Changyun Yu

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.

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.

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.

Metadherin regulation of vascular endothelial growth factor expression is dependent upon the PI3K/Akt pathway in squamous cell carcinoma of the head and neck.

AbstractOur previous study indicated overexpression of metadherin (MTDH) is an adverse prognostic factor in squamous cell carcinoma of the head and neck (SCCHN) and promotes SCCHN cell proliferation and invasion. However, its mechanism remains unclear. Recent studies have indicated that MTDH is a cancer-metastasis-associated molecule that participates in the process of angiogenesis. Therefore, the study is aimed to investigate that whether vascular endothelial growth factor (VEGF), as one of the most potent proangiogenic cytokines, is regulated by MTDH and the role of the phosphatidylinositide 3-kinases/Protein Kinase B (PI3K/Akt) pathway in this process of regulation and the clinical significance of both MTDH and VEGF in SCCHN.Immunohistochemistry was used to assay the expression of MTDH and VEGF in a cohort of 189 SCCHN patients with intact follow-up information. The expression of MTDH was then upregulated or inhibited by lentivirus-mediated MTDH Complementary deoxyribonucleic acid or MTDH short hairpin ribonucleic acid (shRNA) to observe the resulting alterations in VEGF expression and the PI3K/Akt signaling pathway in SCCHN cell lines. In addition, the PI3K/Akt pathway was modulated to observe the resulting changes in the MTDH-mediated expression of VEGF.The immunohistochemistry data showed that MTDH expression is positively correlated with VEGF expression in SCCHN tissues. Moreover, the overexpression of MTDH in SCCHN Tu686 and 5-8F cells led to increases in the expression of VEGF, and this effect was accompanied by activation of the PI3K/Akt pathway. Conversely, shRNA-mediated knockdown of MTDH led to decreased VEGF expression. In addition, inhibition of the Akt signaling pathway reversed the upregulation of VEGF resulting from MTDH overexpression. Moreover, the survival analysis revealed that VEGF is an independent prognostic factor, and a combined survival analysis based on both MTDH and VEGF showed synergistic effects in the prognosis evaluation of SCCHN patients.The findings of the present study demonstrate that MTDH regulates the expression of VEGF via the PI3K/Akt signaling pathway, indicating the potential role of the MTDH-mediated activation of VEGF signaling pathway in SCCHN angiogenesis and metastasis.

Quantitative iTRAQ LC-MS/MS Proteomics Reveals Transcription Factor Crosstalk and Regulatory Networks in Hypopharyngeal Squamous Cell Carcinoma

To date, no effective therapeutic treatments have been developed for hypopharyngeal squamous cell carcinoma (HPSCC), a disease that has a five-year survival rate of approximately 31% because of its late diagnosis and aggressive nature. Despite recent improvements in diagnostic methods, there are no effective measures to prevent or detect HPSCC in an early stage. The goal of the current study was to identify molecular biomarkers and networks that can facilitate the speedy identification of HPSCC patients who could benefit from individualized treatment. Isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed with two-dimensional liquid chromatography-tandem mass spectrometry to identify quantitatively the differentially expressed proteins among three types of HPSCC disease stages. The iTRAQ results were evaluated by literature searches and western blot analysis. For example, FUBP1, one of 412 proteins with significantly altered expression profiles, was confirmed to have elevated expression in fresh HPSCC tissues. Integrin-mediated cell matrix adhesion and actin filament-inducing cytoskeleton remodeling were the cellular events that were the most relevant to HPSCC tumorigenesis and the metastatic process. The construction of transcriptional regulation networks led to the identification of key transcriptional regulators of tumor development and lymph node metastasis of HPSCC, including Sp1, c-Myc and p53. Additionally, our study indicated that the interactions among Sp1, c-Myc and p53 may play vital roles in the carcinogenesis and metastasis of HPSCC.

Analysis of transcriptional factors and regulation networks in laryngeal squamous cell carcinoma patients with lymph node metastasis.

The present study was to identify and quantitate differentially expressed proteins in laryngeal squamous cell carcinoma (LSCC) tissues with or without lymph node metastasis and to explore transcriptional factors and regulation networks associated with the process. Tissue specimens were taken from 20 patients with LSCC, including 10 cases of LSCC without metastasis LSCC (N0) and 10 cases of LSCC with metastasis LSCC (Nx). Among the 643 unique proteins identified by using iTRAQ labeling and quantitative proteomic technology, 389 proteins showed an abundance change in LSCC (Nx) as compared to LSCC (N0). Cytoskeleton remodeling, cell adhesion, and immune response activation were found to be the main processes in LSCC metastasis. The construction of transcription regulation networks identified key transcription regulators for lymph node metastasis of LSCC, including Sp1, c-myc, and p53, which may affect LSCC metastasis through the epithelial-mesenchymal transition. Furthermore, our results suggest that ubiquitination may be a critical factor in the networks. The present study provides insights into transcriptional factors and regulation networks involved in LSCC metastasis, which may lead to new strategies for treatment of LSCC metastasis.

LncRNA PVT1 promotes malignant progression in squamous cell carcinoma of the head and neck

Long non-coding RNAs (lncRNAs) are potentially critical regulators of cancer malignant behaviours. Aberrant expression and dysfunction of lncRNA PVT1 have been reported in multiple human cancers. However, its role in squamous cell carcinoma of the head and neck (SCCHN) remains largely unknown. Our current study demonstrated that PVT1 expression was increased in SCCHN. High PVT1 expression was positively correlated with SCCHN clinical parameters including T classification, clinical stages and cervical lymph node metastasis. More importantly, high PVT1 expression predicted a poor prognosis in SCCHN patients. Gain-of function and loss-of function studies further indicated that PVT1 promoted the proliferation and invasion of SCCHN both in vitro and in vivo, which was accompanied by epithelial-mesenchymal transition and enhanced cancer stem cell-like properties. Further mechanistic investigation revealed that PVT1 activated Wnt/β-catenin signalling pathway, and inhibition of Wnt/β-catenin signalling reversed the malignant progression caused by PVT1 overexpression. Together, our study reveals that PVT1 accelerates the malignant progression of SCCHN and represents a potential biomarker and therapeutic target in SCCHN.