Donghai Huang

Elevated expression of HMGB1 in squamous-cell carcinoma of the head and neck and its clinical significance.

PURPOSE HMGB1 overexpression has been reported in a variety of human cancers. However, the role of HMGB1 in squamous-cell carcinoma of the head and neck (SCCHN) remains unclear. The aim of the present investigation was to analyse HMGB1 protein expression in both SCCHN tissue and cell levels and to assess its prognostic significance in SCCHN. METHODS HMGB1 protein expression in 103 primary SCCHN tissue specimens was analysed by immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Additionally, HMGB1 protein expression was evaluated in cell level by Western blotting. RESULTS By Western blotting analysis, all the 5 SCCHN cell lines overexpressed HMGB1 protein, whereas the non-transformed immortalised cell line NP-69 had relatively weak HMGB1 protein expression. Immunohistochemical staining revealed that HMGB1 protein was detected in 91 (91/103, 88.3%) primary tumour samples, but only in 7 (7/16, 43.75%) adjacent non-carcinoma samples (p<0.001); moreover, HMGB1 overexpression was significantly associated with T classification (p=0.001), clinical stage (p<0.001), recurrence (p<0.001) and lymph node metastasis (p<0.001). Survival analysis demonstrated that high HMGB1 expression was significantly associated with shorter disease-free and overall survival (both p<0.001), especially in late patients with SCCHN. When HMGB1 expression and lymph node status were combined, patients with HMGB1 overexpression/lymph node (+) had both poorer disease-free and overall survival than others (both p<0.001). Multivariate analysis further demonstrated that HMGB1 was an independent prognostic factor for patients with SCCHN. CONCLUSIONS HMGB1 protein may contribute to the malignant progression of SCCHN, and present as a novel prognostic marker and a potential therapeutic target for patients with SCCHN.

miR-185-3p regulates nasopharyngeal carcinoma radioresistance by targeting WNT2B in vitro.

Aberrant microRNA (miRNA) expression contributes to a series of malignant cancer behaviors, including radioresistance. Our previous study showed differential expression of miR‐185‐3p in post‐radiation nasopharyngeal carcinoma (NPC) cells. To investigate the role of miR‐185‐3p in NPC radioresistance, CNE‐2 and 5‐8F cells were transfected with miR‐185‐3p mimic and miR‐185‐3p inhibitor, respectively. CCK‐8 assay and colony formation experiment confirmed that the expression of miR‐185‐3p affected the radioresistance of NPC cells. A negative correlation between miR‐185‐3p and WNT2B expression was observed in NPC cells and tissues. Luciferase reporter assays confirmed that miR‐185‐3p directly targeted the coding region of WNT2B. Furthermore, we found radioresistance decreased in WNT2B‐silenced NPC cells. Activation of the WNT2B/β‐catenin pathway was accompanied by epithelial–mesenchymal transition biomarker changes in NPC. We concluded that miR‐185‐3p contributed to the radioresistance of NPC via modulation of WNT2B expression in vitro.

Increased expression of metadherin protein predicts worse disease-free and overall survival in laryngeal squamous cell carcinoma.

Metadherin (MTDH) is involved in tumourigenesis and cancer progression in multiple human malignancies. However, the MTDH protein has rarely been reported in laryngeal squamous cell carcinoma (LSCC). The expression pattern of the MTDH protein in 176 primary archival LSCC and 27 corresponding adjacent noncarcinoma specimens was detected by immunohistochemistry and further correlated with clinicopathological parameters. The results demonstrated that 161 (91.48%) primary LSCC samples stained positive for MTDH; however, staining was barely detectable in all adjacent noncarcinoma samples. Moreover, the expression of the MTDH protein was significantly associated with the primary tumour site (p = 0.021), T classification (p = 0.002), clinical stage (I + II/III + IV; p < 0.001), lymph node metastasis (p < 0.001) and postoperational recurrence (p < 0.001). Kaplan‐Meier analysis revealed that MTDH expression was significantly associated with worse disease‐free survival (DFS) and overall survival (OS) rates in patients with LSCC (both p < 0.001). When lymph node metastasis and MTDH expression were considered together, patients with lymph node metastasis and high MTDH expression had both poorer DFS and OS rates than others (both p < 0.001). Finally, multivariate analysis demonstrated that MTDH expression was an independent prognostic factor for both DFS and OS rates in patients with LSCC. Strong MTDH expression was negatively correlated with a canonical epithelial–mesenchymal transition molecule E‐cadherin (p < 0.001) and positively associated with proangiogenic protein vascular endothelial growth factor (p < 0.001). MTDH overexpression was tightly associated with more aggressive tumour behaviour and a poor prognosis, indicating that MTDH is a valuable molecular biomarker for LSCC progression.

Inhibiting ERp29 expression enhances radiosensitivity in human nasopharyngeal carcinoma cell lines

ERp29 is an endoplasmic reticulum (ER) stress-inducible protein. It was found that ERp29 was highly expressed in several cancers and associated with resistance to oxidative and radiation stress, which may serve as a novel target for nasopharyngeal carcinoma (NPC) anticancer approach. In this study, we used immunohistochemistry to detect ERp29 expression in radioresistant and radiosensitive NPC tissues. As a result, ERp29 was up-regulated in radioresistant NPC tissues compared to radiosensitive NPC tissues. We also found that ERp29 knockdown attenuated radioresistance of NPC CNE-1 cells and ERp29 overexpression enhanced radioresistance of NPC CNE-2 cells. When exposed to radiation, ERp29 knockdown CNE-1 cells increased radiation-induced cell apoptosis and ERp29 overexpression CNE-2 cells reduced radiation-induced cell apoptosis. Further, we demonstrated that ERp29 up-regulated the expression of Hsp27. In conclusion, our study supports ERp29 could potentiate resistance to radiation in NPC cells, targeting of ERp29 is a rational strategy in treating radioresistant NPC.

Differential Gene Expression Profiling of Laryngeal Squamous Cell Carcinoma by Laser Capture Microdissection and Complementary DNA Microarrays

BACKGROUND AND AIMS Genetic alteration associated with initiation and progression of laryngeal squamous cell carcinoma (LSCC) is largely unknown. The aim of this study was to identify genetic changes associated with the disease pathogenesis and pinpoint genes whose expression is impacted by these genetic alterations. METHODS Tumor cells were collected from eight matched pairs of specimens of glottic carcinoma of the larynx and histologically normal epithelium tissues adjacent to the carcinoma by laser capture microdissection (LCM). RNAs prepared from these cells were used for genome-wide transcriptome analysis by probing 16 cDNA microarrays. Real-time quantitative RT-PCR and immunohistochemistry of tissue microarrays were used to validate a group of the differentially expressed genes identified by the cDNA microarrays. RESULTS Hierarchical cluster analysis of the expressed genes showed that 2351 genes were differentially expressed and could distinguish cancerous and noncancerous samples. We also found 761 differentially expressed genes that were consistently different between early stage and later stage specimens. Furthermore, abnormal expression of some relevant genes such as MMP12, HMGA2, and TIMP4 were validated by real-time quantitative RT-PCR and immunohistochemistry. Analysis of gene ontology and pathway distributions then highlighted genes that may be critically important to laryngeal carcinogenesis. CONCLUSIONS Our results suggest that using LCM plus DNA microarray analysis may facilitate the identification of clinical molecular markers for disease and novel potential therapeutic targets for LSCC.

Clinical significance of EphA2 expression in squamous-cell carcinoma of the head and neck

PurposeEphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in a variety of human cancers. The study aimed to assess EphA2 expression and to explore its roles in squamous-cell carcinoma of the head and neck (SCCHN).MethodsEphA2 expression in 98 primary SCCHN tissue specimens was analyzed by immunohistochemistry and correlated with clinicopathological parameters. Additionally, 13 paired SCCHN tissues and 6 SCCHN cell lines were evaluated for EphA2 expression by RT–PCR and immunoblotting.ResultsEphA2 overexpressed in SCCHN tissues and SCCHN cell lines. More importantly, high EphA2 expression was significantly associated with tumor site, T classification, clinical stage, recurrence, and lymph node metastasis, respectively. Patients with high EphA2 expression had both poorer disease-free survival and overall survival than patients with low EphA2 expression. Multivariate Cox regression analysis revealed that EphA2 overexpression was an independent prognostic factor for patients with SCCHN.ConclusionsThese findings suggested that EphA2 may contribute to SCCHN progression and represent a novel prognostic indicator for patients with SCCHN.

Downregulation of EphA2 expression suppresses the growth and metastasis in squamous-cell carcinoma of the head and neck in vitro and in vivo

PurposeOur previous study has revealed that EphA2 overexpression is significantly associated with aggressive behavior and poor prognosis in patients with squamous-cell carcinoma of the head and neck (SCCHN). However, the function of EphA2 in tumorigenesis and cervical lymph node metastasis of SCCHN has never been elucidated in vivo.MethodsEphA2 was knocked down in SCCHN cell lines. CCK-8 assays, fluorescence-activated cell sorting analysis, invasion and migration assays were performed in vitro. In vivo tumorigenicity assays were performed, and the impact on cervical lymph node metastasis was evaluated.ResultsThe present investigation demonstrated that suppression of EphA2 resulted in a significant inhibition of proliferation, migration, invasion of SCCHN cells in vitro and markedly diminished their tumorigenicity and lymph node metastasis in vivo.ConclusionsThese results suggest that EphA2 plays a critical role in SCCHN growth and metastasis and may be a promising therapeutic target to prevent the progression of SCCHN.

TGF-β1 mediates epithelial to mesenchymal transition via the TGF-β/Smad pathway in squamous cell carcinoma of the head and neck

Development of metastasis is a major cause of death for squamous cell carcinoma of the head and neck (SCCHN) patients. Epithelial to mesenchymal transition (EMT) is now regarded as a correlate of tumor metastasis. Given that transforming growth factor-β1 (TGF-β1) is an important inducer of EMT, we examined the effects of TGF-β1 on the human SCCHN cell line Tu686. We found that TGF-β1 mediated cell morphological changes. Phase-contrast microscopy revealed a loss of the adherent phenotype with cellular elongation, decrease in cell-to-cell contact, and the induction of a fibroblast-like state. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that TGF-β1 could induce down-regulation of the epithelial marker E-cadherin and up-regulation of the mesenchymal marker vimentin in Tu686 cells in a concentration- and time-dependent manner. Wound- healing and transwell invasion assay indicated that TGF-β1 promoted Tu686 cell migration and invasion dramatically. In addition, these changes were mediated via canonical TGF-β/Smad signaling with concomitant up-regulation of phosphorylated Smad2. Smad2 RNAi abrogated both expression and functional effects of TGF-β1 on Tu686 cells. In conclusion, the present study demonstrates that TGF-β1 could induce EMT in the SCCHN cell line via the TGF-β/Smad signaling pathway. More importantly, a cell model for EMT was established, which is valuable for future studies on the metastasis of SCCHN.

Increased expression of miR-93 is associated with poor prognosis in head and neck squamous cell carcinoma

MicroRNA-93-5p (miR-93) is a novel oncogenic microRNA (miRNA) and is elevated in diverse human malignancies. Aberrant expression and dysfunction of miR-93 are involved in many types of human tumours. However, the exact role of miR-93 remains unclear in head and neck squamous cell carcinoma (HNSCC). The objective of this study is to determine the expression pattern and clinical significance of miR-93 in HNSCC. MiR-93 expression levels in 103 primary HNSCC tissues and 16 corresponding non-cancerous epithelia were analysed by miRNA in situ hybridisation and correlated with the clinicopathological parameters and patient outcomes. Moreover, the expression of miR-93 was examined in four HNSCC cell lines and 17 pairs of HNSCC tissues and their corresponding adjacent tissues using quantitative real-time PCR (qRT-PCR). The miR-93 levels in HNSCC tissues and cell lines were significantly higher than those in the non-cancerous tissues. Notably, high miR-93 expression was significantly associated with T classification, lymph node metastasis and clinical stage. Kaplan–Meier survival analysis demonstrated that patients with high miR-93 expression had poorer overall survival than patients with low miR-93 expression. Multivariate Cox regression analysis revealed that miR-93 overexpression and lymph node metastasis were independent prognostic factors in patients with HNSCC. This study demonstrated that miR-93 expression was significantly increased in HNSCC tissue samples and cell lines and that miR-93 overexpression was associated with tumour progression, metastasis and poor prognosis in HNSCC patients. These results suggest that miR-93 may play a critical role in the initiation and progression of HNSCC, indicating that miR-93 may be a valuable marker for the prediction of metastasis and prognosis in HNSCC.

Genome-wide analyses of long noncoding RNA expression profiles correlated with radioresistance in nasopharyngeal carcinoma via next-generation deep sequencing

BackgroundRadioresistance is one of the major factors limiting the therapeutic efficacy and prognosis of patients with nasopharyngeal carcinoma (NPC). Accumulating evidence has suggested that aberrant expression of long noncoding RNAs (lncRNAs) contributes to cancer progression. Therefore, here we identified lncRNAs associated with radioresistance in NPC.MethodsThe differential expression profiles of lncRNAs associated with NPC radioresistance were constructed by next-generation deep sequencing by comparing radioresistant NPC cells with their parental cells. LncRNA-related mRNAs were predicted and analyzed using bioinformatics algorithms compared with the mRNA profiles related to radioresistance obtained in our previous study. Several lncRNAs and associated mRNAs were validated in established NPC radioresistant cell models and NPC tissues.ResultsBy comparison between radioresistant CNE-2-Rs and parental CNE-2 cells by next-generation deep sequencing, a total of 781 known lncRNAs and 2054 novel lncRNAs were annotated. The top five upregulated and downregulated known/novel lncRNAs were detected using quantitative real-time reverse transcription-polymerase chain reaction, and 7/10 known lncRNAs and 3/10 novel lncRNAs were demonstrated to have significant differential expression trends that were the same as those predicted by deep sequencing. From the prediction process, 13 pairs of lncRNAs and their associated genes were acquired, and the prediction trends of three pairs were validated in both radioresistant CNE-2-Rs and 6-10B-Rs cell lines, including lncRNA n373932 and SLITRK5, n409627 and PRSS12, and n386034 and RIMKLB. LncRNA n373932 and its related SLITRK5 showed dramatic expression changes in post-irradiation radioresistant cells and a negative expression correlation in NPC tissues (R = −0.595, p < 0.05).ConclusionsOur study provides an overview of the expression profiles of radioresistant lncRNAs and potentially related mRNAs, which will facilitate future investigations into the function of lncRNAs in NPC radioresistance.