCCs) and hypopharyngeal squamous cell carcinomas (HSCCs), and assess correlations between the two as well as with clinical characteristics of patients., Methods: The expression of mir-21 in tumor and adjacent non-tumor tissues was investigated by real-time RT-PCR. Immunohistochemistry (IHC) was carried out to analyze PTEN protein levels., Results: Mir-21 was up-regulated in LSCCs and HSCCs compared to adjacent non-tumor tissues (P < 0.05), and the up-regulated expression of mir-21 was associated with clinical stage (P = 0.001), T classification (P = 0.007), pathologic differentiation (P = 0.025), and lymph node positivity (P = 0.002). In contrast, PTEN IHC staining was notably weaker in tumor tissues than in matched non-tumor tissues (P < 0.05), and the down-regulated expression of PTEN was correlated with tumor staging (P = 0.025), the extent of tumor (P = 0.017), and lymph node positivity (P = 0.040). Furthermore, the level of mir-21 was reversely correlated with PTEN expression (P = 0.006)., Conclusion: mir-21 and PTEN might play important roles in the progression of LSCC and HSCC, the two fcators demonstrating a negative correlation.

Objective: To investigate the effects of caveolin-1 (CAV1) on the growth of hypopharyngeal squamous cell carcinoma (HSCC) FaDu cells in vitro and in vivo., Methods: A CAV1-RNAi-lentivirus construct was transfected into FaDu cells and expression of caveolin-1 was tested by RT-PCR and western blotting analysis. Cell apoptosis was analyzed by transferase-medisated dUTP nick-end labeling (TUNEL) assay. Tumor inhibition effects were investigated by injecting rCAV1-RNAi-lentivirus construct into tumors created with FaDu cells in the HSCC mouse model, with the empty-vector lentivirus as a control. CAV1 expression in xenografts was tested by RT-PCR and immunohistochemistry., Results: RT-PCR and western blot analysis demonstrated successful construction of the CAV1-RNAi-lentivirus construct producing small hairpin RNA. The average weights and volumes of tumor in mice treated with CAV1-RNAi-lentivirus were lower than in mice with control treatment (P < 0.05). RT-PCR revealed weak positive expression of CAV1 in CAV1-construct-treated xenografts and immunohistochemistry confirmed lower CAV1 expression than in controls.(P < 0.05). In addition, downregulation of CAV1 increased cell apoptosis in vitro., Conclusion: The growth of HSCCs could be inhibited by recombinant CAV1-RNAi-lentivirus in vitro and in vivo.

The 5-year survival rate for oral cavity cancer is poorer than for breast, colon or prostate cancer, and has improved only slightly in the last three decades. Hence, new therapeutic strategies are urgently needed. Here we demonstrate by tissue micro array analysis for the first time that RNA-binding protein La is significantly overexpressed in oral squamous cell carcinoma (SCC). Within this study we therefore addressed the question whether siRNA-mediated depletion of the La protein may interfere with known tumor-promoting characteristics of head and neck SCC cells. Our studies demonstrate that the La protein promotes cell proliferation, migration and invasion of lymph node-metastasized hypopharyngeal SCC cells. We also reveal that La is required for the expression of β-catenin as well as matrix metalloproteinase type 2 (MMP-2) within these cells. Taken together these data suggest a so far unknown function of the RNA-binding protein La in promoting tumor progression of head and neck SCC.

At the beginning of the 21st century the hypopharynx and the cervical esophagus cancer represents a major issue for all countries of the world. The epidemiology of the hypopharynx and cervical esophagus cancer deals with the spread of the disease in the human population with regard to sex, age, profession, time and space, as well as risk factors that contribute to these phenomena. The main goal is to investigate the causes and the factors involved in the development of the tumors at the pharyngoesophageal junction, knowledge that contributes to the latest therapeutic assessment through interdisciplinary collaboration (E.N.T. surgeon, general surgeon, radiation oncologist, chemotherapist, and nutritionist). The epidemiology of the hypopharynx and cervical esophagus cancer includes three major areas of interest: descriptive (the study of the spread in mass population), analytical (the study of causal risk factors on the disease) and experimental (that verifies by experiments on animals the prior identified hypothesis).

Background: Hypopharyngeal squamous cell carcinoma (HSCC) is an aggressive malignancy with one of the worst prognoses among all head and neck cancers. Greater understanding of the pertinent molecular oncogenic pathways could help improve diagnosis, therapy, and prevention of this disease. The aim of this study was to identify tumour-suppressive microRNAs (miRNAs), based on miRNA expression signatures from clinical HSCC specimens, and to predict their biological target genes., Methods: Expression levels of 365 human mature miRNAs from 10 HSCC clinical samples were screened using stem-loop real-time quantitative PCR. Downregulated miRNAs were used in cell proliferation assays to identify a tumour-suppressive miRNA. Genome-wide gene expression analyses were then performed to identify the target genes of the tumour-suppressive miRNA., Results: Expression analysis identified 11 upregulated and 31 downregulated miRNAs. Gain-of-function analysis of the downregulated miRNAs revealed that miR-489 inhibited cell growth in all head and neck cancer cell lines examined. The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene. Knockdown of PTPN11 resulted in the inhibition of cell proliferation in head and neck SCC cells., Conclusion: Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.

Objective: To detect the serum proteomic fingerprints in patients with hypopharyngeal squamous cell carcinoma (HPSCC) by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) protein chip array technique., Methods: The serum samples were obtained from 58 HPSCC patients for protein expression analysis using SELDI-TOF Protein Chip technique and cation-exchange (CM10) protein array. All the spectra were compared and the qualified mass peaks with mass-to-charge ratios (m/z) between 1 and 70 kD were autotimatically detected. The tree analysis pattern was generated using Biomarker Patterns Software., Results: The protein profiles of HPSCC serum were analyzed according to the clinical and pathological features of the patients and their treatment response. No significant difference was noted in the serum proteins between HPSCC patients with different statuses of cervical lympha node metastasis (P>0.05), and the difference between well differentiated and poorly differentiated HPSCC was only minor. No significant difference was found in the serum proteins between chemotherapy-sensitive patients and the insensitive patients (P>0.05), but 5 proteins were identified to be overexpressed in the sensitive patients (P < / = 0.05). Radiotherapy-sensitive HPSCC patients were segregated from the insensitive group with a sensitivity of 86.67% and specificity of 100%., Conclusion: The serum protein at the m/z value of 6115.74 is overexpressed in radiotherapy-sensitive HPSCC patients. Serum protein profiling allows the prediction of radiotherapy response in HPSCC patients, and the identified proteins may serve as candidate biomarkers for predicting the radiotherapy sensitivity of HPSCC.

Objective: To retrospectively evaluate the prognostic impact of gene status and protein expression of receptor tyrosine kinases of the HER (human epidermal growth factor receptor related) family in relation to established clinicopathologic parameters in squamous cell carcinomas of the upper respiratory tract., Study Design: Immunohistochemistry and fluorescence in situ hybridization for HER1-4 and the proliferation marker Ki-67 was performed in 219 cases of head and neck squamous cell carcinomas and related to long-term clinical follow-up. Additionally, the prognostic impact of chromosomal instability was analyzed., Results: High expression of HER1 and HER2 was present in 49.4% and 6.6% of tumors, respectively. Expression of HER3 and HER4 appeared negative or inconspicuous. A gene amplification of HER1 occurred in 5.2% of tumors, whereas none of the tumors showed an amplification of HER2-4 loci. In univariate overall survival analysis a negative prognostic impact could be demonstrated for high expression of HER2 (p < 0.01), advanced local tumor growth (p < 0.01), lymph node metastasis (p < 0.01), presence of residual tumor after surgical therapy (p < 0.01), high proliferative activity (Ki-67; p = 0.02) and high chromosomal instability (p = 0.01). According to the multivariate analysis, the strongest negative predictors of survival were advanced tumor growth (p < 0.01), presence of residual tumor (p < 0.01), high expression of HER2 (p < 0.01) and chromosomal instability (p = 0.03)., Conclusion: Overexpression of HER2 and presence of chromosomal instability harbor an additional prognostic impact on disease-specific survival and prove to be independent negative prognostic factors in head and neck squamous cell carcinomas.

High levels of mitotic progression-associated PLK1 and stress-associated HSF1 have been observed in various human cancers. In the present study, we investigated the effects of PLK1 and HSF1 knockdown on the proliferation of oral cancer cells using small interfering RNA. In human oral squamous cell carcinoma (SCC) tissues, the levels of PLK1 and HSF1 were higher compared to normal tissues. The expression levels of PLK1 and HSF1 were also elevated in the human oral SCC cell lines FaDu and HEp-2. Disruption of PLK1 induced cell cycle arrest at G2/M phase as well as apoptosis in oral cancer cells. Interestingly, knockdown of both PLK1 and HSF1 expression decreased cell proliferation while increasing apoptotic cell death in synergistic fashion. These results establish the potential value of PLK1 and HSF1 as targets for oral cancer therapy.