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.

Dual-receptor (EGFR and c-MET) inhibition by tumor-suppressive miR-1 and miR-206 in head and neck squamous cell carcinoma.

Our studies of microRNA (miRNA) expression signatures have shown that microRNA-1 (miR-1) and microRNA-206 (miR-206) were downregulated in head and neck squamous cell carcinoma (HNSCC) clinical specimens. The seed sequences of these miRNAs are identical, suggesting that the identification of the molecular targets regulated by miR-1 and miR-206 will provide new insights into novel mechanisms of HNSCC pathogenesis. Our present data showed that restoration of miR-1 and miR-206 significantly inhibited HNSCC cells' aggressiveness. A combination of gene expression data and in silico analysis revealed that several pathways ('pathway in cancer', 'focal adhesion pathway', 'MAPK signaling pathway', 'regulation of actin cytoskeleton pathway' and 'ECM-receptor interaction pathway') were regulated by miR-1 and miR-206. Among them, we found that two growth factor receptors, epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET), were directly regulated by both miR-1 and miR-206 in HNSCC cells. Also, downstream oncogenic signaling of these receptors was reduced by restoration of miR-1 or miR-206 expression. Moreover, overexpression of EGFR and c-MET was observed in HNSCC clinical specimens. The identification of targets modulated by tumor-suppressive miR-1 and miR-206 may lead to a better understanding of molecular pathogenesis of HNSCC.

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.

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.

Downregulation of the microRNA-1/133a cluster enhances cancer cell migration and invasion in lung-squamous cell carcinoma via regulation of Coronin1C.

Lung cancer is clearly the primary cause of cancer-related deaths worldwide. Recent molecular-targeted strategy has contributed to improvement of the curative effect of adenocarcinoma of the lung. However, such current treatment has not been developed for squamous cell carcinoma (SCC) of the disease. The new genome-wide RNA analysis of lung-SCC may provide new avenues for research and the development of the disease. Our recent microRNA (miRNA) expression signatures of lung-SCC revealed that clustered miRNAs miR-1/133a were significantly reduced in cancer tissues. Here, we found that restoration of both mature miR-1 and miR-133a significantly inhibited cancer cell proliferation, migration and invasion. Coronin-1C (CORO1C) was a common target gene of the miR-1/133a cluster, as shown by the genome-wide gene expression analysis and the luciferase reporter assay. Silencing of CORO1C gene expression inhibited cancer cell proliferation, migration and invasion. Furthermore, CORO1C-regulated molecular pathways were categorized by using si-CORO1C transfectants. Further analysis of novel cancer signaling pathways modulated by the tumor-suppressive cluster miR-1/133a will provide insights into the molecular mechanisms of lung-SCC oncogenesis and metastasis.

[A case of pulmonary toxocariasis successfully treated with ivermectin].

A 36-year-old man who often ate raw chicken meat had abnormal chest radiograph findings on a health check-up in July 2009, and visited our department. A chest computed tomographic scan revealed 6 nodular shadows in both lungs, and a hematologic examination revealed eosinophilia and elevation of IgE. Because of his history of eating raw chicken meat, antiparasite antibody testing was performed which revealed antibodies to Toxocara canis, leading to the diagnosis of pulmonary toxocariasis due to Toxocara canis. Although treatment with albendazole was performed, it was discontinued because of hepatic impairment. However, hematological examination showed that his eosinophil count and IgE increased again, and chest image findings were exacerbated. Therefore, ivermectin, reported as effective in cases outside Japan, was administered, resulting in decreased peripheral eosinophils, normalized IgE level, and disappearance of the shadows on chest images. In Japan, no cases of pulmonary toxocariasis responding well to ivermectin have previously been reported. Administration of ivermectin should be considered when albendazol cannot be used due to hepatic impairment or related problems.

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 (miR­29a, miR­29b and miR­29c) significantly reduced cancer tissues compared to normal tissues. These findings suggest that miR­29s act as tumor-suppressors by targeting several oncogenic genes. The aim of the study was to investigate the functional significance of miR­29s in lung SCC and to identify miR­29s modulating molecular targets in lung SCC cells. Restoration of all mature members of the miR­29s 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 tumor­suppressive miR­29s. 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 miR­29s 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 miR­29s will provide new insights into the potential mechanisms of oncogenesis and metastasis of the disease.

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.

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.

Dual regulation of receptor tyrosine kinase genes EGFR and c-Met by the tumor-suppressive microRNA-23b/27b cluster in bladder cancer.

Recent clinical trials of chemotherapeutics for advanced bladder cancer (BC) have shown limited benefits. Therefore, new prognostic markers and more effective treatment strategies are required. One approach to achieve these goals is through the analysis of RNA networks. Our recent studies of microRNA (miRNA) expression signatures revealed that the microRNA-23b/27b (miR-23b/27b) cluster is frequently downregulated in various types of human cancers. However, the functional role of the miR-23b/27b cluster in BC cells is still unknown. Thus, the aim of the present study was to investigate the functional significance of the miR-23b/27b cluster and its regulated molecular targets, with an emphasis on its contributions to BC oncogenesis and metastasis. The expression levels of the miR-23b/27b cluster were significantly reduced in BC clinical specimens. Restoration of mature miR-23b or miR-27b miRNAs significantly inhibited cancer cell migration and invasion, suggesting that these clustered miRNAs function as tumor suppressors. Gene expression data and in silico analysis demonstrated that the genes coding for the epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met) were potential targets of the miR-23b/27b cluster. Luciferase reporter assays and western blotting demonstrated that EGFR and c-Met receptor trypsine kinases were directly regulated by these clustered miRNAs. We conclude that the decreased expression of the tumor-suppressive miR-23b/27b cluster enhanced cancer cell proliferation, migration and invasion in BC through direct regulation of EGFR and c-Met signaling pathways. Our data on RNA networks regulated by tumor-suppressive miR-23b/27b provide new insights into the potential mechanisms of BC oncogenesis and metastasis.

Tumor-suppressive microRNA-206 as a dual inhibitor of MET and EGFR oncogenic signaling in lung squamous cell carcinoma.

Expression of the oncogene hepatocyte growth factor receptor (MET) and phosphorylation of the MET protein have been associated with both primary and acquired resistance to tyrosine kinase inhibitors (TKIs) used in therapy targeting the epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancers (NSCLCs). Therefore, simultaneous inhibition of both of these receptor tyrosine kinases (RTKs) should improve disease treatment. Our previous study of microRNA (miRNA) expression signatures of lung squamous cell carcinoma (lung-SCC) revealed that microRNA-206 (miR­206) was significantly reduced in lung-SCC tissues, suggesting that miR­206 functions as a tumor suppressor in the disease. Furthermore, putative miR­206 binding sites were annotated in the 3'-UTRs of MET and EGFR RTKs in miRNA databases. The aim of the study was to investigate the functional significance of miR­206 in lung-SCC and to confirm the inhibition of both MET and EGFR oncogenic signaling by expression of miR­206 in cancer cells. We found that restoration of mature miR­206 inhibited cancer cell proliferation, migration, and invasion in EBC-1 cells through downregulation of both mRNA and protein levels of MET and EGFR. Interestingly, phosphorylation of ERK1/2 and AKT signaling were inhibited by restoration of miR­206 in cancer cells. Overexpression of MET and EGFR were observed in clinical specimens of lung-SCC. Tumor-suppressive miR­206 inhibited dual signaling networks activated by MET and EGFR, and these findings will provide new insights into the novel molecular mechanisms of lung-SCC oncogenesis and new therapeutic approaches for the treatment of this disease.

Tumour-suppressive microRNA-24-1 inhibits cancer cell proliferation through targeting FOXM1 in bladder cancer.

Here, we found that microRNA-24-1 (miR-24-1) is significantly reduced in bladder cancer (BC) tissues, suggesting that it functions as a tumour suppressor. Restoration of mature miR-24-1 inhibits cancer cell proliferation and induces apoptosis. Forkhead box protein M1 (FOXM1) is a direct target gene of miR-24-1, as shown by genome-wide gene expression analysis and luciferase reporter assay. Overexpressed FOXM1 is confirmed in BC clinical specimens, and silencing of FOXM1 induces apoptosis in cancer cell lines. Our data demonstrate that the miR-24-1-FOXM1 axis contributes to cancer cell proliferation in BC, and elucidation of downstream signalling will provide new insights into the molecular mechanisms of BC oncogenesis.

Primary leiomyosarcoma of the pulmonary artery: a case of a 20-month survivor after incomplete surgical resection.

We report a 53-year-old man who presented with dyspnea and low-grade fever. Cardiac ultrasound showed pulmonary hypertension and an ill-defined echogenic mass within the pulmonary trunk. Computed tomography scan revealed an inhomogeneous mass which filled the main pulmonary trunk with near-total occlusion, and extended into both pulmonary arteries. Anticoagulant therapy was administered based on a presumptive diagnosis of pulmonary thromboembolism. Positron-emission tomography scan was useful for differentiating the mass, which was determined as a pulmonary artery sarcoma by surgical resection. Although complete resection was impossible, the patient survived for 20 months with adjuvant chemotherapy and medical treatment.

A case of Aspergillus empyema successfully treated with combination therapy of voriconazole and micafungin: excellent penetration of voriconazole and micafungin into pleural fluid.

A 62-year-old man with empyema caused by Aspergillus fumigatus was successfully treated with a combination of voriconazole (VRCZ) and micafungin (MCFG). Data regarding the penetration of antifungal agents into pleural fluid are limited. Thus, we measured the concentration of VRCZ and MCFG in his plasma and pleural fluid. Penetration of VRCZ and MCFG into the pleural fluid was excellent. Therefore, the combination therapy using VRCZ and MCFG may contribute to successful management of Aspergillus empyema.