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Papers - KANAI Yae

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Frequent activating GNAS mutations in villous adenoma of the colorectum

Yamada, Masayoshi, Sekine, Shigeki, Ogawa, Reiko, Taniguchi, Hirokazu, Kushima, Ryoji, Tsuda, Hitoshi, Kanai, Yae

JOURNAL OF PATHOLOGY 228 ( 1 ) 113 - 118 2012.09

Research paper (scientific journal), ISSN 0022-3417

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Single-CpG-resolution methylome analysis identifies clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas

Arai, Eri, Chiku, Suenori, Mori, Taisuke, Gotoh, Masahiro, Nakagawa, Tohru, Fujimoto, Hiroyuki, Kanai, Yae

CARCINOGENESIS (OXFORD UNIV PRESS) 33 ( 8 ) 1487 - 1493 2012.08

Research paper (scientific journal), ISSN 0143-3334

To clarify the significance of DNA methylation alterations during renal carcinogenesis, methylome analysis using single-CpG-resolution Infinium array was performed on 29 normal renal cortex tissue (C) samples, 107 non-cancerous renal cortex tissue (N) samples obtained from patients with clear cell renal cell carcinomas (RCCs) and 109 tumorous tissue (T) samples. DNA methylation levels at 4830 CpG sites were already altered in N samples compared with C samples. Unsupervised hierarchical clustering analysis based on DNA methylation levels at the 801 CpG sites, where DNA methylation alterations had occurred in N samples and were inherited by and strengthened in T samples, clustered clear cell RCCs into Cluster A (n = 90) and Cluster B (n = 14). Clinicopathologically aggressive tumors were accumulated in Cluster B, and the cancer-free and overall survival rates of patients in this cluster were significantly lower than those of patients in Cluster A. Clear cell RCCs in Cluster B were characterized by accumulation of DNA hypermethylation on CpG islands and considered to be CpG island methylator phenotype (CIMP)-positive cancers. DNA hypermethylation of the CpG sites on the FAM150A, GRM6, ZNF540, ZFP42, ZNF154, RIMS4, PCDHAC1, KHDRBS2, ASCL2, KCNQ1, PRAC, WNT3A, TRH, FAM78A, ZNF671, SLC13A5 and NKX6-2 genes became hallmarks of CIMP in RCCs. On the other hand, Cluster A was characterized by genome-wide DNA hypomethylation. These data indicated that DNA methylation alterations at precancerous stages may determine tumor aggressiveness and patient outcome. Accumulation of DNA hypermethylation on CpG islands and genome-wide DNA hypomethylation may each underlie distinct pathways of renal carcinogenesis.

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Fenton Reaction Induced Cancer in Wild Type Rats Recapitulates Genomic Alterations Observed in Human Cancer

Akatsuka, Shinya, Yamashita, Yoriko, Ohara, Hiroki, Liu, Yu-Ting, Izumiya, Masashi, Abe, Koichiro, Ochiai, Masako, Jiang, Li, Nagai, Hirotaka, Okazaki, Yasumasa, Murakami, Hideki, Sekido, Yoshitaka, Arai, Eri, Kanai, Yae, Hino, Okio, Takahashi, Takashi, Nakagama, Hitoshi, Toyokuni, Shinya

PLOS ONE (PUBLIC LIBRARY SCIENCE) 7 ( 8 ) 207 - 213 2012.08

Research paper (scientific journal), ISSN 1932-6203

Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.

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Expression of a splicing variant of the CADM1 specific to small cell lung cancer

Kikuchi, Shinji, Iwai, Miwako, Sakurai-Yageta, Mika, Tsuboi, Yumi, Ito, Takeshi, Maruyama, Tomoko, Tsuda, Hitoshi, Kanai, Yae, Onizuka, Masataka, Sato, Yukio, Murakami, Yoshinori

CANCER SCIENCE (WILEY-BLACKWELL) 103 ( 6 ) 1051 - 1057 2012.06

Research paper (scientific journal), ISSN 1347-9032

CADM1, a member of the immunoglobulin superfamily cell adhesion molecule, acts as a tumor suppressor in a variety of human cancers. CADM1 is also ectopically expressed in adult T-cell leukemia (ATL), conferring an invasive phenotype characteristic to ATL. Therefore, CADM1 plays dual roles in human oncogenesis. Here, we investigate the roles of CADM1 in small cell lung cancer (SCLC). Immunohistochemistry demonstrates that 10 of 35 (29%) primary SCLC tumors express CADM1 protein. Western blotting and RT-PCR analyses reveal that CADM1 is significantly expressed in 11 of 14 SCLC cells growing in suspension cultures but in neither of 2 SCLC cells showing attached growth to plastic dishes, suggesting that CADM1 is involved in anchorage-independent growth in SCLC. In the present study, we demonstrate that SCLC expresses a unique splicing variant of CADM1 (variant 8/9) containing additional extracellular fragments corresponding to exon 9 in addition to variant 8, a common isoform in epithelia. Variant 8/9 of CADM1 is almost exclusively observed in SCLC and testis, although this variant protein localizes along the membrane and shows similar cell aggregation activity to variant 8. Interestingly, both variant 8/9 and variant 8 of CADM1 show enhanced tumorigenicity in nude mice when transfected into SBC5, a SCLC cell lacking CADM1. Inversely, suppression of CADM1 expression by shRNA reduced spheroid-like cell aggregation of NCI-H69, an SCLC cell expressing a high amount of CADM1. These findings suggest that CADM1 enhances the malignant features of SCLC, as is observed in ATL, and could provide a molecular marker specific to SCLC. (Cancer Sci 2012; 103: 10511057)

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Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: Morphological significance of calponin h1

Hayashi, Takuma, Horiuchi, Akiko, Sano, Kenji, Hiraoka, Nobuyoshi, Kasai, Mari, Ichimura, Tomoyuki, Sudo, Tamotsu, Nishimura, Ryuichiro, Ishiko, Osamu, Shiozawa, Tanri, Kanai, Yae, Yaegashi, Nobuo, Aburatani, Hiroyuki, Konishi, Ikuo

FEBS LETTERS (ELSEVIER SCIENCE BV) 586 ( 13 ) 1824 - 1831 2012.06

Research paper (scientific journal), ISSN 0014-5793

Uterine leiomyosarcoma (LMS) is a highly metastatic smooth muscle neoplasm for which calponin h1 is suspected to have a biological role as a tumor-suppressor. We earlier reported that LMP2-null mice spontaneously develop uterine LMS through malignant transformation of the myometrium, thus implicating this protein as an anti-tumorigenic candidate as well. In the present study, we show that LMP2 may negatively regulate LMS independently of its role in the proteasome. Moreover, several lines of evidence indicate that although calponin h1 does not directly influence tumorigenesis, it clearly affects LMP2-induced cellular morphological changes. Modulation of LMP2 may lead to new therapeutic approaches in human uterine LMS. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

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DNA methyltransferase 3B expression is associated with poor outcome of stage I testicular seminoma

Arai, Eri, Nakagawa, Tohru, Wakai-Ushijima, Saori, Fujimoto, Hiroyuki, Kanai, Yae

HISTOPATHOLOGY (WILEY-BLACKWELL) 60 ( 6B ) E12 - E18 2012.05

Research paper (scientific journal), ISSN 0309-0167

Aims: To examine in testicular seminomas the expression of DNA methyltransferase 3B (DNMT3B), which is known to be associated with early embryonic development and carcinogenesis, and to obtain a predictive marker for relapse of stage I seminomas.
Methods and results: Immunohistochemical examination of DNMT3B was performed in 88 cases of seminoma, 35 (39.8%) of which showed widely scattered nuclear immunoreactivity for DNMT3B, and 53 (60.2%) of which were completely negative. The incidence of focal DNMT3B expression was higher in stage III seminomas (5/5, 100%) than in stage I (25/70, 35.7%) or stage II (5/13, 38.5%) seminomas (P = 0.011). In stage I seminomas there were no significant correlations between DNMT3B expression and tumour size, invasion of the rete testis, or lymphatic or vascular involvement. Six of 25 cases (24%) showing DNMT3B expression relapsed, whereas only 3/45 cases (6.7%) lacking such expression did so (P = 0.037). Patients with seminomas showing DNMT3B expression had a significantly lower relapse-free survival rate than patients whose tumours lacked this feature (P = 0.0464).
Conclusions: Patients with seminomas showing focal DNMT3B expression are at increased risk of relapse, and should be followed up carefully.

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Morules in Intraductal Papillary Mucinous Neoplasm With an Associated Invasive Carcinoma of the Pancreas

Tateno, Yoko, Esaki, Minoru, Shimada, Kazuaki, Ojima, Hidenori, Kanai, Yae, Hiraoka, Nobuyoshi

PANCREAS 41 ( 4 ) 651 - 652 2012.05

Research paper (scientific journal), ISSN 0885-3177

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Higher-Order Chromatin Regulation and Differential Gene Expression in the Human Tumor Necrosis Factor/Lymphotoxin Locus in Hepatocellular Carcinoma Cells

Watanabe, Takehisa, Ishihara, Ko, Hirosue, Akiyuki, Watanabe, Sugiko, Hino, Shinjiro, Ojima, Hidenori, Kanai, Yae, Sasaki, Yutaka, Nakao, Mitsuyoshi

MOLECULAR AND CELLULAR BIOLOGY (AMER SOC MICROBIOLOGY) 32 ( 8 ) 1529 - 1541 2012.04

Research paper (scientific journal), ISSN 0270-7306

The three-dimensional context of endogenous chromosomal regions may contribute to the regulation of gene clusters by influencing interactions between transcriptional regulatory elements. In this study, we investigated the effects of tumor necrosis factor (TNF) signaling on spatiotemporal enhancer-promoter interactions in the human tumor necrosis factor (TNF)/lymphotoxin (LT) gene locus, mediated by CCCTC-binding factor (CTCF)-dependent chromatin insulators. The cytokine genes LT alpha, TNF, and LT beta are differentially regulated by NF-kappa B signaling in inflammatory and oncogenic responses. We identified at least four CTCF-enriched sites with enhancer-blocking activities and a TNF-responsive TE2 enhancer in the TNF/LT locus. One of the CTCF-enriched sites is located between the early-inducible LT alpha/TNF promoters and the late-inducible LT beta promoter. Depletion of CTCF reduced TNF expression and accelerated LT beta induction. After TNF stimulation, via intrachromosomal dynamics, these insulators mediated interactions between the enhancer and the LT alpha/TNF promoters, followed by interaction with the LT beta promoter. These results suggest that insulators mediate the spatiotemporal control of enhancer-promoter associations in the TNF/LT gene cluster.

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Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency

Wang, Linghua, Tsutsumi, Shuichi, Kawaguchi, Tokuichi, Nagasaki, Koichi, Tatsuno, Kenji, Yamamoto, Shogo, Sang, Fei, Sonoda, Kohtaro, Sugawara, Minoru, Saiura, Akio, Hirono, Seiko, Yamaue, Hiroki, Miki, Yoshio, Isomura, Minoru, Totoki, Yasushi, Nagae, Genta, Isagawa, Takayuki, Ueda, Hiroki, Murayama-Hosokawa, Satsuki, Shibata, Tatsuhiro, Sakamoto, Hiromi, Kanai, Yae, Kaneda, Atsushi, Noda, Tetsuo, Aburatani, Hiroyuki

GENOME RESEARCH (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT) 22 ( 2 ) 208 - 219 2012.02

Research paper (scientific journal), ISSN 1088-9051

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARIDIC. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

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Prostate Stem Cell Antigen, a Presumable Organ-Dependent Tumor Suppressor Gene, is Down-Regulated in Gallbladder Carcinogenesis

Ono, Hiroe, Hiraoka, Nobuyoshi, Lee, Yeon-Su, Woo, Sang Myung, Lee, Woo Jin, Choi, Il Ju, Saito, Akira, Yanagihara, Kazuyoshi, Kanai, Yae, Ohnami, Sumiko, Chiwaki, Fumiko, Sasaki, Hiroki, Sakamoto, Hiromi, Yoshida, Teruhiko, Saeki, Norihisa

GENES CHROMOSOMES & CANCER (WILEY) 51 ( 1 ) 30 - 41 2012.01

Research paper (scientific journal), ISSN 1045-2257

Gallbladder cancer (GBC) is relatively rare but has a high mortality rate. One candidate molecule which might be involved in GBC development is prostate stem cell antigen (PSCA), a glycosylphosphatidylinositol- anchored cell surface antigen with a tissue- specific pattern of expression in the epithelium of several organs, such as the prostate, stomach, bladder, and gallbladder. It is up- regulated in a number of cancers including prostate, urinary bladder, and pancreatic cancers, while it is down- regulated in esophageal and gastric cancers, suggesting that PSCA has an oncogenic activity in the former but a tumor suppressor activity in the latter. However, the precise function of PSCA and the regulatory mechanism for its expression in normal and cancer cells are yet to be determined. In this study, immunohistochemical analyses with a specific antibody revealed that PSCA is down- regulated in non- neoplastic gallbladder lesions such as cholesterolosis, cholecystolithiasis, and cholecystitis (9/17; 53%), and also in adenocarcinoma (40/44; 91%), a common neoplasm in gallbladder. Analyses of the DNA methylation status in the GBC cell lines by bisulfite- Pyrosequencing and a reporter assay for the PSCA promoter activity suggested that the down- regulation is explained, at least partly, by DNA methylation. Moreover, colony formation assay revealed that PSCA has cell- proliferation inhibition activity in the GBC cell lines, which was also observed in vivo. These lines of in vivo and in vitro evidence suggest that PSCA is acting as a tumor suppressor in GBC development. (C) 2011 Wiley Periodicals, Inc.

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Frequent activating GNAS mutations in villous adenoma of the colorectum.

Yamada M, Sekine S, Ogawa R, Taniguchi H, Kushima R, Tsuda H, Kanai Y.

J Pathol 228 113 - 118 2012

Accepted

Potential role of LMP2 as tumor-suppressor defines new targets for uterine leiomyosarcoma therapy

Hayashi, Takuma, Horiuchi, Akiko, Sano, Kenji, Hiraoka, Nobuyoshi, Kasai, Mari, Ichimura, Tomoyuki, Sudo, Tamotsu, Tagawa, Yoh-ichi, Nishimura, Ryuichiro, Ishiko, Osamu, Kanai, Yae, Yaegashi, Nobuo, Aburatani, Hiroyuki, Shiozawa, Tanri, Konishi, Ikuo

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 1 907 - 912 2011.12

Research paper (scientific journal), ISSN 2045-2322

Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma (LMS) is extremely malignant, with high rates of recurrence and metastasis. We earlier reported that mice with a homozygous deficiency for LMP2, an interferon (IFN)-gamma-inducible factor, spontaneously develop uterine LMS. The IFN-gamma pathway is important for control of tumor growth and invasion and has been implicated in several cancers. In this study, experiments with human and mouse uterine tissues revealed a defective LMP2 expression in human uterine LMS that was traced to the IFN-gamma pathway and the specific effect of JAK-1 somatic mutations on the LMP2 transcriptional activation. Furthermore, analysis of a human uterine LMS cell line clarified the biological significance of LMP2 in malignant myometrium transformation and cell cycle, thus implicating LMP2 as an anti-tumorigenic candidate. This role of LMP2 as a tumor suppressor may lead to new therapeutic targets in human uterine LMS.

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Carcinogenetic risk estimation based on quantification of DNA methylation levels in liver tissue at the precancerous stage

Nagashio, Ryo, Arai, Eri, Ojima, Hidenori, Kosuge, Tomoo, Kondo, Yutaka, Kanai, Yae

INTERNATIONAL JOURNAL OF CANCER (WILEY-BLACKWELL) 129 ( 5 ) 1170 - 1179 2011.09

Research paper (scientific journal), ISSN 0020-7136

For appropriate surveillance of patients at the precancerous stage for hepatocellular carcinomas (HCCs), carcinogenetic risk estimation is advantageous. The aim of our study was to establish criteria for such estimation based on DNA methylation profiling. The DNA methylation status of 203 CpG sites on 25 bacterial artificial chromosome (BAC) clones, whose DNA methylation status had been proven to discriminate samples of noncancerous liver tissue obtained from patients with HCC (N) from normal liver tissue (C) samples by BAC array-based methylated CpG island amplification, was evaluated quantitatively using pyrosequencing. The 45 CpG sites whose DNA methylation levels differed significantly between C and N in the learning cohort (n = 22) were identified. The criteria combining DNA methylation status for the 30 regions including the 45 CpG sites were able to diagnose N as being at high risk of carcinogenesis with 100% sensitivity and specificity in the learning cohort and 95.6% sensitivity and 100% specificity in the validation (n = 90) cohort. DNA methylation status for the 30 regions in N samples was significantly correlated with the outcome of patients with HCCs, indicating that clinicopathologically valid DNA methylation alterations have already accumulated at the precancerous stage. The DNA methylation status of the 30 regions did not depend on the presence or absence of hepatitis virus infection, or the status of noncancerous liver tissue (chronic hepatitis or cirrhosis). These criteria may be applicable for carcinogenetic risk estimation using liver biopsy specimens obtained from patients who are followed up because of chronic liver diseases.

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Expression of SLCO1B3 is associated with intratumoral cholestasis and CTNNB1 mutations in hepatocellular carcinoma

Sekine, Shigeki, Ogawa, Reiko, Ojima, Hidenori, Kanai, Yae

CANCER SCIENCE (WILEY-BLACKWELL) 102 ( 9 ) 1742 - 1747 2011.09

Research paper (scientific journal), ISSN 1347-9032

Recent studies have shown that intratumoral cholestasis is a hallmark of CTNNB1 mutations in hepatocellular carcinomas (HCC). Here, we analyzed the expressions of genes involved in bile acid and bilirubin metabolism and their correlation with the mutational status of CTNNB1 in a series of HCC. The expressions of CYP7A1 and CYP27A1, which encode rate-limiting enzymes in bile acid synthesis, were unaltered or only marginally increased in CTNNB1-mutated HCC compared with those in HCC with wild-type CTNNB1. Among the genes involved in bile acid and bilirubin transport, the expression of SLCO1B3 was significantly elevated in HCC with CTNNB1 mutations, whereas the expression of ABCC4 was elevated in HCC with wild-type CTNNB1. Immunohistochemistry confirmed the frequent expression of SLCO1B3 in CTNNB1-mutated HCC at the protein level, but not in most HCC with wild-type CTNNB1. Immunohistochemistry for MRP4 (encoded by ABCC4) partly agreed with ABCC4 expression, but most cases did not express detectable levels of MRP4. Notably, all HCC with bile accumulation, including those without CTNNB1 mutations, expressed SLCO1B3, suggesting that SLCO1B3 expression, rather than CTNNB1 mutation, is the critical determinant of intratumoral cholestasis. As SLCO1B3 is involved in the uptake of a number of chemotherapeutic and diagnostic agents, SLCO1B3 expression and the status of CTNNB1 mutation might need to be considered in the drug delivery to HCC. (Cancer Sci 2011; 102: 1742-1747)

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Copy number alterations in urothelial carcinomas: their clinicopathological significance and correlation with DNA methylation alterations

Nishiyama, Naotaka, Arai, Eri, Nagashio, Ryo, Fujimoto, Hiroyuki, Hosoda, Fumie, Shibata, Tatsuhiro, Tsukamoto, Taiji, Yokoi, Sana, Imoto, Issei, Inazawa, Johji, Kanai, Yae

CARCINOGENESIS (OXFORD UNIV PRESS) 32 ( 4 ) 462 - 469 2011.04

Research paper (scientific journal), ISSN 0143-3334

The aim of this study was to clarify the genetic backgrounds underlying the clinicopathological characteristics of urothelial carcinomas (UCs). Array comparative genomic hybridization analysis using a 244K oligonucleotide array was performed on 49 samples of UC tissue. Losses of 2q33.3-q37.3, 4p15.2-q13.1 and 5q13.3-q35.3 and gains of 7p11.2-q11.23 and 20q13.12-q13.2 were correlated with higher histological grade, and gain of 7p21.2-p21.12 was correlated with deeper invasion. Losses of 6q14.1-q27 and 17p13.3-q11.1 and gains of 19q13.12-q13.2 and 20q13.12-q13.33 were correlated with lymph vessel involvement. Loss of 16p12.2-p12.1 and gain of 3q26.32-q29 were correlated with vascular involvement. Losses of 5q14.1-q23.1, 6q14.1-q27, 8p22-p21.3, 11q13.5-q14.1 and 15q11.2-q22.2 and gains of 7p11.2-q11.22 and 19q13.12-q13.2 were correlated with the development of aggressive non-papillary UCs. Losses of 1p32.2-p31.3, 10q11.23-q21.1 and 15q21.3 were correlated with tumor recurrence. Unsupervised hierarchical clustering analysis based on copy number alterations clustered UCs into three subclasses: copy number alterations associated with genome-wide DNA hypomethylation, regional DNA hypermethylation on C-type CpG islands and genome-wide DNA hypo- and hypermethylation were accumulated in clusters A, B(1) and B(2), respectively. Tumor-related genes that may encode therapeutic targets and/or indicators useful for the diagnosis and prognostication of UCs should be explored in the above regions. Both genetic and epigenetic events appear to accumulate during urothelial carcinogenesis, reflecting the clinicopathological diversity of UCs.

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Hepatomas with activating Ctnnb1 mutations in 'Ctnnb1-deficient' livers: a tricky aspect of a conditional knockout mouse model

Sekine, Shigeki, Ogawa, Reiko, Kanai, Yae

CARCINOGENESIS (OXFORD UNIV PRESS) 32 ( 4 ) 622 - 628 2011.04

Research paper (scientific journal), ISSN 0143-3334

Conditional knockout mice, based on the Cre-loxP system, are a widely used model for examining organ-specific gene functions. To date, efficient hepatocyte-specific knockout has been reported in many different models, but little attention has been paid to the long-term stability of the recombination efficiency. In the present study, we characterized Alb-Cre;Ctnnb1(flox/flox) 'hepatocyte-specific Ctnnb1 knockout' mice of different ages to test whether efficient recombination is maintained over time. At 2 months of age, the knockout mouse livers achieved efficient deletions of beta-catenin in hepatocytes. However, as the mice aged, the reappearance and expansion of beta-catenin-expressing hepatocytes were observed. In 1-year-old mice, a significant proportion of the pericentral hepatocytes in the knockout mouse livers were replaced with beta-catenin-positive hepatocytes, whereas the periportal hepatocytes mostly remained beta-catenin-negative. Furthermore, most of the 1-year-old mice spontaneously developed hepatocellular adenomas and carcinomas that were positive for beta-catenin and overexpressed glutamine synthetase and Slc1a2, both of which are hallmarks of active beta-catenin signaling. Sequencing analysis revealed that the Ctnnb1 alleles were not inactivated but had activating mutations in these tumors. The present study suggests that recombination efficiency should be carefully examined when hepatocyte-specific knockout mice of different ages are analyzed. In addition, illegitimate deletion mutations should be recognized as potential adverse effects of the Cre-loxP system.

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Overexpression of alpha-methylacyl-CoA racemase is associated with CTNNB1 mutations in hepatocellular carcinomas

Sekine, Shigeki, Ogawa, Reiko, Ojima, Hidenori, Kanai, Yae

HISTOPATHOLOGY (WILEY-BLACKWELL) 58 ( 5 ) 712 - 719 2011.04

Research paper (scientific journal), ISSN 0309-0167

Aims:
alpha-Methylacyl-CoA racemase (AMACR) is expressed in the majority of hepatocellular carcinomas (HCCs) at variable levels, but the significance of AMACR overexpression remains elusive. The aim of this study was to investigate the relationship between AMACR expression and the presence of CTNNB1 mutations in HCCs.
Methods and results:
The expression of AMACR and GLUL, an established downstream target of beta-catenin was examined in HCCs, by quantitative reverse transcription polymerase chain reaction (PCR), and the expression of their protein products by immunohistochemistry. The quantitative reverse transcription PCR analysis showed that the expression of AMACR was significantly higher in HCCs with CTNNB1 mutations than in mutation-negative HCCs or normal livers, like the expression of GLUL. Immunohistochemistry also showed that strong AMACR protein expression was closely correlated with the presence of CTNNB1 mutations. HCCs with CTNNB1 mutations and those with AMACR overexpression frequently exhibited bile production.
Conclusions:
The overexpression of AMACR was closely correlated with the presence of CTNNB1 mutations in HCCs. AMACR is a putative target of beta-catenin as well as an excellent immunohistochemically detectable marker of HCCs with CTNNB1 mutations. As AMACR is physiologically involved in bile acid synthesis, the current observation implies a regulatory role of beta-catenin in bile acid metabolism.

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CXCL17 and ICAM2 Are Associated With a Potential Anti-Tumor Immune Response in Early Intraepithelial Stages of Human Pancreatic Carcinogenesis

Hiraoka, Nobuyoshi, Yamazaki-Itoh, Rie, Ino, Yoshinori, Mizuguchi, Yasunori, Yamada, Tesshi, Hirohashi, Setsuo, Kanai, Yae

GASTROENTEROLOGY (W B SAUNDERS CO-ELSEVIER INC) 140 ( 1 ) 310 - + 2011.01

Research paper (scientific journal), ISSN 0016-5085

BACKGROUND & AIMS: Anti-tumor immunity changes over the course of tumor progression; it is not clear how or when the developing tumor overcomes immune surveillance. Intraductal papillary mucinous neoplasm (IPMN) is an intraepithelial precursor lesion of pancreatic cancer that progresses from adenoma to carcinoma. We investigated when and how the human anti-tumor immune reaction changes during pancreatic tumor development. METHODS: Using immunohistochemical analysis of cells isolated from patients with IPMN, the numbers of tumor-infiltrating lymphocytes and dendritic cells and the maturation state of dendritic cells in the regional lymph nodes were investigated during tumor progression. Gene expression profiles were compared among epithelial neoplastic cells at each stage of tumor development. Biological functions of the selected gene products were analyzed using syngeneic mouse models. RESULTS: The antitumor immune reaction changed from an immune response to immune tolerance between the stages of intraductal papillary mucinous adenoma (IPMA) and intraductal papillary mucinous carcinoma (IPMC). Chemokine (C-X-C motif) ligand 17 (CXCL17) and intercellular adhesion molecule 2 (ICAM2) were involved in immune surveillance during tumor development-their expression levels were up-regulated exclusively in IPMA and disappeared from IPMC. CXCL17 and ICAM2 induced infiltration and accumulation of the tumor epithelial layer by immature myeloid dendritic cells. This was followed by a cellular immune reaction and ICAM2 simultaneously promoted the susceptibility of the tumor cells to cytotoxic T-cell-mediated cytolysis. These processes had a synergistic effect to increase the anti-tumor immune response. CONCLUSIONS: Immune surveillance occurs during the early intraepithelial stages of human pancreatic carcinogenesis and is mediated by expression of CXCL17 and ICAM2.

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Genome-Wide DNA Methylation Profiles in Renal Tumors of Various Histological Subtypes and Non-Tumorous Renal Tissues

Arai, Eri, Wakai-Ushijima, Saori, Fujimoto, Hiroyuki, Hosoda, Fumie, Shibata, Tatsuhiro, Kondo, Tadashi, Yokoi, Sana, Imoto, Issei, Inazawa, Johji, Hirohashi, Setsuo, Kanai, Yae

PATHOBIOLOGY (KARGER) 78 ( 1 ) 1 - 9 2011

Research paper (scientific journal), ISSN 1015-2008

Objective: The aim of this study is to clarify genome-wide DNA methylation profiles in renal tumors of various histological subtypes. Methods: Bacterial artificial chromosome (BAC) array-based methylated CpG island amplification was performed using tissue samples of 17 patients with papillary renal cell carcinomas (RCCs), chromophobe RCCs and oncocytomas, and the results were compared with those from 51 patients with clear cell RCCs. Results: Unsupervised hierarchical clustering analysis based on DNA methylation status clustered type 1 and type 2 papillary RCCs into different subclasses. Although chromophobe RCCs and oncocytomas were clustered into the same subclass, the DNA methylation status of 21 BAC clones was able to discriminate chromophobe RCCs from oncocytomas. The number of BAC clones showing DNA methylation alteration in non-tumorous renal tissue from patients with chronnophobe RCCs and oncocytomas was smaller than that from patients with clear cell RCCs. Biphasic accumulation of DNA methylation alterations was observed in non-tumorous renal tissue from all 68 patients, and patients showing such alterations on more BAC clones had a poorer outcome than patients showing them on fewer BAC clones. Conclusions: DNA methylation profiles determining the histological subtypes of renal tumors developing in individual patients and/or patient outcome may be already established in non-tumorous renal tissue at the precancerous stage. Copyright (c) 2011 S. Karger AG, Basel

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Involvement of proteasome β1i subunit, LMP2, on development of uterin leiomyosarcma.

Hayashi T, Horiuchi A, Sano K, Hiraoka N, Kasai M, Ichimura T, Nagase S, Ishiko O, Shiozawa T, Kanai Y, Yaegashi N, Aburatani H, Tonegawa S, Konishi I.

N Am J Med Sci (North American Journal of Medical Sciences) 3 ( 9 ) 394 - 399 2011

Accepted, ISSN 1947-2714

Background: Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma is extremely malignant, with high rates of recurrence and metastasis. The development of gynecologic tumors is often correlated with secretion of female hormone
however, the development of human uterine leiomyosarcoma is not substantially correlated with hormonal conditions, and the risk factors are unclearly understood. Importantly, a diagnostic-biomarker, which distinguishes malignant human uterine leiomyosarcoma from benign tumor leiomyoma is yet to be established. Aims: It is necessary to analyze risk factors associated with human uterine leiomyosarcoma, in order to establish a diagnostic-biomarker and a clinical treatment method. Patients and Methods: Histology and Immunofluorescence Staining: Uteri obtained from LMP2-/- mice or its parental mice (C57BL/6 mice) were fixed in 10% buffered formalin, incubated in 4% paraformaldehyde for 8 hours, and embedded in paraffin. Tissue sections (5 μm) were prepared and stained with H&amp
E for routine histological examination or were processed further for immunofluorescence staining with appropriate antidodies. Furthermore, a total of 101 patients between 32 and 83 years of age and diagnosed as having smooth muscle tumors of the uterus were selected from pathological files. Immunohistochemistry staining for LMP2 was performed on serial human uterine leiomyosarcoma, leiomyoma and myometrium sections. Results: Homozygous deficient mice for a proteasome β1i subunit, LMP2 spontaneously develop uterine leiomyosarcoma, with a disease prevalence of ~40% by 14 months of age. Defective LMP2 expression in human uterine leiomyosarcoma was demonstrated, but present in human leiomyoma and myometrium. Conclusions: Loss in LMP2 expression may be one of the risk factors for human uterine leiomyosarcoma. LMP2 may be a potential diagnostic-biomarker and targeted-molecule for a new therapeutic approach.

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