Hidekazu Hiramoto

Plasma level of metastasis‐associated lung adenocarcinoma transcript 1 is associated with liver damage and predicts development of hepatocellular carcinoma

Recent studies have shown that metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) was overexpressed in many human solid cancers, however, its roles in plasma of hepatocellular carcinoma (HCC) patients were unclear. The aim of this study was to investigate the significance of plasma MALAT1 levels in HCC patients. Plasma samples were collected from pre‐operative HCC, hepatic disease patients, and healthy controls, and tissue samples from HCC patients and colorectal cancer patients with liver metastasis. Plasma and tissue MALAT1 levels were measured. Plasma MALAT1 levels were progressively and significantly higher in HCC patients than hepatic disease patients, and higher in hepatic disease patients than healthy controls. The expression of MALAT1 in HCC tissue was slightly higher than that in paired non‐cancerous liver tissue, but not significant. The expression of MALAT1 in the non‐cancerous liver tissue of 20 HCC patients was significantly higher than that in normal liver tissue of 13 colorectal cancer patients. In contrast, plasma MALAT1 levels were significantly low in HCC patients with hepatitis B infection, and significantly high in patients with liver damage B or liver cirrhosis. In a receiver–operator curve analysis of HCC and hepatic disease patients, the cut‐off value of plasma MALAT1 was 1.60 and the area under the curve was 0.66. Plasma MALAT1 levels were not correlated with α‐fetoprotein or protein induced by vitamin K absence II, whereas sensitivity and specificity for the detection of HCC with the combination of MALAT1, α‐fetoprotein, and protein induced by vitamin K absence II were 88.6% and 75%, respectively. In conclusion, the plasma MALAT1 level is associated with liver damage, and has clinical utility for predicting development of HCC.

Tumor exosome-mediated promotion of adhesion to mesothelial cells in gastric cancer cells

Background Peritoneal metastasis consists of a highly complex series of steps, and the details of the underlying molecular mechanism remain largely unclear. In this study, the effects of tumor-derived exosomes (TEX) on the progression of gastric cancers were investigated in peritoneal metastasis. Results TEX were internalized in both mesothelial and gastric cancer cells in a cellular origin non-specific manner. Internalization of TEX into mesothelial cells promoted significant adhesion between mesothelial and gastric cancer cells, and TEX internalization into gastric cancer cells significantly promoted migratory ability, while internalization of mesothelial cell-derived exosomes did not. Expression of adhesion-related molecules, such as fibronectin 1 (FN1) and laminin gamma 1 (LAMC1), were increased in mesothelial cells after internalization of TEX from gastric cancer cell line and malignant pleural effusion. Methods TEX were extracted from cell-conditioned medium by ultracentrifugation. The effects of TEX on the malignant potential of gastric cancer were investigated in adhesion, invasion, and proliferation assays. PCR array as well as western blotting were performed to determine the underlying molecular mechanisms. The molecular changes in mesothelial cell after internalization of TEX derived from malignant pleural effusion were also confirmed. Conclusions TEX may play a critical role in the development of peritoneal metastasis of gastric cancer, which may be partially due to inducing increased expression of adhesion molecules in mesothelial cells.

Clinical utility of circulating cell-free Epstein–Barr virus DNA in patients with gastric cancer

Recent comprehensive molecular subtyping of gastric cancer (GC) identified Epstein–Barr virus (EBV)-positive tumors as a subtype with distinct salient molecular and clinical features. In this study, we aimed to determine the potential utility of circulating cell-free EBV DNA as a biomarker for the detection and/or monitoring of therapeutic response in patients with EBV-associated gastric carcinoma (EBVaGC). The EBV genes-to-ribonuclease P RNA component H1 ratios (EBV ratios) in the GC tumors and plasma samples were determined by quantitative real-time polymerase chain reaction in 153 patients with GC, including 14 patients with EBVaGC diagnosed by the conventional method. Circulating cell-free EBV DNA was detected in 14 patients with GC: the sensitivity and specificity of detection were 71.4% (10/14) and 97.1% (135/139), respectively. Plasma EBV ratios were significantly correlated with the size of EBVaGC tumors, and the plasma EBV DNA detected before surgery in EBVaGC cases disappeared after surgery. Patients with EBVaGC may have a better prognosis, but circulating cell-free EBV DNA had no or little impact on prognosis. In addition, repeated assessment of the plasma EBV ratio in EBVaGC showed a decrease and increase in plasma EBV DNA after treatment and during tumor progression/recurrence, respectively. These results suggest the potential utility of circulating cell-free DNA to reveal EBV DNA for the identification of the EBVaGC subtype and/or for real-time monitoring of tumor progression as well as treatment response in patients with EBVaGC.

Genome-wide screening of DNA methylation associated with lymph node metastasis in esophageal squamous cell carcinoma

Lymph node metastasis (LNM) of esophageal squamous cell carcinoma (ESCC) is well-known to be an early event associated with poor prognosis in patients with ESCC. Recently, tumor-specific aberrant DNA methylation of CpG islands around the promoter regions of tumor-related genes has been investigated as a possible biomarker for use in early diagnosis and prediction of prognosis. However, there are few DNA methylation markers able to predict the presence of LNM in ESCC. To identify DNA methylation markers associated with LNM of ESCC, we performed a genome-wide screening of DNA methylation status in a discovery cohort of 67 primary ESCC tissues and their paired normal esophageal tissues using the Illumina Infinium HumanMethylation450 BeadChip. In this screening, we focused on differentially methylated regions (DMRs) that were associated with LNM of ESCC, as prime candidates for DNA methylation markers. We extracted three genes, HOXB2, SLC15A3, and SEPT9, as candidates predicting LNM of ESCC, using pyrosequencing and several statistical analyses in the discovery cohort. We confirmed that HOXB2 and SEPT9 were highly methylated in LNM-positive tumors in 59 ESCC validation samples. These results suggested that HOXB2 and SEPT9 may be useful epigenetic biomarkers for the prediction of the presence of LNM in ESCC.

miR-509-5p and miR-1243 increase the sensitivity to gemcitabine by inhibiting epithelial-mesenchymal transition in pancreatic cancer

The epithelial-mesenchymal transition (EMT) contributes to various processes in cancer progression, such as metastasis and drug resistance. Since we have already established a cell-based reporter system for identifying EMT-suppressive microRNAs (miRNAs) in the pancreatic cancer cell line Panc1, we performed a function-based screening assay by combining this reporter system and a miRNA library composed of 1,090 miRNAs. As a result, we identified miR-509-5p and miR-1243 as EMT-suppressive miRNAs, although the mechanisms for EMT-suppression induced by these miRNAs have yet to be clarified. Herein, we demonstrated that overexpression of miR-509-5p and miR-1243 increased the expression of E-cadherin through the suppression of EMT-related gene expression and that drug sensitivity increased with a combination of each of these miRNAs and gemcitabine. Moreover, miR-509-5p was associated with worse overall survival in patients with pancreatic cancer and was identified as an independently selected predictor of mortality. Our findings suggest that miR-509-5p and miR-1243 might be novel chemotherapeutic targets and serve as biomarkers in pancreatic cancer.

miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

Bromodomain Containing 4 (BRD4) mediates transcriptional elongation of the oncogene MYC by binding to acetylated histones. BRD4 has been shown to play a critical role in tumorigenesis in several cancers, and the BRD4-NUT fusion gene is a driver of NUT midline carcinoma (NMC), a rare but highly lethal cancer. microRNAs (miRNAs) are endogenous small non-coding RNAs that suppress target gene expression by binding to complementary mRNA sequences. Here, we show that miR-3140, which was identified as a novel tumor suppressive miRNA by function-based screening of a library containing 1090 miRNA mimics, directly suppressed BRD4 by binding to its coding sequence (CDS). miR-3140 concurrently downregulated BRD3 by bind to its CDS as well as CDK2 and EGFR by binding to their 3’ untranslated regions. miR-3140 inhibited tumor cell growth in vitro in various cancer cell lines, including EGFR tyrosine kinase inhibitor-resistant cells. Interestingly, we found that miR-3140 downregulated the BRD4-NUT fusion protein and suppressed in vitro tumor cell growth in a NMC cell line, Ty-82 cells. Furthermore, administration of miR-3140 suppressed in vivo tumor growth in a xenograft mouse model. Our results suggest that miR-3140 is a candidate for the development of miRNA-based cancer therapeutics.

Publisher Correction: mi R-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

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Investigation of a Quality Check for Plasma Samples

Background: MicroRNA (miRNA) molecules have been detected in many body fluids and used as biomarkers. However, blood cell-derived miRNA molecules due to hemolysis have been shown to affect the amounts of plasma miRNAs. It is important to check the quality of plasma samples for clinical applications. In the present study, an objective method for quality checking plasma samples was investigated using permitted color tone level and the absorbance at 414 nm. Material and method: An ROC analysis of the macroscopic color tone and the absorbance in 1213 clinical samples was performed. The optimal cut-off absorbance value was validated using the amount of plasma miRNA. Results: AUC for detecting hemolyzed samples was very high (0.986) at a cut-off absorbance value of 1.664. A sensitivity and specificity were 99% and 92%, respectively. For validation study, 3 candidate miRNAs were selected by a miRNA microarray between fresh and hemolyzed plasma samples; the amounts of miR-16 and miR-19b increased in hemolyzed samples, whereas that of miR-223 remained unchanged. The amounts of plasma miR-16 and miR-19b were significantly increased in 5 clinical samples with higher absorbance values (p=0.0001 and p=0.0003, respectively), while the amounts of these miRNAs were not increased in samples with lower absorbance values. Conclusions: A simple method for quality checking plasma samples using color tone and absorbance is very useful and significant.