Hidenori Kage

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.

Identification of G0S2 as a gene frequently methylated in squamous lung cancer by combination of in silico and experimental approaches

Epigenetic changes can lead to abnormal expression of genes in cancer, and several genes have been reported to have aberrant promoter DNA methylation in non‐small‐cell lung cancer (NSCLC). We identified aberrantly methylated genes in NSCLC by combination of in silico and experimental approaches. We first applied bioinformatics, and from microarray datasets, we selected genes with low expression and having functions related to cancer. Next, combined bisulfite restriction analysis was carried out in 10 pooled resected lung cancer tissues to screen for genes that were aberrantly methylated, and the methylation ratio (the fraction of methylated DNA in extracted DNA from a cancer tissue sample) was quantified using quantitative analysis of methylated alleles. We identified 8 methylated genes (ARPC1B, DNAH9, FLRT2, G0S2, IRS2, PKP1, SPOCK1 and UCHL1) previously unreported in NSCLC. Analyses of methylation profiles of 101 resected lung cancer tissue samples revealed quantitatively low methylation in whole, methylation ratios were almost less than 30% even in the methylated samples, and no significant correlation to prognosis after 2 years of follow‐up using hierarchical clustering. DNA methylation of G0S2 gene was significantly more frequent in squamous lung cancer (n = 18, mean of methylation ratios: 15%) compared with nonsquamous lung cancer (n = 83, mean of methylation ratios: 2.6%) (Mann‐Whitney U test, p < 0.001). DNA methylation of G0S2 can be an important biomarker for squamous lung cancer.

CpG island methylation of microRNAs is associated with tumor size and recurrence of non-small-cell lung cancer

We investigated whether the CpG island methylation of certain microRNAs was associated with the clinicopathological features and the prognosis of non‐small‐cell lung cancer. The methylation of mir‐152, ‐9‐3, ‐124‐1, ‐124‐2, and ‐124‐3 was analyzed in 96 non‐small‐cell lung cancer specimens using a combined bisulfite restriction analysis. The median observation period was 49.5 months. The methylation of mir‐9‐3, ‐124‐2, and ‐124‐3 was individually associated with an advanced T factor independent of age, sex, and smoking habit. Moreover, the methylation of multiple microRNA loci was associated with a poorer progression‐free survival in a univariate analysis. Our result enlightens the accumulation of aberrant DNA methylation which occurs in concordance with the tumor progression. (Cancer Sci 2011; 102: 2126–2131)

A second-generation profiling system for quantitative methylation analysis of multiple gene promoters: application to lung cancer.

Cancer-specific gene promoter methylation has been described in many types of cancers, and various semi-quantified results have shown their usefulness. Here, we show a more sensitive and specific second-generation system for profiling the DNA methylation status. This method is based on bisulfite reaction of DNA and real-time PCR using two TaqMan MGB probes labeled with different fluorescence, followed by clustering analysis. Primers were designed with CpG-less sequences, and TaqMan MGB probes were designed to contain three or four CpG sites and to be shorter than conventional TaqMan probes. We have added new criteria for primer and probe design for further specificity. We confirmed the reliability of this system and applied it to analysis of lung cancers. Using 10 promoters, 90 primary lung cancers were clustered into six groups consisting of cases having similar smoking status and pathological findings. EGFR mutation and p16 promoter DNA methylation were exclusive, as previously reported; however, DNA methylation in other genes was unrelated to EGFR mutation. This system was also useful to distinguish double primary lung cancers from a single cancer with intrapulmonary metastasis. As above, our system has widespread availability in clinical use and biological research.

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.

Worse Prognosis for Stage IA Lung Cancer Patients with Smoking History and More Severe Chronic Obstructive Pulmonary Disease

PURPOSE This retrospective study examined whether the severity of chronic obstructive lung disease (COPD) affects surgical outcomes. METHODS The subjects were 243 consecutive patients who underwent lobectomy for clinical stage IA lung cancer from 1999 to 2008 in our hospital. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) grading system was used to classify the severity of COPD in smokers. RESULTS Among the 149 smokers, 62 were diagnosed with COPD (25 as GOLD 1, 33 as GOLD 2, and 4 as GOLD 3). In univariate analysis, postoperative pulmonary complications were associated with male sex and more severe COPD. The frequencies were 17.1% in non-COPD, 24.0% in GOLD 1-COPD, and 46.0% in GOLD 2/3-COPD smokers (p = 0.0006). In univariate analysis, older age, smoking history, higher smoking pack-years and more severe COPD were associated with poor relapse-free survival. Relapse-free survival at five years was 80.7%, 66.9%, and 61.3% in non-COPD, GOLD 1-COPD, and GOLD 2/3-COPD smokers, respectively (p = 0.0005). Multivariate analyses showed that only GOLD 2/3-COPD was associated with postoperative pulmonary complications and relapse-free survival. Inhaled bronchodilators were prescribed preoperatively to 24.3% of the GOLD 2/3-COPD group. CONCLUSION Smokers with GOLD 2/3-COPD are at high risk for pulmonary complications and have an unfavorable long-term prognosis.

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.

PULMONARY FEATURES ASSOCIATED WITH BEING UNDERWEIGHT IN OLDER MEN

ACKNOWLEDGMENTS The authors wish to thank Angers University Hospital for technical support. Conflict of Interest: Prof. Beauchet serves as an unpaid consultant for Ipsen Pharma company and as a board member for Gériatrie, Psychologie et Neuropsychiatrie du Vieillissement. He has no relevant financial interest in this manuscript. Dr. Annweiler serves as an unpaid consultant for Ipsen Pharma Company. He has no relevant financial interest in this manuscript. Author Contributions: Annweiler had full access to the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analyses. Study concept and design: Annweiler and Beauchet. Acquisition of data: Calès, Redureau, and Abraham. Analysis and interpretation of data: Abraham, Calès, Redureau, Annweiler, and Beauchet. Drafting of the manuscript: Annweiler, Abraham, Beauchet, Calès, and Redureau. Critical revision of the manuscript for important intellectual content: Fantino and De Decker. Statistical expertise: Annweiler. Administrative, technical, or material support: Beauchet. Study supervision: Annweiler. Sponsor’s Role: None.

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.