Invasion and metastasis are the major causes of death in patients with esophageal squamous cell carcinoma (ESCC). Epithelial-mesenchymal transition (EMT) is a critical step in tumor progression and transforming growth factor-β1 (TGF-β1) signaling has been shown to play an important role in EMT. In this study, we investigated how TGF-β1 signaling pathways contributed to EMT in three ESCC cell lines as well as 100 patients of nomadic ethnic Kazakhs residing in northwest Xinjiang Province of China. In vitro analyses included Western blotting to detect the expression of TGF-β1/Smad and EMT-associated proteins in Eca109, EC9706 and KYSE150 cell lines following stimulation with recombinant TGF-β1 and SB431542, a potent inhibitor of ALK5 that also inhibits TGF-β type II receptor. TGF-β-activated Smad2/3 signaling in EMT was significantly upregulated as indicated by mesenchymal markers of N-cadherin and Vimentin, and in the meantime, epithelial marker, E-cadherin, was markedly downregulated. In contrast, SB431542 addition downregulated the expression of N-cadherin and Vimentin, but upregulated the expression of E-cadherin. Moreover, the TGF-β1-induced EMT promoted invasion capability of Eca109 cells. Tumor cells undergoing EMT acquire fibroblastoid-like phenotype. Expressed levels of TGF-β1/Smad signaling molecules and EMT-associated proteins were examined using immunohistochemical analyses in 100 ESCC tissues of Kazakh patients and 58 matched noncancerous adjacent tissues. The results showed that ESCC tissues exhibited upregulated expression of TGF-β1/Smad. We also analyzed the relationship between the above proteins and the patients' clinicopathological characteristics. The TGF-β1/Smad signaling pathway in human Eca109 ESCC cells may carry similar features as in Kazakh ESCC patients, suggesting that TGF-β1/Smad signaling pathway may be involved in the regulation of EMT in ethnic Kazakh patients with ESCC from Xinjiang, China.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is associated with enhanced tumorigenicity in many cancers. A recent study has revealed that MT1-MMP induces epithelial-to-mesenchymal transition (EMT) in prostate and breast cancer cells. However, its role in esophageal squamous cell carcinoma (ESCC) has not been studied. Here, we investigated the role of MT1-MMP in the dissemination of ESCC. Expression of MT1-MMP was detected by immunohistochemistry and tissue microarray in 88 Kazakh ESCC patients. Western blotting was performed to detect endogenous and overexpressed exogenous MT1-MMP in the Eca109 and Eca9706 cell lines, respectively. Transwell assay was used to estimate MT1-MMP-induced invasion and metastasis. EMT-associated proteins were detected by immunohistochemistry and western blotting. The associations between the expression of MT1-MMP and EMT-associated proteins with clinicopathologic parameters were analyzed. Overexpression of MT1-MMP was confirmed in Kazakh ESCC patients. MT1-MMP levels were found to be correlated with the depth of tumor infiltration. MT1-MMP induced EMT in ESCC both in vivo and in vitro, N-cadherin and Vimentin expression was upregulated upon MT1-MMP transfection into cells. However, E-cadherin was found to be downregulated. MT1-MMP-induced EMT led to increase migration and invasion in ESCC cell lines. In conclusion, our results suggest that MT1-MMP promotes ESCC invasion and metastasis.

Restenosis or occlusion after vascular procedures is ascribed to intimal hyperplasia. Transforming growth factor (TGF)-β1 is involved in recruitment of mesenchymal stem cells (MSCs) following arterial injury, and its release from latent TGF-binding protein by matrix metalloproteinase (MMP)-14-induced proteolysis contributes to neointima formation. However, the relationship between MMP-14 and TGF-β1 activation in restenosis is unknown. This study investigated the relationship using a rat model of balloon-induced injury. Rats were assigned to vehicle-, SB431542 (SB)-, or recombinant human (rh)TGF-β1-treated groups and examined at various time points after balloon-induced injury for expression of TGF-β1/Smad signalling pathway components, MMP-14 and MSCs markers including Nestin, CD29, and Sca1+CD29+CD11b/c−CD45−. Intimal hyperplasia was reduced in SB- and rhTGF-β1-treated rats. The expression of TGF-β1, TGF-β1RI, and Smad2/3 was decreased, but the levels of phosphorylated Smad2/3 were higher in SB-treated rats than vehicle-treated after 7 days to 14 days. rhTGF-β1 administration decreased the expression of TGF-β1/Smad pathway proteins, except for TGF-β1RI. Nestin and CD29 expression and the number of Sca1+CD29+CD11b−CD45− cells were reduced, whereas MMP-14 expression was increased after SB431542 and rhTGF-β1 administration. These results suggest that TGF-β1/Smad signalling and MMP-14 act to recruit MSCs which differentiate to vascular smooth muscle cells and mesenchymal-like cells that participate in arterial repair/remodelling.