Ichiro Akagi

Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer

Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC.

Combination of protein coding and noncoding gene expression as a robust prognostic classifier in stage I lung adenocarcinoma.

Prognostic tests for patients with early-stage lung cancer may provide needed guidance on postoperative surveillance and therapeutic decisions. We used a novel strategy to develop and validate a prognostic classifier for early-stage lung cancer. Specifically, we focused on 42 genes with roles in lung cancer or cancer prognosis. Expression of these biologically relevant genes and their association with relapse-free survival (RFS) were evaluated using microarray data from 148 patients with stage I lung adenocarcinoma. Seven genes associated with RFS were further examined by quantitative reverse transcription PCR in 291 lung adenocarcinoma tissues from Japan, the United States, and Norway. Only BRCA1, HIF1A, DLC1, and XPO1 were each significantly associated with prognosis in the Japan and US/Norway cohorts. A Cox regression-based classifier was developed using these four genes on the Japan cohort and validated in stage I lung adenocarcinoma from the US/Norway cohort and three publicly available lung adenocarcinoma expression profiling datasets. The results suggest that the classifier is robust across ethnically and geographically diverse populations regardless of the technology used to measure gene expression. We evaluated the combination of the four-gene classifier with miRNA miR-21 (MIR21) expression and found that the combination improved associations with prognosis, which were significant in stratified analyses on stage IA and stage IB patients. Thus, the four coding gene classifier, alone or with miR-21 expression, may provide a clinically useful tool to identify high-risk patients and guide recommendations regarding adjuvant therapy and postoperative surveillance of patients with stage I lung adenocarcinoma.

SnoN Overexpression is Predictive of Poor Survival in Patients with Esophageal Squamous Cell Carcinoma

BackgroundEarlier studies have identified the minimal overlapping region of amplification at 3q26 in esophageal squamous cell carcinoma (ESCC) by comparative genomic hybridization (CGH) analysis. These include PIK3CA which encodes the p110α catalytic subunit of phosphatidylinositol (PI) 3-kinase, a telomerase RNA component (TERC), a squamous cell carcinoma-related oncogene (SCCRO), ecotropic viral integration site-1 (EVI-1), and a Ski-related novel oncogene (SnoN). In the present study, we investigated the mRNA levels of four candidate genes (TERC, SCCRO, EVI-1, and SnoN) to determine whether genes other than PIK3CA are targets for amplification at 3q26 in ESCC. And also, we examined SnoN expression in ESCC samples.MethodsFifty-nine representative cases with ESCC were selected from our archives. We performed quantitative RT-PCR of four candidate genes (TERC, SCCRO, EVI-1, and SnoN) and immunohistochemistry for SnoN. Finally, we correlated these findings with the clinicopathological characteristics to determine their interrelationship.ResultsAmong the four genes we tested, only SnoN mRNA was consistently overexpressed in primary ESCC, compared with those in corresponding nontumorous esophageal epithelia (P < 0.001). Immunoreactive SnoN was detectable in 31 of 59 (52.5%) esophageal squamous cell carcinoma specimens. The levels of SnoN expression were found to correlate with the depth of invasion and recurrence (P < 0.05). Furthermore, patients with positive staining for SnoN displayed more unfavorable outcomes than patients with negative staining (P < 0.05).ConclusionSnoN is likely to be the target of the amplification at 3q26 in ESCC and plays an important role in the development of ESCC, influencing disease-specific survival.

SnoN/SKIL modulates proliferation through control of hsa-miR-720 transcription in esophageal cancer cells.

It is now evident that changes in microRNA are involved in cancer progression, but the mechanisms of transcriptional regulation of miRNAs remain unknown. Ski-related novel gene (SnoN/SKIL), a transcription co-factor, acts as a potential key regulator within a complex network of p53 transcriptional repressors. SnoN has pro- and anti-oncogenic functions in the regulation of cell proliferation, senescence, apoptosis, and differentiation. We characterized the roles of SnoN in miRNA transcriptional regulation and its effects on cell proliferation using esophageal squamous cell carcinoma (ESCC) cells. Silencing of SnoN altered a set of miRNA expression profiles in TE-1cells, and the expression levels of miR-720, miR-1274A, and miR-1274B were modulated by SnoN. The expression of these miRNAs resulted in changes to the target protein p63 and a disintegrin and metalloproteinase domain 9 (ADAM9). Furthermore, silencing of SnoN significantly upregulated cell proliferation in TE-1 cells, indicating a potential anti-oncogenic function. These results support our observation that cancer tissues have lower expression levels of SnoN, miR-720, and miR-1274A compared to adjacent normal tissues from ESCC patients. These data demonstrate a novel mechanism of miRNA regulation, leading to changes in cell proliferation.

Expression of Akt and Mdm2 in human esophageal squamous cell carcinoma.

The Akt-Mdm2 pathway plays an important role in carcinogenesis in a variety of malignant tumors. However, the Akt-Mdm2 pathway in esophageal squamous cell carcinoma (ESCC) has not been fully studied. We investigated the proteins and mRNA expression of Akt and Mdm2 to elucidate the roles of these proteins in ESCC. We also examined the effect of Akt knockdown on Mdm2 expression in ESCC cells. ESCC tissue samples were obtained from 23 individuals who underwent surgical resection with no preoperative treatment. Akt1-3 and Mdm2 gene and protein expression were analyzed. The effect of siRNA-mediated Akt knockdown on Mdm2 expression was also studied, using ESCC cell lines. Akt1 and Mdm2 immunoreactivity was detected in 77.8 and 66.7% of tumor specimen from ESCC patients, respectively. Akt1 and Mdm2 mRNA expressions were correlated and significantly elevated in tumor tissue (p<0.0001 and p<0.05, respectively). The siRNA-targeted reduction of each Akt isoform reduced Mdm2 protein expression. The overexpression of Akt1 and Mdm2 may be related to esophageal carcinogenesis. Furthermore, Akt expression regulates Mdm2 expression, which may in turn regulate the function of wild-type p53. These results may provide the basis for future preventative or clinical therapies for esophageal cancer.

Esophagoscopy-guided thoracoscopic resection of large epiphrenic esophageal diverticulum

A diverticulum in the lower third of the esophagus is rare. A 62-year-old woman presented with symptoms of dysphagia and regurgitation associated with recurrent aspiration. Endoscopic examination disclosed a large epiphrenic diverticulum, for which esophagoscopy-guided thoracoscopic surgery was performed. The postoperative course was uneventful. It is proposed that this method could be a possible alternative to the standard classical thoracotomy approach for a large epiphrenic diverticulum of the esophagus.

Comprehensive Analysis of MicroRNA and mRNA Expression in Normal and Tumorous Human Esophageal Squamous Cell Lines Using Microarray Datasets

Despite the undisputed importance of altered microRNA (miRNA) expression in various cancers, there is limited information on the clinicopathologic significance of cancer-related miRNAs in esophageal squamous cell carcinoma (ESCC). Previously, it was reported that the expression of several miRNAs was dysregulated in ESCC. However, the target genes of these miRNAs have not been identified. Furthermore, additional miRNAs in humans have been discovered recently, indicating that revised miRNA and gene expression profiling for ESCC are necessary. Here, we provide datasets from microarray analyses to identify miRNA and mRNA expression comprehensively in Het-1A, a normal human esophageal squamous cell line, and three human ESCC cell lines.

Pyogenic liver abscess caused by direct invasion of esophageal squamous cell carcinoma to the liver: Report of a case

We report a case of esophageal squamous cell carcinoma (ESCC) directly invading the liver and causing a pyogenic liver abscess. The patient was a 66-year-old man who presented with dysphagia. Esophagography, endoscopic study, and computed tomography (CT) showed a mass lesion in the lower third of the esophagus. A high fever developed on hospital day 17 and another CT scan revealed a liver abscess, 50 × 45 mm, in the left lateral lobe of the liver. Although imaging demonstrated a liver abscess continuous with the tumor, we performed percutaneous transhepatic drainage, followed thereafter by distal esophagectomy and total gastrectomy with a left lateral segmental resection of the liver. The pathological findings confirmed a diagnosis of ESCC with direct invasion (T4N1M0, stage IVa in the TNM classification). The patient had an uneventful postoperative recovery. Microscopic examination of the resected specimen revealed the expansive growth of tumor cells into the hepatocellular tissues. To our knowledge, this is the first report of the direct invasion of esophageal cancer to the liver causing a pyogenic liver abscess; however, it should be borne in mind when a patient with esophageal cancer becomes febrile.

Enterolith Small-Bowel Obstruction Caused by Crohn's Disease

We herein report on the successful surgical treatment of Crohns disease with enteroliths formed in the ileum. A 58-year-old man presented with abdominal colicky pain, and long-standing constipation. He was diagnosed as ileus due to enteroliths and was initially managed conservatively, but improvement was not observed either clinically or on radio imaging. We, therefore, performed a laparotomy, which revealed Crohns disease with enteroliths in the ileum. Because the enteroliths in the ileum were not mobile and could not be broken manually, we carried out a segmental bowel resection with the removal of the enteroliths. The pathology report revealed Crohns disease with enteroliths. Intestinal obstruction caused by enteroliths associated with Crohns disease occurs rarely, and is difficult to be diagnosed and managed.

MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2, a negative regulator of the PI3K/AKT pathway

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is frequently activated in various human cancers and plays essential roles in their development and progression. Accumulating evidence suggests that dysregulated expression of microRNAs (miRNAs) is closely associated with cancer progression and metastasis. Here, we focused on miRNAs that could regulate genes related to the PI3K/AKT pathway in esophageal squamous cell carcinoma (ESCC). To identify upregulated miRNAs and their possible target genes in ESCC, we performed microarray-based integrative analyses of miRNA and mRNA expression levels in three human ESCC cell lines and a normal esophageal epithelial cell line. The miRNA microarray analysis revealed that miR-31-5p, miR-141-3p, miR-200b-3p, miR-200c-3p, and miR-205-5p were expressed at higher levels in the ESCC cell lines than the normal esophageal epithelial cell line. Bioinformatical analyses of mRNA microarray data identified several AKT/PI3K pathway-related genes as candidate targets of these miRNAs, which include tumor suppressors such as DNA-damage-inducible transcript 4 and pleckstrin homology domain leucine-rich repeat protein phosphatase-2 (PHLPP2). To validate the targets of relevant miRNAs experimentally, synthetic mimics of the miRNAs were transfected into the esophageal epithelial cell line. Here, we report that miR-141-3p suppress the expression of PHLPP2, a negative regulators of the AKT/PI3K pathway, as a target in ESCC.