miR-200b suppresses invasiveness and modulates the cytoskeletal and adhesive machinery in esophageal squamous cell carcinoma cells via targeting Kindlin-2.

To further our understanding of the pathobiology of esophageal squamous cell carcinoma (ESCC), we previously performed microRNA profiling that revealed downregulation of miR-200b in ESCC. Using quantitative real-time PCR applied to 88 patient samples, we confirmed that ESCC tumors expressed significantly lower levels of miR-200b compared with the respective adjacent benign tissues (P = 0.003). Importantly, downregulation of miR-200b significantly correlated with shortened survival (P = 0.025), lymph node metastasis (P = 0.002) and advanced clinical stage (P = 0.020) in ESCC patients. Quantitative mass spectrometry identified 57 putative miR-200b targets, including Kindlin-2, previously implicated in the regulation of tumor invasiveness and actin cytoskeleton in other cell types. Enforced expression of miR-200b mimic in ESCC cells led to a decrease of Kindlin-2 expression, whereas transfection of miR-200b inhibitor induced Kindlin-2 expression. Furthermore, transfection of miR-200b mimic or knockdown of Kindlin-2 in ESCC cells decreased cell protrusion and focal adhesion (FA) formation, reduced cell spreading and invasiveness/migration. Enforced expression of Kindlin-2 largely abrogated the inhibitory effects of miR-200b on ESCC cell invasiveness. Mechanistic studies revealed that Rho-family guanosine triphosphatases and FA kinase mediated the biological effects of the miR-200b-Kindlin-2 axis in ESCC cells. To conclude, loss of miR-200b, a frequent biochemical defect in ESCC, correlates with aggressive clinical features. The tumor suppressor effects of miR-200b may be due to its suppression of Kindlin-2, a novel target of miR-200b that modulates actin cytoskeleton, FA formation and the migratory/invasiveness properties of ESCC.

Down-regulated desmocollin-2 promotes cell aggressiveness through redistributing adherens junctions and activating beta-catenin signalling in oesophageal squamous cell carcinoma.

In contrast to the well-recognized loss of adherens junctions in cancer progression, the role of desmosomal components in cancer development has not been well explored. We previously demonstrated that desmocollin-2 (DSC2), a desmosomal cadherin protein, is reduced in oesophageal squamous cell carcinoma (ESCC), and is associated with enhanced tumour metastasis and poor prognosis. Here, we report that restoration of DSC2 in ESCC cells impeded cell migration and invasion both in vitro and in vivo, whereas siRNA-mediated suppression of DSC2 expression increased cell motility. In E-cadherin-expressing ESCC cells, DSC2 restoration strengthened E-cadherin-mediated adherens junctions and promoted the localization of ß-catenin at these junctions, which indirectly inhibited ß-catenin-dependent transcription. These effects of DSC2 were not present in EC109 cells that lacked E-cadherin expression. ESCC patients with tumours that had reduced E-cadherin and negative DSC2 had poorer clinical outcomes than patients with tumours that lacked either E-cadherin or DSC2, implying that the invasive potential of ESCC cells was restricted by both DSC2 and E-cadherin-dependent junctions. Further studies revealed that DSC2 was a downstream target of miR-25. Enhanced miR-25 promoted ESCC cell invasiveness, whereas restoration of DSC2 abolished these effects. Collectively, our work suggests that miR-25-mediated down-regulation of DSC2 promotes ESCC cell aggressiveness through redistributing adherens junctions and activating beta-catenin signalling.

Empirical study on clique-degree distribution of networks.

The community structure and motif-modular-network hierarchy are of great importance for understanding the relationship between structures and functions. We investigate the distribution of clique degrees, which are an extension of degree and can be used to measure the density of cliques in networks. Empirical studies indicate the extensive existence of power-law clique-degree distributions in various real networks, and the power-law exponent decreases with an increase of clique size.

SMYD3 stimulates EZR and LOXL2 transcription to enhance proliferation, migration, and invasion in esophageal squamous cell carcinoma.

Epigenetic alterations, including DNA methylation and histone modifications, are involved in the regulation of cancer initiation and progression. SET and MYND domain-containing protein 3 (SMYD3), a methyltransferase, plays an important role in transcriptional regulation during human cancer progression. However, SMYD3 expression and its function in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, SMYD3 expression was studied by immunohistochemistry in a tumor tissue microarray from 131 cases of ESCC patients. Statistical analysis showed that overall survival of patients with high SMYD3 expressing in primary tumors was significantly lower than that of patients with low SMYD3-expressing tumors (P = .008, log-rank test). Increased expression of SMYD3 was found to be associated with lymph node metastasis in ESCC (P = .036) and was an independent prognostic factor for poor overall survival (P = .025). RNAi-mediated knockdown of SMYD3 suppressed ESCC cell proliferation, migration, and invasion in vitro and inhibited local tumor invasion in vivo. SMYD3 regulated transcription of EZR and LOXL2 by directly binding to the sequences of the promoter regions of these target genes, as demonstrated by a chromatin immunoprecipitation assay. Immunohistochemical staining of ESCC tissues also confirmed that protein levels of EZR and LOXL2 positively correlated with SMYD3 expression, and the Spearman correlation coefficients (rs) were 0.78 (n = 81; P < .01) and 0.637 (n = 103; P < .01), respectively. These results indicate that SMYD3 enhances tumorigenicity in ESCC through enhancing transcription of genes involved in proliferation, migration, and invasion.

Overexpression of Jumonji AT-rich interactive domain 1B and PHD finger protein 2 is involved in the progression of esophageal squamous cell carcinoma.

Jumonji AT-rich interactive domain 1B (JARID1B) and PHD finger protein 2 (PHF2), members of the histone demethylases, have been found to be involved in many types of tumors. However, the expression and prognostic significance of JARID1B and PHF2 in esophageal squamous cell carcinoma (ESCC) still remains unclear. In this study, JARID1B and PHF2 expression were detected on tissue microarrays of ESCC samples in 120 cases using immunohistochemical staining. Our results showed that JARID1B and PHF2 were overexpressed in ESCCs. In addition, a significant correlation was observed between JARID1B nuclear expression level and histological grade (P=0.003). Kaplan-Meier survival analysis showed a tendency that high cytoplasmic expression of JARID1B and PHF2 was associated with decreased overall survival of ESCC patients, whereas JARID1B high expression in the nucleus was associated with high overall survival, although there was no statistical significance. Overall, our data suggest that JARID1B and PHF2 are overexpressed in ESCC and that they may play crucial roles in the course of ESCC initiation and/or progression.

DACT2 is a candidate tumor suppressor and prognostic marker in esophageal squamous cell carcinoma.

In animals ranging from fish to mice, the function of DACT2 as a negative regulator of the TGF-ß/Nodal signal pathway is conserved in evolution, indicating that it might play an important role in human cancer. In this study, we showed that tumors with higher DACT2 protein level were correlated with better differentiation and better survival rate in patients with esophageal squamous cell carcinoma. Restored expression of DACT2 significantly inhibited growth, migration, and invasion of ESCC cells in vitro, and reduced tumorigenicity in vivo. Furthermore, when DACT2 expression was restored, the activity of TGF-ß/SMAD2/3 was suppressed via both proteasome and lysosomal degradation pathways, leading to F-actin rearrangement that might depend on the involvement of cofilin and ezrin-redixin-moesin (ERM) proteins. Taken together, we propose here that DACT2 serves as a prognostic marker that reduces tumor cell malignancy by suppressing TGF-ß signaling and promotes actin rearrangement in ESCC.