Xiu-E Xu

Involvement of CYR61 and CTGF in the fascin-mediated proliferation and invasiveness of esophageal squamous cell carcinomas cells.

Fascin is overexpressed in esophageal squamous cell [corrected] carcinoma (ESCC) and involved in the proliferation and invasiveness of ESCC cells. In this study, we retrospectively examined the expression of fascin in ESCC samples by immunohistochemistry and revealed that overexpression of fascin was related to poor patient survival. RNAi-mediated knockdown of fascin in ESCC cells significantly inhibited cell proliferation and invasiveness, whereas forced expression of fascin in immortalized esophageal epithelial cells accelerated cell proliferation and invasiveness. To explore the underlying mechanism, cDNA microarray was performed to identify the differential gene expression profiles between a fascin-depleted cell line by RNAi and the corresponding control ESCC cells. Results showed that 296 genes were differentially expressed on fascin depletion. In this study, we focused on two down-regulated genes: CYR61 and CTGF. We found that restored expression of either CYR61 or CTGF led to a recovery of the suppression of cellular proliferation and invasiveness induced by down-regulation of fascin expression; the protein level of CYR61 and CTGF were up-regulated in ESCCs and their expression pattern correlated with fascin overexpression. Finally, analysis of signal transduction revealed that fascin affected the expressions of CYR61 and CTGF through transforming growth factor (TGF)-beta pathway. Taken together, we propose that fascin regulates the proliferation and invasiveness of ESCC cells by modulating the expression of CTGF and CYR61 via TGF-beta pathway.

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 β‐catenin at these junctions, which indirectly inhibited β‐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. Copyright

Neutrophil gelatinase-associated lipocalin and its receptor: independent prognostic factors of oesophageal squamous cell carcinoma

Aim Previous studies have shown that neutrophil gelatinase-associated lipocalin (NGAL) is overexpressed in oesophageal squamous cell carcinoma (ESCC) and closely associated with the invasiveness of ESCC cells. Recently, NGAL receptor (NGALR) was identified from ESCC cells, and was also found to be increased in ESCC. The purpose of this study was to reveal the clinical significance of NGAL and/or NGALR in ESCC. Methods Tissue microarray was performed to detect expression of NGAL and NGALR in 222 ESCC specimens. Pearson χ2 test was used to analyse correlations between NGAL and/or NGALR expression and clinicopathological features. Kaplan–Meier survival curves and the Cox proportional hazards regression model were used to evaluate the effect of NGAL and/or NGALR expression on prognosis of patients with ESCC. Results NGAL and NGALR were highly expressed in ESCC. χ2 test results showed no significant correlations between NGAL or NGALR expression and clinicopathological features. However, NGAL/NGALR coexpression correlated with histological differentiation grade (p=0.033). Survival analysis showed that positive expression of NGAL or NGALR was significantly associated with a poor prognosis for patients with ESCC (p=0.000 or p=0.002). Patients with positive expression of both NGAL and NGALR had a shorter survival time than those with negative expression of both (p=0.048). Multivariate analysis showed that both NGAL and NGALR were independent prognostic factors. Conclusion These results indicate that both NGAL and NGALR may be involved in the progression of ESCC and can be considered as independent prognostic factors of ESCC.

Phosphorylation of Fascin Decreases the Risk of Poor Survival in Patients With Esophageal Squamous Cell Carcinoma

Phosphorylation of fascin at serine 39 (phospho-S39-fascin) could inhibit its actin-binding and actin-bundling activities and decrease filopodia formation. However, the relationship between phospho-S39-fascin expression and clinicopathological parameters in tumors is still unknown. Here, Western blot analysis and IHC applied to tissue microarray technology were performed to examine the expression status of non-phosphorylated fascin (fascin) and phospho-S39-fascin and their impacts on the prognosis of patients with esophageal squamous cell carcinoma (ESCC). Fascin and phospho-S39-fascin expressions were tested by cytoplasmic staining. Among the 254 patients, 90 cases showed high expression of fascin and 87 cases showed high expression of phospho-S39-fascin. Survival analysis showed that high expression of fascin was significantly associated with a poor prognosis of the patients with ESCC (p = 0.004). In contrast, high expression of phospho-S39-fascin correlated significantly with an improved outcome of patients (p = 0.020). Multivariate analysis showed that both fascin and phospho-S39-fascin were independent prognostic factors. In a combined analysis, the patients with high expression of fascin and low expression of phospho-S39-fascin tumors had a shorter overall survival than those with high expression of both fascin and phospho-S39-fascin tumors (5-year overall survival rate: 28.7% vs 48.3%, p = 0.068). Our results suggest that high expression of fascin correlates with poor outcome and that high expression of phospho-S39-fascin decreases the risk of poor prognosis in ESCC. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

The expression of CFL1 and N-WASP in esophageal squamous cell carcinoma and its correlation with clinicopathological features

Cofilin1 (CFL1) is an actin-modulating protein, which belongs to the ADF/Cofilin family. Neural Wiskott-Aldrich syndrome protein (N-WASP) is the key regulator of the actin cytoskeleton, a member of Wiskott-Aldrich syndrome protein family. They have been suggested to be involved in cancer cell invasion and metastasis. In this study, the expression patterns of CFL1 and N-WASP in normal esophageal mucosa and esophageal squamous cell carcinoma (ESCC) and their correlation with clinical characteristics were investigated. Immunohistochemical staining showed that CFL1 was expressed in nuclear and cytoplasm of cancer cells. However, N-WASP was mainly found in the cytoplasm of the cancer cells. There were significant evidences that proved that CFL1 is correlated with clinicopathological factors in ESCC, such as infiltration depth, lymph node metastasis and pathological staging (P < 0.05). It is also proved that N-WASP is related to lymph node metastasis and pathological staging in ESCC (P < 0.05). Kaplan-Meier analysis showed that there was no correlation between CFL1 and N-WASP protein expression and survival (P > 0.05). Moreover, the mRNA expression of CFL1 and N-WASP was detected by quantitative real time PCR in 70 tissue specimens. The results showed that CFL1 mRNA level was over-expressed in ESCC tissue (P < 0.05), while N-WASP mRNA expression level was not different between cancerous tissues and adjacent normal esophageal mucosa (P > 0.05). Also, CFL1 mRNA expression was significantly associated with regional lymph node metastasis and pathological staging (P < 0.05). Kaplan-Meier analysis showed that there was no correlation between CFL1 and N-WASP mRNA expression and survival (P > 0.05). Our findings suggested that CFL1 and N-WASP may play an important role in the tumorigenesis of ESCC, and to be the candidate novel biomarkers for the diagnosis and prognosis of ESCC. These findings may have implications for targeted therapies in patients with ESCC.

The PI3K/AKT/c‐MYC Axis Promotes the Acquisition of Cancer Stem‐Like Features in Esophageal Squamous Cell Carcinoma

The importance of intratumoral heterogeneity has been highlighted by the identification and characterization of cancer stem cells (CSCs). Based on the differential responsiveness to a Sox2 reporter, SRR2, we had found a novel dichotomy in esophageal squamous cell carcinoma (ESCC) cells, with reporter‐responsive (RR) cells showing more CSC‐like features than reporter‐unresponsive (RU) cells. Specifically, RR cells exhibited significantly higher tumorsphere formation capacity, proportions of CD44High cells, chemoresistance to cisplatin, and tumorigenic potential in vivo. H2O2, a potent inducer of oxidative stress and reactive oxygen species, was found to induce a conversion from RU to RR cells; importantly, converted RR cells acquired CSC‐like features. The PI3K/AKT/c‐MYC signalling axis is important in this context, since pharmacologic blockade of PI3K‐AKT or siRNA knockdown of c‐MYC effectively inhibited the RR phenotype and its associated CSC‐like features, as well as the H2O2‐induced RU/RR conversion. In a cohort of 188 ESCC patient samples, we found a significant correlation between strong c‐MYC expression and a short overall survival (p = .009). In conclusion, we have described a novel intratumoral heterogeneity in ESCC. The identification of the PI3K/AKT/c‐MYC axis as a driver of CSC‐like features carries therapeutic implications. Stem Cells 2016;34:2040–2051

Reduced nuclear and ectopic cytoplasmic expression of lysyl oxidase-like 2 is associated with lymph node metastasis and poor prognosis in esophageal squamous cell carcinoma ☆

Lysyl oxidase family members have various roles in cancer progression. The aim of this study was to investigate their expression and clinical significance in esophageal squamous cell carcinoma. We examined messenger RNA expression of lysyl oxidase family members including lysyl oxidase and lysyl oxidase-like proteins (lysyl oxidase L) in 10 esophageal squamous cell carcinoma cell lines and 83 pairs of tumor samples by quantitative real-time polymerase chain reaction. All except lysyl oxidase L3 were expressed at high levels in esophageal squamous cell carcinoma, but only lysyl oxidase L2 was associated with lymph node metastasis (P = .034). We examined lysyl oxidase L2 protein further by immunohistochemistry staining in 178 surgically resected esophageal squamous cell carcinoma tissue samples. The protein manifested decreased nuclear expression and increased cytoplasmic expression. Moreover, these 2 events both had significant correlation with the presence of lymph node metastasis (P = .001 and P < .001). Overall survival rates of the patients with esophageal squamous cell carcinoma with decreased nuclear expression or increased cytoplasmic expression of lysyl oxidase L2 were significantly lower than those of the patients with esophageal squamous cell carcinoma with the reverse expression pattern (P = .040 or P = .022). Multivariate analyses revealed that nuclear expression of lysyl oxidase L2 was an independent prognostic factor for esophageal squamous cell carcinoma. These results suggest that lysyl oxidase L2 exerts a critical effect on esophageal squamous cell carcinoma progression and can be a predictive marker of lymph node metastasis and outcome.

Prognostic implication of ezrin expression in esophageal squamous cell carcinoma

Ezrin, a member of the ezrin–radixin–moesin family, was implicated in tumor progression, metastatic dissemination, and adverse outcomes in several cancer types, including esophageal squamous cell carcinoma (ESCC). The purpose of this study was to explore the clinical significance of ezrin expression in ESCC.

Original article: The expression of CFL1 and N‐WASP in esophageal squamous cell carcinoma and its correlation with clinicopathological features

Cofilin1 (CFL1) is an actin-modulating protein, which belongs to the ADF/Cofilin family. Neural Wiskott-Aldrich syndrome protein (N-WASP) is the key regulator of the actin cytoskeleton, a member of Wiskott-Aldrich syndrome protein family. They have been suggested to be involved in cancer cell invasion and metastasis. In this study, the expression patterns of CFL1 and N-WASP in normal esophageal mucosa and esophageal squamous cell carcinoma (ESCC) and their correlation with clinical characteristics were investigated. Immunohistochemical staining showed that CFL1 was expressed in nuclear and cytoplasm of cancer cells. However, N-WASP was mainly found in the cytoplasm of the cancer cells. There were significant evidences that proved that CFL1 is correlated with clinicopathological factors in ESCC, such as infiltration depth, lymph node metastasis and pathological staging (P < 0.05). It is also proved that N-WASP is related to lymph node metastasis and pathological staging in ESCC (P < 0.05). Kaplan-Meier analysis showed that there was no correlation between CFL1 and N-WASP protein expression and survival (P > 0.05). Moreover, the mRNA expression of CFL1 and N-WASP was detected by quantitative real time PCR in 70 tissue specimens. The results showed that CFL1 mRNA level was over-expressed in ESCC tissue (P < 0.05), while N-WASP mRNA expression level was not different between cancerous tissues and adjacent normal esophageal mucosa (P > 0.05). Also, CFL1 mRNA expression was significantly associated with regional lymph node metastasis and pathological staging (P < 0.05). Kaplan-Meier analysis showed that there was no correlation between CFL1 and N-WASP mRNA expression and survival (P > 0.05). Our findings suggested that CFL1 and N-WASP may play an important role in the tumorigenesis of ESCC, and to be the candidate novel biomarkers for the diagnosis and prognosis of ESCC. These findings may have implications for targeted therapies in patients with ESCC.