Yosuke Amano

Histone methylation-mediated silencing of miR-139 enhances invasion of non-small-cell lung cancer

MicroRNA expression is frequently altered in human cancers, and some microRNAs act as oncogenes or tumor suppressors. MiR‐139‐5p (denoted thereafter as miR‐139) has recently been reported to function as a tumor suppressor in several types of human cancer (hepatocellular carcinoma, colorectal cancer, breast cancer, and gastric cancer), but its function in non‐small‐cell lung cancer (NSCLC) and the mechanism of its suppression have not been studied in detail. MiR‐139 was suppressed frequently in primary NSCLCs. MiR‐139 is located within the intron of PDE2A and its expression was significantly correlated with the expression of PDE2A. A chromatin immunoprecipitation assay revealed that miR‐139 was epigenetically silenced by histone H3 lysine 27 trimethylation (H3K27me3) of its host gene PDE2A and this process was independent of promoter DNA methylation. Pharmacological inhibition of both histone methylation and deacetylation‐induced miR‐139 with its host gene PDE2A. Ectopic expression of miR‐139 in lung cancer cell lines did not affect the proliferation nor the migration but significantly suppressed the invasion through the extracellular matrix. In primary NSCLCs, decreased expression of miR‐139 was significantly associated with distant lymph node metastasis and histological invasiveness (lymphatic invasion and vascular invasion) on both univariate and multivariate analyses. Collectively, these results suggest that H3K27me3‐mediated silencing of miR‐139 enhances an invasive and metastatic phenotype of NSCLC.

Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation.

Detection of novel paraja ring finger 2‐fer tyrosine kinase mRNA chimeras is associated with poor postoperative prognosis in non‐small cell lung cancer

Previously, we reported that the overexpression of fer tyrosine kinase (FER), a non‐receptor tyrosine kinase, is correlated with poor postoperative prognosis and cancer‐cell survival in non‐small cell lung cancer (NSCLC). In the present study, we further analyzed FER‐overexpressed NSCLC cases and identified various patterns of chimeric mRNAs, composed of paraja ring finger 2 (PJA2) and FER. We detected no genomic rearrangements between PJA2 and FER and attributed these chimeric mRNAs to alterations at the transcriptome level: i.e., trans‐splicing. Several chimeric patterns were detected concurrently in each patient, and the pattern sets varied among patients, although the pattern in which PJA2 exon 1 was fused to FER exon 3 (designated as Pe1‐Fe3 mRNA) was detected constantly. Therefore, in a wide screening for PJA2‐FER mRNAs in NSCLC, we focused on this chimeric pattern as a representative chimera. In analyses of 167 NSCLC samples, Pe1‐Fe3 mRNA was identified in about 10% of the patients, and the presence of chimeric mRNA was significantly correlated with a high expression level of parental FER mRNA. Furthermore, we found that the detection of Pe1‐Fe3 mRNA was correlated with poor postoperative survival periods in NSCLC, consistent with a previous finding in which FER overexpression was correlated with poor postoperative prognosis in NSCLC. This report is the first to suggest a correlation between chimeric mRNA and the expression level of parental mRNA. Furthermore, our findings may be clinically beneficial, suggesting that PJA2‐FER mRNAs might serve as a novel prognostic biomarker in NSCLC.

Discrimination of wild and cultured Japanese eels based on otolith stable isotope ratios

Discrimination of wild and cultured Japanese eels based on otolith stable isotope ratios Kenzo Kaifu*, Hikaru Itakura, Yosuke Amano, Kotaro Shirai, Kazuki Yokouchi, Ryoshiro Wakiya, Naoko Murakami-Sugihara, Izumi Washitani, and Takashi Yada Faculty of Law, Chuo University, 724-1 Higashinakano, Hachioji-shi, Tokyo 192-0393, Japan Graduate School of Science, Kobe University, 1-1, Rokkoudaichou, Nadaku, Kobe, Hyogo 657-8501, Japan Tohoku National Fisheries Research Institute, Japan Fisheries Research and Education Agency, 3-27-5, Shinhama, Shiogama, Miyagi 985-0001, Japan Atmosphere and Ocean Research Institute, The University of Tokyo 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8564, Japan National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, Yokosuka, Kanagawa 238-0316, Japan Research and Development Initiative, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, Nikko, Tochigi 321-1661, Japan *Corresponding author: tel: þ81 42 674 3243; fax: þ81 42 674 3243; e-mail: kaifu@tamacc.chuo-u.ac.jp

High expression of IRE1 in lung adenocarcinoma is associated with a lower rate of recurrence

High expression of endoplasmic reticulum stress-related genes was observed in patients with lower stages of lung adenocarcinoma and minimal vascular invasion. High inositol-requiring kinase 1 expression was an independent predictor of recurrence-free survival.

Abstract B28: Histone methylation-mediated silencing of mir-139 enhances an aggressive phenotype of non-small cell lung cancer.

MicroRNA expression is frequently altered in human cancers, and some microRNAs act as oncogenes or tumor suppressors. Mir-139 has recently been reported to function as a tumor suppressor in hepatocellular carcinoma and gastric cancer, but its function in non-small cell lung cancer (NSCLC) and the mechanism of its suppression have not been studied in detail. Mir-139 was suppressed frequently in lung cancer cell lines and primary NSCLCs. Mir-139 was silenced with its host gene PDE2A by histone methylation, which was independent of DNA methylation. In primary NSCLCs, decreased expression of mir-139 was significantly associated with distant lymph node metastasis and histological invasiveness (lymphatic invasion and vascular invasion) on both univariate and multivariate analyses. Overexpression of mir-139 suppressed the proliferation of lung cancer cells. Collectively, these results suggest that histone methylation-mediated silencing of mir-139 enhances an aggressive phenotype of lung cancer. Citation Format: Kousuke Watanabe, Mitsuhiro Sunohara, Yosuke Amano, Rie Ishikawa, Junji Ichinose, Jun Nakajima, Masashi Fukayama, Yutaka Yatomi, Takahide Nagase, Nobuya Ohishi, Daiya Takai. Histone methylation-mediated silencing of mir-139 enhances an aggressive phenotype of non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B28.

Successful Treatment of Mediastinal Unicentric Castleman's Disease Using Video-Assisted Thoracoscopic Surgery with Preoperative Embolization

Unicentric Castlemans disease is a rare, benign lymphoproliferative disorder that is curable with surgical resection. However, significant bleeding often occurs during surgery because of tumor hypervascularity. We herein present a case of hyaline-vascular-type mediastinal unicentric Castlemans disease, successfully resected using video-assisted thoracoscopic surgery with preoperative embolization. In the present case, tumor hypervascularity and feeding vessels were revealed by computed tomography (CT), which led us to perform preoperative angiography and embolization to the tumor feeding arteries to reduce intraoperative bleeding. Castlemans disease should be considered in the differential diagnosis of hypervascular mediastinal tumors. Tumor vascularity should be assessed prior to surgery, and preoperative embolization should be considered.

Abstract B39: Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer

microRNA expression is frequently altered in human cancers. Epigenetic silencing, especially DNA methylation, is one of many mechanisms of microRNA suppression in cancer. To search for epigenetically silenced microRNAs in non-small-cell lung cancer (NSCLC), we mapped human microRNAs on autosomal chromosomes and selected in silico 55 microRNAs that met one of the following criteria: (i) microRNAs within CpG islands, (ii) microRNAs within 1 kbp downstream of CpG islands, and (iii) microRNAs within gene introns whose promoters have CpG islands. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 microRNAs. Fourteen microRNAs (mir-375, mir-196b, mir-126, mir-34b, mir-127, mir-203, mir-148a, mir-181c, mir-30e, mir-449a, mir-340, mir-486, mir-483, mir-139) were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. Among the 14 microRNAs, seven (mir-126, mir-34b, mir-203, mir-30e, mir-449a, mir-486, mir-139) were frequently suppressed in primary NSCLCs. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, while mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. A significant enrichment of H3K27me3 was observed in lung cancer cell lines with decreased mir-126 expression, suggesting the involvement of a polycomb complex in the regulation of EGFL7 and mir-126. The overexpression of mir-34b and mir-126 by plasmid vectors decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these microRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41% and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.

Old age and underlying interstitial abnormalities are risk factors for development of ARDS after pleurodesis using limited amount of large particle size talc

Talc pleurodesis is commonly performed to manage refractory pleural effusion or pneumothorax. It is considered as a safe procedure as long as a limited amount of large particle size talc is used. However, acute respiratory distress syndrome (ARDS) is a rare but serious complication after talc pleurodesis. We sought to determine the risk factors for the development of ARDS after pleurodesis using a limited amount of large particle size talc.

Mean platelet volume and lymphocyte-to-monocyte ratio are associated with shorter progression-free survival in EGFR-mutant lung adenocarcinoma treated by EGFR tyrosine kinase inhibitor

Background A growing body of evidence supports the role of platelets in cancer metastasis, escape from immune surveillance, and angiogenesis. Mean platelet volume (MPV), which reflects platelet turnover, is reported routinely as part of automated complete blood count. Accumulating evidence suggests that MPV is a useful biomarker in several diseases including cancer. However, its role in cancer patients receiving molecular targeted therapy has not been described in the literature. Materials and methods We retrospectively analysed the prognostic impact of MPV in advanced or recurrent EGFR mutant lung adenocarcinoma treated with EGFR tyrosine kinase inhibitors (EGFR-TKIs). Lymphocyte-to-monocyte ratio (LMR) has been previously reported to be a poor prognostic factor in EGFR mutant non-small cell lung cancer and was also included as a covariate. Results Using the previously described Cutoff Finder algorithm, the cut-off points for MPV and LMR that best predicted progression free survival (PFS) of EGFR-TKI were determined as 10.3 and 2.8, respectively. The median PFS was 14.7 and 8.2 months in MPV low and high groups (p = 0.013, log-rank test). The median PFS was 13.5 and 6.2 months in LMR high and low groups (p < 0.001, log-rank test). MPV and LMR were independently distributed (chi square test) and the multivariate analysis using Cox’s proportional hazards regression model revealed that high MPV, low LMR, and pleural effusion were significant predictors for shorter PFS. Conclusion MPV and LMR, measured as part of routine complete blood count, can be utilized to predict the outcome of EGFR-TKI therapy with no additional costs. Our results suggest a mechanism of EGFR-TKI resistance which is associated with the functional status of the platelets.