Background:Recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in blood and can serve as useful biomarkers for cancer.Methods:We performed an miRNA array using serum samples obtained from oesophageal squamous cell carcinoma (ESCC) patients or healthy controls. MiR-1246 was the most markedly elevated in ESCC patients. Therefore, miR-1246 was selected as a candidate for further analysis. The serum miR-1246 level in 46 healthy controls and 101 ESCC patients was evaluated and compared among various clinicopathological characteristics. MiR-1246 expressions in tissue, exosomal, and cellular samples were also examined.Results:Serum miR-1246 alone yielded an receiver-operating characteristic curve area of 0.754, with 71.3% sensitivity and 73.9% specificity for distinguishing ESCC patients from healthy controls. Serum miR-1246 was significantly correlated with the TNM stage and showed to be the strongest independent risk factor for poor survival (HR, 4.032; P=0.017). Unlike the tendency shown in previous reports, miR-1246 was not upregulated in ESCC tissue samples. Furthermore, exosomal miR-1246 did not reflect the abundance in the cell of origin.Conclusion:These data support our contention that serum miR-1246 has strong potential as a novel diagnostic and prognostic biomarker in ESCC, and its releasing mechanism is selective and independent of tissue miRNA abundance.

Our study provides a precise assessment of the comprehensive risk of metastasis and feasible predictive markers for T1 ESCC.

Background:FSCN1 and matrix metalloproteinase 14 (MMP14) are both invadopodia-related proteins. We herein elucidate the tumourigenicity of these proteins and identify novel therapeutic agents in esophageal squamous cell carcinoma (ESCC).Methods:FSCN1 and MMP14 were evaluated by immunohistochemistry and quantitative PCR, and microRNA (miR)-133a was also evaluated by PCR in surgical ESCC specimens. The roles of FSCN1, MMP14 and miR-133a were established in ESCC cells.Results:The expression of FSCN1 or MMP14 was an independent poor prognostic factor according to a multivariate analysis of immunohistochemistry, and their co-expression correlated with the poorest overall survival (OS) out of all the examined factors. Additionally, their mRNAs significantly correlated and both inversely correlated with miR-133a in surgical specimens. Transfection of a miR-133a mimic decreased the mRNA and protein levels of both FSCN1 and MMP14 in ESCC cells. The knockdown of FSCN1 or MMP14 and transfection of a miR-133a mimic inhibited the proliferation and invasion of ESCC cells. Patients with a lower miR-133a expression have a significantly poorer OS than those with a higher expression.Conclusion:The combined expression of FSCN1 and MMP14 is associated with a poor prognosis, and miR-133a, which regulates their mRNAs, can serve as a strong tumour suppressor of ESCC.

Abstract. The aim of this study was to determine whether histone acetylation regulates tumor suppressive microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC) and to identify genes which are regulated by these miRNAs. We identified a miRNA that was highly upregulated in an ESCC cell line by cyclic hydroxamic acid-containing peptide 31 (CHAP31), one of the histone deacetylase inhibi-, one of the histone deacetylase inhibitors (HDACIs), using a miRNA array analysis. miR-375 was strongly upregulated by CHAP31 treatment in an ESCC cell line. The expression levels of the most upregulated miRNA, miR-375 were analyzed by quantitative real-time PCR in human ESCC specimens. The tumor suppressive function of miR-375 was revealed by restoration of miR-375 in ESCC cell lines. We performed a microarray analysis to identify target genes of miR-375. The mRNA and protein expression levels of these genes were verified in ESCC clinical specimens. LDHB and AEG-1/MTDH were detected as miR-375-targeted genes. The restoration of miR-375 suppressed the expression of LDHB and AEG-1/MTDH. The ESCC clinical specimens exhibited a high level of LDHB expression at both the mRNA and protein levels. A loss-of-function assay using a siRNA analysis was performed to examine the oncogenic function of the gene. Knockdown of LDHB by RNAi showed a tumor suppressive function in the ESCC cells. The correlation between gene expression and clinicopathological features was investigated by immunohistochemistry for 94 cases of ESCC. The positive staining of LDHB correlated significantly with lymph node metastasis and tumor stage. It also had a tendency to be associated with a poor prognosis. Our results indicate that HDACIs upregulate miRNAs, at least some of which act as tumor suppressors. LDHB, which is regulated by the tumor suppressive miR-375, may therefore act as an oncogene in ESCC.

The expression of microRNA-203 (miR-203) in esophageal squamous cell carcinoma (ESCC) tissues is remarkably lower than that in non‑ESCC tissues. We investigated how miR-203 could influence the development of ESCC cells. Our analyses revealed that miR-203 inhibited the migration and invasion of ESCC cells. Genome-wide gene expression data and target site inhibition assays showed that miR-203 appears to directly regulate LIM and SH3 protein 1 (LASP1). The knockdown of LASP1 resulted in inhibition of the migration and invasion of ESCC cells. Our results suggest that miR-203 and its target LASP1, may be associated with the progression of ESCC. In clinical ESCC specimens, the expression levels of miR-203, which were lower compared to those in normal tissues, were inversely correlated with the mRNA expression levels of LASP1. Moreover, we found that there was a significant correlation between the expression levels of miR-203 and the relapse‑free survival. The identification of a cancer network regulated by miR-203 could provide new insights into the potential mechanisms of the progression of ESCC.

Abstract. The aim of this study was to assess whether modulated electro-hyperthermia (mEHT) can induce an abscopal effect and thereby enhance the antitumor effects of immunotherapy. We used an intratumoral dendritic cell (DC) injection and mEHT to treat C3H/He mice inoculated with squamous cell carcinoma SCCVII cells in the left leg, and we assessed the whole body antitumor effects. Tumors were examined every two or three days in order to assess growth inhibition. The tumor-draining lymph nodes were removed to enable flow cytometric analysis of CD3 + and CD8 + cells, whereas immunohistochemistry was used to assess CD8, S100 and Foxp3 expression in the tumors. Additionally, GP96 expression in the tumors from the different treatment groups was measured. In the control group, the mean tumor volume was larger than that in other groups. These results indicated that the combination therapy of an intratumoral DC injection and mEHT evoked systemic antitumor activity. A larger number of CD3 + and CD8 + cells were detected by flow cytometric analysis in the DC plus mEHT treatment group. Tumor tissue immunostaining showed that CD8 and S100 were more strongly expressed in the DC plus mEHT treatment group, although Foxp3 expression was much higher in the control group. The GP96 gene expression level in the mEHT group was significantly different from the expression level in the control group. An abscopal effect may be induced by mEHT, and the effect of immunotherapy with DCs was strongly enhanced by the overexpression of GP96. GP96 is thought to be one of the molecules explaining the abscopal effect. Direct intratumoral administration of DCs and mEHT may be a feasible future treatment strategy.

Animal studies suggest that pancreatitis-induced acinar-to-ductal metaplasia (ADM) is a key event for pancreatic ductal adenocarcinoma (PDAC) initiation. However, there has not been an adequate system to explore the mechanisms of human ADM induction. We have developed a flow cytometry-based, high resolution lineage tracing method and 3D culture system to analyse ADM in human cells. In this system, well-known mouse ADM inducers did not promote ADM in human cells. In contrast, TGF-β1 efficiently converted human acinar cells to duct-like cells (AD) in a SMAD-dependent manner, highlighting fundamental differences between the species. Functionally, AD cells gained transient proliferative capacity. Furthermore, oncogenic KRAS did not induce acinar cell proliferation, but did sustain the proliferation of AD cells, suggesting that oncogenic KRAS requires ADM-associated-changes to promote PDAC initiation. This ADM model provides a novel platform to explore the mechanisms involved in the development of human pancreatic diseases.

Of the various factors, the number of pathologic metastatic LNs was the strongest indicator to predict the patients' survival.