Lian-Di Liao

MiR‐142‐3p as a potential prognostic biomarker for esophageal squamous cell carcinoma

microRNAs (miRNAs), small non‐coding RNAs, are always aberrantly expressed in many diseases including human cancers. The aim of this study was to examine and determine the clinical significance of hsa‐miR‐31, hsa‐miR‐142‐3p, hsa‐miR‐338‐3p, and hsa‐miR‐1261 expression in esophageal squamous cell carcinoma (ESCC).

MiRNA profile in esophageal squamous cell carcinoma: Downregulation of miR-143 and miR-145

AIM To investigate the expression profile of miRNA in esophageal squamous cell carcinoma (ESCC). METHODS The expression profile of miRNA in ESCC tissues was analyzed by miRNA microarray. The expression levels of miR-143 and miR-145 in 86 ESCC patients were determined by real-time polymerase chain reaction (PCR) using TaqMan assay. The mobility effect was estimated by wound-healing using esophageal carcinoma cells transfected with miRNA expression plasmids. RESULTS A set of miRNAs was found to be deregulated in the ESCC tissues, and the expression levels of miR-143 and -145 were significantly decreased in most of the ESCC tissues examined. Both miR-143 and miR-145 expression correlated with tumor invasion depth. The transfection of human esophageal carcinoma cells with miR-143 and miR-145 expression plasmids resulted in a greater inhibition of cell mobility, however, the protein level of the previously reported target of miR-145, FSCN1, did not show any significant downregulation. CONCLUSION These findings suggest that the deregulation of miRNAs plays an important role in the progression of ESCC. Both miR-143 and miR-145 might act as anti-oncomirs common to ESCC.

A novel alternative spliced variant of neutrophil gelatinase-associated lipocalin receptor in oesophageal carcinoma cells

Recent studies suggest that NGAL (neutrophil gelatinase-associated lipocalin) is a novel iron transporter with functions distinct from that of transferrin and mediates a new iron-delivery pathway. To get a better understanding of NGAL function in oesophageal carcinoma, we analysed the expression of NGAL receptors in oesophageal carcinoma cells and identified a novel spliced variant designated NgalR-3. When expressed in a heterologous system, the protein produced from this novel spliced variant exhibits the biochemical characteristics of interaction and co-localization with NGAL protein in vivo. This new finding suggests that NgalR-3 may act as a potential NGAL receptor and play a role in NGAL-mediated iron transport in oesophageal carcinoma.

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

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.

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.

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.

Dissection of miRNA-miRNA Interaction in Esophageal Squamous Cell Carcinoma

The relationships between miRNAs and their regulatory influences in esophageal carcinoma remain largely unknown. Accumulated evidence suggests that delineation of subpathways within an entire pathway can underlie complex diseases. To analyze the regulation of differentially expressed miRNAs in subpathways of esophageal squamous cell carcinoma (ESCC), we constructed bipartite miRNA and subpathway networks to determine miRNA regulatory influences on subpathways. The miRNA-subpathway network indicated that miRNAs regulate numerous subpathways. Two principal biological networks were derived from the miRNA-subpathway network by the hypergeometric test. This miRNA-miRNA network revealed the co-regulation of subpathways between the upregulated and downregulated miRNAs. Subpathway-subpathway networks characterized scale free, small world, and modular architecture. K-clique analysis revealed co-regulation of subpathways between certain downregulated and upregulated miRNAs. When ESCC patients were grouped according to their expression levels of paired upregulation of miR-31 and downregulation of miR-338-3p, survival time analysis revealed a significant difference based on miR-31-miR-338-3p interaction. These findings can facilitate the understanding of the biological meaning of miRNA-miRNA interactions with either the same or opposite expression trend.

Specificity protein 1 regulates fascin expression in esophageal squamous cell carcinoma as the result of the epidermal growth factor/extracellular signal-regulated kinase signaling pathway activation.

The overexpression of fascin in human carcinomas is associated with aggressive clinical phenotypes and poor prognosis. However, the molecular mechanism underlying the increased expression of fascin in cancer cells is largely unknown. Here, we identified a Sp1 binding element located at −70 to −60 nts of the FSCN1 promoter and validated that Sp1 specifically bound to this element in esophageal carcinoma cells. Fascin expression was enhanced by Sp1 overexpression and blocked by Sp1 RNAi knockdown. Specific inhibition of ERK1/2 decreased phosphorylation levels of Sp1, and thus suppressed the transcription of the FSCN1, resulting in the down-regulation of fascin. Stimulation with EGF could enhance fascin expression via activating the ERK1/2 pathway and increasing phosphorylation levels of Sp1. These data suggest that FSCN1 transcription may be subjected to the regulation of the EGF/EGFR signaling pathway and can be used as a viable biomarker to predict the efficacy of EGFR inhibitors in cancer therapies.