Yongquan Tian

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.

MicroRNA-324-3p regulates nasopharyngeal carcinoma radioresistance by directly targeting WNT2B.

PURPOSE Radioresistance severely restricts the clinical treatment of nasopharyngeal carcinoma (NPC). Accumulating evidence demonstrates that aberrant expression of microRNAs (miRNAs) contributes to cancer progression and sensitivity to radiation. Therefore, we aimed to identify miRNAs associated with radioresistance in NPC. METHODS Aberrant miRNA-324-3p expression in NPC CNE-2 cells with radioresistance (CNE-2-Rs), compared to its parental cells, was screened by high-throughput sequencing technology and determined by quantitative reverse transcription-polymerase chain reaction analysis (qRT-PCR) analysis. Bioinformatic analysis was used to predict the downstream target genes of miRNA-324-3p. Then, functional and mechanical analyses of miRNA-324-3p in NPC radioresistance were performed by overexpression and down-regulation of miRNA-324-3p in CNE-2-Rs cells and its parental cells. Finally, the clinical significance of miRNA-324-3p and WNT2B was investigated in NPC tissues. RESULTS Our data reveal that the expression of miRNA-324-3p is significantly decreased in CNE-2-Rs cells compared to its parental cells, and WNT2B is predicted to be the downstream target of miRNA-324-3p. Both overexpression and down-regulation of miRNA-324-3p following irradiation result in radiosensitivity alterations and protein changes of WNT2B signalling pathway in CNE-2-Rs cells and its parental cells. Importantly, down-regulation of miRNA-324-3p and up-regulation of WNT2B are significantly correlated with advanced clinical stages of NPC and this inverse expression pattern is also observed in NPC tissues before and after irradiation. CONCLUSIONS The present study reveals that miRNA-324-3p contributes to the radioresistance of NPC by regulating the WNT2B signalling pathway. Both miRNA-324-3p and WNT2B are potential biomarkers for radioresistance in NPC, which may serve as valuable targets for reversing radioresistance in the management of NPC.

Genome-Wide Analyses of Radioresistance-Associated miRNA Expression Profile in Nasopharyngeal Carcinoma Using Next Generation Deep Sequencing

Background Rapidly growing evidence suggests that microRNAs (miRNAs) are involved in a wide range of cancer malignant behaviours including radioresistance. Therefore, the present study was designed to investigate miRNA expression patterns associated with radioresistance in NPC. Methods The differential expression profiles of miRNAs and mRNAs associated with NPC radioresistance were constructed. The predicted target mRNAs of miRNAs and their enriched signaling pathways were analyzed via biological informatical algorithms. Finally, partial miRNAs and pathways-correlated target mRNAs were validated in two NPC radioreisitant cell models. Results 50 known and 9 novel miRNAs with significant difference were identified, and their target mRNAs were narrowed down to 53 nasopharyngeal-/NPC-specific mRNAs. Subsequent KEGG analyses demonstrated that the 53 mRNAs were enriched in 37 signaling pathways. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-324-3p, miR-93-3p and miR-4501), 3 up-regulated miRNAs (miR-371a-5p, miR-34c-5p and miR-1323) and 2 novel miRNAs. Additionally, corresponding alterations of pathways-correlated target mRNAs were observed including 5 up-regulated mRNAs (ICAM1, WNT2B, MYC, HLA-F and TGF-β1) and 3 down-regulated mRNAs (CDH1, PTENP1 and HSP90AA1). Conclusions Our study provides an overview of miRNA expression profile and the interactions between miRNA and their target mRNAs, which will deepen our understanding of the important roles of miRNAs in NPC radioresistance.

Bone Morphogenetic Protein-4-induced Epithelial-Mesenchymal Transition and Invasiveness Through Smad1-mediated Signal Pathway in Squamous Cell Carcinoma of the Head and Neck

BACKGROUND AND AIMS Bone morphogenetic proteins (BMPs) have recently been shown to be involved in the genesis and progression of a wide variety of carcinomas. The present study was undertaken to estimate the effect of BMP-4 on squamous cell carcinoma of the head and neck (SCCHN) in tissue and cell levels. METHODS In this study, immunohistochemistry, Western blotting and RT-PCR were utilized to detect the expression of BMP-4, Smad1 and phosphorylated Smad1 in SCCHN tissues or SCCHN cell lines. Those three proteins in tissues were further correlated with prognosis of SCCHN by Kaplan-Meier analysis. The epithelial-mesenchymal transition (EMT)-associated changes in SCCHN cells were detected after stimulation by human BMP-4 recombinant protein and knockdown of Smad1 gene. Meanwhile, the effect on invasiveness and migration was evaluated by invasion and scratch assays, respectively. RESULTS BMP-4 and p-Smad1 protein were overexpressed in SCCHN tissues with cervical lymph node metastasis, which was significantly higher than those without metastasis. The expression of BMP-4 and p-Smad1 protein was negatively correlated with the prognosis of SCCHN. BMP-4 promoted the invasiveness and migration through EMT, which was demonstrated by morphological alterations, loss of E-cadherin, increase of vimentin and activation of the Smad1 signal pathway. Knockdown of Smad1 expression suppressed BMP-4 induced EMT in both cell lines and weakened the invasiveness and migration of Tu686 and Tu212 in vitro. CONCLUSIONS Our results demonstrate that BMP-4 protein may contribute to the malignant metastasis of SCCHN, which presents 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.

Metadherin regulates metastasis of squamous cell carcinoma of the head and neck via AKT signalling pathway-mediated epithelial-mesenchymal transition.

Our recent study suggested that metadherin (MTDH) is overexpressed in laryngeal squamous cell carcinoma. Here, we further investigated its role in promoting metastasis of squamous cell carcinoma of the head and neck (SCCHN). An immunohistochemistry analysis demonstrated that MTDH is elevated and positively correlated with metastasis in 189 primary SCCHN tissues. In vitro experiments demonstrated that MTDH overexpression enhanced the migratory and invasive ability of SCCHN cells. Moreover, MTDH induced epithelial-mesenchymal transition (EMT) by both regulating morphological changes and mediating the expression of the biomolecular makers E-cadherin and vimentin. In addition, MTDH mediated AKT activation, and all of the above effects were nearly completely blocked by the inhibition of AKT. Our results suggested that MTDH might promote the metastasis of SCCHN through AKT signalling pathway mediated-EMT.

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.