Overview of Research Activities

The mission of the Research Institute at the National Cancer Center is to advance our knowledge of cancer prevention, diagnosis and therapy, with the ultimate goal of cancer control. The activities at this institute include a search for environmental carcinogens and chemopreventive agents using animal models, genome-wide approach to the identification of genetic and epigenetic alterations which might be applicable for early cancer diagnosis, and stratification of cancer, in order to achieve more efficient and personalized therapy. Functional analyses of various cancer-related genes are also conducted. To further facilitate cancer research, the institute uses cutting-edge technologies for genome, epigenome, transcriptome and proteome analyses. The Research Institute is now internationally recognized for its major contributions to various aspects of cancer research. An overview of the research activities undertaken at this institute in 2008 is presented below.

*Detailed explanations can be found by clicking on the numbers in parentheses.

The search for environmental cancer causes and investigation of the underlying mechanisms are being conducted. Elevated levels of endogenous DNA adducts, O⁶carboxymethyl-deoxyguanosine (O⁶-CM-dG) and 3-ethanesulfonic acid-deoxycytidine (3-ESA-dC), produced from N-nitroso bile acid conjugates, were observed in the glandular stomach of rats subjected to duodenal content reflux surgery. A nitrite trapping agent, thioproline, reduced the levels of O⁶-CM-dG and 3-ESA-dC. Five mutagenic nitroarenes were found as contaminants in the surface soil in Kyoto and other residential areas.

An in vitro carcinogenesis model for HPV-associated cancers suggested that only one or two genetic changes might be sufficient. The combined transduction of c-myc and activated ras to human cervical keratinocytes expressing E6 and E7 resulted in the creation of highly potent tumor initiating cells. The PDZ domain-binding motif of the E6 proteins is essential for transformation of rodent cells by inducing by degradation of PDZ-domain containing proteins involved in the polarization of epithelial cells.

(4, 5, 6, 7, 8, 9, 10)

Animal models in combination with molecular biological approaches are being utilized to understand how susceptibility factors affect cancer development. A candidate gene for the susceptibility to 2-amino-1-methyl-6-phenylimidazo-[4,5b]pyridine (PhIP)-induced colon carcinogenesis was identified on rat chromosome 16. Polymorphisms in the 3'-UTR of the gene of F344 and ACI strains were considered to be attributable to the differential expression of the gene. In PhIP-induced colon carcinogenesis models of rodents, distinct expression profiles of microRNA, and dysregulation of b-catenin and c-myc expression were observed. Amplification of around the 500-kb region on chromosome 2 occurred commonly in rat intestinal tumors induced by PhIP and/or azoxymethane.

A mouse model using a tobacco-derived carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1 -butanone (NNK), suggested that smoking increases the risk of inflammation-related colon cancer development.

miR-34a is a p53-regulated novel type tumor suppressive miRNA. SIRT1, a deacetylase of histone, p53, and Rb, was isolated as a new target of miR-34a. The down-regulation or heterozygous deletion of the miR-34a gene locus was observed in human colon cancer specimens.

Poly(ADP-ribose) polymerase-1 (Parp-1) deficiency caused an increase in mutations, especially deletion mutations, with aging. Parp-1 is suggested to be involved in the inaccurate end-joining repair process after g-irradiation. Poly (ADP-ribose) glycohydrolase-deficient (Parg^-/-) mouse ES cell lines showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and g-irradiation.

Parp-1 deficiency induces trophoblast differentiation in ES cells accompanying up-regulation of the H19 gene expression. DNA hypomethylation of the imprinting control region in the H19-Igf2 locus was observed in Parp-1^-/- ES cells, suggesting that Parp-1 deficiency induces epigenetic deregulation in the H19-Igf2 locus.

Epidemiological studies suggested that obesity, consumption of a high-fat diet and hyperlipidemia, especially high serum triglyceride levels, are associated with the risk of colon cancer. Plasminogen activator inhibitor-1 (Pai-1) induction associated with hypertriglyceridemia in Apc^min/+ mice may be related to polyp formation. Pai-1 and inducible nitric oxide synthase are indicated as novel targets for colorectal chemopreventive agents. Adiponectin, secreted by adipocytes, is a key hormone responsible for insulin sensitization via activation of AMPK. Adiponectin suppresses colonic epithelial proliferation by inhibition of the rapamycin pathway under a high-fat diet. Metformin activates AMPK and the rapamycin pathway, and suppresses polyp growth in Apc^min/+ mice. Peroxisome proliferator-activated receptor g (PPARg) regulates colonic epithelial cell turnover via inhibition of b-catenin–mediated transcriptional pathways. PPARg additionally suppresses cell motility in liver metastasis of pancreatic cancer cells.

(80, 81, 82, 83, 84, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98)

The Epidemiology and Prevention Division plans and conducts independent and collaborative studies on cancer etiology and prevention, with a special focus on dietary factors, gene-environmental interaction and effective measures for cancer prevention. Lifestyle factors have been implicated in the etiology of cancer occurrence taken as a whole, each result from epidemiologic studies has contributed to the addition of new evidence.

The Japan Public Health Center-based Prospective Study (JPHC Study), a population-based prospective study, is currently ongoing. A total of 30 articles to analyze the relationship between lifestyle or biochemical data and incidence of chronic disease such as cancer, as well as to assess the validity of the data, were published during 2007.

Studies and projects aimed at evidence-based cancer prevention in Japan have been planned and carried out. Systematic literature reviews, pooled analyses of the main Japanese cohort studies and interventional studies are in progress. The results have been made public or updated on the WEB.

In Brazil, case-control studies for breast cancer and colonic adenoma to investigate the ethnic difference and interaction between environmental and genetic factors are in progress.

Some analyses have been come out and reported to evaluate the risk of endocrine-disrupting chemicals on human health.

The Cancer Information Services and Surveillance Division is providing credible cancer information to patients and their families, the public, healthcare professionals, policy makers and researchers, as well as collecting accurate and useful information on cancer statistics in Japan.

The standardization and quality improvements of population-based and hospital-based cancer registries are supported and maintained. National cancer incidence was then estimated based on the collected data.

An indicator to measure quality for 5 major cancer sites and palliative care were developed to evaluate the achievement of equalization, which is in the testing process in real practice settings.

National cancer statistics information was updated and published. Multi-channel cancer information services are provided through the internet, brochures, lectures and public meeting.

Data on smoking and health were collected and analyzed as a WHO Collaborating Center for Reference on Smoking and Health to distribute the information and to promote effective tobacco controls in Japan.

The Tobacco Control Policy Project was established in October 2007 to facilitate these movements with scientific evidence on risk from tobacco and policy development in Japan. To position Japanese progress on tobacco control in a global context, some international reports were translated and countries such as the USA, UK, and Germany were visited. Policy research groups working under Ministry of Health, Labour and Welfare grants were gathered to share common visions and to develop sustainable strategies. On the other hand, data were collected from all 47 prefectures in Japan to assess tobacco control policy and programs.

(1, 14, 15, 18, 19, 39, 40, 41, 42, 46, 48, 58, 59, 60, 61, 62, 80)

Novel oncogenes, tumor suppressor genes and genetic variations have been reported. Somatic mutations of the KEAP1 gene were identified in lung and gall bladder cancers. NRF2 was defined as an oncogene in lung and head/neck cancers. BRG1 was identified as a tumor suppressor gene for lung cancer, and TCF8 was found to play a role as a tumor suppressor gene in adult T-cell leukemia/lymphoma. The actinin-4 expression level was increased in invasive ductal carcinomas of the pancreas and significantly correlated with poor outcome. Amplification of ACTN4 has been reported in pancreatic cancer. SMURF1, encoding an E3 ubiquitin ligase, was identified as an oncogene in the 7q22 amplification of pancreatic cancer. Heterozygous deletions of p16^INK4a occur early in the development of lung adenocarcinoma.

Bacterial artificial chromosome (BAC) array-based comparative genomic hybridization (CGH) analysis of gastric cancers revealed DNA copy number gains at the two loci of 6p21 that were significantly correlated with a poor prognosis and harbored multiple cancer-related genes. A resequencing analysis for kinase domains of the 90 tyrosine-kinase genes in gastric cancer identified 26 non-synonymous alterations.

A genome-wide association study (GWAS) and other SNP-based association studies have been performed to elucidate germline polymorphisms and mutations implicated in genetic susceptibilities to diffuse-type gastric cancer and drug responses. A genotype study revealed that KRAS polymorphisms were associated with risk for atypical adenomatous hyperplasia. Associations of CYP1A1/GSTM1 polymorphisms with lung cancer risk were confirmed.

Epigenetic alterations in precancerous lesions and cancers have been rigorously analyzed. Epigenetic alterations are found to be accumulated earlier in the gastric mucosa of gastric cancer cases, which can be termed as an epigenetic field for cancerization (epigenetic field defect). BAC array-based methylated CpG island amplification (BAMCA) in non-cancerous renal cortex tissue obtained from patients with clear cell renal cell carcinomas (RCCs) revealed that DNA methylation alteration in non-cancerous tissue was inherited by the corresponding clear cell RCCs, and may generate to more malignant tumors and determine patient outcome. Copy number alterations in the same cohort of clear cell RCCs were also associated with the recurrence-free and overall survival rates of patients. Methylation silencing of rat Tgfbr2 was associated with histone H3 lysine 9 trimethylation, whereas decreased expression of human TGFBR2 was mainly due to decreased transcription activity, sometimes in concert with histone deacetylation and H3 lysine 27 trimethylation.

In order to identify new molecular therapeutic and diagnostic targets, signal transduction pathways specific to diffuse-type gastric cancers and esophageal cancers have been explored. Patient age is an important factor contributing to the biology of AML-M4/M5 and the sub-grouping procedure that could be a powerful tool to identify unfavorable risk patients within pediatric AML-M4/M5 was developed. The comparison of gene expression profiles revealed that similar sets of genes were induced by NOR1 and EWS/NOR1 fusion protein.

This research institute is contributing to the international cancer genome/epigenome projects (International Cancer Genome Consortium (ICGC) and the Alliance for Human Epigenome and Disease (AHEAD)).

(12, 16, 17, 21, 29, 30, 31, 32, 33, 35, 36, 87)

Identification of oncoprotein complexes and their modifications and signal networks have been extensively pursued. Cdt1 is strictly regulated and its deregulation leads to chromosomal instability and eventual carcinogenesis. Cdt1-binding proteins were comprehensively identified by a proteomics approach, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and IIa, and GRWD1/WDR28. Chk1 phosphorylation in the checkpoint response is regulated by both ATR and Cdk2.

A liquid chromatography and mass spectrometry (LC-MS)-based proteomics approach revealed that TCF-4 interacted with nuclear pore complex (NPC) proteins. TCF-4 was sumoylated by a NPC protein Ran binding protein-2 (RanBP2). The sumoylation of TCF-4 enhanced the binding between TCF-4 and b-catenin and increased the nuclear import of TCF-4 and b-catenin and their transcriptional activity.

Promyelocytic leukemia (PML), a nuclear protein with transcription function, formed a complex with transcription factors such as AML1, PU.1, C/EBP and p53 as well as their co-activators such as HIPK2 and p300. The PML complex included Fbxo3 (Fbx3), Skp1, and Cullin1. Phosphorylation of PML stimulated C/EBPe- and PU.1-depnendent transcription and subsequent granulocytic differentiation. Novel posttranslational modification of PML, acetylation, was enhanced by p300 and an HDAC inhibitor, and was associated with apoptosis induced by HDAC inhibitor. Phosphorylation of AML1 was important for its transactivation activity and in bone marrow hematopoiesis and thymocyte differentiation. AML1 was involved in the regulation of the nuclear localization of adnovirus E4orf6 directed E1B-55K proteins.

Human ORC was found to bind to TRF2, a telomere sequence-binding protein that protects telomeres, and is involved in telomere homeostasis. Many human cell types undergo senescence by activation of the p16^INK4a/pRb pathway, and inhibition of the p16^INK4a/pRb pathway, either by Bmi-1, HPV16 E7 or p16^INK4a-specific short hairpin RNA, together with the introduction of hTERT can immortalize many human cell types.

Ras and cAMP regulate Epac2 function in a parallel fashion and Ras-Epac2 interaction is required for the cAMP-dependent activation of endogenous Rap1 by Epac2. MYO18B, a candidate tumor suppressor gene, is essential for the development and/or maintenance of myofibrillar structure. BREK, a myosin VI-interacting protein, is critical for the transition of endocytosed vesicles to recycling endosomes.

Alterations of pro-cell death pathways are critical for cancer initiation, progression and therapy. A new pathway of p53-dependent cell death was discovered, which is unlikely to be related to caspase- and transcription activation.

Crude extracts of the cabbage white butterfly, Pieris rapae, have the ability to induce apoptosis in human cancer cell lines. As an apoptosis-inducing protein, pierisin-1 has been isolated from P. rapae and shown to exhibit DNA ADP-ribosylating activity.

Netrin-1 is an axon-guidance molecule involved in the development and differentiation of neural cells. On the other hand, netrin-1 was shown to inhibit p53-induced cell death. In addition, a new receptor X mediated anti-cell death signaling by netrin-1.

Several new p53 functions were identified. A new p53-inducible protein, which is involved in mitochondrial autophagy, has been identified. This finding suggests that p53 might regulate the mitochondrial degradation via the new protein in order to maintain the quality of mitochondria.

A new mechanism for the prevention of aneuploidy has been identified, in which BLNK, a p53-inducible protein, inhibits cytokinesis of the cells containing abnormal chromosome-segregation, leading to a generation of tetarploid cells.

Clathrin heavy chain (CHC), originally identified as a cytosolic protein regulating endocytosis, was present in the nuclei and functions as a co-activator for p53. The combination of a microarray analysis and a chromatin immunoprecipitation assay identified BLNK and NCCRI2 as novel p53-target genes. Axon-guidance molecules, the other p53-target gene products, were found to be associated with apoptosis. Proliferation related genes including MCM genes were negatively regulated by wild-type p53 and p73.

Activation of the hedgehog (Hh) signaling pathway inhibits the accumulation of the tumor suppressor protein p53, and the inhibition of p53 by the Hh pathway is mediated by Mdm2. Therefore, the Hh pathway may be a powerful accelerator of oncogenesis to inhibit the p53 pathway.

Exposure of cancer cells to oxidative stress induced the association of C9orf10 (designated Ossa) with regulatory domains of Src family kinases (SFKs). Tyrosine-phosphorylated Ossa recruits p85 subunits of phosphatidylinositol 3-kinase (PI3-kinase), leating to activation of the AKT-mediated anti-apoptotic pathway.

CDCP1 is associated with and phosphorylated by SFKs, and is involved in the regulation of anchorage independence of lung cancer cell lines. The CDCP expression level is a useful marker for prediction of patients with lung adenocarcinoma. In addition, CDCP1 was shown to play a critical role in the progression of scirrhous gastric cancers.

Cas-L mediates signals from integrin, T-cell receptors, B-cell receptors and TGF-beta, leading to cell movement and division. Cas-L was shown to be one of the targets of inflammatory cytokines and is modulated by the cell adhesion process in neutrophils.

Amplification of anaplastic lymphoma kinase (ALK) causes the hyperphosphorylation of the Shc family docking proteins including ShcA and ShcC. A high level of ShcC expression was observed in neuroblastomas with poor prognostic factors such as advanced stage and MYC amplification.

Multiple endocrine neoplasia type I (MEN1) is a famililar cancer syndrome. In collaboration with many hospitals, germline and somatic mutations of MEN1 have been examined in patients with MEN1 and related disorders.

Epithelial-to-mesenchymal transition (EMT) plays an important role in cancer progression, especially in invasion and metastasis. TGF-b, Hedgehog, and non-canonical WNT signals are involved in EMT, and Hedgehog signals in particular were found to indirectly upregulate EMT regulators, such as SNAI1 (Snail), ZEB1 and ZEB2 (SIP1), through the regulatory networks of TGF-b, Notch, and miRNA. GLI2 is the primary effector of Hedgehog signaling to induce GLI1 upregulation for the positive feedback regulation of Hedgehog signaling cascades and GLI2 expression is regulated based on the balance of TGF-b, Notch, and p53 signaling cascades.

On the other hand, E-cadherin is a master epithelial cell adhesion molecule which regulates tight cell-cell interaction as well as cell polarity. Detailed promoter analysis revealed that RB1 directly associates with and positively regulates the E-cadherin gene promoter. This study showed for the first time that RB1, a well-known tumor suppressor gene product, is implicated in the EMT of human breast cancer.

Stem cells, including cancer stem cells, are defined as cells with the ability to both extensively self-renew and differentiate into progenitor cell types.

Telomerase expression in stem cells may play an important role in preserving the self-renewal potential of stem cells. Cells expressing hTERT and nucleostemin/GNL3L exhibited high expression of CD133, CD44 and the translocation of b-catenin into the nucleus. Moreover, these cells possess an elevated tumorigenic ability as assessed by anchoring independent tumorigenicity and tumor formation in animals. These observations indicates that hTERT in complex with nucleostemin/GNL3L participates in the regulation of cancer stem cell properties independent of telomere maintenance.

Adult stem cells such as mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell-based therapy for diseases. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. Transplantation of AT-MSCs rescued organ injury of mice via anti-inflammatory and trophic effects, suggesting that AT-MSCs may account for their broad therapeutic efficacy in animal models such as liver diseases and in the clinical settings for liver disease treatment.

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as cancer. Two independent rat ES cells were established that have undifferentiated characters such as Nanog and Oct3/4 genes expression and most importantly, the rat ES cells are capable of producing chimera rats for the first time. The establishment of rat ES cells will provide a suitable model system for cancer study.

Simple and practical diagnostic criteria of minimally invasive intraductal papillary-mucinous carcinoma of the pancreas with nonaggressive characteristics were histopathologically defined focusing on infiltrative distance, mucous lakes not associated with mucinous carcinoma components and expansive growth. To predict liver metastasis of colorectal cancers, the scoring formula regarding expression levels and distribution of dysadherin, E-cadherin and matrilysin was immunohistochemically established.

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Comprehensive DNA methylation analysis using the MS-RDA method identified nine promoter CpG islands on which DNA methylation was significantly associated with poor prognosis in patients with CpG islands methylator phenotype-positive neuroblastomas. Treatment of a neuroblastoma cell line with a demethylating agent caused demethylation of multiple promoter CpG islands, suggesting that the CpG islands methylator phenotype causes a poor prognosis by inducing methylation of multiple promoter CpG islands.

Transcriptome analyses identified the DPP4 gene, the mRNA expression of which was significantly correlated with a poor prognosis in patients with gastrointestinal stromal tumors. Immunohistochemistry using an antibody against CD26, the DPP4 gene product, validated its prognostic value. Transcriptome analyses revealed that expression levels of Glypican-3, a component of the Wnt pathway, were coordinated with those of the alpha pheto-protein which is one of the most established diagnostic and prognostic biomarkers in patients with hepatocellular carcinomas. Expression profiles which can predict the sensitivity for chemoradiotherapy with higher than 90% accuracy were identified in patients with esophageal cancers. The power of the mini-chip, which was developed based on transcriptome analyses to predict the recurrence after surgery, is being validated using peritoneal washings from patients with stomach cancers.

Proteomic studies with two-dimensional difference gel electrophoresis (2D-DIGE) identified EB1 as a predictor of recurrence and poor survival in patients with hepatocellular carcinomas. The predictive power of pfetin identified by 2D-DIGE was immunohistochemically validated in patients with gastrointestinal stromal tumors. Proteomic studies were also conducted to identify prognostic factors in patients with esophageal and lung cancers and Ewing’s and synovial sarcomas. The database software for GeMBDBJ Proteomics was refined and 2D-DIGE data of esophageal cancers and Ewing’s sarcomas were up-loaded. To complement the limitation of separation-based proteomics such as 2D-DIGE, antibody-based proteomics using devices for semi-automated Western blotting was launched for biomarker development.

Gene expression analyses regarding dendritic cells trafficking revealed two genes, each of which encodes a chemokine and an adhesion molecule and were highly expressed in early precursor lesions of pancreatic cancers. Overexpression of these two genes independently in mouse fibrosarcoma cells transplanted into syngeneic mice inhibited growth of the tumor. Anti-tumor immune responses may be affected by expression of these molecules in the early steps of multi-stage pancreatic carcinogenesis.

Although allogeneic hematopoietic stem cell transplantation (alloHSCT) can lead to a significant graft-versus-tumor effect, efforts to enhance a donor T cell response to tumor associated antigens are necessary for the augmentation of antitumor activity. The IFN-a protein is a cytokine with immunomodulatory activity. A mouse MHC-matched alloHSCT model showed that an intratumoral IFN-a gene transfer selectively enhanced tumor immunity against colon and renal cancers without exacerbating graft-versus-host disease.

Natural killer T (NKT) cells are a unique immunoregulatory T cell subset. It turned out that the glucocorticoid-induced TNF receptor plays a co-inhibitory role in antigen-induced NKT cell activation. Insufficient expansion of NKT cells in malignant melanoma patients is related to the reduced expression of CD1d on CD14⁺ cells.

With the use of a cell transfection array system combined with siRNA, ribophorin II (RPN2) was found to contribute to the resistance to docetaxel. RPN2 siRNA delivery restored sensitivity to docetaxel and inhibited tumor growth in animal models: these effects were mediated by the diminished glycosylation of MDR1. Based on an atellocollagen-mediated nano-particle delivery system, it was revealed that downregulation of BRCA2 by siRNA causes radio-sensitization of breast cancer cells, and Glutathione S-transferase Pi mediates the proliferation of androgen-independent prostate cancer in the rat prostate model.

miRNAs are small non-coding RNAs that regulate gene expression through mRNA degradation or translation inhibition. Using a human miRNA microarray, it turned out that MIRN210 is a new member of regulatory miRNAs which plays an important role in erythroid maturation.

Glioblastoma frequently overexpressed not only the wild type EGFR but also its deletion mutant form (EGFRvIII). An antibody-dependent cell-mediated cytotoxicity (ADCC) assay suggested that EGFRvIII can be a target of an anti-EGFR monoclonal antibody (cetuximab) and ADCC activity contributes to the antitumor capacity of cetuximab against EGFRvIII-expressing glioma cells.

Several p53-mutants and p53-target genes are known to induce marked apoptosis in cancer cells. Adenovirus-mediated gene transfer of these genes may well become a new therapeutic strategy for the treatment of p53-resistant tumors.

To examine whether the addition of a tumor-targeting ligand to an oncolytic adenovirus enhances its therapeutic index, the adenovirus library was screened for high affinity to a pancreatic cancer cell line, and particular peptide sequences were selected. The adenovirus displaying the selected targeting ligands showed tumor-specific oncolytic activity.