Overview of Research Activities

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 2004 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
(7, 50, 60, 61, 63, 96, 98, 99, 101)

    Cancers are caused by the cooperative effects of environmental and heritable factors. Exposure to various environmental insults results in the induction of both genetic and epigenetic alterations. A research project to evaluate the risks of heterocyclic amines, which are possible human carcinogens produced while cooking meat and fish, has been continuously carried out. Another research project to identify novel mutagens has also been in progress. Phenylbenzotriazole (PBTA)-type mutagens and 4-amino-3,3'- dichloro-5,4'-dinitro-biphenyl (ADDB) that have been identified in river water have been shown to be genotoxic. A co-mutagen aminophenylnorharman (APNH), which is formed from norharman and aniline, was shown to induce hepatocellular carcinomas and colon cancers in the F344 rat. K-ras, b-catenin and Apc mutations were frequently observed in colon cancers. Other co-mutagens, such as APH and AMPH, were also produced from non-mutagenic harman and aniline or o-toluidine in the presence of S9 mix. Furthermore, novel cytotoxic factors, pierisin- 1 and -2, present in cabbage butterflies, were shown to catalyze ADP-ribosylation at the N-2 position of 2'-deoxyguanosine in DNA, and cause the death of mammalian cells.
    To identify environmental epimutagens that induce epigenetic alterations, a novel assay system was established. An EGFP marker gene was introduced in a human colon cancer cell line to be expressed under the control of methylated endogenous promoter CpG islands. Treatment of the cells with a demethylating agent induced expression of the EGFP marker gene. An assay system for methylating agents is under development.
    As for endogenous environmental factors, DNA replication stresses, such as replication fork arrest in damaged DNA, could be partly responsible for the induction of spontaneous mutations. G-rich short tandem repeats, for example, are frequently altered in various human cancers, and UP1/hnRNPA1 was demonstrated to unfold quadruplex structures of d(CAGGG) and telomeric d(TTAGGG) repeats. hnRNPA3 also bound tightly to telomeric repeats and was suspected to protect telomeres in vivo.
    From an epidemiological point of view, statistical analysis using cancer mortality and incidence data can provide us with clues for cancer etiology. The mortality of Japanese immigrants in the state of Sao Paulo showed a pattern similar to Japanese in Japan for stomach and colon cancers, while similar to Brazilians for other cancer sites. To obtain accurate incidence data, the standardization and quality improvements of population-based and hospital-based cancer registries have been promoted through developing data standards.
    Lifestyle factors have also been implicated in the etiology of cancer occurrence. In order to elucidate associations, the Japan Public Health Center-based Prospective Study on Cancer and Cardiovascular Disease (JPHC Study), a population-based prospective study, is currently ongoing, in which approximately 140,000 individuals from 11 public health center areas will be followed-up for 20 years. By avoidance of smoking, 29% of male and 3% of female cancer could be preventable in the Japanese middle-aged population. In men, a U-shaped association between body mass index (BMI) and cancer occurrence was observed, suggesting a very low BMI rather than high BMI has a significant impact on the total cancer risk in populations with a low average BMI. An inverse association between green tea consumption and distal gastric cancer was observed among women.

Animal models of carcinogenesis
(5, 6, 8, 10, 15, 49)

    Animal models are of great use to dissect molecular mechanisms underlying multistage processes for cancer development. In PhIPinduced rat colon cancers, genes encoding matrix proteases and cell cycle regulators were highly expressed, and, in contrast, mucin-like proteins were under-expressed. Combined administration of PhIP and azoxymethane (AOM) induced poorly differentiated colon carcinomas with submucosal invasion. This novel model could be helpful for investigating molecular mechanisms involved in the process of malignant conversion of cancer cells.
    Poly(ADP-ribose) polymerase-1 (Parp-1) recruits XRCC1 and DNA polymerase b after the induction of DNA strand breaks, and Parp-1^-/- cells showed increased phosphorylation of histone H2AX and p53 than in the wild-type, and indicated its active involvement in the DNA repair process. A significant increase of mutations in Parp-1^-/- mice was demonstrated in the liver after a treatment with an alkylating agent.
    Genetic approaches to identify genes responsible for cancer susceptibility have been undertaken. A candidate locus for a susceptibility gene (Sct) to PhIP-induced colon cancers was narrowed down within a few megabase region between D16Wox7 and D16Wox3. Interestingly, b-catenin mutation frequencies in PhIP colon cancers were different between F344 and ACI, and this could partly account for the higher susceptibility of the former. In MNNG-induced gastric cancers, genes differentially expressed in the gastric mucosa between ACI and BUF were identified and could be responsible for the differential susceptibility. Furthermore, a subset of genes showed persistent expression changes after MNNG-treatment, and this may provide a cellular environment that favors malignant transformation of gastric epithelial cells.

Search for molecular targets and relevant methods for cancer prevention
(13, 14, 62, 90, 97, 98, 100, 102, 105)

    In colon cancers, cyclooxygenase (COX)-2 expression and prostaglandin E₂ (PGE₂) synthesis are elevated. A combined treatment of COX-1 and COX-2 selective inhibitors suppressed intestinal tumorigenesis more efficiently than those with either one alone in Apc-knockout mice. Interestingly, PGE₂-mediated signals through EP₁ and EP₄ enhanced AOM-induced colon carcinogenesis, but those through EP₃ played a suppressive role. Oral administration of bovine lactoferrin (bLF) markedly inhibited cancer development in various organs, and metastatic colony formation in the lung. Moreover, bLF inhibited tumor-induced angiogenesis, and enhanced Fas expression and apoptosis. Enhancement of NK cell activity and anti-angiogenesis could be an underlying mechanism for chemoprevention by bLF.
    A randomized controlled trial to assess the effectiveness of vitamin C supplementation for individuals with chronic atrophic gastritis in preventing gastric cancer has progressed into the tenth year. A community-based randomized cross-over trial designed to develop an effective dietary modification tool and system was held in the same area in 1998-2000, and the followup survey after the intervention is proceeding.
    The methods for developing a guideline for cancer screening are standardized and applied to colorectal cancer screening. Strategies to improve screening compliance are also examined. As a WHO Collaborating Center for Reference on Smoking and Health, information and data on smoking and health are collected and analyzed to support implementation of the WHO Framework Convention on Tobacco Control.

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
(16, 53, 59, 79)

    TSLC1 was isolated by functional cloning, and loss of its expression and/or promoter methylation has been observed at a high incidence in various human cancers, including non-small cell lung cancer (NSCLC). In meningiomas, TSLC1 immunoreactivity was frequently absent, and the loss was associated with decreased patient survival. It was also found that TSLC1 protein associates with an actin-binding protein, DAL-1/4.1B, and that DAL-1/4.1B was methylated with a high incidence (60%) in primary NSCLCs.
    MYO18B was isolated by a genome-wide loss-of-heterozygosity analysis, and is frequently deleted, mutated and hypermethylated in NSCLCs. In 6 of 7 cell lines with reduced MYO18B expression, the levels of histone H3/H4 acetylation were lower than those in a cell line with MYO18B expression. Treatment with trichostatin A (TSA) increased the histone acetylation in all 6 cell lines with restoration of MYO18B expression, suggesting that histone deacetylation plays an important role in MYO18B silencing. In ovarian cancers, mutations of the MYO18B gene were detected in 1/4 cell lines and 1/17 primary samples, and the expression was reduced in 4/4 cell lines and 12/17 primary samples.
    For a genome-wide analysis of DNA copy number alterations with a higher resolution, a genome-wide BAC array system (MCG Whole Genome Array-4500) was developed. Using the system, gastric and breast cancer samples prepared by laser-capture microdissection were analyzed. In gastric cancers, the molecular profile of some well-differentiated intestinal type gastric cancers was found to be similar to that of poorly differentiated solid type gastric cancers. Novel homozygous deletions were found at seven loci. Breast cancers showed amplification of 12 loci and homozygous deletion of three loci.
    The recent explosion of genomic and epigenomic data requires functional analysis of a large number of genes within a limited time. To achieve this goal, a basic technique for high-throughput gene transfer and expression screening has been developed by pre-coating a multi-well plate with an Atelocollagen/cDNA complex (cell transfection array).

Epigenetic mechanisms of carcinogenesis
(1, 48, 73, 74)

    It is now recognized that epigenetic alterations are involved in cancer development and progression, along with genetic alterations. To identify novel epigenetic abnormalities, genome-wide screening methods for differently methylated DNA fragments were developed in this Institute, including the methylationsensitive- representational difference analysis (MS-RDA) method and combined use of a methyl-binding domain column and segregation of partly melted molecules. Analysis of pancreatic cancer cell lines by MSRDA revealed 35 CpG islands that were in 5' regions of known genes and were aberrantly methylated, and 13 genes silenced by the aberrant methylation.
    Silencing of nine genes was previously identified in human stomach cancers by MSRDA. After analysis of two candidate tumor-suppressor genes by introducing their cDNA into gastric cancer cell lines, Lysyl oxidase (LOX), but not HRASLS, was found to suppress anchorage-dependent/ independent colony formation and tumorigenicity in nude mice.
    Overexpression of DNA methyltransferase (DNMT1) protein correlated significantly with poorer differentiation and the CpG island methylator phenotype in gastric cancers. DNMT1 overexpression was also associated with Epstein-Barr virus infection but not with the proliferating cell nuclear antigenlabeling index, suggesting that DNMT1 overexpression may not be just a secondary effect of increased cancer cell proliferative activity, but may be associated with Epstein- Barr virus infection and other etiologic factors during gastric carcinogenesis.
    A splice variant of DNA methyltransferase 3b, DNMT3b4, lacks conserved methyltransferase motifs. Its overexpression was found in the early stages of liver carcinogenesis, and was associated with DNA hypomethylation in pericentromeric satellite regions. DNA hypomethylation was expected to induce chromosomal instability. Gene expression microarray analysis revealed that DNMT3b4 overexpression also affected the expression of specific genes, such as STAT1.
    In a subset of cancer cells, the silencing of E-cadherin (CDH1) is associated with hypermethylation of its CpG island. Although the CDH1 gene expression recovered following treatment with 5-aza-dC in a liver cancer cell line, the methylation status of the entire CpG island and acetylation and methylation status of the associated histones were not significantly altered.

Genetic susceptibility to human cancers
(18, 19, 44)

    The mutation suppressive ability of a DNA repair enzyme, OGG1, was compared between two common polymorphic proteins, OGG1-Ser326 and OGG1-Cys326. Mutations by 8OHG were more efficiently suppressed by OGG1-Ser326 than OGG1-Cys326, supporting the results of association studies that Cys326 is a risk allele for several types of human cancers.
    An epidemiological study on male Japanese smokers indicated that CYP2A6 is a principal determinant not only for smoking behavior but also susceptibility to tobaccorelated lung cancer.
    In addition to analyses of specific candidate genes, genome-wide association studies are under way. Careful attention is also paid to social factors, and clinical genetic testing for familial cancer syndrome cases is offered at the outpatient clinic.

Gene expression profile analyses
(69, 70)

    Expression microarray analyses are being performed from various viewpoints. Analysis of 54 pediatric AML patients has identified gene expression profiles specific to t(8;21) and inv(16), respectively. The expression profiles and gene knock-in experiments using AML1-MTG8 and CBFBb -MYH11 chimeric transcription factors suggested that t(8;21) and inv(16) trigger leukemogenesis through a common pathway involving expansion or maintenance of hematopoietic stem cells. Analysis of 59 clinical neuroblastoma samples suggested that molecular event(s) functionally analogous to MYCN amplification occurs in neuroblastomas without MYCN amplification but with a poor prognosis.

Molecules regulating cancer progression and metastasis
(2, 11, 12, 30, 43, 66, 83)

    The expression of dysadherin, a novel membrane glycoprotein, correlated with a poor prognosis in various types of cancers. Dysadherin down-regulates E-cadherin and contributes to the metastatic potential of cancer cells by modulating the actin structure and stimulating cell motility. Cytoplasmic p120ctn also contributed to the invasive phenotype of E-cadherin deficient breast cancer cells. In the E-cadherin deficient colorectal cancer SW480, actinin-4 was identified as a binding partner of b-catenin. Actinin-4 and b-catenin colocalized at the leading edge of motile cells, and might cooperate to regulate cancer invasion and metastasis. Participation of PARP-1 (poly(ADP-robose) polymerase-1) in the TCF-4/b-catenin complex was also demonstrated by mass spectrometry and immunoprecipitation. By applying a systematic approach of bioinformatics to the WNT, Hedgehog, Notch and BMP signaling pathways, numbers of related molecules were identified and characterized.
    Overexpression of a docking molecule ShcC, but not a ShcC mutant lacking the SH2 domain in neuroblastoma cells, caused decreased anchorage independency and in vivo tumorigenicity, suggesting a novel SH2-mediated suppressive effect of ShcC on cell transformation.
    IGF-1 (insulin-like growth factor-1) is mainly produced in the liver and is inactivated via binding with IGF-binding protein 3(IGFBP3). IGF-BP3 was specifically digested by MMP7, a main metalloprotease produced by colon cancer cells, resulting in phosphorylation of IGF-type 1 receptors and activation of AKT signaling in cancer cells. Involvement of IGF-1 signaling in bone and liver metastasis was also indicated.
    CHO cells in which glycosylation of proteins and glycolipids is terminated at Gal or GalNAc, the biological roles of glycosylation of CD82 and other molecules in cell motility and adhesion were demonstrated.

Protein kinases modulating cancer signaling
(3, 29, 31, 72, 89)

    ARK5, a newly identified member of the AMPK family, is directly activated by Akt and activated ARK5 was found to cause phosphorylation and inactivation of caspase-6 leading to resistance to FasL/Fas-dependent apoptosis. It was also shown that ARK5 regulates cell survival and cell migration through activation of MT1-MMP in vitro.
    NLK (Nemo-like kinase) was recently characterized as a suppressor of transcriptional activity of the b-catenin/TCF complex in mammalian cells. Overexpression of NLK induced apoptotic cell death in DLD-1 colon cancer without suppressing TCF activity. It was indicated that NLK suppresses a wide range of transcription factors, possibly through phosphorylation of CBP transcription cofactor.
    Substrates of Src family kinases are involved in the process of cancer progression. Cortactin, a cortical actin-associated protein, was identified as a molecular marker of hepatocellular carcinoma metastasis by expression profiling, and overexpression of cortactin endowed a non-metastatic hepatocellular carcinoma cell line with high motility and metastatic potential. Similarly, overexpression of paxillin found in metastatic osteosarcoma cell lines was shown to be responsible for enhanced cell motility of these cell lines.
    The interaction of the Eph family receptor protein tyrosine kinase and its ligand ephrin family induces bi-directional signaling via cell-to-cell contacts. The interaction of the extra-cellular domain of ephrin-B1 with claudin, a tight junction component, was observed. Ephrin-B1 induced a novel signaling cascade by an Eph receptor-independent mechanism.

Regulation of p53 pathway
(24, 25, 26, 28, 47)

    To uncover the mechanism of p53-mediated tumor suppression, the functions of p53 have been studied and the genes that are directly regulated by p53 have been isolated. Several axon-guidance molecules including DCC, neogenin, UNC5B, and netrin-1 are regulated by p53 and are suggested to regulate apoptosis. p53R2/Rrm2b, which encodes a small subunit of ribonucletide reductase (RR), was shown to play a pivotal role in the dNTP supply for DNA repair. Inhibition of RR induced depletion of intracellular dNTP pools, leading to cell-cycle arrest and apoptosis. The HU-resistance in cancer cells was shown to be due to the elevated expression of R2. It was demonstrated that endoplasmic reticulum (ER) stress inhibits p53-mediated apoptosis. The mechanism of inhibition involved the increased cytoplasmic localization of p53 due to phosphorylation at serines 315 and 376, which is mediated by glycogen synthase kinase-3 beta (GSK-3beta).

Functional analyses of oncogene products
(20, 33, 34, 36, 37, 41, 76, 77, 91)

    The leukemia-associated factor MLL was shown to associate with a cohort of proteins, including a homolog of Ash2 (a Trx-G group protein), HCF-1/2 and Menin, a product of the MEN1 tumor suppressor gene. Oncogenic mutant forms of MLL retained an ability to interact with menin but not other identified complex components. Mutations in the MEN1 gene were identified. The mutant products were less stable than the wild type and were degraded through the ubiquitine-proteasome pathway. The complex of the leukemiaassociated factor AML1 was shown to contain histone acetyltransferases (HATs) such as p300/CBP and MOZ, PML, mSIN3A and a serine/threonine protein kinase (AML1K). The EWSNOR1 oncogenic fusion gene product was shown to affect pre-mRNA splicing and interact with the U1 small nuclear ribonucleoprotein (snRNP)-specific protein U1C. The SYT-SSX oncogenic fusion gene product was shown to act as a transcriptional repressor. NFX1, a transcription repressor, was identified as an human papillomavirus E6-interacting protein. An isoform NFX123 activated but another isoform NFX1-91 repressed human telomerase reverse transcriptase (hTERT). E6 selectively degraded NFX1-91 to activate hTERT. Expression analysis demonstrated that Notch1 acts as a tumor suppressor in cervical cancer. E6 inhibited the expression of Notch1 via inactivation of p53. p32 was shown to be involved in the regulation of Hrk-mediated apoptosis. The tumor suppressor candidate TSC1 did not act as a tumor suppressor in lung adenocarcinoma.

Novel Approaches to Cancer Diagnosis and Treatment
^* Detailed explanations can be found by clicking on the numbers in parentheses.

Innovative methods of cancer diagnosis
(17, 39, 64, 67, 84)

    Multivariate analyses using proteomic data obtained by surface-enhanced laser desorption and ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) have been reported to be highly successful in serum detection of malignancies of various organs, including the ovary. The feasibility of serum proteomics as a means of predicting the sensitivity to chemotherapy or radiotherapy of esophageal cancer was demonstrated. A group of proteins by which early intrahepatic recurrence of hepatocellular carcinoma could be predicted was identified using twodimensional gel electrophoresis. The morphological features of a dominant papillary subtype in lung adenocarcinoma were significantly associated with the clinical response to gefinitib, an EGFR tyrosine kinase inhibitor.
    Through microarray analyses novel markers for the prediction of peritoneal and non-peritoneal recurrence of gastric cancer and for the molecular diagnosis of lung adenocarcinoma were identified. Breast cancer resistant protein (BCRP) was identified as a significant prognostic factor of non-small cell lung cancer (NSCLC).

Gene therapy and immunotherapy
(21, 27, 46, 51, 54, 56, 58)

    To improve p53-based gene therapy, the adenovirus-mediated gene transfer of certain active forms of p53 and target genes of p53 was evaluated. Adenovirus-mediated intratumoral administration of p53 rapidly induced target genes of p53 and apoptosis. Adenovirus-mediated gene transfer of a target gene of p53, p53AIP1, was found to be effective for treatment of p53-resistant tumors. Adenovirus-mediated interferon-a (IFN-a) has been found to induce growth suppression and cell death of pancreatic cancer cells. The efficacy and toxicity of this approach is currently under investigation in animal models. The alteration of adenovirus tropism based on the modification of the viral capsid proteins may realize the cell-specific targeting of gene therapy. A novel screening system was developed to identify tumor-targeting adenovirus vectors.
    Atelocollagen-based non-viral delivery of siRNA was found to be effective for the inhibition of bone metastasis in an animal model. N-Acetylated chitosan was a useful carrier for oral gene delivery to the gastrointestinal tracts.
    A novel epitope located in extradomain 2 of intracellular adhesion molecule-1 (ICAM-1) defined by monoclonal antibody U5A2-13 was important for NKT cells to control tumor metastasis in an animal model.

Anticancer agents and drug delivery systems
(86, 88)

    A new compound, kigamicin D and an anthelminthic, pyrvinium pamoate have been found to be extremely toxic to human pancreatic cancer cell line PANC-1 cells under nutrient-deprived conditions. Both compounds were also found to exert antitumor activity against PANC-1 in vivo.
    Drug delivery systems (DDSs) have been developed based on hypervascularity, an irregular vascular architecture, vascular permeability factors, and the relatively poor drainage within solid tumors. Macromolecular agents can diffuse out of tumor blood vessels, reach the solid tumor tissue, and be retained for a long period because of the enhanced permeability and retention effect. The phase I study of MCC-465, a doxorubicin encapsulated in PEG immunoliposomes, has been conducted in patients with metastatic stomach cancer.

Psychologic distress after disclosure of genetic test results
(93, 94, 95)

    To identify the prevalence rates of psychologic distress after disclosure of genetic test results, 47 probands and unaffected relatives were interviewed immediately after the first genetic counseling session for HNPCC (hereditary non-polyposis colorectal cancer) and 42 participants (89%) completed the 1-month follow-up interview. At the follow-up interview, 3 of 42 participants (7%) met the criteria for minor depression. Disclosure of genetic test results for HNPCC may not cause significant psychologic distress in Japanese probands or relatives.

[Contributors]
Issay Kitabayashi
Hitoshi Nakagama
Ryuichi Sakai
Tomotaka Sobue
Yosuke Uchitomi
Toshikazu Ushijima
Keiji Wakabayashi
Tesshi Yamada
Teruhiko Yoshida