| Charpter1Clinical significance of MTDH expression in squamous cell carcinoma of the head and neckObjective Metadherin(MTDH), as a novel oncogene, is elevated in diverse human malignancies. Aberrant expression and dysfunction of MTDH has been involved in promoting invasion and metastasis in a wide range of solid human tumors. However, the exact role of MTDH is not clear enough in progresssion of squamous cell carcinoma of the head and neck (SCCHN). The aim of the present study is to investigate the expression pattern of MTDH in SCCHN, and its role in progression of SCCHN.Methods MTDH, E-cadherin and vascular endothelial growth factor (VEGF) expression in189primary SCCHN tissues and27corresponding adjacent noncarcinoma specimens were analyzed by immuno-histochemistry and correlated with clinicopathological parameters and patient outcome. In addition, the correlations between MTDH and E-cadherin, VEGF were examined.Results Elevated MTDH protein expression was detected in SCCHN tissues. MTDH expression was significantly correlated with primary tumor site(P=0.033), T classification(P=0.001), clinical stage(P=0.000), lymph node metastasis(P=0.000), postoperational recurrence(P=0.000). Kaplan-Meier survival analysis demonstrated that patients with high MTDH expression had increased risk of overall recurrence and mortality. The5-year overall survival rates(OS) and disease free survival rates(DSF) of high and low MTDH expression groups were37.6%and72.6%,31.8%and68.6%, respectively. Log-rank tests further confirmed that the difference between5-year DSF and OS rates in these2groups was statistically significant(χ2=20.135, P<0.001;χ2=22.058, P<0.001). And multivariate Cox regression analysis demonstrated that MTDH ws the prognostic factor of SCCHN. Spearman rank test indicated that the expression of MTDH was negatively correlated with E-cadherin expression (r=-0.336, P<0.001), and positively correlated with VEGF expression(r=0.319, P<0.001).Conclusion MTDH is elevated in SCCHN and significantly correlated with primary tumor site, T classification, clinical stage, lymph node metastasis, postoperational recurrence. MTDH expression can be a valuable prognostic biomarker of SCCHN. In addition, MTDH expression is negatively correlated with E-cadherin expression, and positively correlated with VEGF expression, which suggests that MTDH may mediate EMT and angiogenesis. Charpter2MTDH mediates invasion of squamous cell carcinoma of the head and neck via EMTObjective Our previous study has demonstrated that MTDH is associated with tumor metastasis and E-cadherin(biomarker of EMT) expression, suggesting the significant role of MTDH in mediating EMT in SCCHN. So we further evaluate the affection of MTDH on invasion and EMT in SCCHN cells.Methods The plasmid lenti-MTDHcDNA and plasmid lenti-MTDH-shRNA were employed to up-and down-regulate MTDH expression in SCCHN cell line Tu686. Then, the morphology change was observed under phase-contrast microscopy. Western blot was conducted to examine the expression of EMT makers (E-cadherin and vimentin). Wound-healing assay and transwell invasion assay were carried out to evaluate the migration and invasion ability of SCCHN cell line.Results Stable cell lines-expressing MTDH or MTDH RNAis and vectors were established and terms as Tu686MTDHpcDNA(+) and Tu686MTDHpcDNA(-), Tu686MTDHRNAi(+) and Tu686MTDHRNAi(-), respectively. Phase-contrast microscopy revealed that Tu686MTDHpcDNA(+) cells undergone morphological change, which accompanying loss of the adherent phenotype with decreased in cell-to-cell contact, and the induction of a fibroblast-like state. Western blot analysis showed that epithelial marker E-cadherin was downregulated while the mesenchymal maker vimentin was upregulated in Tu686MTDHpcDNA(+) cells Wound healing assay and transwell invasion assay indicated that migration and invasion of Tu686MTDHpcDNA(+) cells were greatly enhanced compared with the Tu686MTDHpcDNA(-) cells and Tu686cells(84±15%vs15±14%and18±8%, P<0.05;317±8vs224±15and214±6, P<0.05). Conversely, inhibited expression of MTDH caused an upregulation of E-cadherin, decreased migration and invasion ability(9±4%vs29±5%and28±6%, P<0.05;139±12vs250±20and241±24, P<0.05)。Conclusion MTDH can induce SCCHN cell migration and invasion via EMT. Charpter3MTDH mediates the expression of VEGF in squamous cell carcinoma of the head and neckObjective Angiogenesis is a fundamental process in tumor progression. Our previous study has demonstrated that MTDH expression was positively correlated with VEGF expression, suggesting the close relationship between MTDH and angiogenesis. So we further examined the affection of MTDH on VEGF expression in SCCHN.Methods Stable cell lines-expressing MTDH or MTDH RNAis and vectors were established in previous study. Then, the expression level of VEGF was investigated by western blot and ELISA assays.Results Western blot analysis showed that VEGF expression was upregulated in Tu686MTDHpcDNA(+) cells and downregulated in Tu686MTDHRNAi(+) cells. In addition, quantitation of secreted VEGF was increased in Tu686MTDHpcDNA(+) cells and decreased in Tu686MTDHRNAi(+) cells. The concentration (pg/ml) of VEGF protein in supernatants of Tu686MTDHpcDNA(+) cells, Tu686MTDHpcDNA(-) cells and Tu686cells were498.65±32.19,324.14±33.66and334.93±42.26, respectively. The concentration(pg/ml) of VEGF protein in supernatants of Tu686MTDHRNAi(+) cell,Tu686MTDHRNAi(-) cells,and Tu686cells were267.71±41.39,427.19±60.47,412.48±35.72, respectively.Conclusion MTDH can mediate the expression of VEGF, which suggests that MTDH may induce angiogenesis in SCCHN. Charpter4MTDH mediates EMT and expression of VEGF in squamous cell carcinoma of the head and neck via AKT signaling pathwayObjective To examine the mechanism of MTDH mediates EMT and VEGF expression in SCCHN.Methods Stable cell lines-expressing MTDH or MTDH RNAis and vectors were established in previous study. And western blot was conducted to detect the expression of AKT and p-AKT. siRNA was employed to knock down the AKT expression in. Then, the expression of EMT biomakers(E-cadherin and vimentin) was measured by western blot assay. Wound-healing assay and transwell invasion assay were carried out to evaluate the migration and invasion ability of Tu686MTDHpcDNA(+) cells. In addition, the expression of VEGF was investigated by western blot assay and ELISA assay, too.Results Ectopic expression of MTDH in Tu686cells could induce AKT and p-AKT upregulation, inhibited expression of MTDH caused downregulation of AKT and p-AKT. Downregulation of E-cadherin and upregulation of vimentin induced by MTDH was obviously attenuated by AKT RNAi. Also, migration and invasion induced by MTDH were blocked when AKT inhibited (78±15%vs11±5%, P<0.05;274±22vs157±22, P<0.05). Besides, the expression of VEGF was downregulated as well. The concentration(pg/ml) of VEGF protein in supernatants of Tu686MTDHRNAi(+) cells,Tu686MTDHRNAi(-) AKT RNAi cells were535.27±47.72,255.60±39.89, respectively(P<0.05).Conclusion MTDH can active AKT signaling pathway. Moreover, MTDH can mediate EMT and expression of VEGF in SCCHN via AKT signaling pathway. |
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