Akio Ooyama

Down regulation of c-Myc and induction of an angiogenesis inhibitor, thrombospondin-1, by 5-FU in human colon cancer KM12C cells

5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA.

Low expression of γ-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype

The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of γ-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin.

Molecular Basis for the Induction of an Angiogenesis Inhibitor, Thrombospondin-1, by 5-Fluorouracil

5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein.

Genome‐wide screening of loci associated with drug resistance to 5‐fluorouracil‐based drugs

Resistance to chemotherapeutic agents represents the chief cause of mortality in cancer patients with advanced disease. Chromosomal aberration and altered gene expression are the main genetic mechanisms of tumor chemoresistance. In this study, we have established an algorithm to calculate DNA copy number using the Affymetrix 10K array, and performed a genome‐wide correlation analysis between DNA copy number and antitumor activity against 5‐fluorouracil (5‐FU)‐based drugs (S‐1, tegafur + uracil [UFT], 5′‐DFUR and capecitabine) to screen for loci influencing drug resistance using 27 human cancer xenografts. A correlation analysis confirmed that the single nuceotide polymorphism (SNP) showing significant associations with drug sensitivity were concentrated in some cytogenetic regions (18p, 17p13.2, 17p12, 11q14.1, 11q11 and 11p11.12), and we identified some genes that have been indicated their relations to drug sensitivity. Among these regions, 18p11.32 at the location of the thymidylate synthase gene (TYMS) was strongly associated with resistance to 5‐FU‐based drugs. A change in copy number of the TYMS gene was reflected in the TYMS expression level, and showed a significant negative correlation with sensitivity against 5‐FU‐based drugs. These results suggest that amplification of the TYMS gene is associated with innate resistance, supporting the possibility that TYMS copy number might be a predictive marker of drug sensitivity to fluoropyrimidines. Further study is necessary to clarify the functional roles of other genes coded in significant cytogenetic regions. These promising data suggest that a comprehensive DNA copy number analysis might aid in the quest for optimal markers of drug response. (Cancer Sci 2007; 98: 577–583)

Gene expression analysis using human cancer xenografts to identify novel predictive marker genes for the efficacy of 5-fluorouracil-based drugs

The development of a diagnostic method for predicting the therapeutic efficacy or toxicity of anticancer drugs is a critical issue. We carried out a gene expression analysis to identify genes whose expression profiles were correlated with the sensitivity of 30 human tumor xenografts to 5‐fluorouracil (5‐FU)‐based drugs (tegafur + uracil [UFT], tegafur + gimeracil + oteracil [S‐1], 5′‐deoxy‐5‐fluorouridine [5′‐DFUR], and N4‐pentyloxycarbonyl‐5′‐deoxy‐5‐fluorocytidine [capecitabine]), as well as three other drugs (cisplatin [CDDP], irinotecan hydrochloride [CPT‐11], and paclitaxel) that have different modes of action. In the present study, we focused especially on the fluoropyrimidines. The efficacy of all anticancer drugs was assayed using human tumor xenografts in nude mice. The mRNA expression profile of each of these xenografts was analyzed using a Human Focus array. Correlation analysis between the gene expression profiles and the chemosensitivities of seven drugs identified 39 genes whose expression levels were correlated significantly with multidrug sensitivity, and we suggest that the angiogenic pathway plays a pivotal role in resistance to fluoropyrimidines. Furthermore, many genes showing specific correlations with each drug were also identified. Among the candidate genes associated with 5‐FU resistance, the dihydropyrimidine dehydrogenase mRNA expression profiles of the tumors showed a significant negative correlation with chemosensitivity to all of the 5‐FU based drugs except for S‐1. Therefore, the administration of S‐1 might be an effective strategy for the treatment of high dihydropyrimidine dehydrogenase‐expressing tumors. The results of the present study may enhance the prediction of tumor response to anticancer drugs and contribute to the development of tailor‐made chemotherapy. (Cancer Sci 2006; 97: 510 – 522)

VEGF expression is augmented by hypoxia‑induced PGIS in human fibroblasts

Prostacyclin synthase (PGIS or PTGIS) is an enzyme that catalyses the conversion of prostaglandin H2 (PGH2) to prostaglandin I2 (PGI2). PGI2 promotes cancer growth by activating peroxisome proliferator-activated receptor δ (PPARδ), and increases the expression levels of the pro-angiogenic factor vascular endothelial growth factor (VEGF). We found that the expression of the PGIS gene was enhanced in WI-38, TIG-3-20 and HEL human lung fibroblast cells and two cancer cell lines (NB-1 and G361) under hypoxic conditions. The main localization of PGIS changed from the cytoplasm to the nucleus by hypoxia in WI-38 cells. The induced PGIS had an enzymatic activity since the intracellular level of 6-keto-prostaglandin, a useful marker of PGI2 biosynthesis in vivo, was increased with the increasing levels of PGIS. Expression of VEGF was increased in parallel with PGIS induction under hypoxic conditions. PGIS knockdown resulted in the decreased expression of VEGF mRNA. Since VEGF is a known PPARδ target gene, we examined the effects of siRNAs targeting PPARδ on the expression of VEGF under hypoxic conditions. Knockdown of PPARδ suppressed the expression of VEGF under hypoxic conditions in WI-38 cells. These findings suggest that PGIS is induced by hypoxia and regulates the expression of VEGF in fibroblasts. Fibroblasts in the hypoxic area of tumors may have an important role in tumor growth and angiogenesis.

Gene expression of ferredoxin reductase predicts outcome in patients with metastatic colorectal cancer treated by 5-fluorouracil plus leucovorin

Purpose: Ferredoxin reductase (FDXR) is a putative contributor to p53-mediated apoptosis from 5-fluorouracil (5-FU) through the generation of oxidative stress in the mitochondria. However, the influence of FDXR gene expression levels on the outcome of 5-FU chemotherapy has been relatively little studied. The aim of this study is to investigate the association between FDXR gene expressions and the clinical outcome when treated by 5-FU chemotherapy, as well as the correlation of FDXR gene expressions and p53 mutation. Methods: Pre-chemotherapeutic fresh frozen samples of 33 patients with metastatic colorectal cancer, who received bolus 5-FU and leucovorin (LV) as first line chemotherapy, were studied. FDXR gene expression and p53 mutation were evaluated by real-time RT-PCR and direct sequencing, respectively. Results: FDXR gene expression was significantly higher in responding tumors compared with non-responding ones (P=0.0379). Patients with FDXR values above the cutoff value of 13.52 had a statistically longer survival than those with FDXR gene expressions below the cutoff value (P=0.0148). The 9 tumors with wild-type p53 had statistically higher FDXR gene expressions than the 14 tumors with mutant-type p53 which had sequence alterations within the “hot spot” codons, the L2–L3 loops, or frameshift (P=0.0463). Conclusions: FDXR gene expression did not affect clinical outcome in patients with wild-type p53 tumors, whereas, among patients with p53 mutant-type tumors, patients with tumors with low FDXR gene expression had a worse outcome than those with a high FDXR gene expression (P=0.0200). FDXR gene expression, which is regulated at least in part by p53, is associated with both response and survival when metastatic colorectal cancer is treated with 5-FU plus LV. In addition, analysis of p53 mutation combined with FDXR gene expression might be useful in estimating the outcome in 5-FU-treated patients.