Hyo Sung Jeon

Gender and telomere length: Systematic review and meta-analysis

BACKGROUND It is widely believed that females have longer telomeres than males, although results from studies have been contradictory. METHODS We carried out a systematic review and meta-analyses to test the hypothesis that in humans, females have longer telomeres than males and that this association becomes stronger with increasing age. Searches were conducted in EMBASE and MEDLINE (by November 2009) and additional datasets were obtained from study investigators. Eligible observational studies measured telomeres for both females and males of any age, had a minimum sample size of 100 and included participants not part of a diseased group. We calculated summary estimates using random-effects meta-analyses. Heterogeneity between studies was investigated using sub-group analysis and meta-regression. RESULTS Meta-analyses from 36 cohorts (36,230 participants) showed that on average females had longer telomeres than males (standardised difference in telomere length between females and males 0.090, 95% CI 0.015, 0.166; age-adjusted). There was little evidence that these associations varied by age group (p=1.00) or cell type (p=0.29). However, the size of this difference did vary by measurement methods, with only Southern blot but neither real-time PCR nor Flow-FISH showing a significant difference. This difference was not associated with random measurement error. CONCLUSIONS Telomere length is longer in females than males, although this difference was not universally found in studies that did not use Southern blot methods. Further research on explanations for the methodological differences is required.

Somatic mutations in epidermal growth factor receptor signaling pathway genes in non-small cell lung cancers.

Introduction: Epidermal growth factor receptor (EGFR) signaling pathway plays a crucial role in the development and progression of lung cancer. We searched for mutations of EGFR pathway genes in non-small cell lung cancers (NSCLCs) and analyzed their relationship with clinicopathologic features. Methods: Mutations of EGFR, ERBB2, ERBB3, ERBB4, KRAS, NRAS, BRAF, PTEN, PIK3CA, LKB1, and AKT1 genes were determined by direct sequencing in 173 surgically resected NSCLCs—56 squamous cell carcinomas (SCCs) and 117 adenocarcinomas (ACs). Results: Of the 173 NSCLCs, a total of 65 mutations were detected in 63 (36.4%) tumors—10 (17.9%) in SCCs and 53 (45.3%) in ACs. Mutations in EGFR pathway genes were significantly more frequent in women and ACs than in women and SCCs (p = 0.02 and p < 0.001, respectively). The mutations occurred in a mutually exclusive pattern. When the genes were divided into three subgroups according to their roles in the signaling cascade, mutations in the EGFR/ERBB2 and KRAS/BRAF genes were more frequent in ACs than in SCCs (p < 0.001 and p = 0.01, respectively). In marked contrast, mutations in the PIK3CA/PTEN were more frequent in SCCs than in ACs (p = 0.002). Furthermore, mutations in the PIK3CA/PTEN genes were more frequent in smokers (p = 0.04). Discussion: Our study demonstrates that mutations in each part of the EGFR pathway were associated with different clinicopathologic features in patients with NSCLCs.

Increased miR-708 Expression in NSCLC and Its Association with Poor Survival in Lung Adenocarcinoma from Never Smokers

Purpose: miRNA plays an important role in human disease and cancer. We seek to investigate the expression status, clinical relevance, and functional role of miRNA in non–small cell lung cancer. Experimental Design: We conducted miRNA expression profiling in matched lung adenocarcinoma and uninvolved lung using 56 pairs of fresh-frozen (FF) and 47 pairs of formalin-fixed, paraffin-embedded (FFPE) samples from never smokers. The most differentially expressed miRNA genes were evaluated by Cox analysis and log-rank test. Among the best candidate, miR-708 was further examined for differential expression in two independent cohorts. Functional significance of miR-708 expression in lung cancer was examined by identifying its candidate mRNA target and through manipulating its expression levels in cultured cells. Results: Among the 20 miRNAs most differentially expressed between tested tumor and normal samples, high expression level of miR-708 in the tumors was most strongly associated with an increased risk of death after adjustments for all clinically significant factors including age, sex, and tumor stage (FF cohort: HR, 1.90; 95% CI, 1.08–3.35; P = 0.025 and FFPE cohort: HR, 1.93; 95% CI, 1.02–3.63; P = 0.042). The transcript for TMEM88 gene has a miR-708 binding site in its 3′ UTR and was significantly reduced in tumors high of miR-708. Forced miR-708 expression reduced TMEM88 transcript levels and increased the rate of cell proliferation, invasion, and migration in culture. Conclusions: miRNA-708 acts as an oncogene contributing to tumor growth and disease progression by directly downregulating TMEM88, a negative regulator of the Wnt signaling pathway in lung cancer. Clin Cancer Res; 18(13); 3658–67. ©2012 AACR.

TGF-² Signaling and the Role of Inhibitory Smads in Non-small Cell Lung Cancer

The signaling pathway mediated by transforming growth factor-&bgr; (TGF-&bgr;) participates in various biologic processes, including cell growth, differentiation, angiogenesis, apoptosis, and extracellular matrix remodeling. In the context of cancer, TGF-&bgr; signaling can inhibit tumor growth in early-stage tumors. However, in late-stage tumors, the very same pathway promotes tumor invasiveness and metastasis. This paradoxical effect is mediated through similar to mothers against decapentaplegic or Smad protein dependent and independent mechanisms and provides an opportunity for targeted cancer therapy. This review summarizes the molecular process of TGF-&bgr; signaling and the changes in inhibitory Smads that contribute to lung cancer progression. We also present current approaches for rational therapies that target the TGF-&bgr; signaling pathway in cancer.

Common Oncogene Mutations and Novel SND1-BRAF Transcript Fusion in Lung Adenocarcinoma from Never Smokers

Lung adenocarcinomas from never smokers account for approximately 15 to 20% of all lung cancers and these tumors often carry genetic alterations that are responsive to targeted therapy. Here we examined mutation status in 10 oncogenes among 89 lung adenocarcinomas from never smokers. We also screened for oncogene fusion transcripts in 20 of the 89 tumors by RNA-Seq. In total, 62 tumors had mutations in at least one of the 10 oncogenes, including EGFR (49 cases, 55%), K-ras (5 cases, 6%), BRAF (4 cases, 5%), PIK3CA (3 cases, 3%), and ERBB2 (4 cases, 5%). In addition to ALK fusions identified by IHC/FISH in four cases, two previously known fusions involving EZR- ROS1 and KIF5B-RET were identified by RNA-Seq as well as a third novel fusion transcript that was formed between exons 1–9 of SND1 and exons 2 to 3′ end of BRAF. This in-frame fusion was observed in 3/89 tested tumors and 2/64 additional never smoker lung adenocarcinoma samples. Ectopic expression of SND1-BRAF in H1299 cells increased phosphorylation levels of MEK/ERK, cell proliferation, and spheroid formation compared to parental mock-transfected control. Jointly, our results suggest a potential role of the novel BRAF fusion in lung cancer development and therapy.

TP53 Mutations in Korean Patients with Non-small Cell Lung Cancer

Although TP53 mutations have been widely studied in lung cancer, the majority of studies have focused on exons 5-8 of the gene. In addition, TP53 mutations in Korean patients with lung cancers have not been investigated. We searched for mutations in the entire coding exons, including splice sites of the gene, in Korean patients with non-small cell lung cancer (NSCLC). Mutations of the gene were determined by direct sequencing in 176 NSCLCs. Sixty-nine mutations (62 different mutations) were identified in 65 tumors. Of the 62 mutations, 12 were novel mutations. TP53 mutations were more frequent in males, ever-smokers and squamous cell carcinomas than in females, never-smokers and adenocarcinomas, respectively (all comparisons, P<0.001). Missense mutations were most common (52.2%), but frameshift, nonsense, and splice-site mutations were frequently observed at frequencies of 18.8%, 15.9% and 10.1%, respectively. Of the 69 mutations, 9 (13.0%) were found in the oligomerization domain. In addition, the proportion of mutations in the oligomerization domain was significantly higher in adenocarcinomas than in squamous cell carcinomas (23.5% vs. 2.9%, P=0.01). Our study provides clinical and molecular characteristics of TP53 mutations in Korean patients with NSCLCs.

A genetic variation in microRNA target site of KRT81 gene is associated with survival in early stage non-small cell lung cancer

BACKGROUND MicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenoms MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs. RESULTS Of the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues. CONCLUSION The rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.BACKGROUND MicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenoms MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs. RESULTS Of the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues. CONCLUSION The rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.

EML4-ALK Fusion Gene in Korean Non-Small Cell Lung Cancer

A fusion gene between echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) has been identified in non-small cell lung cancers (NSCLCs). Although a few studies have evaluated EML4-ALK fusion genes in Korean NSCLCs, the prevalence of different EML4-ALK fusion variants has yet to be clearly assessed. Herein, we have examined the profiles of EML4-ALK fusion gene variants in Korean patients of NSCLCs. EML4-ALK fusion genes have been detected in 10 (6.0%) of 167 patients of NSCLCs and in 9 (7.4%) of 121 patients of adenocarcinoma. Of the 10 patients with fusion genes identified, 8 (80%) were E13;A20 (variant 1) and 2 (20%) were E6;A20, with an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). These results indicate that the profiles of EML4-ALK fusion gene variants in Korean patients of NSCLC may differ from those in other ethnic populations. Herein, we describe for the first time the profiles of EML4-ALK fusion variants of Korean patients with NSCLCs.

Homeobox A9 directly targeted by miR-196b regulates aggressiveness through nuclear Factor-kappa B activity in non-small cell lung cancer cells.

MicroRNAs (miRNAs) are recognized as crucial posttranscriptional regulators of gene expression, and play critical roles as oncogenes or tumor suppressors in various cancers. Here, we show that miR‐196b is upregulated in mesenchymal‐like‐state non‐small cell lung cancer (NSCLC) cells and lung cancer tissues. Moreover, miR‐196b upregulation stimulates cell invasion and a change in cell morphology to a spindle shape via loss of cell‐to‐cell contacts. We identified homeobox A9 (HOXA9) as a target gene of miR‐196b by using public databases such as TargetScan, miRDB, and microRNA.org. HOXA9 expression is inversely correlated with miR‐196b levels in clinical NSCLC samples as compared to that in corresponding control samples, and with the migration and invasion of NSCLC cells. Ectopic expression of HOXA9 resulted in a suppression of miR‐196b‐induced cell invasion, and HOXA9 reexpression increased E‐cadherin expression. Furthermore, HOXA9 potently attenuated the expression of snail family zinc finger 2 (SNAI2/SLUG) and matrix metallopeptidase 9 (MMP9) by controlling the binding of nuclear factor–kappa B to the promoter of SLUG and MMP9 genes, respectively. Therefore, we suggest that HOXA9 plays a central role in controlling the aggressive behavior of lung cancer cells and that miR‐196b can serve as a potential target for developing anticancer agents.

MicroRNA-146a inhibits epithelial mesenchymal transition in non-small cell lung cancer by targeting insulin receptor substrate 2.

During cancer progression, some tumor cells show changes in their plasticity by morphological and phenotypical conversions, as an expression of mesenchymal markers and loss of epithelial markers, collectively referred to as epithelial-mesenchymal transition (EMT). EMT has been increasingly recognized as a critical phenomenon in lung cancer progression. The goal of this study was to identify microRNAs involved in lung cancer progression. A microarray and qRT-PCR were performed to investigate the miRNA expression profiles in mesenchymal-like lung cancer cells. The role of miR‑146a in lung cancer progression was measured by invasion and migration assays in vitro. Bioinformatics and luciferase report assays were used to identify the target of miR‑146a. The expression of miR‑146a was reduced in mesenchymal-like lung cancer cell lines. The overexpression of miR‑146a induced a marked reduction of the mesenchymal marker and increase the epithelial marker in lung cancer cell lines. Moreover, the overexpression of miR‑146a suppressed lung cancer cell migration and invasion. Co-treatment with miR‑146a and gefitinib treatment showed a significant reduction of invasion in the resistant lung cancer cells induced by EMT. The expression of miR‑146a was downregulated in advanced lung cancer tissues. Insulin receptor substrate 2 (IRS2), an adaptor protein that modulates normal growth, metabolism, survival, and differentiation, was identified as a target of miR‑146a. miR‑146a regulated the expression of IRS2 at the mRNA and protein levels. These data demonstrate for the first time that miR‑146a suppresses lung cancer progression by repressing IRS2 expression. This provides new insight into the post-transcriptional regulation of lung cancer progression by miRNAs, a potential approach for the treatment of lung cancer.