Overview of Research Activities

The mission of the Research Institute at the National Cancer Center is to advance our knowledge of cancer prevention, diagnosis and therapy, with the ultimate goal of cancer control. The activities at this institute include a search for environmental carcinogens and chemopreventive agents using animal models, genome-wide identification of genetic and epigenetic alterations which might be applicable for early cancer diagnosis, and stratification of cancer, in order to achieve more efficient therapy. Functional analyses of various cancer-related genes are also conducted. To further facilitate cancer research, the institute uses cutting-edge technologies for genome, epigenome, transcriptome and proteome analyses. The Research Institute is now internationally recognized for its major contributions to various aspects of cancer research. An overview of the research activities undertaken at this institute in 2007 is presented below.

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

Search for environmental and endogenous factors involved in human cancer development has long been conducted. Aminophenylnorharman (APNH) was demonstrated to be a novel endogenous mutagen/carcinogen. It is formed from norharman and aniline in the presence of CYP3A4/1A2. APNH was detected widely in human urine samples.

4-Amino-3,3'-dichloro-5,4'-dinitrobiphenyl (ADDB) was found to be a novel environmental direct-acting mutagen. ADDB is suggested to form during the process of wastewater treatment of drainage water containing 3,3f-dichlorobenzidine (DCB) from chemical plants.

Expression of E6 and E7 protein is observed in cervical cancers associated with high-risk human papilloma viruses (HPVs). DLG4/PSD95, which contains PDZ domains, was identified as a novel E6 target, besides p53. The role of ErbB2 upregulation by HPV16 E6 through p53 degradation is suggested to be involved in oncogenic transformation of human cervical keratinocytes. The Notch1 gene, a determinant of keratinocyte differentiation and a tumor suppressor, was identified as a novel target of p53. Notch1 inactivation suppressed keratinocyte differentiation.

( 5, 6, 20, 40, 55 )

To identify germline polymorphisms and mutations implicated in susceptibility to cancer or drug responses, several studies were carried out using animal models and clinical samples. One of the candidate loci for the susceptibility gene (Sct) for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced colon carcinogenesis of rat was narrowed down to a 0.8-Mb region around D16Rat129 using aberrant crypt foci as a surrogate biomarker. One gene within this region was differentially expressed in the colon between two rat strains not trated with PhIP. Array-based comparative genomic hybridization analysis identified the amplified genes and regions in the genome in colon and renal carcinogenesis models of rodents.

The mouse Atf2 gene was shown to act as a susceptibility gene for mammary tumors by activating a group of target genes, including Maspin and Gadd45a.

The deletion mutation frequency at non-run sequences was elevated in the liver of poly(ADP-ribose) polymerase-1 (Parp-1)-knockout mice of advanced age, supporting the suggestion in that Parp-1 is involved in suppressing imprecise non-homologous end-joining repair of DNA damage.

Parp-1 deficiency also induces trophoblast differentiation of mouse ES cells during teratocarcinoma formation. Expression of the genes involved in the pathway of extraembryonic tissue development is augmented in Parp-1^-/- ES cells, including H19.

A DNA ADP-ribosylating enzyme, pierisin-1, isolated from the cabbage butterfly, induces apoptosis in cancer cell lines and its properties were further characterized.

Factors affecting cancer development have been studied using animal models. Colon tumor development in Apc-deficient Min mice treated with dextran sulfate was suppressed by an inducible nitric oxide synthase inhibitor, ONO-1714. Min mice show an age-dependent hyperlipidemic state along with downregulation of the mRNA expression of lipoprotein lipase (Lpl), which catalyzes the hydrolysis of triglycerides (TG). Treatment of Min mice with indomethacin, which is known to suppress polyp formation, reduced the serum TG levels in a dose-dependent manner, along with induction of Lpl and modification of expression of sterol regulatory element binding protein-1c-regulated genes.

The hyperlipidemic state in hamsters was improved by a PPARg agonist, pioglitazone, accompanied by suppression of invasive pancreatic adenocarcinomas induced by an alkylating agent, N-nitrosobis(2-oxopropyl)amine. A diabetic/hyperlipidemic state was shown to enhance carcinogenesis of the thyroid gland in rat model of non-insulin-dependent diabetes mellitus.

Lifestyle factors have been implicated in the etiology of cancer. Each of various epidemiologic studies has contributed new evidence. The Japan Public Health Center-based Prospective Study (JPHC Study), a population-based prospective study, is currently ongoing. Relations between physical activity, intake of vitamin B6, coffee consumption and colorectal cancer risk, the past-one-year history of colorectal cancer screening and colorectal cancer mortality, history of diabetes, current smoking, body mass index (BMI), height, physical activity, coffee or green tea consumption and pancreatic cancer risk, height, BMI, reproductive factors and breast cancer risk, and isoflavone intake and prostate cancer risk were investigated in this study. Effects of plasma C-peptide and 25(OH) vitamin D on colorectal cancer risk were investigated in 2 case-control studies using cryopreserved blood samples. Results on cerebral infarction, myocardial infarction and age-related cataracts were also reported from the study.

Case-control studies for breast cancer and colonic adenoma to investigate the ethnic differences and interactions between environmental and genetic factors are in progress in Brazil.

Endometriosis risk in association with serum organochlorines and cytochrome P450 gene polymorphisms, urinary isoflavone levels and urinary bisphenol-A was investigated.

Studies and projects aimed at evidence-based cancer prevention have been planned and carried out. The relation between alcohol drinking and the overall, lung, and breast cancer risk was systematically reviewed and evaluated. Other epidemiologic analyses for disease control have been made.

Standardization and quality improvements of population-based and hospital-based cancer registries are promoted and maintained. National cancer incidence was then estimated based on the collected data. An indicator to measure quality of care for 5 major cancer sites and palliative care is under development, to evaluate the achievement of equalization.

Descriptive analyses of cancer incidence and mortalities were made to evaluate the association of various risk factors with cancer incidence. A new cohort of breast cancer patients is being established.

National cancer statistics information was updated. Data collection, integrated management, analysis and dissemination of cancer information at the national level have been conducted, providing multi-channel cancer information services through internet, brochures, lectures and public meetings.

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

( 1, 16, 17, 32, 62, 65 )

Whole genome scanning with several platforms identified novel genomic regions of homozygous deletions (HDs) and amplifications (AMPs) in several human cancers. A HD of the CTGF gene was identified in ovarian cancer cell lines. In primary ovarian cancers, DNA methylation of the CTGF gene was frequently observed and was inversely correlated with its mRNA expression. Silencing of the CTGF gene due to DNA hypermethylation may contribute to ovarian carcinogenesis in a stage-dependent and/or histological subtype-dependent manner. Unsupervised clustering analyses of HD and AMP profiles revealed that hepatocellular carcinomas (HCCs) could be divided into 2 major groups based on the pathological features, HBV/HCV infection of the background liver and clinical outcome of the patients. An amplified 17q region contained a putative therapeutic target, RPS6KB1 (a signal transducer of the mTOR pathway). An in vitro experiment raised the possibility that rapamycin, an inhibitor of the mTOR pathway, could inhibit the proliferative activity of HCC cells with RPS6KB1 AMP. The 6p21 AMP region in gastric cancer cells contains over 50 upregulated genes, and downregulation of 13 of these by siRNA exerted an inhibitory effect on cancer cell growth, suggesting that this region harbors several genes involved in the proliferation of gastric cancer cells. Expression profiling of amplified genes in pancreatic cancers identified Smurf1 and TRRAP at 7q22.1, BCAS1 at 20q13.2-3, and VCL at 10q22.1, as potential novel oncogenes in pancreatic cancer.

Germline LKB1 mutations cause Peutz-Jeghers syndrome, a hereditary disorder that predisposes to gastrointestinal polyposis and several malignant tumors. A large number of lung cancer cell lines and lung adenocarcinoma (AdC) specimens were examined for genetic alterations of LKB1. LKB1 alterations were frequently detected in the cell lines (21/70), and were significantly more frequent in those with KRAS mutations. Among lung AdC specimens, LKB1 mutations were found in 7/91 (8%) male smokers, but in none of 64 females and/or nonsmokers, and were significantly more frequent in poorly differentiated tumors. The results indicate that genetic alterations of LKB1 may occur preferentially in a subset of poorly differentiated lung AdCs that appear to be associated with male gender and smoking. Several models of cancer progression, including clonal evolution, parallel evolution, and same-gene models, have been proposed. To investigate the authenticity of these models, a whole-genome allelic imbalance (AI) scanning and mutational analysis of the p53, EGFR and KRAS genes were performed on primary and metastatic lung cancers. Accumulated genetic alterations in each case indicated that all three models are applicable to lung cancer progression. According to the clonal and parallel evolution models, a metastasis suppressor gene(s) for lung cancer was suggested to be present on chromosome 11p.

The AML1 transcription factor complex is the most frequent target of leukemia-associated chromosomal translocations. Homeodomain-interacting protein kinase 2 (HIPK2) is a component of the AML1 complex and activates AML1-mediated transcription. Among 50 cases of acute myeloid leukemia and 80 cases of myelodysplastic syndrome, two missense mutations (R868W and N958I) were detected in the speckle-retention signal (SRS) domain of HIPK2, which is required for localization of HIPK2 in nuclear speckles; indeed, these mutants showed impaired localization of HIPK2 in the nuclear speckles. The mutations impaired the overlapping localization of AML1 and HIPK2. HIPK2 interactions result in the phosphorylation of AML1 and p300, leading to stimulation of AML1-mediated transcription. The R868W and N958I mutants showed decreased activities and a dominant-negative function over wild-type protein of AML1- and p53-dependent transcription. Deletion analysis of HIPK2 indicated that the SRS domain is required for HIPK2 to stimulate AML1-mediated transcription activation. These data suggest that HIPK2 mutations may play a role in the pathogenesis of leukemia, by destabilizing the localization of HIPK2, leading to dysfunction of the AML1 transcription factor complex.

Methylation of CpG islands (CGIs) is closely involved in human carcinogenesis. Identification of aberrantly methylated DNA fragments leads to identification of novel tumor suppressor genes and development of prognostic markers. Methylation-sensitive representational difference analysis (MS-RDA) of ovarian cancer cell lines identified 33 methylated CGIs in putative promoter regions. Among these, several genes were actually silenced or methylated in primary ovarian cancers. Microarray-based gene expression analysis of melanoma cell lines treated with a demethylating agent identified 18 silenced genes, including TFPI-2. TFPI-2 was methylated in some metastases but not in primary tumors, suggesting that TFPI-2 silencing is involved in melanoma metastasis.

A subset of cancer cells show a high incidence of aberrant DNA methylation, which is known as the CpG island methylator phenotype (CIMP). While aberrant DNA methylation is known to be widely involved in human cancers, very limited information is available on the mechanisms of how aberrant DNA methylation might be induced. Some cancer cell lines show increased rates of methylation of scattered CpG sites within a CGI, denoted as "methylation seeds". It was shown that increased production of methylation seeds was associated with a larger number of methylation-silenced genes. DNA methyltransferase 1 (DNMT1) overexpression is significantly correlated with CIMP. Immunohistochemical analysis of cervical cancer revealed that DNMT1 expression was increased in low-grade neoplasia as compared with that in normal squamous epithelium, and that it was increased even further in higher-grade neoplasia. Progressively increasing expression of DNMT1 was associated with an early step of multistage cervical carcinogenesis.

( 8, 37, 39, 65, 72, 74 )

Gene expression profiles of malignant tumors reflect their genetic and epigenetic alterations, and include several pieces of information that are useful in diagnosis and therapeutics. Microarray-based gene expression analyses were performed in various tumors, including colorectal cancer, gastric cancer, acute myeloid leukemia (AML), and soft-tissue sarcoma (STS). Analysis of pediatric AML revealed age-associated differences of gene expression and their correlation to prognosis. To utilize this finding for improvement of risk stratification, a classification method was developed. Analysis of 8 types of STSs revealed that malignant fibrous histiocytoma (MFH) is heterogeneous as compared to other types of STSs. By developing a method to quantify the distance in gene expression, it was shown that more than 70% of MFH samples could be reclassified. Synovial sarcoma (SS) was an STS bearing the t(X;18) chromosomal translocation, which resulted in production of the SYT-SSX fusion protein. Analysis of SSs revealed that they could be separated into 3 subgroups, highly correlated with the histological subtypes. Target gene search of SYT-SSX led to detection of the COM1 gene. COM1 downregulation appears to play an important role in SS growth.

Altered expression of miRNA was also found in human cancers. miR-34a was isolated as an upregulated gene after treatment of the HCT116 colon cancer cell line with adriamycin. miR-34a was regulated by p53, and forced expression of miR-34a induced growth arrest in colon cancer cell lines. miR-34a expression was repressed in colon cancer specimens as compared with that in matched normal counterparts. These results suggest that miR-34a may be a novel type of tumor suppressor.

( 3, 11, 12, 13, 29, 30, 54, 61 )

Several functional components of the b-catenin/T-cell factor 4 (TCF4) complex were identified. Among them, Splicing factor-1 (SF1) was shown to be a negative regulator of the oncogenic activity of the TCF4/ b-catenin complex, while topoisomerase IIa (Topo II a) enhanced the TCF transcriptional activity. Ku70 and poly (ADP-ribose) polymerase-1 competitively interact with the b-catenin/TCF4 complex, and transcriptional activity of TCF4 was inhibited by association with Ku70.

Expression of actinin-4, an actin-binding protein, is significantly lower in prostate cancer cells, and restoration of actinin-4 causes inhibition of cell proliferation. Actinin-4 forms a complex with several endocytosis-related proteins, suggesting that it may cause aberrations in intercellular trafficking.

Ephrin-B1, a ligand of the EphB tyrosine kinases, induced secretion of MMP-8 when activated by the EphB receptor. Invasiveness of tumor cells expressing epherin-B1 was also promoted through MMP-8 activation, suggesting that stromal cells expressing EphB can promote invasion of ephrin-B1-expressing cancer cells by activating a specific set of metalloproteases.

High expression of the CXC chemokine, CXXL-12, in pancreatic endocrine tumors was found to be an unfavorable prognostic indicator and to be associated with hematogenous tumor spread, low microvessel density, high endothelial cell proliferation index and presence of large solid nests.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme degrading poly(ADP-ribose). Parg^-/- ES cells show enhanced sensitivity to g-irradiation. It was suggested that Parg is involved in DNA damage response and that it might be a useful target for sensitization to chemo- and radiotherapy.

CUB-domain containing protein 1 (CDCP1) was identified as a prominent substrate of the Src family kinases (SFK) and as a regulator of anchorage-independent growth of lung cancer cells. PKCd was shown to be essential for anchorage independence downstream of CDCP1. Suppression of CDCP1 expression also inhibited the metastatic potential of A549 cells in vivo

Overexpression of the type I collagen gene (HCOL1A1) in malignant glioma cells significantly suppressed invasion and tumor growth both in vitro and in vivo, and it was suggested that apoptosis may participate in the mechanism underlying the suppression of malignant characteristics of T98G glioma cells.

Integrative genomic analyses and meta analyses on genome-wide association studies (GWAS) were carried out on the genes encoding the stem cell signaling molecules in relation to the risk of gastric cancer. It was concluded that dysregulation of the stem cell signaling network due to various factors gives rise to gastric cancer.

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) exhibited high hepatic differentiation ability under an adherent monoculture condition after incubation with specific growth factors. The CD105fraction of AT-MSCs exhibited high hepatic differentiation ability and hepatocyte-like cells derived from these cells revealed several liver-specific markers and functions, and were incorporated into the parenchyma of the liver after transplantation into mice.

The functional roles of telomerase/TERT in the self-renewal potential of cancer stem cells are being investigated through affinity purification of the protein complex containing hTERT and several critical molecules associated with the maintenance of stem cells were identified.

( 18, 26, 43, 45, 46 )

The p53 tumor suppressor plays a major role in maintaining genomic stability mainly by acting as a transcription activator. A p53-target gene, p53R2, was found to play an essential role in DNA synthesis of mitochondria, and its mutations caused congenital mitochondrial diseases. This finding suggests that p53 might regulate the biosynthesis of mitochondria to prevent tumor initiation and progression.

Two transactivation domains (TADs) located at the amino-terminus of p53 are required for transcription activation, and the activity of these TADs is tightly regulated by post-translational modifications, such as phosphorylation. p53 carrying point mutations at all serine residues within the two TADs failed to activate approximately 80% of genes, including 29 previously reported p53 target genes, such as Hdm2 and Bax upregulated by wild-type p53, emphasizing the importance of phosphorylation within the TADs.

In neuronal cells, MDMX was found to act as an antiapoptotic factor, whose degradation is induced by various stresses and results in the activation of p53 and E2F-1 during neuronal apoptosis. Forced downregulation of MDMX by shRNA induced apoptosis, suggesting that MDMX is required for the survival of neurons.

The retinoblastoma tumor suppressor protein (pRB) is inactivated by phosphorylation by CDKs. However, Ser612 of pRB was found to be phosphorylated by Chk1/2 after DNA damage, leading to the formation of pRB-E2F-1.

HOMER2, a Homer/Ves1 family protein, was identified as a binding partner of MYO18B, a candidate tumor suppressor. These proteins co-localized in the regions of membrane protrusion and stress fibers. The biological effects of HOMERS and MYO18B in cultured cells suggested that they act as tumor suppressors not only in lung cancer, but also in malignant mesothelioma.

A novel role of PML-RARA in the pathogenesis of APL was clarified. The type IV isoform of PML interacted with PU.1 and p300 to enhanced PU.1-dependent transcription of the C/EBPe gene, which is essential for granulocytic differentiation. Four serine residues in the PML C-terminal region, highly phosphorylated in a myeloid cell line, were required for these processes. PML-RARA dissociated the PU.1/PML IV/p300 complex and inhibited PU.1-induced transcription, suggesting that PML-RARA may act as a dominant-negative inhibitor of PML-induced transcription.

Cdt1 plays a central role in DNA replication. Novel Cdt1-binding proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), were identified by a proteomics approach. Further analyses demonstrated that in addition to SCF^Skp2 and cullin4-based ubiquitin ligases, APC/C^Cdh1 is a third ubiquitin ligase that may play a crucial role in proteolytic regulation of Cdt1.

( 4, 14, 19, 37, 48, 73, 76, 77, 78, 79, 80 )

Translational researches have proved the diagnostic and prognostic utilities of molecular aberrations in cancer. CLASS-11 was identified in stage I lung adenocarcinoma patients with a poor prognosis, and OCIAD2, in invasive lung adenocarcinoma associated with a favorable prognosis among tumors with bronchial alveolar carcinoma components. The CpG island methylator phenotype was found to be associated with poor overall survival of patients with neuroblastoma (NBL) in Japan and Germany. NBL patients with unfavorable prognosis shared similar gene expression profiles, regardless of the MYCN amplification status. Missense MENIN mutations can predict the prognosis in patients with the familial cancer syndrome, multiple endocrine neoplasia types 1 (MEN1). These observations suggest that aberrant methylation, expression and/or mutation of specific genes in cancers may serve as efficient diagnostic indicators.

Global gene expression studies are also producing practical diagnostic tools. An RT-PCR-based mini-chip assay was established for multiple marker genes. Mini-chip assay for 10 marker genes in the peritoneal fluid showed a diagnostic potential for gastric cancer. Because the sensitivity of the mini-chip assay is higher than that of peritoneal cytology, it may serve to identify novel prognostic markers. Mini-chip assay was also useful for fecal RNA-based colorectal cancer screening. Mini-chip assay for six genes included in the 85 cancer patient-derived colonocyte-specific genes was shown to be of diagnostic value in colorectal patients, even at an early stage.

Recent progress in proteomics has also identified candidate proteins and peptides for diagnosis. Mass spectrometry identified three serum-albumin-associated peptides expressed at different levels between endometrial cancer patients and controls. Such peptides are expected to be candidates for diagnostic biomarkers. Combination of multi-dimensional chromatography with two-dimensional difference gel electrophoresis (2D-DIGE) identified leucine-rich alpha-2-glycoprotein (LRG) as plasma diagnostic proteins in pancreatic cancer patients. 2D-DIGE also identified 23 unique proteins in primary tumors of hepatocellular carcinoma, by which early intrahepatic recurrence could be predicted. The predictive performance of the 23 proteins was validated in an independent patient group, suggesting the prognostic usefulness of these proteins. Proteomic study with 2D-DIGE led to the identification of nine unique proteins to predict the response to treatment with gefitinib, an anti-cancer drug against non-small cell lung carcinoma, in patients with lung adnocarcinoma. The predictive performance of the nine identified proteins was successfully validated in additional cases. The results of such proteomic studies using clinical samples have been integrated with clinico-pathological information in the proteome database, as a part of the Genome Medicine Database of Japan (GeMDBJ). The database will contribute to the discovery and development of cancer biomarkers.

Histopathological examinations have revealed that esophageal cardiac glands and columnar epithelial islands may play a role in the development of short-segment Barrett's esophagus. Other clinicopathological studies were also conducted to further our understanding of the pathogenesis and promote early diagnosis and treatment of various tumors.

( 2, 15, 56, 57, 82 )

Tumor-infiltrating lymphocytes represent the host immune response to cancer. The prevalence of Foxp3+ Treg cells was significantly higher in the stroma of hepatocellular carcinoma than in the nontumorous parenchyma. The patient group with a high count of infiltrating Treg cells showed a significantly lower survival rate. The mechanism of IL-2 gene repression by Foxp3 has been investigated. Foxp3 and AML1 have been demonstrated to bind to the IL-2 gene promoter and regulate IL-2 gene expression. This interaction resulted in the transcriptional repression of not only IL-2, but also of interferon-gamma, and in the upregulation of Treg-associated molecules, thereby mediating the immunosuppressive activity of Treg.

In autologous hematopoietic stem cell transplantation (HSCT), lymphopenia-induced homeostatic proliferation of T cells is driven by the recognition of self-antigens, and there is an opportunity to skew the T-cell repertoire during T cell recovery by engaging tumor-associated antigens, to produce a break of tolerance against tumors. The efficacy and safety of integrating intratumoral alloMHC gene transfer with an autologous HSCT for the treatment of solid cancers has been experimentally proposed.

The efficiency of in vitro expansion of NKT cells is determined by the ratio of production of interleukin-4 and IFN-g by the NKT cells. Effective expansion of NKT cells was shown to require T helper type 2-biased culture conditions. Using a syngenic pancreatic cancer model in hamsters, local IFN-a gene therapy was demonstrated as a promising therapeutic strategy for pancreatic cancer, due to the multiple mechanisms underlying the antitumor activities and lack of significant toxicity.

( 15, 27, 52, 58, 64 )

Atelocollagen-mediated nanoparticle delivery systems to deliver siRNAs and miRNAs to metastatic animal models of human cancers were investigated and these RNAi strategies effectively inhibited metastasis from human prostate and breast tumors in mice.

A unique adenovirus library system, which was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob, was established. To improve p53 gene therapy, adenovirus-mediated gene transfer of active forms of p53 or apoptotic p53-target genes in cancers has been investigated.