Overview of Research Activities

The mission of the Research Institute at the National Cancer Center is to advance knowledge in relation to cancer prevention, diagnosis and therapy, with the ultimate aim of for cancer control. The activities at this institute include the search for carcinogens and chemopreventive agents using animal models, identification and functional analysis of cancer-related genes, and development of cancer diagnosis and therapy. To promote cancer research, cutting-edge technologies in genome and proteome analysis are being utilized, and the Research Institute is making major contributions to various aspects of worldwide cancer research. An overview of the research activities at this institute in 2006 is shown below.

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

Mutagenic heterocyclic amines (HCAs) form DNA adducts, mainly at the C8-position of guanine bases. Synthesis of dG-C8 adducts with HCAs was achieved via the Buchwald-Hartwig arylamination reaction. Oligonucleotide DNA with PhIP-C8-guanyl adducts caused complete arrest of DNA synthesis, which could be repaired by translesional DNA polymerases and lead to base-substitution type mutations at the PhIP-adducted sites. Novel mutagenic compounds, such as APNH, AMPNH and APH, formed from non-mutagenic b-carbolines and aromatic amines, were shown to be activated mainly by CYP1A2 and NAT2. Non-CIPBTA-type mutagens were newly found to be produced from azo dyes during industrial processes in dyeing factories.

Helicobacter pylori infection was demonstrated to induce aberrant DNA methylation and a field defect for gastric cancer. Gastric cancer risk can thereby be estimated from the severity of the defect using DNA methylation as a marker. Development of convenient assay systems for the detection of compounds that induce epigenetic abnormalities is also currently underway.

Search for heritable factors determining the susceptibility to carcinogenesis has been conducted by several research groups at this institute, and a candidate locus for colon cancer susceptibility was narrowed down to a 0.8 Mb region on rat chromosome 16, and one gene reacted differently to PhIP administration between the two rat strains. Lung cancer susceptibility genes were also investigated among 36 genes involved in DNA repair pathways by a case-control study, and an SNP (Val83Met) in the coding region of the MTH1 gene and another SNP in the 5'-UTR leading to alternative translation initiation showed a significant association with the risk of small cell lung carcinoma in humans. The Ser326Cys polymorphism of the OGG1 gene was also indicated as a risk allele for lung adenocarcinoma. A search for genetic susceptibility to gastric cancer is also being conducted using rat model of MNNG-induced gastric cancer.

Animal models provide substantial contributions to cancer research and pharmaceutical research. The rat model of HCA-induced colon cancer has been used to dissect genetic and epigenetic events during cancer development. An approximately 500 kb region on chromosome 2 was commonly amplified in PhIP-induced colon tumors by array CGH analysis. SND1, a component of the RNA-induced silencing complex (RISC), was demonstrated to be overexpressed in aberrant crypt foci (ACF), preneoplastic lesions of the colon. SND1 modulates Apc protein levels in some post-transcriptional manner and could be implicated in the early stages of colon carcinogenesis. Targeted activation of Ha-rasG12V in the pancreatic ducts of rats induced tubular adenocarcinoma.

Investigation on the role of the ADP- ribosylation reaction in carcinogenesis has long been conducted. Poly (ADP-ribose)-polymerase-1 (Parp-1)-knockout mice show increased susceptibility to carcinogenesis induced by alkylating agents, and Parp-1 is required to maintain transcriptional regulation of a wide variety of genes. Pierisin-1 and -2 from cabbage butterflies catalyze transfer of the ADP-ribose moiety of NAD at the N2 position of 2-deoxyguanosine in DNA. Pierisin-1 treatment of HeLa cells induced an intra-S-phase arrest through both ATM and ATR pathways. Another DNA ADP-ribosylating proteins, CARP-1, was also isolated from the hard clam and showed homology with pierisin-1 and -2.

Expression of inducible nitric oxide (iNOS) and cycloocygenases (COX)-2 is frequently enhanced in colon cancers, and the iNOS inhibitor, ONO-1714, and a COX-1-selective inhibitor, mofezolac, suppressed the development of colon cancer induced by azoxymethane (AOM) in the rat. A new flavone derivative, chafuroside, in oolong tea suppressed intestinal polyp formation in Apc -deficient Min mice and also AOM-induced ACF formation in the rat. Interestingly, Min mice showed age-dependent hyperlipidemia along with low lipoprotein lipase (LPL) levels. The PPARg agonist pioglitazone and the LPL inducer NO-1886 suppressed hyperlipidemia and the development of intestinal polyps in the Min mice.

( 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 )

Lifestyle factors have been implicated in the occurence of cance. The Japan Public Health Center-based Prospective Study (JPHC Study) is investigating several population-based prospective studies; for example, relation between a history of diabetes mellitus and the total cancer risk, family history of lung cancer and lung cancer risk, body mass index (BMI)/height and prostate cancer risk, and dietary fiber intake and colorectal cancer risk. Effects of plasma C-reactive protein on the colorectal cancer risk, and of Helicobacter pylori infection on the gastric cancer risk in combination with CagA and the pepsinogen status were also investigated in two case-control studies. Results on coronary heart disease, suicide, and validation of the data were reported from the study. In Brazil, case-control studies are in progress to investigate the ethnic differences and gene-environment interactions in the incidence of breast cancer and colonic adenoma.

Relations between fish intake and serum organochlorines, urinary mono-n-butyl phthalate, mono-2-ethylhexyl phthalate and serum free testosterone were investigated to evaluate the risk of endocrine-disrupting chemicals (EDC) on human health.

The relation between tobacco smoking and the risk of colorectal, lung, breast, stomach and liver cancer was systematically reviewed and evaluated, aimed at the establishment of evidence-based cancer prevention. Evidence for alcohol drinking and the colorectal cancer risk was reviewed, and the standardization and quality improvements of population-based and hospital-based cancer registries are supported and maintained.

Guidelines for colorectal, gastric and lung cancer screening have been developed based on a standardized process. The guideline for prostate cancer screening is being revised.

Data analysis was made for health services research in cancer care. Information and data on smoking and health were collected for analysis to support implementation of the WHO Framework Convention on Tobacco Control. Other data collection, integrated management, analyses and dissemination of cancer information have been conducted at the national level.

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

( 15, 18, 35, 37, 38, 62 )

Alterations in specific genes, including oncogenes and tumor suppressor genes, are involved in the multistage carcinogenesis of human tumors. Examination of epidermal growth factor receptor (EGFR) in lung adenocarcinoma (ADC) revealed the frequent occurrence of EGFR mutations in non-invasive bronchioloalveolar carcinoma (60%) as well as in metastases to the brain from this cancer. Mutant EGFR (delE746-A750), but not a wild-type EGFR, phosphorylated AKT and STAT3 after serum starvation in an in vitro study. The MYO18B gene was previously isolated as a candidate lung tumor suppressor gene at this institute. Yeast two-hybrid screening identified Sug1, a 19S regulator subunit of the 26S proteasome, as a binding partner of MYO18B, suggesting that MYO18B may be a substrate for proteasomal degradation.

To elucidate the genetic and genomic alterations, oligonucleotide array-based CGH (comparative genomic hybridization) was applied to various human cancers. A homozygous deletion at the DOCK8 locus at chromosomal region 9p24 was detected in a lung cancer cell line, yielding another candidate lung tumor suppressor gene. Analysis of nodule-in-nodule-type hepatocellular carcinoma (HCC) newly revealed genetic inactivation of the APC gene in HCC. Analysis of esophageal squamous cell carcinoma (ESCC) revealed amplification of the CCND1 gene and homozygous deletion of the p16 gene associated with ESCC progression. Somatic mutation of KEAP1, whose product induces phase II enzymes, was also detected in lung cancer, in collaboration with other groups.

Non-homologous end joining (NHEJ) repair and homologous recombinational repair (HRR) are the two main modes of repair of DNA double-strand breaks (DSBs) in vertebrates. Therefore, a plasmid-based system to assess both NHEJ and HRR was developed to investigate the role of these repair activities in human carcinogenesis. The germline polymorphisms and mutations implicated in cancer susceptibility or drug responses were analyzed and novel methods for genomic data analysis were developed in collaboration with the National Cancer Center Hospital and other institutes. A part of the single nucleotide polymorphism (SNP) data was published in a database (http://gemdbj.nibio.go.jp).

( 1, 46, 48, 63, 81 )

Methylation of CpG islands (CGIs) is closely involved in human carcinogenesis. Some cancer cells show a high incidence of aberrant DNA mehylation, known as the CpG island methylator phenotype (CIMP), and the finding of increased rates of de novo methylation in some cancer cells that lead to induction of dense methylation in CGIs would provide important information in understanding the mechanisms of CIMP. In liver carcinogenesis, epigenetic-instability-dependent HCC, showing frequent epigenetic aberrations but no chromosomal instability, was newly recognized. Methylation-sensitive-representational difference analysis (MS-RDA), which was originally developed at this institute, identified peroxiredoxin 2 (PRDX2 ), a negative regulator of platelet-derived growth factor signaling, as one of the silenced genes in melanoma. Combinatorial analyses of chemical genomic screening using a demethylating agent, 5-aza-2'-deoxycytidine, with an expression microarray was also useful to identify genes methylated and silenced in various cancers. These include the very low density lipoprotein receptor (VLDLR ) and the MTSS1, a metastasis receptor, in gastric cancer, and the UCHL1, a regulator of cellular ubiquitin levels, in colorectal cancer.

The significance of aberrant DNA methylation in multistage carcinogenesis was also investigated in an organ-specific manner. In the kidney, the incidence of methylated CpG islands was significantly higher in the non-tumorous renal tissue of patients with renal cell carcinoma (RCC) than in the normal renal tissue of patients without RCC. The degree of CpG methylation in the non-tumorous renal tissue of RCC patients was correlated with the histological grade of the corresponding RCCs. Furthermore, the degree of CpG methylation in RCCs was correlated with various malignant features of the RCCs and also the prognosis of the patients. In the pancreas, the incidence and degree of methylation in the tumor-related genes were significantly higher in pancreatic duct epithelia with inflammation or pancreatic intraepithelial neoplasias (PanINs) than in pancreatic duct epithelia without inflammation. Ductal carcinomas frequently showed methylation of the BRCA1, APC, p16 and TIMP-3 genes, although considerable heterogeneity in the degree of methylation was observed. In addition, the degree of DNA methylation was correlated with the expression level of DNA methyltransferase 1 (DNMT1). These findings indicate that aberrant DNA methylation is involved in the multistage carcinogenetic process from a precancerous condition to malignant progression in the kidney and pancreas. In the human stomach, MS-RDA was applied to intestinal metaplasia (IM) and the ZIK1, ZNF141, KAL1, and FGF14 genes were identified as methylated and downregulated genes in glands with IM. An independent ZIK1 methylation in physically distant glands in IM suggests that methylation of specific genes could also play a role in disorders of polyclonal origin.

Methylation analyses of specific tumor suppressor genes revealed that the tumor suppressor gene TSLC1/IGSF4, previously identified at this institute, was inactivated by methylation in 44% of lung ADCs, serving as a possible indicator of poor prognosis. Another candidate tumor suppressor gene, DAL-1/4.1B, that binds to TSLC1/IGSF4, was also found to be frequently methylated in lung ADC as well as RCC. These results, taken into consideration with other findings, suggest that the cascade including TSLC1/IGSF4 and DAL-1/4/1B as essential components may be involved in various cancers as well as in spermatogenesis.

Expression microarray analyses in combination with laser capture microdissection were performed in various human cancers to categorize tumors into several subtypes or to identify novel tumor markers as well as possible therapeutic targets. Analysis of AML harboring chromosomal translocations revealed that t(8;21)-AML and inv(16)-AML showed very similar gene expression profiles, suggesting that AML1-MTG8 and CBFb-MYH11 chimeric proteins may affect a common cascade of leukemogenesis. Expression profiling of pretreatment biopsy specimens from 33 ESCC patients identified 57 and 120 genes that were correlated with short-term and long-term survival, respectively. An advanced microarray data analysis using a boosted fuzzy classifier could identify ESCC with intramural metastasis, a subtype with a poorer prognosis, and identified CDK6 as a possible novel diagnostic marker of ESCC. Gene expression analyses were also performed in surgical specimens from lung ADC, neuroblastoma, soft tissue sarcoma, and gastric cancer to identify profiles specific to each disease. A part of the expression profile data is published in a database (http://gemdbj.nibio.go.jp).

Since altered expression of microRNA (miRNA) may represent another molecular mechanism important in human carcinogenesis, the expression profile of miRNA was examined in lung and colon cancers, and possible diagnostic and prognostic markers were identified in the lung cancers.

( 3, 23, 24, 26, 27, 53, 78, 79 )

Expression of dysadherin, a cell membrane glycoprotein, was found to be correlated with a higher clinical stage, poorer prognosis, or lymph node metastasis in various cancers, including colorectal cancers, head and neck squamous carcinomas and epithelioid carcinomas. Dysadherin-positive cells are usually gathered around and infiltrating small intratumoral lymphatic vessels.

Solid-pseudopapillary neoplasms of the pancreas are uncommon and occur preferentially in young women, and nuclear accumulation of b-catenin was found to be a useful marker of this disease.

A group of axon-guidance molecules were inactivated in human cancers and directly regulated by the tumor suppressor gene, p53, suggesting possible common functioning in tumorigenesis. Among them, netrin-1 was shown to inhibit transcriptional activation of p53-targets and p53-dependent cell death.

CDCP1 was identified as a major substrate of the Src family kinases in a suspension culture of lung cancer cells. RNAi analysis suggested that CDCP1 is a molecule regulating anchorage independency under the control of the Src family kinases in a subset of lung cancer cells.

A new function of ephrin-B1 in the secretion of matrix metalloprotease was discovered through analysis of receptor- mediated cleavage of ephrin-B1 in cancer of the pancreas.

It was demonstrated that fibroblast growth factor 4 (FGF4) is expressed in regions of the brain where adult neurogenesis is continuously occurring, and that it has the ability to promote neural stem cell proliferation and neuronal differentiation in the postnatal brain.

NOR1, an orphan nuclear receptor, has been implicated in the neural, endocrine and immune systems. NOR1 expression is rapidly induced in smooth muscle cells by stimulation with platelet-derived growth factor (PDGF), and analysis of NOR1-deficient mice revealed that NOR1 plays a key role in the transcriptional regulation of smooth muscle cell proliferation. EWS/NOR1 and EWS/FLI-1 fusion proteins found in human sarcomas were shown to interact with the U1 small nuclear ribonucleoprotein-specific protein, U1C, and affect pre-mRNA splicing.

( 13, 39, 73, 74, 76 )

The b-catenin / TCF-4 complex is thought to be involved in colorectal carcinogenesis. A colorectal cancer cell line with inducible expression of dominant-negative TCF-4 was established and used to identify proteins regulated by the b-catenin / TCF-4 complex. Splicing factor-1 (SF1) was identified as one of the proteins that was negatively regulated by the complex.

Molecules transcriptionally activated by canonical and non-canonical signaling cascades downstream of WNT signals were analyzed and interaction among these molecules were further investigated using comparative proteomics and comparative genomics.

Proteomic signatures corresponding to the histology of soft-tissue sarcomas and esophageal cancers were analyzed using the 2D-DIGE system. Proteins associated with the histology and prognosis of these malignancies were identified by mass spectrometry. To develop novel clinical applications, a proteome database based on quantitative protein expression profiles using 2D-DIGE is being constructed.

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The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to DNA double-strand breaks (DSBs) are regulated by the ATM protein kinase. ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx are mediated by phosphorylation of Hdmx. S403 is targeted directly by ATM. Phosphorylation of S367 and S342, but not S403, which is directly targeted by ATM, contributes to the 14-3-3 interaction and nuclear accumulation of Hdmx.

In response to DNA damage, KAP-1 is phosphorylated in an ATM-dependent manner on Ser 824. KAP-1 is phosphorylated exclusively at the site of damage, from which phosphorylated KAP-1 spreads rapidly throughout the chromatin. These results suggest that KAP-1 functions as a primary mediator of chromatin relaxation at sites of DNAdamage.

As transcriptional targets of p53, Notch1, DFNA5, and a few novel genes have been identified. UV irradiation induced p53, Notch1, and keratinocyte differentiation in wild-type mice, but not in p53-defficient mice, indicating a novel function of p53 in regulating cell differentiation under genotoxic stress.

Unexpectedly, clathrin heavy chain (CHC) was found to bind to p53 and to contribute to p53-mediated transcription. CHC binds to the p53-responsive promoter in vivo and stabilizes the p53-p300 interaction to promote p53-mediated transcription.

The leukemia-associated factor AML1 forms a complex with p300, MOZ and the HIPK2 protein kinase. HIPK2 was found to phosphorylate AML1 and to stimulate AML1-mediated transcription. AML1 interacts with both p300 and HIPK2, thus, AML1 induces phosphorylation of p300 by HIPK2. Phosphorylation of AML1 at Ser249/276 is critical for this activity.

Phosphorylation of p300 is also induced by other transcription factors, and inhibited by co-expression of dominant-negative HIPK2. Thus, HIPK-mediated phosphorylation of p300 seems to be a common event in transcriptional regulation by a range of transcription factors.

HIPK1/HIPK2 double-deficient embryos exhibited phenotypes partially similar to those observed in p300- and CBP-deficient mice. Phosphorylation of p300 was inhibited in the Hipk1/Hipk2 -double deficient embryos. These findings suggest that phosphorylation of p300/CBP by HIPK1/HIPK2 is essential for the function of p300 and CBP in embryonic development.

Analyses of MOZ-null mice show that MOZ is required for the maintenance of hematopoietic stem cells and that it plays a role in the differentiation of erythroid and myeloid cells.

Strict regulation of Cdt1 activity was further demonstrated by a couple of novel pathways that enhance the degradation of Cdt1. Cullin 4-DDB1 during the S phase and the APC/CCdh1 during G1/G0 phase are involved in the proteolytic regulation of Cdt1.

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

Proteomic profiling coupled with machine learning is clearly a promising alternative method, but technological innovations are crucial for this growing science. A powerful novel system was developed for the detection, visualization, quantification, and comparison of LC-MS data generated from unlabeled and unpurified clinical protein samples, and designated as 2-Dimensional Image Converted Analysis of Liquid chromatography (2DICAL) and mass spectrometry. Protein identification by tandem mass spectrometry (MS/MS) can be performed on the peaks of interest in the preparatory LC run.

Another novel proteomics method that has been developed is based on multi-dimensional liquid chromatography, 2D-DIGE, and mass spectrometry, to enable more comprehensive and sensitive identification of differentialy expressed plasma proteins, which may contain abundant but non-informative proteins, such as albumin. The plasma proteome of lung cancer patients and healthy donors were compared using this approach. Among 3890 protein spots observed, 364 protein spots from 58 genes showed significantly different intensities. This method is now being optimized for plasma biomarker development in patients of lung cancer and pancreatic cancer.

Just like other omics-based biomarker search, a validation phase is critical for proteomics research. To validate the proteomic profile identified in our institute last year as a possible new diagnostic marker of pancreatic cancer, prospective collection of a large number of serum/plasma samples has been started with the participation of 6 medical institutions.

In the germline genetic testing for the patients with multiple endocrine neoplasia type 1 (MEN1), routine sequencing analysis has occasionally failed to detect mutations. One reason is the heterozygous large deletion in the MEN1 gene. A quantitative PCR-based new method has been developed, which has successfully identified the causative mutation in an affected family in which the mutation was not detected by the routine analysis. This method was also shown to be useful for the diagnosis of hereditary retinoblastoma and familial adenomatous polyposis.

Tolerance is one of the major hurdles against successful immune therapy. Anti-FOXP3 antibodies were raised and used in immunohistochemical studies of the FOXP3+CD4+CD25+ regulatory T cells (TR). A significance increase in the prevalence of TR was found during the progression of premalignant lesions and was correlated with the prognosis in pancreatic cancer. TR may play a role in the regulation of the immune response against pancreatic ductal carcinomas from the premalignant stage to the stage of established cancer.

A unique immunoregulatory T-cell population, the natural killer T (NKT) cells, are considered to be promising effector cells for cancer immunotherapy. Factors influencing the in vitro expansion of NKT cells from mobilized peripheral blood mononuclear cells were determined to aid in the development of an efficient and practical expansion protocol for adoptive immunotherapy with NKT cells.

Prevention of acute graft-versus-host disease (GVHD) depends on interleukin-4 production by NKT cells, suggesting that adoptive transfer of in vitro-cultured NKT cells may help prevent acute GVHD after allogeneic hematopoietic stem cell transplantation (alloHSCT).

To enhance the graft-versus-tumor effect of alloHSCT, direct intratumoral injection of the allogeneic major histocompatibility complex (alloMHC) gene was examined as a component of combination therapy in a mouse colon cancer xenograft model. The alloMHC gene transfer enhanced the antitumor effects of alloHSCT without exacerbating GVHD, suggesting that the combination strategy may have important implications in the development of therapeutic strategies against human solid cancers.

Some p53-mutants show enhanced apoptosis-inducing activity, and several p53 target genes were found to induce marked apoptosis in cancer cells. Among these candidate therapeutic genes, adenovirus-mediated gene transfer of a p53-46F mutant was suggested to be useful for the development of cancer therapy.

There has been a growing interest in RNA interference (RNAi) as a possible new therapeutic strategy for various diseases, including cancers. Delivery is one of the key issues, and methods for the systemic administration of the interfering RNAs have been investigated. Atelocollagen has been developed at our institute as a versatile delivery system for both local and systemic delivery of nucleic acids, including siRNAs and antisense oligonucleotides. The atelocollagen-siRNA complex showed a gene silencing effect and inhibition of tumor growth in vivo, suggesting its potential as a novel agent in cancer therapeutics.

In the field of regenerative medicine, recent observations suggest that stem cell-based therapy may be a potential alternative to liver transplantation. Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) were prepared from clinical samples, and it was found that incubation with specific growth factors (HGF, FGF1, and FGF4) induced the production of functioning hepatic cells from the CD105+ fraction of AT-MSCs. Adipose tissue may be a good source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes.

The modes of action of cytotoxic and target-based drugs, including tyrosine kinase inhibitors, have been demonstrated. Expression profiling by microarray and other methods has been found to be useful to select candidate biomarkers of sensitivity and resistance to target-based drugs in clinical samples.

The JCOG (Japan Clinical Oncology Group) Data Coordinating Center has been supporting a number of nationwide multi-institutional and multi-modality cooperative studies and is currently managing approximately 70 clinical trials. A new web-based clinical data management system is now under evaluation for improvement of the data management performance and data quality. Final results of a phase II study, have been reported. Methodological studies are being conducted in the realms of data management and regulatory sciences.