Overview of Research Activities     The mission of the Research Institute in the National Cancer Center is to advance knowledge in cancer prevention, diagnosis and therapy for cancer control. Activities include a search for carcinogens and chemopreventive agents using animal models, identification and functional analysis of cancer-related genes and development of cancer diagnosis and therapy. For promotion of 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 in 2002 is shown below. Research on The Causes and Prevention of Cancer Environmental cancer causes (2, 3, 4, 9, 10, 17, 23, 24)     Exposure to various endogenous and exogenous environmental insults is considered to cause genetic and/or epigenetic alterations in humans. Heterocyclic amines (HCAs), produced while cooking meat and fish, induce various kind of cancers in rodents, and are therefore considered to be potential human carcinogens. In addition to HCAs, significant numbers of mutagens have been found in water samples from several rivers in Japan and some of them were suspected to originate from textile dyes. Mutagens formed from norharman with aromatic amines were identified as aminophenylnorharman, and the effects on DNA were investigated. Endocrine disrupting chemicals were suggested to be present in fish and meat in an international collaborative study. High levels of bisphenol A were found among epoxy resin workers. Using GST-P positive lesions in liver and lungs of wild rodents as environmental monitors, no linear dose-response was observed at a very low dose range of carcinogens in rats. Viral infection is another cause for several human cancers, including hepatocellular carcinoma and cervical cancers. HCV RNA replicon-replicating cells were generated, and the effects of HCV specific RNAi are currently being investigated using RNAi expressing viral vectors.     The Japan Public Health Center-based Prospective study on Cancer and Cardiovascular Disease (JPHC study) is currently ongoing, in which approximately 140,000 individuals from 11 public health centers will be followed-up for 20 years. Several basic investigations have been conducted to validate the quality of the JPHC study. Lifestyle factors in a baseline questionnaire survey, such as cigarette smoking, alcohol consumption, being under- or over-weight, and food intake, were examined with relation to subsequent risk of premature deaths and cancer incidence, such as stomach and lung cancer.     Genetic polymorphisms of CYP2A6, NQ01 and GSTT1 were investigated with regards to susceptibility to lung cancer, especially adenocarcinoma in humans. Case-control studies of gastric cancer and breast cancer were conducted in Japanese Brazilian and Brazilians not of Japanese ancestry in Sao Paulo, in order to clarify the role of genetic susceptibility and environmental carcinogenesis. Among the patients with squamous cell carcinoma (SCC) in head and neck, who often develop second primary esophageal SCC, strong gene-environmental interactions between the ALDH2-2 allele and alcohol drinking were revealed. Animal models of carcinogenesis (2, 3, 13)     Animal models are relevant tools to elucidate the carcinogenic activities of various compounds, to screen chemopreventive and modifying agents, and to detect multistage genetic alterations, especially at early phases in carcinogenesis. PhIP is one of the most abundant HCAs, and induces colon and mammary tumors in male and female rats, respectively. Some of the dysplastic aberrant crypt foci (ACF) in the colon harbored genetic alterations in Apc and b-catenin genes, and mutation spectra in dysplastic ACF were quite similar to those observed in colon tumors. AOM-induced colon cancers in mice and MNNG-induced gastric cancers in rats also well represented human lesions with regards to histopathological features and/or genetic alterations. Human Ha-RAS transgenic rats were generated, and were highly susceptible to chemical-induced mammary and bladder carcinogenesis. An increase in the numbers of terminal endbuds, higher levels of c-myc, cyclin D1, cyclin D2 and active MAP kinase in mammary glands have been noted. Poly(ADP-ribose) polymerase-1-disrupted mice became highly sensitive to various DNA damaging agents.     The genetic backgrounds of individuals have substantial impacts on cancer development. A genetic trait on chromosome 16 in F344 rats, named Sct, conferred higher susceptibility to the development of ACF by PhIP. For an MNNG-induced gastric cancer model, congenic rat strains harboring respective susceptible and resistant loci were constructed, and their cancer susceptibility is being tested. Search for cancer prevention methods (3, 17, 23)     Selective inhibitors for COX-1 and COX-2 suppressed ACF formation in the colon, and antagonists of prostanoid receptors EP1 and EP4 could be good candidates for chemopreventive agents. Urease inhibitors and garlic extracts suppressed H. pylori-induced gastritis in Mongolian gerbils. Bovine lactoferrin (bLF) markedly inhibits carcinogenesis and lung metastatic colony formation. The elevation of caspase-1 activity induced by bLF may be important for IL-18 production, which potentiates the killing activity of T- and NK-cells.     A randomized controlled trial to assess the effectiveness of vitamin C supplementation on gastric cancer has progressed using individuals with chronic atrophic gastritis. Another randomized controlled trial for assessment of dietary modification was completed and analysis is now underway. Research on The Mechanisms of Cancer Development Genetic aberration of human cancers (4, 11, 13, 16, 21)     Functional complementation based on the suppression of in vivo tumorigenicity identified a novel tumor suppressor gene, TSLC1. Two-hit inactivation by promoter methylation and allelic loss was observed in 44% of primary non-small cell lung cancers. The gene was also inactivated in various other cancers, and its restoration also suppressed metastasis of cancer cell lines. TSLC1 has homology with NCAM, presumably acting as a cell adhesion molecule, interacts with the actin filament through DAL-1, and is suggested to be involved in cell motility.     MYO18B is a novel myosin family candidate tumor suppressor gene identified at chromosome 22q12.1. It is deleted, mutated, and hypermethylated frequently in lung cancers. Restoration of MYO18B expression in lung carcinoma cells suppressed anchorage-independent growth.     Based on in-house physical and bacterial clone contigs, minimal deleted regions were identified for neuroblastoma, endometrial and esophageal cancers. In the latter two cancers, candidate tumor suppressor genes showing somatic mutations at significant frequencies have been identified.     In lymphoid leukemia, 9p21 deletions inactivate the p16 INK4a and p15 ARF tumor suppressor genes. Analysis of the breakpoints revealed that the majority of deletions were mediated by illegitimate V(D)J recombination targeted at ectopic recombination signal sequences widely distributed in 9p21.     Gene amplification at eight loci was found in esophageal cancers by DNA array CGH. CAB2 was identified at 17q12, as a human homologue of the yeast COS16 required for the repair of DNA double-strand breaks.     A microarray system harboring 5,000 human BAC DNAs was established in collaboration with Tokyo Medical and Dental University. These human BAC DNAs are FISH-mapped, sequence-tagged, and cover the human genome at about 0.6-Mb resolution. A genome-wide survey for gross genetic alterations is about to enter a new stage.     Single nucleotide instability (SNI), a new type of genetic instability in which spontaneous point mutation rates increase without microsatellite instability, was discovered. Using a characteristic increase of A:T to C:G transversions as a clue, its potential mechanism was investigated.     Both candidate gene analysis and genome scan are underway to identify germline polymorphisms associated with cancer susceptibility. Moreover, some single nucleotide polymorphisms (SNPs) were analyzed for their functional significance. Functional analyses of cancer related genes (2, 8, 14, 16, 20)     The AML1-MTG8 fusion transcription factor was identified in AML. Microarray analysis unveiled many genes previously not associated with AML1-MTG8 signal transduction, including those involved in granulocyte differentiation. Microarray technology was also applied to analyze the expression profile of pediatric patients with AML. A supervised approach selected 35 genes associated with prognosis. Most of the 35 genes were not correlated with morphological subtypes or karyotypes and may be useful to provide therapeutic direction for individual risk-adapted therapy for pediatric AML patients.     cyclin D1 conditional transgenic mice were established for in vivo functional analyses. The skin-specific overexpression in this model was associated with increased tumor formation in vivo, and resistance to calcium-induced terminal differentiation and growth arrest in vitro. EWS/ NOR1, a fusion gene observed in extraskeletal myxoid chondrosarcomas, was shown to gain a novel activity affecting pre-mRNA splicing.     Chromosomal instability is one of the hallmarks of cancer cells, and is caused by perturbation of mitotic checkpoints, alteration of recombination repair systems and abrogation of DNA replication. The tandem repeat of d(CAGGG)n was demonstrated to form an intramolecular quadruplex structure and to cause arrest of DNA synthesis at the d(GGG)n site in vitro. Epigenetic mechanisms of carcinogenesis (1, 13, 19)     Two methodologies for genome-scannings of aberrant methylations in cancers, methylation-sensitive-representational difference analysis (MS-RDA) and methyl-CpG binding domain/ segregation of partly melted molecules (MBD/SPM), were developed. By the MS-RDA method, HTR1B silencing in human lung cancers and novel silencing of nine genes in human stomach cancers have been discovered. Among the nine genes, three genes were with reported growth-suppressive activities. By the MBD/SPM method, CpG islands specifically methylated in human lung adenocarcinomas were comprehensively isolated. Although methylated CpG islands were generally distributed "randomly" in the genome, clusters of CpG islands at the HOXA and HOXD loci were methylated in human lung adenocarcinomas.     Molecular mechanisms leading to occurrence of these aberrant methylations are also under investigation. Overexpression of DNMT3b4, a splice variant of DNA methyltransferase DNMT3b, was shown to induce DNA hypomethylation on satellite 2. In breast cancers, hypomethylation on satellite 2 was associated with the accumulation of chromosome alterations and the acquisition of aggressive histological features. The DNMT1 expression was found to increase with stages in hepatocarcinogenesis, and to become a biological predictor of both HCC recurrence and a poor prognosis for HCC patients. Factors involved in metastatic and invasive potential of cancer cells (1, 4, 22)     Starting from Hu09 osteosarcoma cell lines and A549 lung adenocarcinoma cell lines, several new sublines having different metastatic potential were established by in vivo and in vitro selection systems. They are useful tools for analyzing the regulation mechanisms of cancer metastasis.     A new membrane protein, dysadherin, was identified as a suppressor of E-cadherin-mediated cell adhesion. It shows a compensatory expression pattern with E-cadherin during the progression of cancers, and shows metastasis promoting activity by overexpression in hepatocellular carcinoma cell lines. On the other hand, E-cadherin-mediated cell adhesion was promoted by inhibition of Src-family kinases or Ras. Expression levels of laminin g2 were investigated in pancreas adenocarcinomas. Overexpression of cytoplasmic laminin g2 chain correlated with invasive potential and metastatic potential of cancers. Similarly, cytoplasmic expression of g2 chain of laminin5 was associated with a poor prognosis in tongue and colon cancers. Signaling pathways modulating malignant characteristics of tumors (1, 6, 7, 10, 11, 17, 22, 24)     Non-apoptotic programmed cell death which is defined by autophagic degeneration without caspase activation was observed in neuroblastoma tissues with overexpression of Ras protein. Expression of Ras in vitro also caused similar non-apoptotic cell death suggesting that this type of cell death is involved in spontaneous regression of neuroblastomas. A potent apoptosis-inducing substance, pierisin-1, was originally isolated from butterflies. It induced activation of caspases and release of cytochrome c in vitro and showed significant anti-cancer activity in nude mice.     Cas is a docking protein connecting the integrin signal with cellular proteins like Src, Fak and Crk. Cas-negative fibroblasts derived from knockout mice are defective in anchorage-independent growth by activated Src and in cell migration. By expressing deletion mutants of Cas in these fibroblasts, it was found that the central repeats of tyrosine motifs are critical for cell migration and the C-terminal Src-binding domain is important in Src-mediated transformation.     The Wnt / b-catenin pathway is known to regulate a wide range of biological events including cellular differentiation and carcino-genesis. Numbers of Wnt pathway proteins were cloned and their expression in human cancers was investigated. By the somatic gene targeting method, wild and mutant alleles of b-catenin were disrupted in a colon cancer cell line, which caused no change in growth rate. It was suggested that b-catenin mutations contribute to colon carcinogenesis in aspects other than cell proliferation.     The prostate specific antigen (PSA) plays a key role in osteoplastic changes of prostate cancer at metastatic sites. It was shown that PSA acts as a serine protease which activates TGF-b secreted from osteoblasts by partial cleavage, resulting in apoptosis of osteoclast precursor cells. Modes of action of the Lon protease, which degrades abnormal proteins such as denatured proteins, were also analyzed using an Escherichia coli expression system.     Involvement of AMPK, the 5'-AMP activated protein kinase, in the induction of the tolerance to glucose starvation during hypoxia was demonstrated using the AMPK antisense vector. It was shown that a novel member of AMPK, ARK5, was directly activated by Akt causing ATM phosphorylation during nutrient starvation.     E6 and E7 of human papillomavirus can cooperatively immortalize normal human epithelial cells. Both inactivation of the RB pathway by E7 and activation of telomerase by E6 are required for immortalization of these cell types. On the other hand, the PDZ domain-binding motif of E6 and the ability to bind an ubiquitinligase, E6AP, are known to be important for the transforming activity of E6. The enhanced degradation of possible tumor suppressors D1g and Scribble, both of which contain the PDZ domains, could be the mechanism of transformation by E6. Functional analysis of tumor-associated transcription factors (8, 12)     p53 is stabilized and activated by its phosphorylation/acetylation to induce cell-cycle arrest and/or apoptosis. Previous studies have shown that phosphorylation of Ser46 on p53 mediates induction of an apoptosis gene, p53AIP1. Analysis of Ser46-kinase suggested that at least two types of protein complexes, one containing casein kinase 2 and the other containing HIPK2, are involved in the phosphorylation of p53 at Ser46. Mdm2 binds p53 and stimulates its degradation. Cyclin G, a p53 target, formed a quaternary complex with phosphatase 2A (PP2A) as well as Mdm2 and markedly stimulated the ability of PP2A to dephosphorylate Mdm2 at Thr216. Thus, cyclin G recruits PP2A to modulate the phosphorylation of Mdm2 and thereby to stimulate the degradation of p53.     The AML1 transcription factor is the most frequent target of chromosome translocation found in human acute leukemia. Purification of the AML1 complex indicated that MOZ as well as p300 and CBP form complexes with AML1. Purification of the MOZ complex indicated that MOZ also forms a complex with p53. Forced expression of the MOZ-induced expression of p21/Waf1 but not those of BAX and mdm2, suggesting that MOZ is involved in the specific regulation of the expression of p53-target genes. The leukemia-associated fusion MOZ-CBP inhibited both AML1- and p53-mediated transcription, suggesting that MOZ-CBP might induce leukemia by antagonizing the function of the AML1 and p53. Research in Cancer Diagnosis and Therapy Transcriptome, proteome and peptidome (7, 11, 18)     The SELDI or ProteinChip(tm) system is a powerful tool to identify peptide biomarkers for cancer detection. Global transcriptomic and proteomic analyses using the 2D-DIGE and GeneChip(tm) oligonucleotide microarray systems were extensively performed as a means of dissecting a population of proteins and genes involved in various aspects of carcinogenesis and cancer progression. For rapid identification of proteins in 2-dimensional gels a large in-house database was constructed. New approaches of cancer therapy (5, 7, 14, 15, 22, 24)     Tyrosine kinases have been considered as promising targets for anti-cancer drug therapy. EGFR-TKI (epidermal growth factor receptor-tyrosine kinase inhibitor), Iressa (ZD1839), is an orally active and clinically proven promising anti-cancer drug against various human cancers. The anti-cancer activity of ZD1839 was proven even in drug-refractory cancer cells. TZT-1027 is another anti-cancer drug that targets microtubules. Because of the interaction with anti-tumor cytokines, TZT-1027 may have a mode of action other than antimitotic mechanisms.     The use of dendritic cells (DCs) loaded with tumor antigen is one of the most attractive approaches in cancer immunotherapy. The anti-tumor activity of primed DCs was confirmed in vivo and ex vivo. The Effects of prostaglandin E(2) (PGE(2)) on the function of dendritic cells (DCs), T-cell polarization, and expression of chemokine receptors was explored.     The potentials of the adenoviral gene transfer system for gene therapy of human cancer were explored. Formation of the double strand RNA by the induction of exogenous antisense RNA may be applicable for target gene silencing. The formation of a DNA and collagen complex was found to prolong the period of gene expression and suppression. This system was considered useful for analyses of gene function.     An objective of studies of DDS in cancer chemotherapy is to find methods by which anticancer agents selectively target solid tumors. The DDS agents such as liposomal or micellar drugs were verified to diffuse out of tumor blood vessels, reach the solid tumor tissue effectively and be retained for a long period due to the EPR (enhanced permeability and retention) effect.     In order to predict sensitivity for treatment in esophageal cancer patients given chemoradiotherapy, the relationships between the clinical course of the patients and histopathological factors in biopsy specimens were investigated. It was suggested that the microvessel density of the tumor had significant predictive power for sensitivity in esophageal cancer patients after chemoradiotherapy. Osteopontin (OPN) was shown to be involved in angiogenesis by upregulating endothelial cell migration in cooperation with the vascular endothelial cell growth factor (VEGF). Neuropsychological approach to cancer patients (25)     Although depression is well known to be among the most common expressions of psychological distress in cancer patients and their families, it is poorly recognized and not appropriately treated. A nationwide survey, using questionnaires mailed to 1436 Japanese oncologists and palliative care physicians, revealed that only 39 % considered psychiatric treatment to be a strong possibility in vignettes in which they were asked whether they use sedation for a patient with depression. The results suggested that training and education for physicians in regard to end-of-life care and valid clinical guideline for palliative sedation therapy for terminally ill cancer patients with depression are necessary. Database for clinical studies (9)     A cancer patient database has been developed in the National Cancer Center hospital and trends in the 5-year survival rates were reported. The Japan Clinical Oncology Group (JCOG) data center is currently managing approximately 60 clinical trials. The final results have been published on several phase II and III trials. [Contributors] Issay Kitabayashi Hitoshi Nakagama Ryuichi Sakai Tomotaka Sobue Yosuke Uchitomi Toshikazu Ushijima Keiji Wakabayashi Tesshi Yamada Teruhiko Yoshida Numbers in parentheses indicate Division, Section or Project involved in research activities. Detailed explanations can be found by clicking on the number of the Division, Section or Project listed below.  1. Pathology Division  2. Biochemistry Division  3. Experimental Pathology and Chemotherapy Division  4. Biology Division  5. Pharmacology Division  6. Cancer Medicine and Biophysics Division  7. Growth Factor Division  8. Molecular Oncology Division  9. Cancer Information and Epidemiology Division 10. Virology Division 11. Genetics Division 12. Radiobiology Division 13. Carcinogenesis Division 14. Section for Studies on Metastasis 15. Section for Studies on Host-Immune Response 16. Cancer Genomics Project 17. Cancer Prevention Basic Research Project 18. Cancer Proteomics Project 19. DNA Methylation and Genome Function Project 20. Tumor Endocrinology Project 21. Tumor Suppression and Functional Genomics Project 22. Pathology Division, East 23. Epidemiology and Biostatistics Division 24. Investigative Treatment Division 25. Psycho-Oncology Division