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 2003 is shown below. Research on the Causes and Prevention of Cancer * Detailed explanations can be found by clicking on the numbers in parentheses. Environmental cancer causes (5, 6, 14, 37, 38, 40, 42, 60, 69, 70, 72, 94, 96, 97)     Exposure to various environmental insults results in the induction of genetic and/or epigenetic alterations, and cancers develop as a consequence. Heterocyclic amines (HCAs), produced while cooking meat and fish, induce cancers in various organs in rodents, and are considered to be potential human carcinogens. Numbers of other mutagens have also been identified. Five PBTA-type mutagens, PBTA-1-4 and 6, were found in water samples taken downstream of sewage treatment plants. A novel cytotoxic factor present in butterflies, pierisin-1, produced ADP-ribosylated DNA adducts at the N-2 position of the guanine base in DNA and induced mutations at the HPRT locus of CHL cells. In addition to genotoxic agents, an assay system to search for environmental agents inducing epigenetic alterations is being conducted.     Co-mutagenicity is another intriguing phenomenon to be clarified. Mutagenic aminophenylnorharman (APNH) is formed from non-mutagenic norharman and aniline. The gpt mutant frequency of mice fed APNH increased 10- and 5-fold in the liver and colon, respectively. Viral infection is also an important causative factor for human cancers. A replicon library system was established to obtain diverse clones of the HCV replicon, including an interferon resistant replicon, and HCV variants with diverse phenotypes were obtained.     Analysis using cancer mortality and incidence data can provide clues for cancer etiology. Time trend analyses of lung cancer incidence using data from Osaka and the US showed an increase of adenocarcinoma in both countries. Lung cancer mortality will double during the next three decades in Japan. The Japan Public Health Center-based Prospective Study on Cancer and Cardiovascular Disease (JPHC Study) has been conducting a population-based prospective study. Approximately 140,000 individuals from 11 public health center areas will be followed-up for 20 years. To date, frequent miso soup and isoflavone consumption was revealed to be associated with a reduced risk of breast cancer, and approximately half of all colorectal cancer cases could be preventable by controlling tobacco and alcohol consumption in middle-aged and elderly Japanese men.     A case-control study of gastric cancer in Japanese Brazilians and Brazilians not of Japanese ancestry in Sao Paulo showed the CagA-Ab of Helicobacter pyroli (H. pylori) could be a useful marker for non-cardiac gastric cancer. Cruciferous vegetables were suggested to decrease the risk of both stomach and colorectal cancers. Mushrooms were associated with a decreased risk of stomach cancer, and frequent fresh fish consumption, irrespective of the cooking method, may reduce the risk of lung cancer. Animal models of carcinogenesis (5, 6, 10, 11, 59, 60)     Animal models are useful to dissect the molecular mechanisms underlying multistage processes for cancer development. In PhIP-induced colon cancers, genes encoding Paneth cell-specific proteins, such as the defensin family and lysozyme genes, were over-expressed. The presence of Paneth cells was also evidenced in high-grade dysplastic ACF, which indicated dysplastic ACF could be preneoplastic lesions of the colon. A novel method to detect dysplastic ACF easily and effectively was also established. In MNNG-induced gastric cancers, growth factor genes and genes involved in extracellular matrix remodeling were up-regulated, and those involved in the differentiated phenotype of the stomach were down-regulated. Poly(ADP-ribose) polymerase-1 (Parp-1) is involved in DNA damage repair, apoptosis and maintenance of chromosomal stability. Tumorigenicity in Parp-1-/- mice was significantly enhanced in the colon and liver when treated with AOM and BHP, respectively. The frequency of deletion-type mutations was enhanced in Parp-1-/- mice. A transgenic rat line (Hras128) carrying 3-copies of the human c-Ha-ras proto-oncogene is highly susceptible to the induction of cancers of various organs induced by chemical carcinogens. A single injection of MNU into mammary ducts caused mutations in codon 12 of the human c-Ha-ras transgene in terminal end buds (TEB). Elevated expression of the c-Ha-ras proto-oncogene was sufficient to cause a highly proliferative phenotype of mammary alveoli. Using TGFa transgenic rats, TGFa was demonstrated to have an important role at the initial stage of liver carcinogenesis.     Genetic backgrounds of individuals have substantial impacts on carcinogenesis. A genetic trait for the susceptibility to PhIP colon carcinogenesis (Sct) was mapped within a 10 Mb region on rat chromosome 16, and the induction of N-acetyltransferase 2 (Nat2) by PhIP was demonstrated to be regulated by Sct. As for a mechanism of varying susceptibilities to MNNG stomach carcinogenesis, differential induction of cell proliferation after mucosal damage was focused on. Twenty-two genes were differentially expressed among six rat strains in accordance with the extent of cell proliferation in the stomach epithelium. Search for cancer suppressing methods (12, 13, 71, 95)     Cladosiphon fucoidan and green tea extract were effective to control H. pylori infection and may be useful for gastric cancer prevention. In colon cancers, prostaglandin E2 (PGE2) receptor subtypes EP1 and EP4 were shown to be good targets for cancer prevention. In rat colon cancer, an activating K-ras mutation elevated iNOS expression in the presence of inflammatory stimuli. One of the tetrasulfophthalocyanine metal complexes with zinc, (Zn(II)TSPC), firmly associated with MeIQx and inhibited MeIQx from incorporating into cells. These complexes could be promising chemopreventive agents for hepato-carcinogenesis by HCAs. Bovine lactoferrin (bLF) markedly inhibited carcinogenesis and lung metastatic colony formation. bLF was demonstrated to participate as a regulator of angiogenesis, possibly by abrogating endothelial function and inducing IL-18 production, and the anti-tumorgenic activity of bLF may thus be partly mediated by angiogenesis inhibition.     A randomized controlled trial to assess the effectiveness of vitamin C supplementation on gastric cancer is now in its ninth year for individuals with chronic atrophic gastritis, and vitamin C supplementation may protect against the progression of gastric mucosal atrophy. Another randomized controlled trial revealed dietary modification to be effective in decreasing sodium and increasing carotene and vitamin C intake, the former being more distinct. Research on The Mechanisms of Cancer Development * Detailed explanations can be found by clicking on the numbers in parentheses. Genetic alterations and their functional significance (3, 7, 68, 87, 88)     Chromosomal imbalances are often associated with important tumor-related genes, to scan for which in a genome-wide and high-resolution manner, a BAC array for comparative genomic hybridization (CGH) analysis was developed. Application of the system to gastric cancers revealed novel small hemizygous losses and amplifications, in addition to previously known ones.     Wnt-signaling mediated by b-catenin plays crucial roles in the development of human cancers. A protein interaction screening identified EBP50, a cytoplasmic protein with 2 PDZ domains, to form the EBP50/b-catenin complex, which promotes Wnt-signaling in hepatocarcinogenesis. A potential target of Wnt-signaling was identified by screening of differentially expressed genes in human hepatocellular carcinomas (HCCs). An orphan G -protein-coupled receptor, Gpr49, was frequently overexpressed in HCCs with b-catenin mutations.     Inactivation mechanisms of the TSLC1 tumor suppressor gene, which was originally identified in this Institute, were found to be (i) promoter methylation and loss of the other allele and (ii) biallelic promoter methylation. Loss of TSLC1 expression was correlated with advanced stages in esophageal squamous cell carcinoma. TSLC1 and its paralogues, TSLL1 and TSLL2, form a subfamily in the immunoglobulin superfamily molecules. Expression of TSLL1 and TSLL2 was reduced in human glioma cell lines and prostate cancer cell lines. Murine orthologues of the Tslc1, Tsll1, and Tsll2 genes were isolated to generate gene knock-out mice.     The cancer genome shows a range of genomic instability, including an instability of minisatellite DNA sequences (MNs). The tandem repeat of d(CAGGG)n in the mouse hypervariable MNPc-1 forms an intramolecular quadruplex structure, which can cause arrest of DNA synthesis at the d(GGG)n site. UP1 and p100 were identified as single-stranded MN Pc-1 binding proteins. Epigenetic mechanisms of carcinogenesis (1, 58)     Promoter methylation is one of the major epigenetic events during carcinogenesis, causing silencing of the downstream genes. Methylation-sensitive-representational difference analysis (MS-RDA), which was developed in this Institute, allows a genome-wide search for differentially methylated DNA fragments, and successfully identified genes silenced in human lung and stomach cancers. This year, MS-RDA was applied to human breast cancers, and revealed silencing of the Monocarboxylate transporter 1 (MCT-1) gene and the heparan sulfate D-glucosaminyl 3-O-sulfotransferase-2 (3-OST-2) gene.     A genome-wide analysis of aberrant methylation indicated that some cancer cells have decreased fidelity in replicating DNA methylation patterns. A system to measure the fidelity was established by combination of single cell culture and bisulfite sequencing. The assay on human mammary epithelial cells showed that the fidelity was high in CpG islands in promoter regions, but relatively low in CpG islands outside promoter regions, and that maintenance of the methylated status is more reliable than maintenance of the unmethylated status.     Although dysregulation of the DNA methyltransferase 1 (DNMT1) gene could be involved in aberrant methylation in various human cancers, mutational inactivation of the gene appeared to be a rare event. On the other hand, progressive increase in DNMT1 expression was observed in the course of the malignant progression of HCCs, and the increase could serve as a biological predictor of both HCC recurrence and a poor prognosis for HCC patients. Similar activation of DNMT1 was observed in the early stage of urothelial carcinogenesis, which was in accordance with frequent hypermethylation of the E-cadherin gene in flat carcinomas in situ of the urinary bladder. Gene expression alterations (3)     Gene expression is characteristically altered during cancer development and progression. New diagnostic and therapeutic targets are being identified by powerful cDNA microarray techniques. Profiling of early components and progressed components within HCCs identified heat-shock protein 70 as a sensitive marker for early HCCs. Profiling of renal cell carcinomas of different histologies identified the oncogene KIT as a useful marker for chromophobe renal cell carcinomas. Search for genes differentially expressed in clear cell carcinomas (CCCs) of the ovary revealed that a transcription factor, hepatocyte nuclear factor-1 b (HNF-1 b), was frequently up-regulated in CCCs and that its down-regulation induced apoptosis.     High expression of LI-cadherin was observed in well-differentiated pancreatic ductal adenocarcinomas, and correlated with good survival. Genetic susceptibility of human cancers (18, 19)     Although smoking is a well-established risk factor for lung cancer, other major factors may remain to be identified for adenocarcinoma, which constitutes the largest fraction of lung cancers in Japan. A systematic survey of the human genome with an average resolution of 10 cM was performed to search for a susceptibility gene by genotyping 322 microsatellite loci. The result indicated that the 19q13.3 region contains a gene associated with lung adenocarcinoma risk.     Researchers of the Institute previously reported a polymorphism of the OGG1 gene is significantly associated with the risk of squamous cell carcinoma of the lung. The abilities of OGG1, MYH and APE1 proteins, which are components of a base excision repair pathway, to suppress G:C to T:A transversions caused by 8-hydroxyguanine (8OHG) or benzo[a]pyrene-diol-epoxide (BPDE) were evaluated by a bacterial assay. The results indicated that OGG1 and MYH function as suppressors for G:C to T:A transversions by 8OHG but not by BPDE in human cells. Factors involved in metastasis and invasion of cancer cells (2, 20, 73, 91)     Using Hu09 osteosarcoma sublines with different metastatic potentials, cell motility and adhesive properties were demonstrated to be key determinants of lung metastasis of osteosarcoma. A new animal model for bone metastasis of human prostate cancer to human bone has been established using NOD (non-obese diabetes)-SCID mice. This model was utilized to show that inhibition of osteoclastic bone resorption by OCIF (osteoclastogenesis inhibitory factor) results in suppression of the growth of LNCaP prostate in human adult bone.     Increased expression of dysadherin, a new membrane glycoprotein, was associated with the metastatic and/or invasive potentials of colorectal carcinomas, pancreatic ductal adenocarcinomas and thyroid cancers. It was indicated that dysadherin expression may become a biological predictor of the aggressiveness of tumors and poor prognosis in human cancers.     Actinin-4 was identified as a new biomarker for cell motility and cancer invasion of breast cancers. Expression of actinin-4 was elevated in colorectal carcinomas, especially in cancer cells invading into fibrous stroma. Inducible expression of actinin-4 in a colorectal cancer DLD-1, caused elevated cell movement and showed higher invasion and lymph node metastasis in SCID mice. Signals modulating progression of tumors (8, 15, 29, 34, 88, 102)     Tumor survival under nutrient starvation requires both Akt and AMPK. ARK5, a newly identified member of the AMPK family, was directly activated by Akt and the activated ARK5 caused phosphorylation of ATM and suppression of cell death during nutrient starvation.     Src family kinases are potent regulators of metastasis. Phosphorylation at tyrosine 253 of Cas, a major substrate of Src, was shown to promote cell migration. The complex formation between Fyn, a member of Src family kinases, and its substrate cortactin was identified as a key modulator of metastatic potential using K-1735 murine melanoma cell lines.     NLK (Nemo-like kinase) was recently characterized as a suppressor of the tran-scriptional activity of the b-catenin/TCF (T-cell factor) complex in mammalian cells. Overexpression of NLK in DLD-1 colon cancer suppressed cell growth and induced apoptotic cell death, suggesting a target other than TCF because dominant-negative TCF did not cause apoptosis in the same cells.     Over-expression of a novel serine/ threonine protein phosphatase PP5 in Hela cells inhibited cell cycle progression at mitosis, suggesting that PP5 is required for mitosis progression. SHIP (the SH2 domain-containing inositol 5'-phosphatase) is involved in the regulation of PI3 kinase activity in myeloid linage cells. Analysis of the promoter region of SHIP revealed that its expression is controlled by a transcriptional factor PU.1.     TSLC1 and MPP3, products of tumor suppressor genes which are frequently lost in non-small cell lung cancer (NSCLC), physically associate with each other at the cell-cell attachment sites. It was suggested these two molecules are involved in lung tumor formation using the same signal cascade of cell-cell interaction. Functional analysis of oncogene and tumor suppressor gene products (24, 25, 26, 33, 41, 43, 44, 53, 55, 56, 84)     A microarray analysis and immunoprecipitation assay identified new p53-target genes such as p53RDL1, hCDC46b, p53RFP and DUSP5. p53RDL1 mediated p53-dependent apoptosis wherease the apoptosis was blocked when p53RDL1 interacted with its ligand Netrin-1. The previously identified p53-target gene p53R2, which encodes a small subunit of ribonucleotide reductase, was shown to play a pivotal role in dNTP supply for DNA repair. In most cancers, in which p53 is inactivated, expression of p53R2 is dysregulated, implying attenuation of dNTP pools for DNA repair and the resulting accumulation of spontaneous mutations in cancer cells. p53 is regulated by mdm2. Ser407 of mdm2 was shown to be phosphorylated by DNA replication inhibition, mediated by the ATR and reduced Mdm2-dependent export of p53 from nuclei to the cytoplasm. Expression of p73, a member of the p53 family, was shown to be regulated during the cell cycle. p73 can interact with variouis cyclins and it can be phosphorylated in vitro by cycline A/cdk1/2, cycline B/cdk1/2 and cycline E/cdk2 at threonine. The thr86 phosphorylation was regulated in a cell-cycle dependent manner.     Cdt1, an origin recognition factor complex, was found to be phosphorylated by cyclin A-dependent kinases. SKP2 bound to Cdt1 in a phosphorylation-dependent manner and inhibited DNA-binding activity of Cdt1.     Both inactivation of the p16/RB pathway and activation of telomerase are required for E6 and E7 of the human papillomavirus to immortalize normal human epitherial cells. These activities were found to be substituted by hTERT and a polycomb group gene, bmi-1, which suppressed transcription of p16.     AML1, PML and MLL are the frequent targets of chromosomal translocations in acute leukemia. PML interacted with AML1 and activated AML1-mediated transcription and differentiation of myeloid cells. A germline mutation of the ataxia telangiectasia (ATM) was found in childhood acute leukemia with MLL gene rearrangement, suggesting that the altered function of ATM plays some pathogenic roles in the development of leukemia with MLL rearrangement. To identify molecules that are important for supporting hematopoietic stem/progenitor cells (HSPCs), secreted and membrane molecules have been isolated from OP9 stromal cells. One of them, mKirre was shown to be involved in the hematopoietic supportive capacity of stromal cells.     Germline and somatic mutations of the MEN1 gene were identified in patients with multiple endocrine neoplasia type 1. Mutants of menin, the products of the MEN1 gene, were shown to be less stable than the wild-type menin and were liable to be degraded through the ubiquitine-proteasome pathway. Novel Approaches to Cancer Diagnosis and Treatment * Detailed explanations can be found by clicking on the numbers in parentheses. Innovative methods of cancer diagnosis (21, 23, 32, 75, 76, 77, 78, 91, 103)     The GeneChip, 2D-DIGE and ProteinChip systems have been introduced in the Institute as the methods of global gene and protein expression profiling. The main signaling pathways targeted by molecular therapy were defined by gene expression profiling. To obtain variable information useful for clinical uses from these large-volume data sets, statistical analyses seem to be powerful. The statistical analyses identified gene expression patterns associated with the FAB subtypes and chromosomal translocation of AML, and the unfavorable prognosis of neuroblastoma. Supervised analyses identified 35 genes useful for prediction of the prognosis of AML patients. Statistical approaches using multivariate and machine learning methods were also found to be useful to identify protein expression signatures associated with the origin, histology and metastatic potentials of various tumors.     Microvessel density (MVD) was found to be the most significant single patho-logical finding useful for the prediction of radiosensivity of esophageal cancer. The level of matrix metalloproteinase-9 in portal and peripheral blood was found to be a predictior of liver metastasis from colorectal cancer.     A novel optical visualization technique, the narrow band imaging (NBI) system, was developed. NBI seems to be powerful for discovering head and neck squamous cell carcinoma at the early stage during routine endoscopic examination. Gene- and immunotherapy (21, 22, 23, 27, 62, 63, 64, 65, 66, 67)     Atelocollagen is a powerful transporter of various nucleic acids into mammalian cells. Antisense oligos against HST-1/FGF-4 mixed with atelocollagen was found to show anticancer activity against experimental germ cell tumors. Antitumor effects against pancreatic cancer of an intratumoral injection of an INF-a -expressing adenovirus vector were investigated in an animal model. To improve cell and tissue-specific targeting, fiber-modified adenovirus vectors were constructed. Adenovirus-mediated gene transfer of mutant p53 with enhanced apoptosis-induction or apoptotic p53-target genes seems to be a promising alternative to gene therapy using the wild-type p53.     Va24+ T cells kill a variety of tumors, but large-scale cultivation is required for effective adaptive immunotherapy. A new efficient method for cultivation of Va24+ T cells was developed. An early inducible cell surface antigen during lymphoid activation was identified. Drug delivery system (99)     Macromolecular anticancer agents such as liposomal or micellar drugs have long plasma half-lives because they are too large to pass through the normal vessel walls unless they are trapped by the reticuloendothelial system in various organs. Such macromolecular agents could diffuse out of tumor blood vessels, reach the solid tumor tissue effectively and be retained for a long period due to the EPR effect. Several DDS drugs are being investigated basically or clinically. Screening for psychological distress (104, 105)     To develop a new, brief screening tool for depression, a total of 275 cancer patients completed the newly developed One-Question Interview; the Distress Thermometer; and the Hospital Anxiety and Depression Scale. The results suggest that One-Question Interview had a good sensitivity (84%) and specificity (61%). Its performance was inferior to that of the HADS but comparable to that of the Distress Thermometer. The One-Question Interview may be suitable for widespread use in routine care and this valid tool, with appropriate psychotherapeutic and psychopharmacological interventions, may be a promising strategy for depression in cancer patients. [Contributors] Issay Kitabayashi Hitoshi Nakagama Ryuichi Sakai Tomotaka Sobue Yosuke Uchitomi Toshikazu Ushijima Keiji Wakabayashi Tesshi Yamada Teruhiko Yoshida