Ryouichi Tsunedomi

Integrin α2 Mediates Selective Metastasis to the Liver

Cancers display distinct patterns of organ-specific metastasis. Comparative analysis of a broad array of cell membrane molecules on a liver-metastasizing subline of B16 melanoma versus the parental B16-F0 revealed unique up-regulation of integrin alpha2. The direct role of integrin alpha2 in hepatic metastasis was shown by comparison of high versus low-expressing populations, antibody blockade, and ectopic expression. Integrin alpha2-mediated binding to collagen type IV (highly exposed in the liver sinusoids) and collagen type IV-dependent activation of focal adhesion kinase are both known to be important in the metastatic process. Analysis of primary colorectal cancers as well as coexisting liver and lung metastases from individual patients suggests that integrin alpha2 expression contributes to liver metastasis in human colorectal cancer. These findings define integrin alpha2 as a molecule conferring selective potential for formation of hepatic metastasis, as well as a possible target to prevent their formation.

Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs.

BackgroundCancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs.MethodsThe human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed.ResultsPoorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P < 0.05). The cell cycle of SK-spheres was arrested at the G0/G1 phase compared to SK cells. SK-spheres showed higher ABCG2 and HIF1A mRNA expression and lower ROS production compared to parental cells.ConclusionOur novel method successfully induced cancer stem-like cells, which possessed chemoresistance that was related to the cell cycle, drug efflux, and ROS.

Involvement of c-myc-regulated genes in hepatocellular carcinoma related to genotype-C hepatitis B virus

Purpose: The purpose of this study was to elucidate the molecular basis of hepatocellular carcinoma (HCC) caused by genotype-C hepatitis B virus (HBV). Methods: We compared molecular profiles of 15 HCCs and five non-tumorous livers, all of which were associated with genotype-C HBV infection, using DNA microarray technology. Results: Our supervised learning identified 237 genes whose expression differed between HCCs and non-tumorous livers. This result was validated by a false discovery rate of 0%. Levels of expression of 35 and 202 genes were higher and lower, respectively, in HCCs than in non-tumorous livers. Among the 237 genes, we highlighted the top 35 upregulated and top 35 downregulated genes in tumor. Interestingly, when overlapping genes were excluded, 12 (e.g., NM23-H2, MCM7, PARP1, YWHAH, HSPB1, and MSH2) of the top 34 upregulated genes and five (e.g., MT1A and MT3) of the top 33 downregulated genes were c-myc-regulated genes. The microarray data for five randomly selected genes (MCM7, UBE2L3, PPIA, CXCL12, and ASS) were confirmed by quantitative real-time reverse transcription-polymerase chain reaction. Conclusions: Our results indicate that many c-myc-regulated genes are involved in genotype-C-HBV-related HCC, suggesting that c-myc is related to the hepatocarcinogenic activity of genotype-C HBV.

The Activator of GntII Genes for Gluconate Metabolism, GntH, Exerts Negative Control of GntR-Regulated GntI Genes in Escherichia coli

Gluconate is one of the preferred carbon sources of Escherichia coli, and two sets of gnt genes (encoding the GntI and GntII systems) are involved in its transport and metabolism. GntR represses the GntI genes gntKU and gntT, whereas GntH was previously suggested to be an activator for the GntII genes gntV and idnDO-gntWH. The helix-turn-helix residues of the two regulators GntR and GntH exhibit extensive homologies. The similarity between the two regulators prompted analysis of the cross-regulation of the GntI genes by GntH. Repression of gntKU and gntT by GntH, as well as GntR, was indeed observed using transcriptional fusions and RNA analysis. High GntH expression, from cloned gntH or induced through 5-ketogluconate, was required to observe repression of GntI genes. Two GntR-binding elements were identified in the promoter-operator region of gntKU and were also shown to be the target sites of GntH by mutational analysis. However, the GntI genes were not induced by gluconate in the presence of enhanced amounts of GntH, whereas repression by GntR was relieved by gluconate. The repression of GntI genes by GntH is thus unusual in that it is not relieved by the availability of substrate. These results led us to propose that GntH activates GntII and represses the GntI genes in the presence of metabolites derived from gluconate, allowing the organism to switch from the GntI to the GntII system. This cross-regulation may explain the progressive changes in gnt gene expression along with phases of cell growth in the presence of gluconate.

Decreased ID2 Promotes Metastatic Potentials of Hepatocellular Carcinoma by Altering Secretion of Vascular Endothelial Growth Factor

Purpose: We aimed to explore the molecular and biological functions of Inhibitor of DNA binding/differentiation 2 (ID2), which was found to be responsible for portal vein invasion of hepatocellular carcinoma (HCC). Experimental Design: We measured ID2 mRNA levels in 92 HCC patients by real-time reverse transcription-PCR and examined the relation to clinicopathologic features. To clarify the precise roles of ID2, we did in vitro analysis with expression vectors and small interfering RNAs. Effects of ID2 on cell invasive potential and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α were analyzed by Matrigel-coated invasion chamber, ELISA, and Western blot analysis, respectively. Results:ID2 mRNA level correlated inversely with portal vein invasion (P < 0.001), tumor-node-metastasis stage (P < 0.001), tumor size (P < 0.001), and early intrahepatic recurrence (P < 0.05). When limited to a cohort of hepatitis C virus–related HCCs, patients with low levels of ID2 had significantly shorter disease-free survival time than those with high levels of ID2. Invasive potential of cells transfected with ID2 expression vector was lower than that of empty vector–transfected cells. Cells overexpressing ID2 also showed decreased VEGF secretion and hypoxia-inducible factor-1α protein levels. The results of ID2-knockdown experiments were opposite to those of ID2 overexpression experiments. Conclusions: On the basis of our clinical and in vitro data, we suggest that ID2 plays a significant role in the metastatic process during progression of HCC. This action might be explained, at least in part, by altered cell mobility due to decreased secretion of VEGF.

UGT1A1*6, 1A7*3, and 1A9*22 genotypes predict severe neutropenia in FOLFIRI-treated metastatic colorectal cancer in two prospective studies in Japan

Retrospective studies have suggested that UDP‐glucuronosyltransferase (UGT)1A1, UGT1A7, and UGT1A9 predict severe toxicity and efficacy of irinotecan‐containing regimens. We prospectively evaluated the impact of UGT1A genotypes and haplotypes on severe toxicity and efficacy in patients treated with fluorouracil, leucovorin, and irinotecan combination chemotherapy (FOLFIRI) for metastatic colorectal cancer (mCRC) from the two prospective multicenter phase II studies in Japan. The FLIGHT1 study was a first‐line FOLFIRI trial, and FLIGHT2 was a FOLFOX‐refractory, second‐line FOLFIRI trial. A total of 73 patients agreed to additional analysis, and were genotyped for UGT1A polymorphisms, UGT1A1*28 (TA6>TA7), UGT1A1*6 (211G>A), UGT1A1*27 (686C>A), UGT1A1*60 (−3279T>G), UGT1A1*93 (−3156G>A), UGT1A7 (−57T>G), UGT1A7*3 (387T>G, 622T>C), and UGT1A9*22 (T9>T10). Of 73 patients, 34 developed G3/4 severe hematological toxicities. The toxicities were significantly more frequent in patients with UGT1A1*6 (211A), UGT1A7 (387G), and UGT1A9*22 reference alleles (T9). Haplotype I, which consists of all favorable alleles, was associated with a significant reduction in hematologic toxicity (P = 0.031). In contrast, haplotype II, which contains four high‐risk alleles, showed significantly higher hematologic toxicity than the other haplotypes (P = 0.010). Six out of seven patients who were homozygous for UGT1A1*28 or *6 experienced severe hematological toxicity despite the fact that their response rate was not impaired (42.9%). We concluded that UGT1A polymorphisms, especially UGT1A1*6, are important for the prediction of severe toxicity of FOLFIRI in northeast Asian populations. In this regard, haplotype analyses should substantially impact the prediction of severe hematological toxicities of FOLFIRI. (Clinical Trial Registration: UMIN000002388 and UMIN000002476).

Methylated cyclin D2 gene circulating in the blood as a prognosis predictor of hepatocellular carcinoma.

BACKGROUND Prognosis of hepatocellular carcinoma (HCC) remains poor because of high recurrence rate. We examined preoperatively the methylated CCND2 gene levels present in the serum following release from HCC cells as a prognosis predictor in patients undergoing curative hepatectomy. METHODS Quantitative real-time RT-PCR and quantitative methylation-specific PCR were used to measure methylated CCND2 gene and its mRNA levels. RESULTS The CCND2 mRNA levels were down-regulated in HCC with early intrahepatic recurrence (IHR) within 1year of curative hepatectomy. We also identified that this down-regulation was due to promoter hypermethylation. In 70 HCC patients who underwent curative hepatectomy, 39 patients sero-positive for the methylated CCND2 gene (>70pg/ml serum) exhibited a significantly shorter disease-free survival (DFS) period (P=0.02) than the 31 patients who were sero-negative for the methylated CCND2 gene. None of the sero-negative patients demonstrated early IHR, and this method of serum testing did not produce any false-negative predictions for early IHR. Multivariate analysis showed that the serum level of methylated CCND2 was an independent risk factor for DFS (hazard ratio of 1.866, 95% CI: 1.106-3.149). CONCLUSION Methylated CCND2 gene in the serum serves as a prognosis predictor of HCC after curative hepatectomy.

Divergent Promoter Organization May Be a Preferred Structure for Gene Control in Escherichia coli

The prokaryote possesses intriguing promoter arrangements called divergent promoters, which transcribe adjacent genes in opposite directions. The genome analysis in Escherichia coli revealed the occurrence of a large number of divergent promoters, whose total number was estimated to be about 1.6-fold larger than that of single promoters. Surprisingly, more than 65% divergent promoters transcribe functionally unrelated genes. Transcription from one promoter has been proposed and demonstrated to influence that from the other promoter via change in superhelicity in the divergent promoter region. Therefore, the organism may have acquired divergent promoters as a common structure for ingenious gene controls including transcription coupling.

MicroRNA-6826 and -6875 in plasma are valuable non‑invasive biomarkers that predict the efficacy of vaccine treatment against metastatic colorectal cancer

Various vaccine treatments against metastatic colorectal cancer have been developed and applied. However, to improve the efficacy of immunotherapy, biomarkers that can predict the effects are needed. It has been reported that various microRNAs (miRNAs) in peripheral blood may be useful as non-invasive biomarkers. In this study, miRNAs influencing the efficacy of vaccine treatment were screened for in a microarray analysis of 13 plasma samples that were obtained from patients prior to vaccine treatment. To validate the screening results, real-time RT-PCR was performed using 93 plasma samples obtained from patients prior to vaccine treatment. Four candidate miRNAs were selected according to the results of the comprehensive analysis of miRNA expression, which were ranked using the Fisher criterion and the absolute value of the log2 ratio in the screening analysis. The validation analysis showed that in the HLA-A*2402-matched patient group (vaccine-treated group), patients with a high expression of plasma miR-6826 had a poorer prognosis than those with a low expression (P=0.048). In contrast, in the HLA-A*2402-unmatched patient group (control group), there was no difference between the patients with high or low plasma miR-6826 expression (P=0.168). Similar results were obtained in the analysis of miR-6875 (P=0.029 and P=0.754, respectively). Moreover, multivariate analysis of the Cox regression model indicated that the expression of miR-6826 was the most significant predictor for overall survival (P=0.003, hazard ratio, 3.670). In conclusion, plasma miR-6826 and miR-6875 may be predictive biomarkers for a poor response to vaccine treatment. Although further clarification is needed regarding the functions of miR-6826 and miR-6875 and their relationship to immune-related molecules, plasma miR-6826 and miR-6875 may be useful negative biomarkers for predicting the efficacy of vaccine treatment.

A novel system for predicting the toxicity of irinotecan based on statistical pattern recognition with UGT1A genotypes

To predict precisely severe toxicity of irinotecan, we evaluated the association of UGT1A variants, haplotypes and the combination of UGT1A genotypes to severe toxicity of irinotecan. UGT1A1*6 (211G>A), UGT1A1*28 (TA6>TA7), UGT1A1*60 (−3279T>G), UGT1A7 (387T>G), UGT1A7 (622T>C), and UGT1A9*1b (−118T9>T10, also named *22) were genotyped in 123 patients with metastatic colorectal cancer who had received irinotecan-based chemotherapy. Among the 123 patients, 73 were enrolled in either of two phase II studies of the FOLFIRI (leucovorin, 5-fluorouracil and irinotecan) regimen; these patients constituted the training population, which was used to construct the predicting system. The other 50 patients constituted the validation population; these 50 patients either had participated in a phase II study of irinotecan/5′-deoxy-5-fluorouridine or were among consecutive patients who received FOLFIRI therapy. This prediction system used sequential forward floating selection based on statistical pattern recognition using UGT1A genotypes, gender and age. Several UGT1A genotypes [UGT1A1*6, UGT1A7 (387T>G), UGT1A7 (622T>C) and UGT1A9*1b] were associated with the irinotecan toxicity. Among the haplotypes, haplotype-I (UGT1A1: −3279T, TA6, 211G; UGT1A7: 387T, 622T; UGT1A9: T10) and haplotype-II (UGT1A1: −3279T, TA6, 211A; UGT1A7: 387G, 622C; UGT1A9: T9) were also associated with irinotecan toxicity. Furthermore, our new system for predicting the risk of irinotecan toxicity was 83.9% accurate with the training population and 72.1% accurate with the validation population. Our novel prediction system using statistical pattern recognition depend on genotypes in UGT1A, age and gender; moreover, it showed high predictive performance even though the treatment regimens differed among the training and validation patients.