論文 - 金井　弥栄

自閉症スペクトラムのエピゲノム異常（和訳）

下田将之, 金井弥栄

実験医学 34   1662 - 1668 2016年

TERT promoter mutations are frequent and show association with MED12 mutations in phyllodes tumors of the breast

金井 弥栄

British Journal of Cancer （British Journal of Cancer）  113 （ 8 ） 1244 - 1248 2015年10月

研究論文（学術雑誌）,  ISSN  0007-0920

　概要を見る

© 2015 Cancer Research UK. All rights reserved. Background:Phyllodes tumors are rare fibroepithelial neoplasms of the breast, which carry the potential risk of local recurrence and metastasis. Phyllodes tumors share several histological features with fibroadenomas, and no widely accepted markers for distinguishing these lesions have been identified.Methods:We analyzed molecular abnormalities related to telomere elongation in tumors, including TERT promoter mutations, as well as loss of expression of ATRX and DAXX, in a total of 104 phyllodes tumors and fibroadenomas.Results:Sequencing analyses showed that TERT promoter mutations were frequent in phyllodes tumors (30/46, 65%), but rare in fibroadenomas (4/58, 7%). Among phyllodes tumors, the mutations were more frequent in borderline tumors (13/15, 87%), but were also common in benign (9/18, 50%) and malignant tumors (8/13, 62%). Remarkably, all but one TERT promoter-mutated tumor also contained MED12 mutations, indicating that these mutations are strongly associated (P=8.4 × 10 -6). Expression of ATRX and DAXX, as evaluated by immunohistochemistry, was retained in all tumors.Conclusions:Our observations suggest a critical role of TERT promoter mutations, in cooperation with MED12 mutations, in the development of phyllodes tumors. Because TERT promoter mutations are rare among fibroadenomas, their detection may be of potential use in discriminating between phyllodes tumors and fibroadenomas.

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Prognostic implication of PTPRH hypomethylation in non-small cell lung cancer

金井 弥栄

Oncology Reports （Oncology Reports）  34 （ 3 ） 1137 - 1145 2015年09月

研究論文（学術雑誌）,  ISSN  1021-335X

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PTPRH is a receptor-type protein tyrosine phosphatase thought to be a potential regulator of tumorigenesis. The aim of the present study was to clarify the significance of PTPRH expression and its regulation by DNA methylation in non-small cell lung cancer (NSCLC), especially in lung adenocarcinoma (LADC). PTPRH mRNA expression was examined in 89 NSCLC and corresponding non-cancerous tissues. The correlation between DNA methylation and PTPRH gene expression was investigated in another cohort that consisted of 145 patients with LADC, a major NSCLC subtype. Gene regulation by DNA methylation was assessed using a DNA methylation inhibitor. PTPRH mRNA expression was significantly upregulated in NSCLC. PTPRH DNA methylation was reduced in LADC samples and inversely correlated with mRNA expression. 5-Aza-2'-deoxycytidine treatment of lung cancer cell lines with low PTPRH expression, restored mRNA PTPRH expression levels. Furthermore, low PTPRH methylation was associated with shorter recurrence-free survival (P=1.64x10-4) and overall survival (P=5.54x10-5). Multivariate analysis revealed that PTPRH DNA methylation was an independent prognostic factor (P=6.88x10-3). It was confirmed that PTPRH is overexpressed in NSCLC. Furthermore, we determined that PTPRH is epigenetically regulated by DNA hypomethylation, with prognostic implications for LADC.

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Heat shock protein 90 is a potential therapeutic target in cholangiocarcinoma

金井 弥栄

Molecular Cancer Therapeutics （Molecular Cancer Therapeutics）  14 （ 9 ） 1985 - 1993 2015年09月

研究論文（学術雑誌）,  ISSN  1535-7163

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©2015 AACR. Cholangiocarcinoma is an aggressive malignancy with a poor prognosis, with no effective therapy other than surgical resection. Heat shock protein 90 (HSP90) is a key component of a multichaperone complex involved in the posttranslational folding of a number of client proteins, many of which play essential roles in tumorigenesis. Here, we attempted to clarify its prognostic significance and potential utility as a therapeutic target in cholangiocarcinoma. Immunohistochemical expression of HSP90 was assessed retrospectively in 399 cholangiocarcinoma cases and 17 human cholangiocarcinoma cell lines, along with the effect of a small-molecule HSP90 inhibitor (NVP-AUY922) on cholangiocarcinoma tumor growth and angiogenesis in human cholangiocarcinoma cell lines and xenografts. The positivity of HSP90 was 44.6% in intrahepatic cholangiocarcinoma (IHCC) and 32.8% in extrahepatic cholangiocarcinoma (EHCC), respectively. HSP90 expression was significantly associated with the 5-year survival rate for IHCC (P < 0.001) and EHCC (P < 0.001). HSP90 inhibition showed potent antiproliferative activity and reduced growth-associated signaling in human cholangiocarcinoma cells in vitro. Furthermore, treatment of cholangiocarcinoma xenograft-bearing mice with NVP-AUY922 significantly inhibited growth at doses far below the maximum-tolerated dose. HSP90 overexpression is a prognostic marker for cholangiocarcinoma. HSP90-targeted therapy may be an option for a subset of cholangiocarcinoma.

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An integrated prognostic classifier for Stage I lung adenocarcinoma based on mRNA, microRNA, and DNA methylation biomarkers

金井 弥栄

Journal of Thoracic Oncology （Journal of Thoracic Oncology）  10 （ 7 ） 1037 - 1048 2015年07月

研究論文（学術雑誌）,  ISSN  1556-0864

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© 2015 by the International Association for the Study of Lung Cancer. Introduction: Up to 30% stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers. Methods: Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts. Results: Promoters of genes marked by polycomb in embryonic stem cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in stage I tumors (p < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (hazard ratio [HR], 2.6; p = 0.02) and recurrence-free survival (HR, 3.0; p = 0.01), and identified high-risk patients in stratified analysis of stages IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1α, and DLC1), miR-21 expression, and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; p = 0.002; HR, 2.3; p = 0.01; and HR, 2.4; p = 0.005, respectively), and when combined, identified high-risk, therapy naive, stage I patients (HR, 10.2; p = 3 × 10-5). All associations were confirmed in two independently collected cohorts. Conclusion: A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of stage I lung cancer patients at high risk of recurrence.

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Epigenetic clustering of gastric carcinomas based on dna methylation profiles at the precancerous stage: Its correlation with tumor aggressiveness and patient outcome

金井 弥栄

Carcinogenesis （Carcinogenesis）  36 （ 5 ） 509 - 520 2015年05月

研究論文（学術雑誌）,  ISSN  0143-3334

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© The Author 2015. Published by Oxford University Press. The aim of this study was to clarify the significance of DNA methylation alterations during gastric carcinogenesis. Single-CpG resolution genome-wide DNA methylation analysis using the Infinium assay was performed on 109 samples of non-cancerous gastric mucosa (N) and 105 samples of tumorous tissue (T). DNA methylation alterations in T samples relative to N samples were evident for 3861 probes. Since N can be at the precancerous stage according to the field cancerization concept, unsupervised hierarchical clustering based on DNA methylation levels was performed on N samples (β<inf>N</inf>) using the 3861 probes. This divided the 109 patients into three clusters: A (n = 20), B1 (n = 20), and B2 (n = 69). Gastric carcinomas belonging to Cluster B1 showed tumor aggressiveness more frequently than those belonging to Clusters A and B2. The recurrence-free and overall survival rates of patients in Cluster B1 were lower than those of patients in Clusters A and B2. Sixty hallmark genes for which β<inf>N</inf> characterized the epigenetic clustering were identified. We then focused on DNA methylation levels in T samples (β<inf>T</inf>) of the 60 hallmark genes. In 48 of them, including the ADAM23, OLFM4, AMER2, GPSM1, CCL28, DTX1 and COL23A1 genes, β<inf>T</inf> was again significantly correlated with tumor aggressiveness, and the recurrence-free and/or overall survival rates. Multivariate analyses revealed that β<inf>T</inf> was a significant prognostic factor, being independent of clinicopathological parameters. These data indicate that DNA methylation profiles at the precancerous stage may be inherited by gastric carcinomas themselves, thus determining tumor aggressiveness and patient outcome.

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Tumour multifocality and grade predict intravesical recurrence after nephroureterectomy in patients with upper urinary tract urothelial carcinoma without a history of bladder cancer

金井 弥栄

Japanese Journal of Clinical Oncology （Japanese Journal of Clinical Oncology）  45 （ 5 ） 488 - 493 2015年05月

研究論文（学術雑誌）,  ISSN  0368-2811

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© The Author 2015. Published by Oxford University Press. All rights reserved. Objective: Patients with upper urinary tract urothelial carcinoma (UUT-UC) without a history of bladder cancer have a different natural history of intravesical recurrence after nephroureterectomy compared with those with a history of bladder cancer. The aim of this study was to identify predictive factors for post-operative intravesical recurrence in patients with non-metastatic upper urinary tract-localized urothelial carcinoma without a history of bladder cancer and who were not taking medication during the perioperative period. Methods: This retrospective study included 133 patients who were treated between 1995 and 2012. Univariate and multivariate analyses were used to evaluate the clinical and pathological factors associated with the cumulative incidence of bladder cancer. Results: Of the 133 patients, 51 (38.3%) developed intravesical recurrence during a median follow-up of 71 months (range, 0.8-210.8). In the multivariate analysis, multifocality (P = 0.03) and high tumour grade (P = 0.007) were significantly associated with the cumulative incidence of bladder cancer. We constructed a prediction classification model on the basis of the total number of risk factors. The 2-year cumulative incidence rates were 5.6, 34.8 and 50.0% in individuals with no, one and two risk factors, respectively. There was a significant difference between patients with no risk factors and those with two risk factors (P = 0.01). Conclusions: Although this retrospective study had several limitations, tumour multifocality and tumour grade were found to be potential risk factors for intravesical recurrence in our cases.

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Activating GNAS and KRAS mutations in gastric foveolar metaplasia, gastric heterotopia, and adenocarcinoma of the duodenum

Matsubara A., Ogawa R., Suzuki H., Oda I., Taniguchi H., Kanai Y., Kushima R., Sekine S.

British Journal of Cancer （British Journal of Cancer）  112 （ 8 ） 1398 - 1404 2015年04月

査読有り,  ISSN  00070920

　概要を見る

© 2015 Cancer Research UK. All rights reserved. Background:Heterotopic gastric-type epithelium, including gastric foveolar metaplasia (GFM) and gastric heterotopia (GH), is a common finding in duodenal biopsy specimens; however, there is still controversy regarding their histogenetic backgrounds.Methods:We analysed a total of 177 duodenal lesions, including 66 GFM lesions, 81 GH lesions, and 30 adenocarcinomas, for the presence of GNAS, KRAS, and BRAF mutations.Results:Activating GNAS mutations were identified in 27 GFM lesions (41%) and 23 GH lesions (28%). The KRAS mutations were found in 17 GFM lesions (26%) and 2 GH lesions (2%). A BRAF mutation was found in only one GFM lesion (2%). These mutations were absent in all 32 normal duodenal mucosa specimens that were examined, suggesting a somatic nature. Among the GFM lesions, GNAS mutations were more common in lesions without active inflammation. Analyses of adenocarcinomas identified GNAS and KRAS mutations in 5 (17%) and 11 lesions (37%), respectively. Immunohistochemically, all the GNAS-mutated adenocarcinomas diffusely expressed MUC5AC, indicating gastric epithelial differentiation.Conclusions:A significant proportion of GFM and GH harbours GNAS and/or KRAS mutations. The common presence of these mutations in duodenal adenoma and adenocarcinoma with a gastric epithelial phenotype implies that GFM and GH might be precursors of these tumours.

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Lysine demethylase LSD1 coordinates glycolytic and mitochondrial metabolism in hepatocellular carcinoma cells

金井 弥栄

Cancer Research （Cancer Research）  75 （ 7 ） 1445 - 1456 2015年04月

研究論文（学術雑誌）,  ISSN  0008-5472

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© 2015 American Association for Cancer Research. The hallmark of most cancer cells is the metabolic shift from mitochondrial to glycolytic metabolism for adapting to the surrounding environment. Although epigenetic modification is intimately linked to cancer, the molecular mechanism, by which epigenetic factors regulate cancer metabolism, is poorly understood. Here, we show that lysine-specific demethylase-1 (LSD1, KDM1A) has an essential role in maintaining the metabolic shift in human hepatocellular carcinoma cells. Inhibition of LSD1 reduced glucose uptake and glycolytic activity, with a concurrent activation of mitochondrial respiration. These metabolic changes coexisted with the inactivation of the hypoxia-inducible factor HIF1α, resulting in a decreased expression of GLUT1 and glycolytic enzymes. In contrast, during LSD1 inhibition, a set of mitochondrial metabolism genes was activated with the concomitant increase of methylated histone H3 at lysine 4 in the promoter regions. Consistently, both LSD1 and GLUT1 were significantly overexpressed in carcinoma tissues. These findings demonstrate the epigenetic plasticity of cancer cell metabolism, which involves an LSD1-mediated mechanism.

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The excision repair cross-complementation group 1

Sakamoto Y., Ojima H., Morizane C., Kanai Y., Okusaka T.

Nihon rinsho. Japanese journal of clinical medicine （Nihon rinsho. Japanese journal of clinical medicine）  73   485 - 490 2015年03月

ISSN  00471852

家族性大腸腺腫症に伴う幽門腺腺腫と散発性幽門腺腺腫は共通の遺伝子変異を有する

橋本 大輝, 小川 玲子, 松原 亜季子, 谷口 浩和, 菅野 康吉, 牛尼 美年子, 吉田 輝彦, 金井 弥栄, 関根 茂樹

日本病理学会会誌 （(一社)日本病理学会）  104 （ 1 ） 305 - 305 2015年03月

ISSN  0300-9181

Long-term exposure to gefitinib induces acquired resistance through DNA methylation changes in the EGFR-mutant PC9 lung cancer cell line

金井 弥栄

International Journal of Oncology （International Journal of Oncology）  46 （ 1 ） 430 - 436 2015年01月

研究論文（学術雑誌）,  ISSN  1019-6439

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This study was designed to identify epigenetically regulated genes and to clarify the contribution of epigenetic alteration to acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-T KIs). We established a gefitinib-resistant lung cancer cell line, PC9, which was originally gefitinib-sensitive, by serial long-term exposure to gefitinib. RNA and DNA were collected from both gefitinib-sensitive and -resistant PC9 cells, and comprehensive DNA methylation and mRNA expression analyses were performed using Infinium HumanMethylation27 Bead Arrays and Agilent SurePrint G3 Human Gene Expression 8x60K Array, respectively. DNA methylation was increased in 640 genes in gefitinib-resistant cells compared to parental cells. Among them, we selected 29 candidate genes that presented a decrease in mRNA expression in resistant PC9. We further studied four of the selected genes (C10orf116, IGFBP3, KL, and S100P) and found that KL or S100P silencing by siRNA induced a decrease in gefitinib sensitivity compared to that in the negative control in PC9. In conclusion, KL and S100P could be potential targets to overcome resistance to EGFR-TKIs.

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Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas

金井 弥栄

International Journal of Cancer （International Journal of Cancer）  137 （ 11 ） 2589 - 2606 2015年

査読有り,  ISSN  00207136

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© 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC. CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP-positive renal carcinogenesis. Genome (whole-exome and copy number), transcriptome and proteome (two-dimensional image converted analysis of liquid chromatography-mass spectrometry) analyses were performed using tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and corresponding specimens of non-cancerous renal cortex. Genes encoding microtubule-associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non-synonymous single-nucleotide mutations and insertions/deletions) in CIMP-positive RCCs, whereas CIMP-negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP-positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the "The metaphase checkpoint (p = 1.427 × 10-6)," "Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10-6)" and "Spindle assembly and chromosome separation (p = 9.260 × 10-6)" pathways. Quantitative RT-PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP-positive than in CIMP-negative RCCs. All CIMP-positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP-positive RCCs. What's new? CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by an accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness, and poor patient outcome. The molecular pathways responsible for generating CIMP-positive clear cell RCCs, however, remain unclear. Based on an integrated multilayer omics approach including genome, transcriptome, and proteome analyses, here the authors show that abnormalities in the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis. The results also suggest the aurora kinases AURKA and AURKB as potential therapeutic targets in more aggressive CIMP-positive RCCs.

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Biological characterization of soft tissue sarcomas.

金井 弥栄

Ann Transl Med 3   368 2015年

査読有り

Frequent MED12 mutations in phyllodes tumours of the breast

金井 弥栄

British Journal of Cancer （British Journal of Cancer）  112: （ 10 ） 1703 - 1708 2015年

査読有り,  ISSN  00070920

　概要を見る

© 2015 Cancer Research UK. All rights reserved. Background:Phyllodes tumours are rare fibroepithelial tumours of the breast, that include benign, borderline, and malignant lesions. Although the molecular basis of phyllodes tumours largely remains unknown, a recent exome study identified MED12 mutations as a sole recurrent genetic alteration in fibroadenoma, a common benign fibroepithelial tumour that shares some histological features with the phyllodes tumour.Methods:Forty-six phyllodes tumours and 58 fibroadenomas of the breast were analysed for MED12 mutations by using Sanger sequencing.Results:MED12 mutations were identified in 37 out of the 46 phyllodes tumours (80%). The prevalence of MED12 mutations was similar among benign (15/18, 83%), borderline (12/15, 80%), and malignant tumours (10/13, 77%). MED12 mutations were also identified in 36 of the 58 fibroadenomas (62%). The mutations were frequent among intracanalicular-type (24/32, 75%) and complex-type lesions (4/6, 67%), but were significantly less common among the pericanalicular-type lesions (8/20, 40%). A microdissection-based analysis showed that MED12 mutations were confined to the stromal components in both phyllodes tumours and fibroadenomas.Conclusions:MED12 mutations were frequent among the phyllodes tumours of the breast, regardless of the tumour grade. Phyllodes tumours and fibroadenomas share, at least in part, a common genetic background.

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Intratumoral tertiary lymphoid organ is a favourable prognosticator in patients with pancreatic cancer

金井 弥栄

British Journal of Cancer （British Journal of Cancer）  112 （ 11 ） 1782 - 1790 2015年

査読有り,  ISSN  00070920

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© 2015 Cancer Research UK. Background:Host immunity has critical roles in tumour surveillance. Tertiary lymphoid organs (TLOs) are induced in various inflamed tissues. The aim of this study was to investigate the clinicopathological and pathobiological characteristics of tumour microenvironment in pancreatic ductal carcinoma (PDC) with TLOs.Methods:We examined 534 PDCs to investigate the clinicopathological impact of TLOs and their association with tumour-infiltrating immune cells, the cytokine milieu, and tissue characteristics.Results:There were two different localisations of PDC-associated TLOs, intratumoral and peritumoral. A better outcome was observed in patients with intratumoral TLOs, and this was independent of other survival factors. The PDC tissues with intratumoral TLOs showed significantly higher infiltration of T and B cells and lower infiltration of immunosuppressive cells, as well as significantly higher expression of Th1- and Th17-related genes. Tertiary lymphoid organs developed with an association with arterioles, venules, and nerves. These structures were reduced in an association with cancer invasion in PDC tissues, except for those with intratumoral TLOs. The PDC tissues with intratumoral TLOs had capillaries consisting of mature endothelial cells covered by pericytes.Conclusions:Our results suggest that the presence of intratumoral TLOs represents a microenvironment that has an active immune reaction, and shows a relatively intact vascular network retained.

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Activating GNAS and KRAS mutations in gastric foveolar metaplasia, gastric heterotopia and adenocarcinoma of the duodenum.

金井 弥栄

Br J Cancer 112   1398 - 1404 2015年

査読有り

Familial adenomatous polyposis-associated and sporadic pyloric gland adenomas of the upper gastrointestinal tract share common genetic features

金井 弥栄

Histopathology （Histopathology）  67 （ 5 ） 689 - 698 2015年

査読有り,  ISSN  03090167

　概要を見る

© 2015 John Wiley & Sons Ltd. Aims: Familial adenomatous polyposis (FAP) is a hereditary cancer predisposition syndrome caused by a germline APC mutation. A recent study showed the enrichment of pyloric gland adenomas (PGAs) of the stomach, in addition to fundic gland polyps (FGPs) and foveolar-type adenomas (FAs), in patients with FAP. In the present study, we analysed the genetic alterations in these FAP-associated gastric lesions. Methods and results: Mutational statuses of GNAS and KRAS, which are frequently mutated in sporadic PGAs, as well as those of APC, were examined in PGAs, FAs and FGPs in patients with FAP using Sanger sequencing. Our analysis identified GNAS mutations in five of six PGAs (83%), but in none of the three FAs or the 40 FGPs examined. KRAS mutations were identified in four PGAs (67%), one FA (33%) and one FGP (3%). Somatic truncating APC mutations were found in all PGAs (100%), two FAs (67%) and 14 FGPs (47%). We additionally analysed sporadic PGAs of the stomach and duodenum and identified truncating APC mutations in 11 of 25 lesions (44%). Conclusions: FAP-associated and sporadic PGAs not only show similar morphologies, but also share common genetic aberrations, including mutations of GNAS, KRAS and APC.

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The importance of multifocality and tumor grade for predicting bladder cancer recurrence after nephroureterectomy in patients with non-metastatic upper urinary tract cancer without a history of bladder cancer.

金井 弥栄

Int J Urol 45   488 - 493 2015年

査読有り

多層的オミックス解析に基づくコンパニオン診断マーカー開発と治療標的同定

金井 弥栄, 新井 恵吏

日本分子腫瘍マーカー研究会誌 （日本分子腫瘍マーカー研究会）  31   15 - 16 2015年

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