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 the search for carcinogens and chemopreventive agents using animal models; identification and functional analysis of cancer-related genes; and development of cancer diagnosis and therapy. To promote cancer research, cutting-edge technologies in genome and proteome analysis are being utilized, and the Research Institute is making major contributions to various aspects of worldwide cancer research. An overview of the research activities in 2004 is shown below.

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

Cancers are caused by the complex interaction of environmental and heritable factors, and exposure to environmental insults is considered to result in the induction of both genetic and epigenetic alterations in cells. A research project to evaluate the risks of heterocyclic amines (HCAs) produced while cooking meat and fish as exogenous human carcinogens has been conducted. A novel type of HCAs, APNH, was found to be formed from two non-mutagenic compounds, norharman and aniline, in the presence of S9 mix, and induced hepatocellular carcinoma in mice.

As for endogenous factors, N-nitorosotaurocholic acid, N-nitroso taurodeoxycholic acid, and cholyl adenylate in bile acid were found to react with DNA to form several DNA adducts, suggesting that these compounds may have some possibility as DNA modifiers in vivo. DNA replication stresses could also be responsible for spontaneous mutations. The formation of unusual DNA structures at the G-rich repetitive sequences causes inhibition of DNA replication at the repeats in E. coli, and could be responsible for the induction of genomic instability at G-rich repetitive sequences in mammalian cells.

Animal models are useful tools to elucidate the multi-step molecular mechanisms of cancer development, especially in the early stages, and genetic bases underlying differential susceptibility to carcinogenesis among various strains. In colon cancer models, preneoplastic lesions in the colon were differentially and efficiently contrasted by a modified methylene blue staining. Furthermore, the combined administration of PhIP and AOM showed a synergistic effect on cancer induction and induced poorly differentiated colon carcinomas featuring prominent submucosal invasion in rats. The colonic inflammation associated with Th2-dominant cytokine response also enhanced the formation of colon cancer in mice.

Involvement of the polyADP-ribosylation reaction in carcinogenesis has also been investigated. Poly(ADP-ribose) polymerase-1 (Parp-1) knockout mice showed increased susceptibility to carcinogenesis induced by alkylating agents, but the incidences of tumors were not changed by heterocyclic amine treatment. This suggests that Parp-1 could be involved in a particular type of DNA repair. The main enzyme for polyADP-ribose degradation, Parg, was also suggested to be involved in the induction of apoptosis after DNA damage.

In colon cancers, PGE2 expression produced by COX-2 during inflammation exerts its biological activity through binding to its membrane receptors, EPs. EP3 plays a suppressive role in later steps in contrast to the promotive role of EP1, EP2 and EP4. A ligand for peroxisome proliferators-activated receptor g (PPARg), which is expressed in several types of human cancers, reduced the incidence of gastric cancer in wild, but not in the PPAR g-deficient mice, suggesting its potential usefulness as chemopreventive agent for gastric cancer.

Epidemiologically, consumption of a high fat diet and high serum levels of triglycerides and cholesterol were suggesed to be associated with the risk of colon cancer. Apc-deficient mice show an age-dependent hyperlipidemic state along with suppressed lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides. Treatment with an LPL inducer, NO-1886 without PPAR agonistic activity, suppressed both hyperlipidemia and intestinal polyp formation in the mice with induction of LPL mRNA and suppression of COX-2 mRNA. This suggests that the suppression of serum lipid levels by increasing LPL activity may contribute to a reduction of intestinal polyp formation with Apc-deficiency.

( 96, 97, 99, 101, 102, 103 )

Lifestyle factors have been implicated in the etiology of cancer occurrence. Each result from the epidemiologic studies contributed to add new evidence to be summed up. The Japan Public Health Center-based Prospective Study (JPHC Study), a population-based prospective study, is currently ongoing. Nearly 13% of cancers among males in this study were due to heavy drinking, to which smoking substantially contributed. Those who drank more coffee had a lower hepatocellular carcinoma risk. Smoking women, as well as premenopausal women with never-active but passive smoking, had a higher breast cancer risk. Both endogenous and extraneous estrogen might increase the risk of lung cancer in women. Vegetables and fruit intake was not associated with the reduced risk of colorectal cancer, while a higher BMI in men and the traditional and the Western dietary pattern in women increased the risk. Results on diabetes and suicide were also reported from the study. Risk of endocrine-disputing chemicals (EDC) for endometriosis among Japanese women was examined in a case-control study, which found no association between the serum levels and the increased risk.

Studies and projects aimed at evidence-based cancer control have been planned and carried out. Establishment of a cancer surveillance system in Japan is urgently required to comprehend facts and figures and discuss cancer control strategy. Up-to-date information on cancer registry-related issues is disseminated through the websites. Some descriptive analysis on cancer incidences and mortalities, and international comparisons were performed. The effects of smoking on the future mortality trend are evaluated. A systematic literature review and meta-analysis showed that current tobacco smoking moderately increases the risk (1.5 times) of total cancer in the Japanese population.

An appropriate screening strategy for early cancer detection shares a central role of cancer control with primary prevention. The guidelines for cancer screening have been updated based on new research, and the guideline development process was standardized, and applied to colorectal and gastric cancer screening. The guideline for lung cancer screening is being revised.

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

( 17, 61, 62, 63, 64 )

Several types of genome-wide screening methods were applied to identify genomic regions showing copy number alterations of chromosomal regions in human cancers. Microarray-based comparative genomic hybridization analysis led to the detection of a homozygous deletion (HD) at the cyclic AMP response element binding protein-binding protein (CBP) locus in a lung cancer cell line. Moreover, point mutations of the CBP gene were detected in 5-10% of lung cancer cases, indicating that genetic CBP alterations are involved in the genesis and/or progression of a subset of lung cancers. Genome scanning at a 1-Mb resolution using a high-resolution SNP array revealed a high incidence of HDs at the CDKN2A locus on 9p21 (26%) and at the PTPRD locus on 9p23 (10%) in lung cancers, suggesting that PTPRD is an additional candidate tumor suppressor gene for lung cancer. A BAC array-based CGH (comparative genomic hybridization) was also applied to the analysis of hepatocellular carcinoma (HCC), lung cancer, gastric cancer and breast cancer. HCCs with HBV infection showed significantly more frequent chromosomal alterations than those with HCV infection or those without viral infection, suggesting that HBV might induce a chromosomal instability phenotype. The analyses also identified 95 loci with amplification and 34 loci with HD in gastric cancer, and 42 loci with amplification and 7 loci with HD in breast cancer.

Poly(ADP-ribose) polymerase-1 (PARP-1) deficiency induces trophoblast differentiation from mouse embryonic stem cell during teratocarcinoma formation. Analyses of 16 human germ cell tumors identified possible germline mutations causing amino acid substitutions that could affect PARP-1 function, including Glu251Lys and Met1229Thr. These findings suggest that genetic and functional aberrations of PARP-1 influence the development and phenotypes of human germ cell tumors.

( 1, 17, 37, 49, 80 )

The methylation of CpG islands (CGIs) is deeply involved in human carcinogenesis. Aberrantly methylated CGIs are also useful for diagnostic markers of human tumors. Methylation-sensitive-representational difference analysis (MS-RDA) was developed in this institute and used to identify novel DNA fragments that are differentially methylated in cancer. A number of tumor suppressors and candidates have been identified using this method, including lysyl Oxidase and Thrombomodulin. In neuroblastomas, methylation of multiple specific CGIs (CGI methylator phenotype: CIMP) was shown to be a very strong prognostic marker with a hazard ratio of 22. Tumor suppressor genes and candidates previously identified in this institute were also analyzed for their epigenetic inactivation. Promoter methylation of the TSLC1/IGSF4 and the DAL-1/4.1B genes were observed respectively in 44% and 59% of primary non-small cell lung cancers (NSCLCs), providing markers of poor prognosis in lung adenocarcinoma. Moreover, histone deacetylation of the MYO18B gene was detected in a considerable fraction of colorectal cancer as was in lung cancer. Studies on the molecular mechamisms of methylation revealed that most of the promoter CGIs methylated in cancer were derived from genes with little transcription in normal tissues and that methylation of sparse CpG sites within the promoter CGIs could act as seeds to promote methylation of CGIs.

Molecular pathological studies have shown that aberrant DNA methylation could participate in multistage carcinogenesis of human tumors, even at the precancerous stage. In the urinary bladder, concurrent DNA hypermethylation on multiple CGIs, as well as the enhanced immunoreactivity of DNA methyltarnsferase 1 (DNMT1), was preferentially observed in non-cancerous urothelia from patients with urinary bladder cancers than in normal urothelia. In the pancreas, nuclear DNMT1 immunoreactivity was already detected in peripheral pancreatic duct epithelia with an inflammatory background (possible precancerous lesions) and its incidence increased progressively in pancreatic intraepithelial neoplasias and in invasive ductal carcinomas of the pancreas.

On the other hand, DNA hypomethylation on pericentromeric satellite regions was shown to participate in the development and progression of urothelial carcinomas through enhanced genomic instability. Cancer-linked hypomethylation was also important to activate specific genes. R-RAS was identified as one of the genes that were silenced by DNA methylation in the stomach mucosa but were activated by demethylation in a subset of gastric cancer. Functional studies of R-RAS by RNAi suggested that blockade of this cascade was a potential target for therapy for gastric cancer.

Fifty single nucleotide polymorphisms (SNPs) in 36 DNA repair genes were identified and were assessed for associations with lung cancer risk by a case-control study. Four SNPs in the TP53, REV, LIG4 and POLI genes were suggested to function as genetic factors underlying lung cancer susceptibility by modulating activities to maintain the genome integrity of each individual. A genome-wide screening was carried out, as a collaborative project with various other groups, to search for regions of the human genome containing gastric cancer susceptibility genes in 170 affected sibpairs from 142 families using 392 microsatellite markers. The strongest LOD score was observed on chromosome 2q33-q35, suggesting the presence of a gastric cancer susceptibility locus on this chromosome arm. Collaborative studies on SNPs implicated in the susceptibility to common diseases were performed and a part of the SNP data has been publicized in the Genome Medicine Database of Japan (www.gemdbj.jp).

Expression microarray analyses were performed in various human tumors. An analysis of 50 pediatric AML patients revealed that t(8;21)-AML and inv(16)-AML shared specific expression profiles. Expression profiling of 58 neuroblastomas (NBL) suggested that molecular event(s) functionally analogous to MYCN amplification occurs in NBL without MYCN amplification but with a poor prognosis. Further studies demonstrated that MYCN expression was inversely correlated with expression of genes associated with neuronal differentiation.

To uncover the mechanism of p53-mediated tumor suppression and to develop novel therapies for cancer, the genes directly regulated by p53 have been isolated using a microarray technology in combination with chromatin immunoprecipitation. This year, the SEMA3F and DFNA5 have been identified as novel p53-target genes. Physiological functions of the p53-target molecules previously identified by the same methods revealed that DCC, neogenin, UNC5B, and netrin-1 were involved in axon-guidance, suggesting that there are common underlying mechanisms between axon-guidance and tumorigenesis.

( 2, 12, 13, 27, 73, 75, 81, 84 )

Increased expression of dysadherin, a newly identified cell membrane glycoprotein, was shown to be a significant predictor of poor survival in patients with cutaneous malignant melanoma. Tissue factor, identified as an antigen expressed in the invasive front, was also a significant predictor of poor survival in ductal carcinoma of pancreas. Akt phosphorylation is a risk factor for early recurrence and poor prognosis in patients with hepatocellular carcinomas.

Actinin-4 was identified as a binding partner of b-catenin protein in nuclear extracts of SW480 colorectal cancer cells. Overexpression of Actinin-4 induces cell motility and specifically promotes lymph node metastasis by colorectal cancer. The TCF-4/b-catenin complex was found to physically interact with poly(ADP-ribose) polymerase-1 (PARP-1), FUS and various classes of RNA-binding proteins. PARP-1 functioned as a co-activator of b-catenin evoked gene transactivation, while the expression of FUS was closely associated with the accumulation of b-catenin and with the undifferentiated status of intestinal epithelial cells.

Analysis of phosphotyrosines-containing proteins in low- and high-metastatic human osteosarcoma HuO9 sublines suggested that the enhanced activity of Src family kinases and overexpression of paxillin synergistically contribute to the high metastatic potential of osteosarcoma cells through the hyper-phosphorylation of paxillin.

A proteomic study for the comprehensive understanding of the protein network behind cancer phenotypes is being performed using the 2D-DIGE system. An analysis of proteomic signatures corresponding to the histology of lung cancer, and the differential protein expression during liver carcinogenesis are among current targets of this study.

Loss of function of a cell adhesion molecule, TSLC1/IGSF4 could induce invasion and metastasis in cancer cells. Intervention of epithelial-mesenchymal transition (EMT) by expression of TSLC1/IGSF4 was observed in an in vitro model, suggesting another mechanism of tumor suppression by TSLC1/IGSF4.

IGF-1 belongs to a growth factor for human cancers. Treatment of a neutralizing antibody to active IGF-1 led to significant tumor growth inhibition and longer survival in late phase of hepatic metastasis of human colon cancer cells in the SCID mouse model.

While NLK (Nemo-like kinase) has been considered as a suppressor of the b-catenin/TCF signaling, it can positively regulate Wnt/b-catenin signaling in neuroblastoma and osteosarcoma cell lines.

Interaction of the extra-cellular domain of ephrin-B1 with claudin, a component of the tight junction, suggested that ephrin-B1 induces a novel signaling cascade via an Eph receptor-independent mechanism, indicating the possible involvement of the Eph-ephrin system in aberrant cell-cell adhesion observed in cancer cells.

It was revealed that AMPK-a, Akt and ARK5, a member of AMPK family, are activated by TGF-b induced under hypoxic conditions. In primary myeloma samples, a higher expression of ARK5 was closely associated with c-MAF and MAFB expression which is an obvious poor prognostic factor for multiple myeloma.

( 23, 24, 33, 34, 44, 47 )

The mechanisms of p53-mediated tumor suppression include both transcriptional regulation of target genes and transcription-independent induction of apoptosis. As transcriptional targets of p53, Notch1, p53R2 and several axon-guidance molecules including DCC, neogenin, UNC5B, and netrin-1 have been identified. Notch1 induced keratinocyte differentiation and functions as a tumor suppressor in keratinocytes, indicating a novel function of p53 in regulating cell differentiation of genotoxic stress. A small subunit of ribonucleotide reductase (RR), P53R2, was shown to play a pivotal role in the dNTP supply for DNA repair, and thus dysregulation of p53R2 in most cancers, in which p53 is inactivated, might enhance accumulation of spontaneous mutations. The axon-guidance molecules were suggested to regulate apoptosis. As a transcriptional activation-independent function of p53, alpha-amanitin induced apoptosis was found to be mediated by the translocation of p53 into mitochondria.

The target-specificity of p53 can be determined by different mechanisms including post-translational modifications and binding partners. MOZ forms a complex with p53 and specifically enhance the transcription of p21Waf1, but not Bax and Mdm2. MOZ-deficient MEF abrogated G1 arrest upon DNA damage. The complex formation of MOZ and p53 increased upon moderate DNA damage, but decreased upon severe damage, indicating a critical role of MOZ in determining the response to DNA damage.

A novel mechanism activating p53 by enhanced degradation of Mdmx was identified. It was proposed that Mdmx phosphorylation at S367 induced by DNA damage triggers Mdm2-dependent degradation of Mdmx and thus leads to p53 accumulation.

( 29, 30, 33, 36, 78, 79 )

MOZ and p300/CBP are found in the complex of the leukemia-associated factor AML1. MOZ-deficient mice were embryonically lethal due to reduced hematopoietic stem cells and lineage-committed progenitor cells. As the phenotypes were similar to those of PU.1-defficient mice, MOZ plays an essential role in the maintenance of hematopoietic stem cells and differentiation of erythroid and myeloid cells as a co-activator for PU.1 and AML. Homeodomain-interacting protein kinase-2 (HIPK2) forms a ternary complex with AML1 and p300, and phosphorylates them to stimulate their activities. The observed phenotypes of Hipk1/2 double-deficient mouse embryos suggested the importance of AML1 and p300 phosphorylation by HIPK1/2 in primitive/definitive hematopoiesis.

The EWS-NOR1 and EWS/FLI-1 oncogenic fusion gene products found in soft tissue malignancies and an isoform of CARM1 were shown to interact with the U1 small nuclear ribonucleoprotein (snRNP)-specific protein U1C and may participate in the aberrant splicing in cancer cells.

The activity of Cdt1 is negatively regulated by multiple pathways including ubiquitination by Skp2. Overexpression of Cdt1, which can be generally observed in cancer cells, induced chromosomal damage, suggesting the importance of the strict regulation of Cdt1.

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

Recently, mass spectrometry (MS)-based proteomic approaches have gained much attention as effective modalities for identifying new biomarkers of various diseases. Surface-enhanced laser disorption/ionization (SELDI)-MS can generate a comprehensive proteomic profile from 20 ml of serum/plasma samples. An original software program to detect and align peaks from spectra obtained using a high-resolution hybrid quadrupole time of flight mass spectrometry instrument was developed and used for the identification of biomarkers of various cancers.

A discriminating plasma proteomic pattern was extracted from a training cohort of 71 pancreatic cancer patients and 71 healthy controls. This pattern was able to distinguish cancer patients from healthy controls in an independent validation cohort with a sensitivity of 90.9% (30/33) and specificity of 91.1% (41/45) in a blinded manner. The combination of CA19-9 and SELDI-Qq-TOF-MS could detect 100% (29/29) of pancreatic cancer patients including those with stage I and II disease.

The proteomic profiles of serum samples were also examined for their potential to predict the efficacy of combined preoperative chemoradiotherapy (PCRT) against esophageal cancer. The combination of 4 peaks could perfectly distinguish responders from non-responders in the training set of 27 patients. This set of peaks, i.e., the classifier, correctly diagnosed chemoradiosensitivity in 93.3% (14/15) of the cases in the independent validation set in a blinded manner.

Colon cancer is known to be almost 100% curable by operation if detected early enough. A new method has been developed to isolate exfoliated colorectal cancer cells effectively from naturally voided feces. The cell recovering method and apparatus allow the analysis of a variety of biomarkers, such as gene mutation detection, to enhance the specificity and sensitivity of the current colorectal cancer screening methods.

Silencing gene expression by siRNAs has been attracting a great deal of attention, not only as a powerful tool for experimental analysis but also as a novel therapeutic agent, which is already being examined in a clinical trial. However, much still remain to be desired in the development of optimal siRNA delivery strategies. Atelocollagen is a highly purified type I collagen that is modified to have low immunogenicity. siRNA complexed with atelocollagen is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing in vivo. The atelocollagen system was used to deliver siRNAs of EZH2 (a gene overexpressed in metastatic prostate cancer) and p110 alpha (a gene regulating cell survival, proliferation, and migration) to the metastatic prostate cancer animal models. A significant decrease in metastasis was observed, and siRNA/atelocollagen treatment did not elicit an interferon immune response in the mice. Thus, for clinical applications of siRNA, an atelocollagen-based delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.

The IFN-a gene transfer was found to induce more prominent growth suppression and cell death in pancreatic cancer cells than in other types of cancers and normal cells. When a mouse IFN-a adenovirus was injected into a subcutaneous tumor xenograft, all mice showed significant tumor inhibition, an effect that was dependent on the stimulation of natural killer cells. Moreover, the tumor regression was observed not only for the injected subcutaneous tumors but also for untreated tumors at distant sites. The IFN-a protein concentration was markedly increased in the injected tumor, but leakage of the cytokine into the systemic blood circulation was minimal. Local IFN-a gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its dual mechanisms of antitumor activities, the direct local cytotoxicity and an indirect systemic immunological antitumor effect, as well as the lack of significant toxicity.

An objective of DDS in cancer chemotherapy is to achieve a passive targeting of anticancer agents to solid tumors. Macromolecular anticancer agents such as liposomal or micellar drug are too large to pass through the normal vessel walls but can diffuse out of tumor blood vessels, reach the solid tumor tissue effectively and be retained for a long period due to the EPR effect. Paclitaxel or cisplatin-incorporated micelle nanoparticles have been developed and have been shown to extend in vivo antitumor activity and reduce the neurotoxicity of the parent drug.

Depression is the most common expression of psychological distress in cancer patients. Since depression is poorly recognized and not appropriately treated, screening cancer patients for depression is important. The Distress and Impact Thermometer (DIT), a 2-item questionnaire, was developed as a brief screening tool for adjustment disorders and/or major depression. Two hundred ninety-five cancer patients completed the DIT, and were assessed on DSM-IV. Using cutoff points on "distress" and "impact" scores, the sensitivity and specificity were 0.82 and 0.82, respectively. Its brevity and good performance suggest that the DIT is an effective tool for routine screening in clinical oncology settings.

Even if depression is recognized, it is usually not appropriately referred to the Psychiatry service. The nurse-assisted screening and psychiatric referral program (NASPRP) has been introduced in clinical practice. The program consists of two stages. In the first stage, the DIT is administered to patients newly admitted to the Oncology/Hematology Unit by nurses as a brief screening tool for major depression and adjustment disorders. In the second stage, the nurses recommend psychiatric referral to patients with scores above the cutoff point. During the program period, 86.0% (135/157) completed the DIT and results were positive in 49.6% (67/135), but only 28.2% (19/67) accepted psychiatric referral. Ultimately, 11.5% of newly admitted patients were diagnosed as having major depression or adjustment disorders and treated by the psychiatric service, a significantly higher proportion than during the preceding 3-month period (2.5%; P<0.001).