Mitsuhiro Sunohara

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

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non‐small‐cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. 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 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5‐aza‐CdR treatment. 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, whereas 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. The overexpression of mir‐34b and mir‐126 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 miRNAs 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.

Paradoxical association between body mass index and in-hospital mortality in elderly patients with chronic obstructive pulmonary disease in Japan

Background and objective The prevalence and mortality of chronic obstructive pulmonary disease (COPD) in elderly patients are increasing worldwide. Low body mass index (BMI) is a well-known prognostic factor for COPD. However, the obesity paradox in elderly patients with COPD has not been well elucidated. We investigated the association between BMI and in-hospital mortality in elderly COPD patients. Methods Using the Diagnosis Procedure Combination database in Japan, we retrospectively collected data for elderly patients (>65 years) with COPD who were hospitalized between July 2010 and March 2013. We performed multivariable logistic regression analysis to compare all-cause in-hospital mortality between patients with BMI of <18.5 kg/m2 (underweight), 18.5–22.9 kg/m2 (low–normal weight), 23.0–24.9 kg/m2 (high–normal weight), 25.0–29.9 kg/m2 (overweight), and ≥30.0 kg/m2 (obesity) with adjustment for patient backgrounds. Results In all, 263,940 eligible patients were identified. In-hospital mortality was 14.3%, 7.3%, 4.9%, 4.3%, and 4.4%, respectively, in underweight, low–normal weight, high–normal weight, overweight, and obese patients. Underweight patients had a significantly higher mortality than low–normal weight patients (odds ratio [OR]: 1.55, 95% confidence interval [CI]: 1.48–1.63), whereas lower mortality was associated with high–normal weight (OR: 0.76, CI: 0.70–0.82), overweight (OR: 0.73, CI: 0.66–0.80), and obesity (OR: 0.67, CI: 0.52–0.86). Higher mortality was significantly associated with older age, male sex, more severe dyspnea, lower level of consciousness, and lower activities of daily living. Conclusion Overweight and obese patients had a lower mortality than low–normal weight patients, which supports the obesity paradox.

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.

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis.

DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 degrees C or 65 degrees C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

Clinical features of 280 hospitalized patients with lymphangioleiomyomatosis in Japan

Lymphangioleiomyomatosis (LAM) is rare, but potentially life threatening owing to respiratory failure. However, knowledge is limited about the condition of hospitalized LAM patients. The objectives of this study were to investigate patient characteristics, comorbidities and causes of death among hospitalized LAM patients in Japan.

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.

Performance of severity scores for home care-based patients suffering from pneumonia.

The Pneumonia Severity Index (PSI) is used to determine the prognosis of community‐acquired pneumonia (CAP). The concept of nursing‐ and healthcare‐associated pneumonia (NHCAP) has recently been established in Japan. The present study aims to examine whether the PSI can predict the prognosis of home care‐based patients diagnosed with NHCAP.

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.

Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).

Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5′- and 3′-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.