Tokuzo Arao

Kinki University, Ōsaka, Ōsaka, Japan

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Publications (100)542.8 Total impact

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    ABSTRACT: An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as "imaging biomarkers", with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 µm2 and (2) nucleus area larger than 90 µm2 were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 µm2 were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers-cluster area, nuclei area, nuclei number, and ratio of perimeter-can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.
    PLoS ONE 08/2014; 9(8):e104372. DOI:10.1371/journal.pone.0104372 · 3.53 Impact Factor
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    ABSTRACT: Chromosomal band 11q13 seems to be one of the most frequently amplified lesions in human cancer, including esophageal squamous cell cancer (ESCC). The oral cancer overexpressed 1 (ORAOV1) gene has been identified within this region, but its detailed biological function in human ESCC remains largely unclear. In our clinical samples of stage III ESCC, ORAOV1 amplification was observed in 49 of 94 cases (53%). ORAOV1 amplification was significantly associated with a poorly differentiated histology and tumors located in the upper or middle esophagus. Patients with ORAOV1 amplification tended to have a shorter survival period, although the difference was not significant. To investigate the function of ORAOV1, we created ORAOV1--overexpressed ESCC cell lines that exhibited increased cellular proliferation and colony formation, compared with in vitro controls. In vivo, ORAOV1-overexpressed cells exhibited a significantly increased tumorigenicity and a significantly larger tumor volume and poorer differentiation than controls. The peptide mass fingerprinting technique demonstrated that ORAOV1 bound to pyrroline-5-carboxylate reductase (PYCR), which is associated with proline metabolism and reactive oxygen species (ROS) production. Then, ORAOV1-overexpressed cell lines were resistant to stress treatment, which was cancelled by PYCR-knockdown. In addition, the ORAOV1-overexpressed cell line had a higher intracellular proline concentration and a lower ROS level. Our findings indicate that the ORAOV1 gene is frequently amplified in ESCC, enhances tumorigenicity and tumor growth, and is associated with a poorly differentiated tumor histology via proline metabolism and ROS production. ORAOV1 could be a novel target for the treatment of ESCC.
    Oncotarget 05/2014; 5(10):2962-73. · 6.63 Impact Factor
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    ABSTRACT: Molecular markers for predicting or monitoring the efficacy of bevacizumab in patients with metastatic colorectal cancer (mCRC) remain to be identified. We have now measured the serum concentrations of 25 angiogenesis-related molecules with antibody suspension bead array systems for 25 mCRC patients both before and during treatment in a previously reported phase II trial of FOLFIRI chemotherapy plus bevacizumab. The serum concentration of vascular endothelial growth factor-A (VEGF-A) decreased after the onset of treatment (P < 0.0001), whereas that of placental growth factor increased (P < 0.0001). Significant differences in the levels of several factors (such as VEGF-A, soluble VEGF receptor-2, and interleukin-8) were apparent between responders and nonresponders during treatment. The rapid and pronounced decrease in serum VEGF-A level after treatment onset was apparent in all subjects and was independent of the baseline concentration. However, four of nine nonresponders showed a subsequent early increase in the serum VEGF-A level. Our results thus suggest that an early increase in the serum VEGF-A concentration after the initial decrease is a potential predictive marker of a poor response and reactive resistance to bevacizumab plus chemotherapy.
    Oncotarget 03/2014; 5(9). · 6.63 Impact Factor
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    ABSTRACT: POU5F1B (POU domain class 5 transcription factor 1B), a processed pseudogene that is highly homologous to OCT4, was recently shown to be transcribed in cancer cells, but its clinical relevance and biological function have remained unclear. We now show that POU5F1B, which is located adjacent to MYC on human chromosome 8q24, is frequently amplified in gastric cancer (GC) cell lines. POU5F1B, but not OCT4, was also found to be expressed at a high level in GC cell lines and clinical specimens. In addition, the DNA copy number and mRNA abundance for POU5F1B showed a positive correlation in both cancer cell lines and GC specimens. Overexpression of POU5F1B in GC cells promoted colony formation in vitro as well as both tumorigenicity and tumor growth in vivo, and these effects were enhanced in the additional presence of MYC overexpression. Furthermore, knockdown of POU5F1B expression with a short hairpin RNA confirmed a role for the endogenous pseudogene in the promotion of cancer cell growth in vitro and tumor growth in vivo. POU5F1B overexpression induced upregulation of various growth factors in GC cells as well as exhibited mitogenic, angiogenic and antiapoptotic effects in GC xenografts. Finally, amplification of POU5F1B was detected in 17 (12%) of 145 cases of GC and was a significant predictor of poor prognosis in patients with stage IV disease. In conclusion, we found that the POU5F1B pseudogene is amplified and expressed at a high level in, as well as confers an aggressive phenotype on, GC, and that POU5F1B amplification is associated with a poor prognosis in GC patients.Oncogene advance online publication, 23 December 2013; doi:10.1038/onc.2013.547.
    Oncogene 12/2013; 34(2). DOI:10.1038/onc.2013.547 · 8.56 Impact Factor
  • Annals of Oncology 11/2013; 24(suppl 9):ix60-ix60. DOI:10.1093/annonc/mdt459.131 · 6.58 Impact Factor
  • Annals of Oncology; 11/2013
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    ABSTRACT: The heparan sulfate sulfotransferase gene family catalyzes the transfer of sulfate groups to heparan sulfate and regulates various growth factor-receptor signaling pathways. However, the involvement of this gene family in cancer biology has not been elucidated. It was demonstrated that the heparan sulfate D-glucosaminyl 6-O-sulfotransferase-2 (HS6ST2) gene is overexpressed in colorectal cancer (CRC) and its clinical significance in patients with CRC was investigated. The mRNA levels of HS6ST2 in clinical CRC samples and various cancer cell lines were assessed using a microarray analysis and quantitative RT-PCR, respectively. An immunohistochemical (IHC) analysis of the HS6ST2 protein was performed using 102 surgical specimens of CRC. The correlations between the HS6ST2 expression status and clinicopathological characteristics were then evaluated. HS6ST2 mRNA was significantly overexpressed by 37-fold in CRC samples compared to paired colonic mucosa. High levels of HS6ST2 mRNA expression were also observed in colorectal, esophageal and lung cancer cell lines. The IHC analysis demonstrated that HS6ST2 was expressed in the cytoplasmic region of CRC cells, but not in normal colonic mucosal cells. Positive staining for HS6ST2 was detected in 40 patients (39.2%). There was no significant association between the clinicopathological characteristics and HS6ST2 expression. However, positive staining for HS6ST2 was associated with a poor survival (P=0.074, log-rank test). In conclusion, HS6ST2 was found to be overexpressed in CRC and its expression tended to be a poor prognostic factor, although the correlation was not significant. These findings indicate that HS6ST2 may be a novel cancer-related marker that may provide insight into the glycobiology of CRC.
    Molecular and Clinical Oncology 09/2013; 1(5):845-850. DOI:10.3892/mco.2013.151
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    ABSTRACT: The biological basis for cancer of unknown primary (CUP) at the molecular level remains largely unknown, with no evidence of whether a common biological entity exists. Here, we assessed the possibility of identifying a common diagnostic biomarker for CUP using a microarray gene expression analysis. Tumor mRNA samples from 60 patients with CUP were analyzed using the Affymetrix U133A Plus 2.0 GeneChip and were normalized by asinh (hyperbolic arc sine) transformation to construct a mean gene-expression profile specific to CUP. A gene-expression profile specific to non-CUP group was constructed using publicly available raw microarray datasets. The t-tests were performed to compare the CUP with non-CUP groups and the top 59 CUP specific genes with the highest fold change were selected (p-value<0.001). Among the 44 genes that were up-regulated in the CUP group, 6 genes for ribosomal proteins were identified. Two of these genes (RPS7 and RPL11) are known to be involved in the Mdm2-p53 pathway. We also identified several genes related to metastasis and apoptosis, suggesting a biological attribute of CUP. The protein products of the up-regulated and down-regulated genes identified in this study may be clinically useful as unique biomarkers for CUP.
    PLoS ONE 05/2013; 8(5):e63249. DOI:10.1371/journal.pone.0063249 · 3.53 Impact Factor
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    ABSTRACT: Molecular targeted therapy is expected to be a promising therapeutic approach for the treatment of esophageal squamous cell carcinoma (ESCC); however, the gene amplification status of molecular targeted genes in ESCC remains largely unclear. The gene amplification of EGFR, HER2, FGFR2 and MET was examined using a real-time PCR-based copy number assay of 245 ESCC surgical specimens of formalin-fixed, paraffin-embedded samples. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization analyses verified the results of the copy number assay. EGFR mutation was detected using the Scorpions-ARMS method. The EGFR status and drug sensitivity to an EGFR tyrosine kinase inhibitor was then evaluated in vitro. Gene amplification of EGFR and HER2 was observed in 7% (16/244) and 11% (27/245) of the ESCC specimens. A multivariate analysis revealed that HER2 amplification was a significant predictor of a poor prognosis in patients with stage III post-operative ESCC. The L861Q type of EGFR mutation with hypersensitivity to EGFR tyrosine kinase inhibitor was found in one of the eight ESCC cell lines and one del745 type of EGFR mutation was identified in 107 clinical samples. In addition, we demonstrated for the first time that FGFR2 amplification was observed in 4% (8/196) of the ESCC specimens. MET amplification was observed in 1% (2/196). In conclusion, the frequent gene amplification of EGFR, HER2 and FGFR2 and the presence of active EGFR mutations were observed in ESCC specimens. Our results strongly encourage the development of molecular targeted therapy for ESCC.
    International Journal of Oncology 04/2013; 42(4):1151-8. DOI:10.3892/ijo.2013.1830 · 2.77 Impact Factor
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    ABSTRACT: Transcription factor Slug/SNAI2 (snail homolog 2) plays a key role in the induction of the epithelial mesenchymal transition in cancer cells; however, whether the overexpression of Slug mediates the malignant phenotype and alters drug sensitivity in lung cancer cells remains largely unclear. We investigated Slug focusing on its biological function and involvement in drug sensitivity in lung cancer cells. Stable Slug transfectants showed typical morphological changes compared with control cells. Slug overexpression did not change the cellular proliferations; however, migration activity and anchorage-independent growth activity with an antiapoptotic effect were increased. Interestingly, stable Slug overexpression increased drug sensitivity to tubulin-binding agents including vinorelbine, vincristine, and paclitaxel (5.8- to 8.9-fold increase) in several lung cancer cell lines but did not increase sensitivity to agents other than tubulin-binding agents. Real-time RT-PCR (polymerase chain reaction) and western blotting revealed that Slug overexpression downregulated the expression of βIII and βIVa-tubulin, which is considered to be a major factor determining sensitivity to tubulin-binding agents. A luciferase reporter assay confirmed that Slug suppressed the promoter activity of βIVa-tubulin at a transcriptional level. Slug overexpression enhanced tumor growth, whereas Slug overexpression increased drug sensitivity to vinorelbine with the downregulation of βIII and βIV-tubulin in vivo. Immunohistochemistry of Slug with clinical lung cancer samples showed that Slug overexpression tended to be involved in response to tubulin-binding agents. In conclusion, our data indicate that Slug mediates an aggressive phenotype including enhanced migration activity, anoikis suppression, and tumor growth, but increases sensitivity to tubulin-binding agents via the downregulation of βIII and βIVa-tubulin in lung cancer cells.
    Cancer Medicine 04/2013; 2(2):144-54. DOI:10.1002/cam4.68
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    ABSTRACT: The response rate to sorafenib in hepatocellular carcinoma (HCC) is relatively low (0.7 - 3%), however, rapid and drastic tumor regression is occasionally observed. The molecular backgrounds and clinicopathological features of these responders remain largely unclear. We analyzed the clinical and molecular backgrounds of thirteen responders to sorafenib with significant tumor shrinkage in a retrospective study. A comparative genomic hybridization analysis using one frozen HCC sample from a responder demonstrated that the 11q13 region, a rare amplicon in HCC including the loci for FGF3 and FGF4, was highly amplified. A real-time PCR-based copy number assay revealed that FGF3/FGF4 amplification was observed in three of the ten HCC samples from responders in which DNA was evaluable, whereas amplification was not observed in 38 patients with stable or progressive disease (p=0.006). A fluorescence in situ hybridization analysis confirmed FGF3 amplification. In addition, the clinicopathological features showed that multiple lung metastases (5/13, p=0.006) and a poorly differentiated histological type (5/13, p=0.13) were frequently observed in responders. A growth inhibitory assay showed that only one FGF3/FGF4-amplified and three FGFR2-amplified cancer cell lines exhibited hyper-sensitivity to sorafenib in vitro. Finally, an in vivo study revealed that treatment with a low dose of sorafenib was partially effective for stably and exogenously expressed FGF4 tumors, while being less effective in tumors expressing EGFP or FGF3. Conclusion: FGF3/FGF4 amplification was observed in around 2% of HCCs. Although the sample size was relatively small, FGF3/FGF4 amplification, a poorly differentiated histological type, and multiple lung metastases were frequently observed in responders to sorafenib. Our findings may provide a novel insight into the molecular background of HCC and sorafenib responders, warranting further prospective biomarker studies. (HEPATOLOGY 2012.).
    Hepatology 04/2013; 57(4). DOI:10.1002/hep.25956 · 11.19 Impact Factor
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    ABSTRACT: Previously, we have shown that an AP-1 family member, FRA-2, is constitutively expressed in adult T-cell leukemia/lymphoma (ATL) and, together with JUND, upregulates CCR4 and promotes ATL cell growth. Among the identified potential target genes of FRA-2/JUND was SOX4. Here, we examine the expression and function of SOX4 in ATL. SOX4 was indeed consistently expressed in primary ATL cells. FRA-2/JUND efficiently activated the SOX4 promoter via an AP-1 site. Knockdown of SOX4 expression by small interfering RNA (siRNA) strongly suppressed cell growth of ATL cell lines. Microarray analyses revealed that SOX4 knockdown reduced the expression of genes such as germinal center kinase related (GCKR), NAK-associated protein 1 (NAP1), and histone deacetylase 8 (HDAC8). We confirmed consistent expression of GCKR, NAP1, and HDAC8 in primary ATL cells. We also showed direct activation of the HDAC8 promoter by SOX4. Furthermore, siRNA knockdown of GCKR, NAP1, and HDAC8 each significantly suppressed cell growth of ATL cell lines. Taken together, we have revealed an important oncogenic cascade involving FRA-2/JUND and SOX4 in ATL, which leads to the expression of genes such as GCKR, NAP1, and HDAC8.
    Blood 03/2013; 121(18). DOI:10.1182/blood-2012-07-441022 · 9.78 Impact Factor
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    ABSTRACT: The gene amplification of ribosomal protein S6 kinase 1 and 2 (S6K1 and S6K2) and its clinical relevance in gastric cancer remain unclear. A comparative genomic hybridization analysis and DNA copy number assay were performed for nine cancer cell lines. The gene amplification of S6K1 and S6K2 were determined using a DNA copy number assay of 213 gastric cancer tissues. S6K1 and S6K2 amplifications were observed in one and three cancer cell lines, respectively. No amplification of S6K1 was detected in the gastric cancer tissues, while S6K2 amplification was observed in 4.7% of the gastric carcinoma tissues. Patients with stage IV gastric cancer whose tumors exhibited amplification had a significantly shorter overall survival. S6K2 amplification was frequently observed in gastric cancer and was related to a poor prognosis. Our findings may provide novel insight into the dysregulation of mammalian target of rapamycin signaling by S6K2 amplification in gastric cancer.
    Anticancer research 02/2013; 33(2):469-75. · 1.87 Impact Factor
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    ABSTRACT: Our aim was to investigate both the prevalence of MET amplification in gastric cancer as well as the potential of this genetic alteration to serve as a therapeutic target in gastric cancer. MET amplification was assessed by initial screening with a PCR-based copy number assay followed by confirmatory FISH analysis in formalin-fixed, paraffin-embedded specimens of gastric cancer obtained at surgery. The effects of MET tyrosine kinase inhibitors (MET-TKIs) in gastric cancer cells with or without MET amplification were also examined. The median MET copy number in 266 cases of gastric cancer was 1.7, with a range of 0.41 to 21.3. We performed FISH analysis for the 15 cases with the highest MET copy numbers. MET amplification was confirmed in the four assessable cases with a MET copy number of at least 4, whereas MET amplification was not detected in those with a gene copy number of less than 4. The prevalence of MET amplification was thus 1.5% (4 out of 266 cases). Inhibition of MET by MET-TKIs resulted in the induction of apoptosis accompanied by attenuation of downstream MET signaling in gastric cancer cell lines with MET amplification but not in those without this genetic change. MET amplification identifies a small but clinically important subgroup of gastric cancer patients who are likely to respond to MET-TKIs. Furthermore, screening with a PCR-based copy number assay is an efficient way to reduce the number of patients requiring confirmation of MET amplification by FISH analysis.
    Oncotarget 11/2012; 4(1). DOI:10.1158/1538-7445.AM2013-4657 · 6.63 Impact Factor
  • Tokuzo Arao, Kazuto Nishio
    Nippon rinsho. Japanese journal of clinical medicine 11/2012; 70 Suppl 8:27-30.
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    ABSTRACT: OTS11101 is a novel peptide vaccine that acts as an angiogenesis inhibitor by inducing cytotoxic T lymphocyte (CTL) cells that specifically target vascular endothelial cells expressing vascular endothelial growth factor (VEGF) receptor 1. We conducted a phase I study to evaluate the safety, tolerability, maximum tolerated dose, and pharmacodynamic biomarker status of this vaccine. Nine patients with advanced solid tumors received 1.0 mg, 2.0 mg, or 3.0 mg of OTS11101 subcutaneously, once a week in a 28-day cycle. Three patients experienced grade 1 injection site reactions, which were the most frequent adverse events. Grade 2 proteinuria and hypertension each occurred in one patient. As other toxicities were generally mild, the maximum tolerated dose was not reached. Furthermore, we explored the induction of specific activated CTLs, and biomarkers related to angiogenesis. A pharmacodynamics study revealed that induction of specific CTLs was observed for a dose of 2.0 and 3.0 mg. The serum concentrations of soluble VEGF receptor 1 and 2 after vaccination increased significantly compared with baseline. A microarray was performed to give a comprehensive analysis of gene expression, suggesting that OTS11101 vaccination resulted in T cell activation in a clinical setting. In conclusion, OTS11101 was well tolerated in patients up to 3.0 mg once weekly and our biomarker analysis suggested that this anti-angiogenesis vaccine is biologically active.
    Cancer Science 09/2012; 104(1). DOI:10.1111/cas.12034 · 3.53 Impact Factor
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    ABSTRACT: Targeting of tumor angiogenesis with vaccines is a potentially valuable approach to cancer treatment. Elpamotide is an immunogenic peptide derived from vascular endothelial growth factor receptor 2, which is expressed at a high level in vascular endothelial cells. We have now carried out a phase I study to evaluate safety, the maximum tolerated dose, and potential pharmacodynamic biomarkers for this vaccine. Ten HLA-A*24:02-positive patients with advanced refractory solid tumors received elpamotide s.c. at dose levels of 0.5, 1.0, or 2.0 mg once a week on a 28-day cycle. Five patients experienced an injection site reaction of grade 1 and 2, which was the most frequent adverse event. In the 1.0 mg cohort, one patient experienced proteinuria of grade 1 and another patient developed both hypertension and proteinuria of grade 1. No adverse events of grade 3 or higher were observed, and the maximum tolerated dose was therefore not achieved. The serum concentration of soluble vascular endothelial growth factor receptor 2 decreased significantly after elpamotide vaccination. Microarray analysis of gene expression in PBMCs indicated that several pathways related to T cell function and angiogenesis were affected by elpamotide vaccination, supporting the notion that this peptide induces an immune response that targets angiogenesis in the clinical setting. In conclusion, elpamotide is well tolerated and our biomarker analysis indicates that this anti-angiogenic vaccine is biologically active. Clinical trial registration no. UMIN000008336.
    Cancer Science 09/2012; DOI:10.1111/cas.12014 · 3.53 Impact Factor
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    ABSTRACT: : Approximately 50% of lung cancer patients with epidermal growth factor receptor (EGFR)-mutations (deletion in exon 19 or L858R) who develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) reportedly carry a secondary EGFR T790M mutation. This mutation has been suggested to be present in tumor cells before EGFR-TKI treatment in a small population of individuals. Here, we use a highly sensitive colony hybridization technique in an attempt to evaluate the actual incidence of T790M in pretreatment tumor specimens. : DNA was extracted from surgically resected tumor tissues of 38 patients with the EGFR mutation and examined for the presence of T790M, using a standard polymerase chain reaction based method followed by a modified colony hybridization (CH) technique with an analytical sensitivity of approximately 0.01%. Associations between the T790M status and clinical characteristics including time to treatment failure (TTF) for EGFR-TKI were evaluated. : The T790M mutation analysis of the specimens from the 38 patients detected 30 mutants (79%). The median TTF was 9 months for the patients with pretreatment T790M and 7 months for the patients without the T790M mutation (p = 0.44). When the patients with T790M were divided into strongly positive and modestly positive subgroups in terms of the frequency of positive signals observed using CH technique, the 7 patients with strong positivity had a TTF that was significantly longer than that of the 8 patients without T790M (p = 0.0097) and of the 23 patients with modest positivity (p = 0.0019). : Our highly sensitive CH method showed that a subgroup of non-small-cell lung cancer patients with the EGFR mutation harbored the rare T790M allele before EGFR-TKI treatment. A high proportion of T790M allele may define a clinical subset with a relatively favorable prognosis.
    Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 08/2012; 7(11):1640-4. DOI:10.1097/JTO.0b013e3182653d7f · 5.80 Impact Factor
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    ABSTRACT: Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.
    PLoS ONE 07/2012; 7(7):e41312. DOI:10.1371/journal.pone.0041312 · 3.53 Impact Factor
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    ABSTRACT: Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.
    PLoS ONE 07/2012; 7(7):e41017. DOI:10.1371/journal.pone.0041017 · 3.53 Impact Factor

Publication Stats

1k Citations
542.80 Total Impact Points

Institutions

  • 2007–2014
    • Kinki University
      • Faculty of Medicine
      Ōsaka, Ōsaka, Japan
  • 2010
    • Kanazawa University
      • Department of Respiratory Medicine
      Kanazawa, Ishikawa, Japan
  • 2005–2006
    • National Hospital Organization Kyushu Cancer Center
      Hukuoka, Fukuoka, Japan
    • National Cancer Center, Japan
      Edo, Tōkyō, Japan