Takeru Zama

TNFalpha-induced ATF3 expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.

ATF3 (Activating transcription factor 3), a member of the CREB/ATF family, can be induced by stress and growth factors in mammalian cells, and is thought to play an important role in the cardiovascular system. However, little is currently known about how the induction of ATF3 is regulated, except that the JNK pathway is involved. Here, we investigated the differential roles of the MAPK pathways involved in TNFα (tumour necrosis factor α)‐induced ATF3 expression in vascular endothelial cells. In human umbilical vein endothelial cells, the expression of constitutively active MKK7 (MAPK kinase 7) increased the number of ATF3‐positive cells, and dominant negative MKK7 suppressed the TNFα‐induced expression of ATF3, indicating a requirement for the JNK pathway. In contrast, the expression of constitutively active or dominant negative MEK1/2 (MAPK/ERK kinase 1/2) suppressed or enhanced TNFα‐mediated induction of ATF3, respectively. In support of this, the MEK1/2 specific inhibitor U0126 enhanced the expression of ATF3 induced by TNFα. Furthermore, the ERK pathway inhibits the TNFα‐mediated induction of ATF3 mRNA, but not its stability, suggesting the involvement of ERK activity in the transcriptional regulation of the ATF3 gene. Our results suggest that TNFα‐induced ATF3 gene expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.

MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer

Background MicroRNA (miRNA) is an emerging subclass of small non-coding RNAs that regulates gene expression and has a pivotal role for many physiological processes including cancer development. Recent reports revealed the role of miRNAs as ideal biomarkers and therapeutic targets due to their tissue- or disease-specific nature. Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, and laryngeal cancer has the highest incidence in it. However, the molecular mechanisms involved in laryngeal cancer development remain to be known and highly sensitive biomarkers and novel promising therapy is necessary. Methodology/Principal Findings To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. The resultant differentially expressed miRNAs were further tested by using quantitative real time PCR (qRT-PCR) on 43 laryngeal tissue samples including cancers, noncancerous counterparts, benign diseases and precancerous dysplasias. Significant expressional differences between matched pairs were reproduced in miR-133b, miR-455-5p, and miR-196a, among which miR-196a being the most promising cancer biomarker as validated by qRT-PCR analyses on additional 84 tissue samples. Deep sequencing analysis revealed both quantitative and qualitative deviation of miR-196a isomiR expression in laryngeal cancer. In situ hybridization confirmed laryngeal cancer-specific expression of miR-196a in both cancer and cancer stroma cells. Finally, inhibition of miR-196a counteracted cancer cell proliferation in both laryngeal cancer-derived cells and mouse xenograft model. Conclusions/Significance Our study provided the possibilities that miR-196a might be very useful in diagnosing and treating laryngeal cancer.

A family with hereditary factor X deficiency with a point mutation Gla32 to Gln in the Gla domain (factor X Tokyo).

We report a new family with hereditary factor X deficiency. The propositus had a markedly prolonged prothrombin time, a mild prolongation of activated partial thromboplastin time and a clotting time activated by Russells viper venom. Factor X activity in plasma was 3u2003u/dl (normal range 56–138u2003u/dl). Factor X antigen level was 61u2003u/dl. Molecular analysis revealed a homozygous mutation, Glu (GAG) to Gln (CAG) at residue 32 which normally undergoes γ‐carboxylation within the γ‐carboxyglutamic acid rich domain. The genotypes of family members completely correlated with their factor X activities. It is suggested that the Glu32 to Gln mutation is the molecular basis for the abnormal factor X in this family.

Polymorphisms in the UGT1A1 gene predict adverse effects of irinotecan in the treatment of gynecologic cancer in Japanese patients

Irinotecan is a key chemotherapeutic drug used to treat many tumors, including cervical and ovarian cancers; however, irinotecan can cause toxicity, particularly in the presence of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene polymorphisms, which are associated with reduced enzyme activity. Here, we investigated the prevalence of three different variants of UGT1A1 (UGT1A1*6, UGT1A1*27 and UGT1A1*28) and their relationships with irinotecan-induced adverse events in patients with gynecologic cancer, who are treated with lower doses of irinotecan than patients with other types of solid tumors. Fifty-three female patients treated with irinotecan and 362 female patients not treated with irinotecan were screened for UGT1A1*6, UGT1A1*27 and UGT1A1*28. Homozygosity for UGT1A1*6 or heterozygosity for UGT1A1*6/*28 was associated with a high risk of severe absolute neutrophil count decrease or diarrhea (odds ratios: 16.03 and 31.33, respectively). In contrast, serum bilirubin levels were not associated with irinotecan toxicity. Homozygosity for UGT1A1*6/*6 and heterozygosity for UGT1A1*6/*28 were associated with an increased risk of absolute neutrophil count and/or diarrhea in Japanese gynecologic cancer patients, despite the lower doses of irinotecan used in these patients. UGT1A1*6 and UGT1A1*28 are potential predictors of severe absolute neutrophil decrease and diarrhea caused by low-dose irinotecan in gynecologic cancer patients.

Abstract 4039: miR-196a is a novel diagnostic biomarker and therapeutic target for laryngeal cancer

Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, comprising roughly 6% of all malignancies worldwide, and laryngeal cancer has the highest incidence in it. At present, laryngeal cancer is treated with surgery and/or (chemo)radiation, each of which can have devastating consequences on speech and swallowing function. Even with the combined treatment approaches as mentioned above, laryngeal cancers with advanced stages have poor prognosis, therefore in need of novel less invasive treatments against their high morbidity. However, although to date its relevance to smoking has been well documented, the molecular mechanism involved in laryngeal cancer development and its highly sensitive biomarkers remain to be known. Thus, we have focused on microRNAs (miRNAs) showing highly tissue-or disease-specific patterns as potential novel biomarkers or therapeutic targets with clinical applicability. In this study, to screen the miRNAs highly sensitive to laryngeal cancer as putative biomarkers, we first examined the expression of 723 human miRNAs in 8 laryngeal tissues (1 polyp, 2 dysplasias, 3 laryngeal cancers and 2 neighboring normal tissues matched to laryngeal cancers) using microarrays (Agilent Human miRNA V2). The result showed the up-regulation of 6 miRNAs and the down-regulation of 3 miRNAs in laryngeal cancer compared to others. Subsequently, to confirm these findings and quantitate cancer-specific miRNAs, we performed quantitative RT-PCR (qRT-PCR) analysis (TaqMan miRNA assays, Applied Biosystems) using 48 laryngeal tissue samples including 16 cancers on 5 miRNAs (miR-196a, miR-130b*, miR-133b, miR-375, miR-455-5p), which showed markedly differential expression in cancers compared with others on microarrays. The results were consistent with those from microarrays, especially when cancer samples were compared with matched laryngeal tissues as normal controls. Of the 5 miRNAs examined, miR-196a showed significant increase in cancers when compared to either benign tissues or dysplasias. Next, to examine the utility of miR-196a as a promising biomarker for laryngeal cancer, we performed qRT-PCR on miR-196a using 83 tissue samples and 68 serum samples and found that miR-196a could be very useful in diagnosing the disease. We also analyzed miR-196a expression in FFPE sections by in situ hybridization and the result showed its robust expression in cancers but barely detectable in benign tissues Finally, to explore whether miR-196a can promote tumor growth in vitro or in vivo, we treated human laryngeal cancer cells or xenografted nude mice, respectively, with either miR-196a inhibitor or negative control inhibitor, and found that miR-196a inhibition suppresses tumorigenic properties of laryngeal cancer. Together with these findings, our study provided the first demonstration of miR-196a as a putative diagnostic biomarker and therapeutic target for laryngeal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4039.

Abstract 4089: Regulation of cancer-specific microRNA by aberrant DNA methylation in laryngeal cancer

DNA methylation plays an essential role in the regulation of gene expression during development and differentiation, and furthermore has been reported to be involved in various diseases such as diabetes, schizophrenia and cancers. The majority of CpG islands are normally unmethylated, but a sizeable fraction is prone to become methylated in various cell types and pathological situations. The down-regulation of tumor suppressor genes has been reported to be linked to the hypermethylation of CpG sites located within CpG islands with promoter activity. The same mechanism could play an important role in silencing of tumor-suppressive microRNAs (miRNAs) in tumors. Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, comprising roughly 6% of all malignancies worldwide, and laryngeal cancer has the highest incidence in it. However, although to date its relevance to smoking has been well documented, the molecular mechanism involved in laryngeal cancer development and its highly sensitive biomarkers as well as promising therapeutic targets remain to be known. Thus, we have focused on the down-regulated miRNAs in laryngeal cancers and their neighboring methylated DNA sites on the genome as potential novel biomarkers or therapeutic targets with clinical applicability. In this study, we used an approach with a series of sequential analyses using clinical samples to clarify DNA methylation loci and examine the relationship between miRNA down-regulation and DNA methylation in laryngeal cancer. First, we analyzed the methylation levels at 27,578 CpG dinucleotides in 14,495 genes in 8 laryngeal tissues (1 dysplasia, 4 laryngeal cancers) using HumanMethylation27 BeadChip® (Illumina), in which AVG_Beta Methylation Values were used to quantify methylation levels at specific loci on the genome of each patient. We have compared dysplasia samples with laryngeal cancer samples and found that 198 hypermethylation loci within CpG islands were specific for laryngeal cancers, and also clarified 72 miRNAs on/around these CpG sites using the human genome database. Next, we have examined the expression patterns of 723 human miRNAs in 8 laryngeal tissues (1 polyp, 2 dysplasias, 3 laryngeal cancers and 2 neighboring normal tissues matched to laryngeal cancers) using microarrays (Agilent Human miRNA V2) and found 88 miRNAs down-regulated in laryngeal cancers compared to others. Finally, we combined the results of these two analyses to reveal that 11 miRNAs were down-regulated together with DNA methylation status on/around their neighboring CpG islands. Our study suggested that tumor-specific DNA methylation could be an important molecular mechanism for the down-regulation of miRNA expression. These novel findings may lead to the identification of methylated DNAs associated with down-regulated miRNAs specific to laryngeal cancer and the future elucidation of the molecular mechanism of HNC development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4089.

MicroRNA Expression Signature in Laryngeal Cancer

OBJECTIVES: In this study, we examined the microRNA (miRNA) expression profile in laryngeal cancer. We further investigated the role of miR-133b and miR-375 in the growth of head and neck cancer (HNC) in vitro. METHODS: Expression profile of 723 human miRNAs in 8 laryngeal tissues (polyp, dysplasia, cancer, neighboring normal tissue matched to laryngeal cancer) was examined using microarrays. Subsequently, qRT-PCR was performed to confirm the microarray data using 48 laryngeal tissues including 16 laryngeal cancers on 3 miRNAs (miR-130b*, miR-133b, miR375), which showed distinctive expression in cancer on microarray. Six cancer samples had matched laryngeal tissues next to the tumor. Pairwise Mann-Whitney’s U test and Wilcoxon signed ranks test were utilized for statistical analyses. Furthermore, impact of miR-133b or miR-375 transfection on the growth of HNC cell lines was examined using WST-based assay. RESULTS: Microarray analysis revealed the up-regulation of 8 miRNAs, including miR-130b* and the down-regulation of 3 miRNAs, including miR-133b and miR-375. The results obtained from qRT-PCR were consistent with those obtained from microarray. Especially, the difference of miR-133b expression level between matched samples was significant (p 0.0277). MiR-133b suppressed the growth of SAS cell line and miR-375 suppressed the growth of JHU-011 cell line 5 days after transfection compared to control. CONCLUSIONS: We demonstrated the miRNA expression signature in laryngeal cancer and the role of miRNAs on the growth of HNC cell lines. Our novel findings may pave the way to elucidate the molecular mechanism of oncogenesis in HNC.