Tomohiro Kumamoto

Dual-strand tumor-suppressor microRNA-145 ( miR-145-5p and miR-145-3p ) coordinately targeted MTDH in lung squamous cell carcinoma

Patients with lung adenocarcinoma may benefit from recently developed molecular targeted therapies. However, analogous advanced treatments are not available for patients with lung squamous cell carcinoma (lung SCC). The survival rate of patients with the advanced stage of lung SCC remains poor. Exploration of novel lung SCC oncogenic pathways might lead to new treatment protocols for the disease. Based on this concept, we have identified microRNA- (miRNA) mediated oncogenic pathways in lung SCC. It is well known that miR-145-5p (the guide strand) functions as a tumor suppressor in several types of cancer. However, the impact of miR-145-3p (the passenger strand) on cancer cells is still ambiguous. Expression levels of miR-145-5p and miR-145-3p were markedly reduced in cancer tissues, and ectopic expression of these miRNAs inhibited cancer cell aggressiveness, suggesting that both miR-145-3p as well as miR-145-5p acted as antitumor miRNAs. We identified seven putative target genes (MTDH, EPN3, TPD52, CYP27B1, LMAN1, STAT1 and TXNDC12) that were coordinately regulated by miR-145-5p and miR-145-3p in lung SCC. Among the seven genes, we found that metadherin (MTDH) was a direct target of these miRNAs. Kaplan–Meier survival curves showed that high expression of MTDH predicted reduced survival of lung SCC patients. We investigated pathways downstream from MTDH by using genome-wide gene expression analysis. Our data showed that several anti-apoptosis and pro-proliferation genes were involved in pathways downstream from MTDH in lung SCC. Taken together, both strands of miR-145, miR-145-5p and miR-145-3p are functional and play pivotal roles as antitumor miRNAs in lung SCC.

Tumor-suppressive microRNA-29 family inhibits cancer cell migration and invasion directly targeting LOXL2 in lung squamous cell carcinoma

Lung cancer remains the most frequent cause of cancer-related death in developed countries. A recent molecular-targeted strategy has contributed to improvement of the remarkable effect of adenocarcinoma of the lung. However, such treatment has not been developed for squamous cell carcinoma (SCC) of the disease. Our recent studies of microRNA (miRNA) expression signatures of human cancers showed that the microRNA-29 family (miR-29a, miR-29b and miR-29c) significantly reduced cancer tissues compared to normal tissues. These findings suggest that miR-29s act as tumor-suppressors by targeting several oncogenic genes. The aim of the study was to investigate the functional significance of miR-29s in lung SCC and to identify miR-29s modulating molecular targets in lung SCC cells. Restoration of all mature members of the miR-29s inhibited cancer cell migration and invasion. Gene expression data combined in silico analysis and luciferase reporter assays demonstrated that the lysyl oxidase-like 2 (LOXL2) gene was a direct regulator of tumor-suppressive miR-29s. Moreover, overexpressed LOXL2 was confirmed in lung SCC clinical specimens, and silencing of LOXL2 inhibited cancer cell migration and invasion in lung SCC cell lines. Our present data suggested that loss of tumor-suppressive miR-29s enhanced cancer cell invasion in lung SCC through direct regulation of oncogenic LOXL2. Elucidation of the novel lung SCC molecular pathways and targets regulated by tumor-suppressive miR-29s will provide new insights into the potential mechanisms of oncogenesis and metastasis of the disease.

MicroRNAs in non-small cell lung cancer and idiopathic pulmonary fibrosis

In spite of advances in the diagnosis and current molecular target therapies of lung cancer, this disease remains the most common cause of cancer-related death worldwide. Approximately 80% of lung cancers is non-small cell lung cancer (NSCLC), and 5-year survival rate of the disease is ~20%. On the other hand, idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology. IPF is refractory to treatment and has a very low survival rate. Moreover, IPF is frequently associated with lung cancer. However, the common mechanisms shared by these two diseases remain poorly understood. In the post-genome sequence era, the discovery of noncoding RNAs, particularly microRNAs (miRNAs), has had a major impact on most biomedical fields, and these small molecules have been shown to contribute to the pathogenesis of NSCLC and IPF. Investigation of novel RNA networks mediated by miRNAs has improved our understanding of the molecular mechanisms of these diseases. This review summarizes our current knowledge on aberrantly expressed miRNAs regulating NSCLC and IPF based on miRNA expression signatures.

Regulation of LOXL2 and SERPINH1 by antitumor microRNA-29a in lung cancer with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease that is refractory to treatment and carries a high mortality rate. IPF is frequently associated with lung cancer. Identification of molecular targets involved in both diseases may elucidate novel molecular mechanisms contributing to their pathology. Recent studies of microRNA (miRNA) expression signatures showed that microRNA-29a (miR-29a) was downregulated in IPF and lung cancer. The aim of this study was to investigate the functional significance of miR-29a in lung cancer cells (A549 and EBC-1) and lung fibroblasts (MRC-5) and to identify molecular targets modulated by miR-29a in these cells. We confirmed the downregulation of miR-29a in clinical specimens of IPF and lung cancer. Restoration of miR-29a suppressed cancer cell aggressiveness and fibroblast migration. A combination of gene expression data and in silico analysis showed that a total of 24 genes were putative targets of miR-29a. Among them, lysyl oxidase-like 2 (LOXL2) and serpin peptidase inhibitor clade H, member 1 (SERPINH1) were direct targets of miR-29a by luciferase reporter assays. The functions of LOXL2 and SERPINH1 contribute significantly to collagen biosynthesis. Overexpression of LOXL2 and SERPINH1 was observed in clinical specimens of lung cancer and fibrotic lesions. Downregulation of miR-29a caused overexpression of LOXL2 and SERPINH1 in lung cancer and IPF, suggesting that these genes are involved in the pathogenesis of these two diseases.

Regulation of TPD52 by antitumor microRNA-218 suppresses cancer cell migration and invasion in lung squamous cell carcinoma

The development of targeted molecular therapies has greatly benefited patients with lung adenocarcinomas. In contrast, these treatments have had little benefit in the management of lung squamous cell carcinoma (lung SCC). Therefore, new treatment options based on current genomic approaches are needed for lung SCC. Aberrant microRNA (miRNA) expression has been shown to promote lung cancer development and aggressiveness. Downregulation of microRNA-218 (miR-218) was frequently observed in our miRNA expression signatures of cancers, and previous studies have shown an antitumor function of miR-218 in several types of cancers. However, the impact of miR-218 on lung SCC is still ambiguous. The present study investigated the antitumor roles of miR-218 in lung SCC to identify the target genes regulated by this miRNA. Ectopic expression of miR-218 greatly inhibited cancer cell migration and invasion in the lung SCC cell lines EBC-1 and SK-MES-1. Through a combination of in silico analysis and gene expression data searching, tumor protein D52 (TPD52) was selected as a putative target of miR-218 regulation. Moreover, direct binding of miR-218 to the 3′-UTR of TPD52 was observed by dual luciferase reporter assay. Overexpression of TPD52 was observed in lung SCC clinical specimens, and knockdown of TPD52 significantly suppressed cancer cell migration and invasion in lung SCC cell lines. Furthermore, the downstream pathways mediated by TPD52 involved critical regulators of genomic stability and mitotic checkpoint genes. Taken together, our data showed that downregulation of miR-218 enhances overexpression of TPD52 in lung SCC cells, promoting cancer cell aggressiveness. Identification of tumor-suppressive miRNA-mediated RNA networks of lung SCC will provide new insights into the potential mechanisms of the molecular pathogenesis of the disease.

The microRNA expression signature of small cell lung cancer: tumor suppressors of miR-27a-5p and miR-34b-3p and their targeted oncogenes

Small cell lung cancer (SCLC) constitutes approximately 15% of all diagnosed lung cancers. SCLC is a particularly lethal malignancy, as the 2-year survival rate after appropriate treatment is less than 5%. The patients with SCLC have not been received a benefit of the recently developed molecular targeted treatment. Therefore, a new treatment strategy is necessary for the patients. The molecular mechanisms underlying the aggressiveness of SCLC cells and their development of treatment-resistance are still ambiguous. In this study, we newly constructed a microRNA (miRNA) expression signature of SCLC by analysis of autopsy specimens. Based on the resultant signature, four miRNAs (miR-27a-5p, miR-485-3p, miR-34-5p and miR-574-3p) were found to be candidate anti-tumor miRNAs. To investigate their functional importance, we first validated the downregulation of miR-27a-5p and miR-34b-3p in SCLC clinical specimens. Next, we demonstrated that ectopic expression of both miR-27a-5p and miR-34b-3p significantly inhibited cancer cell aggressiveness. Our in silico analyses showed that four genes (topoisomerase 2 alpha (TOP2A), maternal embryonic leucine zipper kinase (MELK), centromere protein F (CENPF) and SRY-box 1 (SOX1) were identified as miR-27a-5p- and miR-34b-3p-regulated genes. Based on immunohistochemical analysis, TOP2A, MELK and CENPF were involved in SCLC pathogenesis. These genes might contribute to high proliferation and early metastatic spread of SCLC cells. Elucidation of differentially expressed miRNA-mediated cancer pathways based on SCLC signature may provide new insights into the mechanisms of SCLC pathogenesis.

Comparison of the COPD Population Screener and International Primary Care Airway Group questionnaires in a general Japanese population: the Hisayama study.

Background The incidence of chronic obstructive pulmonary disease (COPD) is increasing worldwide. In Japan and other countries, epidemiological studies have found that many patients with COPD are underdiagnosed and untreated, and thus, early detection and treatment of COPD has been emphasized. Screening questionnaires may have utility in the initial detection of COPD. Objective This study aimed to validate and compare the COPD Population Screener (COPD-PS) and the International Primary Care Airway Group (IPAG) questionnaires in a general Japanese population. Patients and methods Eligible subjects 40 years of age and older living in the town of Hisayama were solicited to participate in a health checkup in 2012. All subjects 40–79 years of age without physician-diagnosed asthma or lung resection were recruited, and 2,336 subjects who fully completed both questionnaires and who had valid spirometry measurements were analyzed. Persistent airflow obstruction (AO) was defined by a postbronchodilator forced expiratory volume in 1 second/forced vital capacity <0.70. Receiver operating characteristic curves, net reclassification improvement, and integrated discrimination improvement were used to examine the ability of the COPD-PS and IPAG questionnaires to discriminate between subjects with and without AO. Results The overall area under the receiver operating characteristic curve for the COPD-PS questionnaire was 0.747 (95% confidence interval [CI], 0.707–0.788) and for the IPAG was 0.775 (95% CI, 0.735–0.816), with no significant difference (P=0.09). The net reclassification improvement and integrated discrimination improvement were −0.107 (95% CI, −0.273–0.058; P=0.203) and −0.014 (95% CI, −0.033–0.006; P=0.182), respectively. Conclusion The five-item COPD-PS questionnaire was comparable to the eight-item IPAG for discriminating between subjects with and without AO. The COPD-PS is a simple and useful screening questionnaire for persistent AO.

Serum B cell–activating factor (BAFF) level in connective tissue disease associated interstitial lung disease

BackgroundInterstitial lung diseases (ILDs) are common in patients with connective tissue diseases (CTDs). Although the diagnosis of an underlying CTD in ILD (CTD-ILD) affects both prognosis and treatment, it is sometimes difficult to distinguish CTD-ILD from chronic fibrosing interstitial pneumonia (CFIP). B cell–activating factor belonging to the tumour necrosis factor family (BAFF) plays a crucial role in B cell development, survival, and antibody production.MethodsWe examined serum levels of BAFF, surfactant protein D (SP-D), and Krebs von den Lungen-6 (KL-6) in 33 patients with CTD-ILD, 16 patients with undifferentiated CTD-ILD, 19 patients with CFIP, and 26 healthy volunteers. And we analysed the relationship between serum BAFF levels and pulmonary function, as well as the expression of BAFF in the lung tissue of patients with CTD-ILD.ResultsSerum levels of BAFF were significantly higher in CTD-ILD patients compared to healthy subjects and CFIP patients. However, there were no significant differences in serum levels of SP-D and KL-6. Furthermore, serum BAFF levels in CTD-ILD patients were inversely correlated with pulmonary function. BAFF was strongly expressed in the lungs of CTD-ILD patients, but weakly in normal lungs.DiscussionThis is the first study to demonstrate that serum BAFF levels were significantly higher in CTD-ILD patients compared to healthy subjects and CFIP patients. Furthermore, serum BAFF levels were correlated with pulmonary function. We consider that serum BAFF levels in patients with CTD-ILD reflect the presence of ILDs disease activity and severity.ConclusionThese finding suggest that BAFF may be a useful marker for distinguishing CTD-ILD from CFIP.

Dual strands of the miR-145 duplex ( miR-145-5p and miR-145-3p ) regulate oncogenes in lung adenocarcinoma pathogenesis

Our original microRNA (miRNA) expression signatures (based on RNA sequencing) revealed that both strands of the miR-145 duplex (miR-145-5p, the guide strand, and miR-145-3p, the passenger strand) were downregulated in several types of cancer tissues. Involvement of passenger strands of miRNAs in cancer pathogenesis is a new concept in miRNA biogenesis. In our continuing analysis of lung adenocarcinoma (LUAD) pathogenesis, we aimed here to identify important oncogenes that were controlled by miR-145-5p and miR-145-3p. Downregulation of miR-145-5p and miR-145-3p was confirmed in LUAD clinical specimens. Functional assays showed that miR-145-3p significantly blocked the malignant abilities in LUAD cells, e.g., cancer cell proliferation, migration and invasion. Thus, the data showed that expression of the passenger strand of the miR-145-duplex acted as an anti-tumor miRNA. In LUAD cells, we identified four possible target genes (LMNB2, NLN, SIX4, and DDC) that might be regulated by both strands of miR-145. Among the possible targets, high expression of LMNB2 predicted a significantly poorer prognosis of LUAD patients (disease-free survival, p = 0.0353 and overall survival, p = 0.0017). Overexpression of LMNB2 was detected in LUAD clinical specimens and its aberrant expression promoted malignant transformation of LUAD cells. Genes regulated by anti-tumor miR-145-5p and miR-145-3p are closely involved in the molecular pathogenesis of LUAD. We suggest that they are promising prognostic markers for this disease. Our approach, based on the roles of anti-tumor miRNAs, will contribute to improved understanding of the molecular pathogenesis of LUAD.

Napsin A levels in epithelial lining fluid as a diagnostic biomarker of primary lung adenocarcinoma

BackgroundIt is crucial to develop novel diagnostic approaches for determining if peripheral lung nodules are malignant, as such nodules are frequently detected due to the increased use of chest computed tomography scans. To this end, we evaluated levels of napsin A in epithelial lining fluid (ELF), since napsin A has been reported to be an immunohistochemical biomarker for histological diagnosis of primary lung adenocarcinoma.MethodsIn consecutive patients with indeterminate peripheral lung nodules, ELF samples were obtained using a bronchoscopic microsampling (BMS) technique. The levels of napsin A and carcinoembryonic antigen (CEA) in ELF at the nodule site were compared with those at the contralateral site. A final diagnosis of primary lung adenocarcinoma was established by surgical resection.ResultsWe performed BMS in 43 consecutive patients. Among patients with primary lung adenocarcinoma, the napsin A levels in ELF at the nodule site were markedly higher than those at the contralateral site, while there were no significant differences in CEA levels. Furthermore, in 18 patients who were undiagnosed by bronchoscopy and finally diagnosed by surgery, the napsin A levels in ELF at the nodule site were identically significantly higher than those at the contralateral site. In patients with non-adenocarcinoma, there were no differences in napsin A levels in ELF. The area under the receiver operator characteristic curve for identifying primary lung adenocarcinoma was 0.840 for napsin A and 0.542 for CEA.ConclusionEvaluation of napsin A levels in ELF may be useful for distinguishing primary lung adenocarcinoma.