Hirotaka Osada

A Polycistronic MicroRNA Cluster, miR-17-92, Is Overexpressed in Human Lung Cancers and Enhances Cell Proliferation

MicroRNAs (miRNAs) are small noncoding RNAs, thought to be involved in physiologic and developmental processes by negatively regulating expression of target genes. We have previously reported frequent down-regulation of the let-7 miRNA family in lung cancers and, in the present study, assessed alteration in a panel of 19 lung cancer cell lines. As a result, we found for the first time that the miR-17-92 cluster, which comprises seven miRNAs and resides in intron 3 of the C13orf25 gene at 13q31.3, is markedly overexpressed in lung cancers, especially with small-cell lung cancer histology. Southern blot analysis revealed the presence of increased gene copy numbers of the miRNA cluster in a fraction of lung cancer cell lines with overexpression. In addition, we were able to show predominant localization of C13orf25 transcripts within the nucleus and introduction of the expression construct of the miR-17-92 cluster, but not the putative open reading frame of C13orf25, enhancing lung cancer cell growth. These findings clearly suggest that marked overexpression of the miR-17-92 cluster with occasional gene amplification may play a role in the development of lung cancers, especially in their most aggressive form, small-cell lung cancer, and that the C13orf25 gene may well be serving as a vehicle in this regard.

Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer

Lung cancer has become the leading cause of cancer death in many economically well-developed countries. Recent molecular biological studies have revealed that overt lung cancers frequently develop through sequential morphological steps, with the accumulation of multiple genetic and epigenetic alterations affecting both tumor suppressor genes and dominant oncogenes. Cell cycle progression needs to be properly regulated, while cells have built-in complex and minute mechanisms such as cell cycle checkpoints to maintain genomic integrity. Genes in the p16INK4A-RB and p14ARF-p53 pathways appear to be a major target for genetic alterations involved in the pathogenesis of lung cancer. Several oncogenes are also known to be altered in lung cancer, leading to the stimulation of autocrine/paracrine loops and activation of multiple signaling pathways. It is widely acknowledged that carcinogens in cigarette smoke are deeply involved in these multiple genetic alterations, mainly through the formation of DNA adducts. A current understanding of the molecular mechanisms of lung cancer pathogenesis and progression is presented in relation to cigarette smoking, an absolute major risk factor for lung cancer development, by reviewing genetic alterations of various tumor suppressor genes and oncogenes thus far identified in lung cancer, with brief summaries of their functions and regulation.

Reduced expression of class II histone deacetylase genes is associated with poor prognosis in lung cancer patients.

HDAC genes are thought to be involved in gene expression through the regulation of chromatin structure, alterations of which may cause abnormal gene silencing in cancers. To clarify the possible role of HDAC genes during tumor development and progression, we studied their expression and influence on clinical features. Expression levels of HDAC class I and class II genes in cancer tissues resected from 72 patients with NSCLC were measured with real‐time RT‐PCR. Their association with clinicopathologic features was statistically investigated. Reduced expression of each class II HDAC gene was significantly associated with poor prognosis and an independent predictor of poor prognosis. Of all the genes, HDAC10 was the strongest predictor of poor prognosis. Hierarchical clustering analysis showed that lung cancer tissues could be divided into 3 groups based on the expression level of class I and class II HDAC genes. The group with reduced expression of class II HDACs showed poor prognosis. These results suggest that class II HDACs may repress critical genes and that low expression of these genes may play a role in lung cancer progression. Results of clustering analyses imply that class II HDAC genes may be regulated by a similar mechanism and deregulated during cancer development.

Frequent and histological type-specific inactivation of 14-3-3σ in human lung cancers

One isoform of the 14-3-3 family, 14-3-3σ, plays a crucial role in the G2 checkpoint by sequestering Cdc2-cyclinB1 in the cytoplasm, and the expression of 14-3-3σ is frequently lost in breast cancers. This loss of expression is thought to cause a G2 checkpoint defect, resulting in chromosomal aberrations. Since lung cancers frequently carry numerous chromosomal aberrations, we examined the DNA methylation status and expression level of the 14-3-3σ gene in 37 lung cancer cell lines and 30 primary lung tumor specimens. We found that small cell lung cancer (SCLC) cell lines frequently showed DNA hypermethylation (9 of 13 lines, 69%), and subsequent silencing of the 14-3-3σ gene. Among non-small cell lung cancers (NSCLC), large cell lung cancer cell lines showed frequent hypermethylation and silencing of 14-3-3σ (4 or 7 lines, 57%). In contrast, in other NSCLC cell lines, hypermethylation occurred very rarely (1 of 17 lines, 6%). All eight primary SCLC specimens examined also showed a loss or significant reduction in 14-3-3σ expression in vivo, while a loss or reduction of 14-3-3σ expression was very rare in primary NSCLC specimens (1 of 22 tissues, 5%). This is the first description that indicates lung cancers frequently show significant inactivation of the 14-3-3σ gene mainly due to DNA hypermethylation in SCLC, but rarely in NSCLC, suggesting involvement of the 14-3-3σ gene in lung tumorigenesis in a histological type-specific manner.

Identification of frequent impairment of the mitotic checkpoint and molecular analysis of the mitotic checkpoint genes, hsMAD2 and p55CDC, in human lung cancers.

The mitotic checkpoint is thought to be essential for ensuring accurate chromosome segregation by implementing mitotic delay in response to a spindle defect. To date, however, very little data has become available on the defects of the mitotic checkpoint in human cancer cells. In the present study, impaired mitotic checkpoint was found in four (44%) of nine human lung cancer cell lines. To our knowledge, this is the first demonstration of frequent impairment of the mitotic checkpoint in this leading cause of cancer deaths. As an initial step towards elucidation of the underlying mechanism, we further undertook a search for mutations in a key component of the mitotic checkpoint, known as hsMAD2, and its immediate downstream molecule, p55CDC. No such mutations were found, however, in either 21 lung cancer cell lines or 25 primary lung cancer cases, although we could identify silent polymorphisms and the transcribed and processed hsMAD2 pseudogene that was subsequently mapped at 14q21-q23. The present observations appear to warrant further investigations, such as search for alterations in other components, to better understand the molecular pathogenesis of this fatal disease, and warn against potential misinterpretation when performing mutational analyses for other cancer types based on cDNA templates.

Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers.

The CHFR gene, which was recently cloned by Scolnick and Halazonetis in search for a novel mitotic checkpoint gene with fork-head association motifs, has been suggested to play a key role in the mitotic prophase checkpoint. In this study, we demonstrated tumor-specific aberrant hypermethylation of the promoter region of the CHFR gene in a significant fraction of lung cancers in association with loss of detectable levels of CHFR transcripts. Aberrant hypermethylation was observed in seven of 37 primary lung cancer cases. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored expression of the CHFR gene in lung cancer cell lines exhibiting aberrant hypermethylation and loss of its expression. In contrast, genetic alterations were found to be infrequent in lung cancers. This is the first description of aberrant hypermethylation of the CHFR gene in any type of human cancer, and provides further evidence of the involvement of multiple checkpoint alterations in lung cancer.

Clinicopathologic study of large cell anaplastic lymphoma (ki‐1‐positive large cell lymphoma) among the japanese

The clinical, prognostic, phenotypic, and genotypic findings of 30 patients with large cell anaplastic lymphoma (Ki‐1‐positive large cell lymphoma) were analyzed. There were 13 male and 17 female patients (male‐female ratio, 0.8) whose ages ranged from 3 to 81 years of age (mean, 28 years of age; 67% of the patients younger than 30 years of age). The 5‐year survival rate was 52%; this was better than that of other types of high‐grade peripheral T‐cell lymphoma. Histologic examination showed distinctive morphologic features such as tumor cell pleomorphism, sinus infiltration, fibrosis, partial lymph node involvement, sparing of B‐cell regions, and occasional plasma cell infiltrates. Eighty percent of the cases were of T‐cell phenotype, and others expressed neither B‐cell nor T‐cell markers. The tumors were frequently positive for a histocompatibility antigen (HLA‐DR), CD25 (the interleukin‐2 receptor), and epithelial membrane antigen. Rearrangements of the T‐cell receptor beta gene were observed in nine of 13 cases (69%). These findings indicated that many of the tumors had the phenotype and genotype of activated T‐cells. This study also showed that large cell anaplastic lymphoma has a survival figure intermediate between Hodgkins disease and low‐grade peripheral T‐cell lymphoma.

Counterbalance between RB inactivation and miR-17-92 overexpression in reactive oxygen species and DNA damage induction in lung cancers.

Small-cell lung cancer (SCLC) is a highly aggressive disease that exhibits rapid growth and genetic instability. We found earlier frequent overexpression of the miR-17–92 microRNA cluster, and showed that SCLC cells were addicted to continued expressions of miR-17–5p and miR-20a, major components of this microRNA cluster. In this study, we identified the frequent presence of constitutively phosphorylated H2AX (γ-H2AX), which reflects continuing DNA damage, preferentially in SCLC. Knockdown of RB induced γ-H2AX foci formation in non-small cell lung cancer (NSCLC) cells with wild-type RB, in association with growth inhibition and reactive oxygen species (ROS) generation, which was canceled by overexpression of miR-17–92. Conversely, induction of γ-H2AX was observed in a miR-17–92-overexpressing SCLC cell line with miR-20a antisense oligonucleotides. These findings suggest that miR-17–92 overexpression may serve as a fine-tuning influence to counterbalance the generation of DNA damage in RB-inactivated SCLC cells, thus reducing excessive DNA damage to a tolerable level and consequently leading to genetic instability. Therefore, miR-17–92 may be an excellent therapeutic target candidate to elicit excessive DNA damage in combination with DNA-damaging chemotherapeutics.

Multi-faceted analyses of a highly metastatic human lung cancer cell line NCI-H460-LNM35 suggest mimicry of inflammatory cells in metastasis.

This study established and characterized low-metastatic revertant and parental clones of a highly metastatic human lung cancer cell line, NCI-H460-LNM35 (hereafter referred to as LNM35). Expression-profiling analysis revealed that up-regulation of various proinflammatory cytokines and angiogenic chemotactic chemokines was present in LNM35. Further, while COX-2 itself is known to be inducible in inflammation, COX-2 expression levels correlated well with the capabilities of these clones for not only in vitro motility and invasion but also in vivo metastasis, and COX-2 inhibitors were shown for the first time to reduce lung cancer metastasis in vivo. These findings suggest that lung cancer cells may mimic inflammatory cells in the process of metastasis.

Molecular analysis of the mitotic checkpoint genes BUB1, BUBR1 and BUB3 in human lung cancers

Our previous studies showed that mitotic checkpoint impairment is present in about 40% of human lung cancer cell lines but that mutations in the MAD mitotic checkpoint genes are infrequent. In the present study, we examined 44 lung cancer cases for the potential involvement of the other gene family involved in the mitotic checkpoint, i.e. BUB. We found that the BUB gene family members including BUB1, BUBR1 and BUB3 are not frequent targets for mitotic checkpoint defects in lung cancers, if present at all. Further studies are thus warranted to elucidate the molecular basis for the acquisition of mitotic checkpoint defects in order to better understand the molecular pathogenesis of lung cancers.