Yoshihiko Fujita

Publications (25)89.52 Total impact

• Article: Gene amplification of EGFR, HER2, FGFR2 and MET in esophageal squamous cell carcinoma.

  Hiroaki Kato, Tokuzo Arao, Kazuko Matsumoto, Yoshihiko Fujita, Hideharu Kimura, Hidetoshi Hayashi, Kouhei Nishiki, Mitsuru Iwama, Osamu Shiraishi, Atsushi Yasuda, Masayuki Shinkai, Motohiro Imano, Haruhiko Imamoto, Takushi Yasuda, Kiyotaka Okuno, Hitoshi Shiozaki, Kazuto Nishio
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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. · 2.40 Impact Factor
• Article: Slug increases sensitivity to tubulin-binding agents via the downregulation of βIII and βIVa-tubulin in lung cancer cells.

  Daisuke Tamura, Tokuzo Arao, Tomoyuki Nagai, Hiroyasu Kaneda, Keiichi Aomatsu, Yoshihiko Fujita, Kazuko Matsumoto, Marco A De Velasco, Hiroaki Kato, Hidetoshi Hayashi, Shuhei Yoshida, Hideharu Kimura, Yoshimasa Maniwa, Wataru Nishio, Yasuhiro Sakai, Chiho Ohbayashi, Yoshikazu Kotani, Yoshihiro Nishimura, Kazuto Nishio
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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.
• Article: Highly Sensitive Detection of EGFR T790M Mutation Using Colony Hybridization Predicts Favorable Prognosis of Patients with Lung Cancer Harboring Activating EGFR Mutation.

  Yoshihiko Fujita, Kenichi Suda, Hideharu Kimura, Kazuko Matsumoto, Tokuzo Arao, Tomoyuki Nagai, Nagahiro Saijo, Yasushi Yatabe, Tetsuya Mitsudomi, Kazuto Nishio
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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. · 4.55 Impact Factor
• Article: Expression changes in arrestin β 1 and genetic variation in catechol-O-methyltransferase are biomarkers for the response to morphine treatment in cancer patients.

  Hiromichi Matsuoka, Tokuzo Arao, Chihiro Makimura, Masayuki Takeda, Hidemi Kiyota, Junji Tsurutani, Yoshihiko Fujita, Kazuko Matsumoto, Hideharu Kimura, Masatomo Otsuka, Atsuko Koyama, Chiyo K Imamura, Yusuke Tanigawara, Takeharu Yamanaka, Kyoko Tanaka, Kazuto Nishio, Kazuhiko Nakagawa
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  ABSTRACT: Genetic differences in individuals with regard to opioid-receptor signaling create clinical difficulties for opioid treatment; consequently, useful pharmacodynamic and predictive biomarkers are needed. In this prospective study, we studied gene expression changes in peripheral blood leukocytes using a microarray and real-time RT-PCR analysis to identify pharmacodynamic biomarkers for monitoring the effect of morphine in a cohort of opioid-treatment-naïve cancer patients. We also examined genetic variations in opioid receptor mu 1 (OPRM1, 118A→G) and catechol-O-methyltransferase (COMT, 472G→A) to evaluate predictive biomarkers of the treatment outcome of morphine. The plasma concentration of morphine was measured using a liquid chromatography-tandem mass spectrometry method. Microarray analysis revealed that the mRNA expression levels of arrestin β 1 (ARRB1) were significantly down-regulated by morphine treatment. Real-time RT-PCR analysis against independent samples confirmed the results (P=0.003) and changes during treatment were negatively correlated with the plasma morphine concentration (R=-0.42). No correlation was observed between the genotype of OPRM1 and morphine treatment; however, the plasma concentration of morphine and the required dose of morphine were significantly lower for the A/A genotype of COMT (vs. A/G+G/G, P=0.008 and 0.03). We found that changes in the expression of ARRB1 may be a novel pharmacodynamic biomarker and the COMT 472G→A genotype may be a predictive biomarker of the response to morphine treatment.
  Oncology Reports 05/2012; 27(5):1393-9. · 1.84 Impact Factor
• Article: A novel mass spectrometry-based assay for diagnosis of EML4-ALK-positive non-small cell lung cancer.

  Kazuko Sakai, Isamu Okamoto, Ken Takezawa, Tomonori Hirashima, Hiroyasu Kaneda, Masayuki Takeda, Kazuko Matsumoto, Hideharu Kimura, Yoshihiko Fujita, Kazuhiko Nakagawa, Tokuzo Arao, Kazuto Nishio
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  ABSTRACT: The presence of the transforming fusion gene echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) in non-small-cell lung cancer (NSCLC) is a predictive marker for the efficacy of anaplastic lymphoma kinase inhibitors. However, the currently available assays for the detection of the different variants of EML4-ALK have limitations. We developed an assay system for the detection of EML4-ALK variants 1, 2, 3a, 3b, 4, 5a, 5b, 6, or 7 transcripts in total RNA obtained from formalin-fixed, paraffin-embedded (FFPE) specimens of NSCLC tissue. The assay is based on region-specific polymerase chain reaction amplification of EML4-ALK complementary DNA followed by specific single-base primer extension and analysis of the extension products by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The assay was validated by fluorescence in situ hybridization and the results confirmed by subcloning and sequencing of polymerase chain reaction products. Evaluation of the analytic sensitivity of the assay with serial dilutions of plasmids containing EML4-ALK complementary DNA sequences revealed it to be capable of the reliable detection of one copy of each plasmid per reaction. The assay also detected EML4-ALK variants 1 or 3 in three FFPE samples of surgically resected NSCLC shown to be positive for anaplastic lymphoma kinase rearrangement by fluorescence in situ hybridization. Furthermore, the assay identified variant 1 of EML4-ALK in 3 of 20 FFPE biopsy samples from patients with advanced NSCLC. All positive samples were confirmed by subcloning and sequencing. Our novel assay is highly sensitive and effective for the detection of EML4-ALK in FFPE specimens.
  Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 05/2012; 7(5):913-8. · 4.55 Impact Factor
• Article: Integrated analysis of whole genome exon array and array-comparative genomic hybridization in gastric and colorectal cancer cells.

  Kazuyuki Furuta, Tokuzo Arao, Kazuko Sakai, Hideharu Kimura, Tomoyuki Nagai, Daisuke Tamura, Keiichi Aomatsu, Kanae Kudo, Hiroyasu Kaneda, Yoshihiko Fujita, Kazuko Matsumoto, Yasuhide Yamada, Kazuyoshi Yanagihara, Masaru Sekijima, Kazuto Nishio
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  ABSTRACT: Whole genome-scale integrated analyses of exon array and array-comparative genomic hybridization are expected to enable the identification of unknown genetic features of cancer cells. Here, we evaluated this approach in 22 gastric and colorectal cancer cell lines, focusing on protein kinase genes and genes belonging to the cadherin-catenin family. Regarding alternative splicing patterns, several cancer cell lines predominantly expressed isoform 1 of protein kinase A catalytic subunit beta (PRKACB). Paired gastric cancer specimens demonstrated that isoform 1 of PRKACB was a novel cancer-related variant transcript in gastric cancers. In addition, the exon array analysis clearly identified exon 3 or exon 3-4 skipping in catenin beta 1, a short intron insertion with exon 9 skipping in CDH1, and a deletional transcript of CDH13. These abnormal transcripts were shown to have arisen from small genomic deletions. Meanwhile, an integrated analysis of 11 gastric cancer cell lines revealed that four cell lines amplified fibroblast growth factor receptor 2, with truncated forms observed in two of the cell lines. Gene amplification, and not the truncated form, was found to determine the sensitivity to a fibroblast growth factor receptor inhibitor, indicating that our cell line panel might be useful for cell-based evaluations of specific inhibitors. Using an integrated analysis, we identified several abnormal transcripts and genomic alterations in gastric and colorectal cancer cells. Our approach might enable genetic changes to be identified more efficiently, and the present results warrant further investigation using clinical samples and integrated analyses.
  Cancer Science 02/2012; 103(2):221-7. · 3.33 Impact Factor
• Article: Slug is upregulated during wound healing and regulates cellular phenotypes in corneal epithelial cells.

  Keiichi Aomatsu, Tokuzo Arao, Kosuke Abe, Aya Kodama, Koji Sugioka, Kazuko Matsumoto, Kanae Kudo, Hideharu Kimura, Yoshihiko Fujita, Hidetoshi Hayashi, Tomoyuki Nagai, Yoshikazu Shimomura, Kazuto Nishio
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  ABSTRACT: The involvement of the epithelial mesenchymal transition (EMT) in the process of corneal wound healing remains largely unclear. The purpose of the present study was to gain insight into Slug expression and corneal wound healing. Slug expression during wound healing in the murine cornea was evaluated using fluorescence staining in vivo. Slug or Snail was stably introduced into human corneal epithelial cells (HCECs). These stable transfectants were evaluated for the induction of the EMT, cellular growth, migration activity, and expression changes in differentiation-related molecules. Slug, but not Snail, was clearly expressed in the nuclei of corneal epithelial cells in basal lesion of the corneal epithelium during wound healing in vivo. The overexpression of Slug or Snail induced an EMT-like cellular morphology and cadherin switching in HCECs, indicating that these transcription factors were able to mediate the typical EMT in HCECs. The overexpression of Slug or Snail suppressed cellular proliferation but enhanced the migration activity. Furthermore, ABCG2, TP63, and keratin 19, which are known as stemness-related molecules, were downregulated in these transfectants. It was found that Slug is upregulated during corneal wound healing in vivo. The overexpression of Slug mediated a change in the cellular phenotype affecting proliferation, migration, and expression levels of differentiation-related molecules. This is the first evidence that Slug is regulated during the process of corneal wound healing in the corneal epithelium in vivo, providing a novel insight into the EMT and Slug expression in corneal wound healing.
  Investigative ophthalmology & visual science 02/2012; 53(2):751-6. · 3.43 Impact Factor
• Article: SRPX2 is a novel chondroitin sulfate proteoglycan that is overexpressed in gastrointestinal cancer.

  Kaoru Tanaka, Tokuzo Arao, Daisuke Tamura, Keiichi Aomatsu, Kazuyuki Furuta, Kazuko Matsumoto, Hiroyasu Kaneda, Kanae Kudo, Yoshihiko Fujita, Hideharu Kimura, Kazuyoshi Yanagihara, Yasuhide Yamada, Isamu Okamoto, Kazuhiko Nakagawa, Kazuto Nishio
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  ABSTRACT: SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.
  PLoS ONE 01/2012; 7(1):e27922. · 4.09 Impact Factor
• Article: Prospective study evaluating the plasma concentrations of twenty-six cytokines and response to morphine treatment in cancer patients.

  Chihiro Makimura, Tokuzo Arao, Hiromichi Matsuoka, Masayuki Takeda, Hidemi Kiyota, Junji Tsurutani, Yoshihiko Fujita, Kazuko Matsumoto, Hideharu Kimura, Masatomo Otsuka, Atsuko Koyama, Chiyo K Imamura, Takeharu Yamanaka, Kyoko Tanaka, Kazuto Nishio, Kazuhiko Nakagawa
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  ABSTRACT: Cytokine signaling is involved in pain and opioid-receptor signaling. In this prospective study, we studied the plasma cytokine levels in order to identify candidate biomarkers for predicting resistance to morphine treatment in a cohort of opioid-treatment-naïve cancer patients. We analyzed pain rating and the plasma concentrations of 26 cytokines at baseline and after morphine treatment using a multiplex immunoassay system for the following cytokines: eotaxin, colony stimulating factor, granulocyte (G-CSF), colony stimulating factor granulocyte-macrophage (GM-CSF), interferon α2 (IFN-α2), IFN-γ, interleukin 1α (IL-1α), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, tumor necrosis factor-α (TNF-α) and TNF-β. No correlation was observed between the clinical characteristics and the numerical rating scale for pain at baseline or among patients who developed resistance to morphine treatment. Interestingly, the plasma concentration of MIP-1α significantly decreased during morphine treatment (day 8 vs. baseline, p=0.03). Regarding the baseline plasma cytokine concentrations, none of the cytokine levels were correlated with the numerical rating scale for pain at baseline; however, the baseline plasma concentrations of eotaxin, IL-8, IL-12 (p40), IL-12 (p70), MIP-1α and MIP-1β were significantly lower in patients who required a high dose of morphine or who developed resistance to morphine treatment. In conclusion, this is the first report revealing that the plasma concentrations of several cytokines were significantly modulated during treatment and were correlated with treatment outcome of morphine. Our results suggest that plasma cytokine levels may be promising biomarkers for morphine treatment and that they warrant further study.
  Anticancer research 12/2011; 31(12):4561-8. · 1.73 Impact Factor
• Article: Aza-derivatives of resveratrol are potent macrophage migration inhibitory factor inhibitors.

  Yoshihiko Fujita, Rafiqul Islam, Kazuko Sakai, Hiroyasu Kaneda, Kanae Kudo, Daisuke Tamura, Keiichi Aomatsu, Tomoyuki Nagai, Hidekazu Kimura, Kazuko Matsumoto, Marco A de Velasco, Tokuzo Arao, Tadashi Okawara, Kazuto Nishio
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  ABSTRACT: Resveratrol (3, 4', 5-trihydroxy-trans-stilbene), a natural phytoalexin found in grapes and wine, has anti-proliferative activity on human-derived cancer cells. In our study, we used a conventional condensation reaction between aldehydes and amines to provide a number of aza-resveratrol (3, 4', 5-trihydroxy-trans- aza-stilbene) derivatives in an attempt to screen for compounds with resveratrol's action but with increased potency. Aza-resveratrol and its hydroxylated derivative (3, 4, 4', 5-tetrahydroxy-trans- aza-stilbene) showed a more enhanced anti-proliferative effect than resveratrol in an MCF-7 breast carcinoma cell line. To identify the cellular targets of the aza derivatives of resveratrol, we conjugated the latter aza-stilbene compound with epoxy-activated agarose and performed affinity purification. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, was identified as a major target protein in MCF-7 cell lysates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). The aza-resveratrol and its hydroxylated derivative, but not resveratrol, were also found to be potent inhibitors of MIF tautomerase activity, which may be associated with their inhibitory effects on MIF bioactivity for cell growth.
  Investigational New Drugs 09/2011; 30(5):1878-86. · 3.36 Impact Factor
• Article: Acquired drug resistance to vascular endothelial growth factor receptor 2 tyrosine kinase inhibitor in human vascular endothelial cells.

  Tokuzo Arao, Kazuko Matsumoto, Kazuyuki Furuta, Kanae Kudo, Hiroyasu Kaneda, Tomoyuki Nagai, Kazuko Sakai, Yoshihiko Fujita, Daisuke Tamura, Keiichi Aomatsu, Fumiaki Koizumi, Kazuto Nishio
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  ABSTRACT: Acquired resistance to antiangiogenic drugs has emerged as a potentially important issue in clinical settings; however, the underlying molecular and cellular mechanism of resistance to vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitor (TKI) remains largely unclear. We evaluated the cellular characteristics of human umbilical vein endothelial cell (HUVEC) clones, which are resistant to VEGFR2-TKI (Ki8751) to elucidate this mechanism of resistance to antiangiogenic drugs. Resistant HUVEC clones were 10-fold more resistant to VEGFR2-TKI than the parental cells and they exhibited an almost complete absence of VEGF-mediated cellular proliferation. The mRNA expression analysis revealed that expression of VEGFR1, VEGFR2 and VEGFR3 was lower in resistant clones, while that of several angiogenic ligands was increased. The protein expression of VEGFR2 was markedly down-regulated in two (R5 and R6 clone) out of five resistant clones. Focusing on the R5 clone, VEGF stimulation did not increase the phosphorylation of VEGFR2 or the dimerization of VEGFR2. The inhibition of phospho-AKT by VEGFR2-TKI was also weakened more than 10-fold in the R5 clone. Finally, a microarray analysis revealed that some angiogenesis-associated, and some angiogenesis-specific genes, including platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31, homeobox A9 (HOXA9), and endothelial cell-specific molecule 1 (ESM1), were remarkably down-regulated in all the resistant clones compared with the parental cells. HUVEC clones resistant to VEGFR2-TKI exhibited down-regulation of VEGFR2, a decreased signal response to VEGF stimulation, and the loss of vascular endothelial markers. These results strongly suggest that an escape from VEGFR2 signaling-dependency is one of the cellular mechanisms of resistance to VEGFR2-TKI in vascular endothelial cells.
  Anticancer research 09/2011; 31(9):2787-96. · 1.73 Impact Factor
• Article: An early event of EGFR mutation in pleomorphic carcinoma of the lung.

  Miyako Saitoh, Mafumi Niijima, Yuichi Takiguchi, Kenzo Hiroshima, Yoshihiko Fujita, Kazuto Nishio, Koichiro Tatsumi
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  ABSTRACT: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are effective in prolonging progression-free survival time of patients with non-small cell lung carcinoma, typically adenocarcinoma, bearing some active EGFR mutations in their tumors. However, the close relationship between the EGFR mutations and pleomorphic carcinoma of the lung, which is a very rare type of primary lung cancer, has never been elucidated. We present a 60-year-old Japanese woman with pleomorphic carcinoma of the lung that became resistant to cytotoxic chemotherapies including platinum-based chemotherapy, and her general condition seriously deteriorated. Thereafter, treatment with gefitinib was started and resulted in significant tumor shrinkage and a dramatic improvement in her general condition for up to 8.5 months. Analyses of the EGFR mutation in separately microdissected specimens from adenocarcinoma and spindle cell components revealed that both components possessed the L858R point mutation. These findings gave us some insight into the carcinogenesis of pleomorphic carcinoma of the lung in relation to EGFR gene alteration. Testing for EGFR mutation may be important in patients with advanced pleomorphic carcinoma including adenocarcinoma component that is usually chemoresistant.
  International Journal of Clinical Oncology 05/2011; 16(6):770-3. · 1.41 Impact Factor
• Article: Sorafenib inhibits the hepatocyte growth factor-mediated epithelial mesenchymal transition in hepatocellular carcinoma.

  Tomoyuki Nagai, Tokuzo Arao, Kazuyuki Furuta, Kazuko Sakai, Kanae Kudo, Hiroyasu Kaneda, Daisuke Tamura, Keiichi Aomatsu, Hideharu Kimura, Yoshihiko Fujita, Kazuko Matsumoto, Nagahiro Saijo, Masatoshi Kudo, Kazuto Nishio
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  ABSTRACT: The epithelial mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Sorafenib, a VEGFR inhibitor with activity against RAF kinase, is active against hepatocellular carcinoma (HCC); however, the possible involvement of sorafenib in the EMT remains unclear. Here, we examined the effect of sorafenib on the EMT. Hepatocyte growth factor (HGF) induced EMT-like morphologic changes and the upregulation of SNAI1 and N-cadherin expression. The downregulation of E-cadherin expression in HepG2 and Huh7 HCC cell lines shows that HGF mediates the EMT in HCC. The knockdown of SNAI1 using siRNA canceled the HGF-mediated morphologic changes and cadherin switching, indicating that SNAI1 is required for the HGF-mediated EMT in HCC. Interestingly, sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced morphologic changes, SNAI1 upregulation, and cadherin switching, whereas the PI3 kinase inhibitor wortmannin did not. Collectively, these findings indicate that sorafenib downregulates SNAI1 expression by inhibiting mitogen-activated protein kinase (MAPK) signaling, thereby inhibiting the EMT in HCC cells. In fact, a wound healing and migration assay revealed that sorafenib completely canceled the HGF-mediated cellular migration in HCC cells. In conclusion, we found that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in HCC. Our findings may provide a novel insight into the anti-EMT effect of tyrosine kinase inhibitors in cancer cells.
  Molecular Cancer Therapeutics 01/2011; 10(1):169-77. · 5.23 Impact Factor
• Article: TGF-β induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways in a human corneal epithelial cell line.

  Keiichi Aomatsu, Tokuzo Arao, Koji Sugioka, Kazuko Matsumoto, Daisuke Tamura, Kanae Kudo, Hiroyasu Kaneda, Kaoru Tanaka, Yoshihiko Fujita, Yoshikazu Shimomura, Kazuto Nishio
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  ABSTRACT: The aim of this study was to investigate the expression changes of epithelial mesenchymal transition (EMT)-related molecules induced by TGF-β signaling in a human corneal epithelial cell line (HCECs). The cellular response to TGF-β was evaluated by immunoblotting, quantitative real-time RT-PCR, and immunofluorescence microscopy in HCECs. TGF-β significantly increased mRNA expression of SNAI1, SNAI2, VIM, and FN1, but not TWIST1 through Smad and non-Smad pathways in HCECs. Protein expression of a mesenchymal marker N-cadherin was dose-dependently increased and that of an epithelial marker of E-cadherin was decreased by TGF-β. TGF-β, but not EGF, mediated the EMT-like morphologic changes. Both TGF-β and EGF were capable of upregulating SNAI1 and SNAI2 by about two-fold within a short response time. However, a detailed time course analysis revealed drastically different expression patterns, with TGF-β mediating a sustained upregulation of SNAI1 and SNAI2 for at least for 6 days and EGF allowing a return to the baseline expression values after 8 ∼ 12 h. These data indicate that TGF-β, but not EGF, induces sustained upregulation of SNAI1 and SNAI2 in HCECs. TGF-β induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways, EMT-like morphologic changes, downregulation of E-cadherin, and upregulation of N-cadherin in HCECs. The authors' findings provide insight into the TGF-β signaling and the temporal expression patterns of EMT-inducible transcription factors in HCECs.
  Investigative ophthalmology & visual science 12/2010; 52(5):2437-43. · 3.43 Impact Factor
• Article: Antitumor activity of BIBF 1120, a triple angiokinase inhibitor, and use of VEGFR2+pTyr+ peripheral blood leukocytes as a pharmacodynamic biomarker in vivo.

  Kanae Kudo, Tokuzo Arao, Kaoru Tanaka, Tomoyuki Nagai, Kazuyuki Furuta, Kazuko Sakai, Hiroyasu Kaneda, Kazuko Matsumoto, Daisuke Tamura, Keiichi Aomatsu, Marco A De Velasco, Yoshihiko Fujita, Nagahiro Saijo, Masatoshi Kudo, Kazuto Nishio
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  ABSTRACT: BIBF 1120 is a potent, orally available triple angiokinase inhibitor that inhibits VEGF receptors (VEGFR) 1, 2, and 3, fibroblast growth factor receptors, and platelet-derived growth factor receptors. This study examined the antitumor effects of BIBF 1120 on hepatocellular carcinoma (HCC) and attempted to identify a pharmacodynamic biomarker for use in early clinical trials. We evaluated the antitumor and antiangiogenic effects of BIBF 1120 against HCC cell line both in vitro and in vivo. For the pharmacodynamic study, the phosphorylation levels of VEGFR2 in VEGF-stimulated peripheral blood leukocytes (PBL) were evaluated in mice inoculated with HCC cells and treated with BIBF 1120. BIBF 1120 (0.01 μmol/L) clearly inhibited the VEGFR2 signaling in vitro. The direct growth inhibitory effects of BIBF 1120 on four HCC cell lines were relatively mild in vitro (IC(50) values: 2-5 μmol/L); however, the oral administration of BIBF 1120 (50 or 100 mg/kg/d) significantly inhibited the tumor growth and angiogenesis in a HepG2 xenograft model. A flow cytometric analysis revealed that BIBF 1120 significantly decreased the phosphotyrosine (pTyr) levels of VEGFR2(+)CD45(dim) PBLs and the percentage of VEGFR2(+)pTyr(+) PBLs in vivo; the latter parameter seemed to be a more feasible pharmacodynamic biomarker. We found that BIBF 1120 exhibited potent antitumor and antiangiogenic activity against HCC and identified VEGFR2(+)pTyr(+) PBLs as a feasible and noninvasive pharmacodynamic biomarker in vivo.
  Clinical Cancer Research 12/2010; 17(6):1373-81. · 7.74 Impact Factor
• Article: De novo resistance to epidermal growth factor receptor-tyrosine kinase inhibitors in EGFR mutation-positive patients with non-small cell lung cancer.

  Masayuki Takeda, Isamu Okamoto, Yoshihiko Fujita, Tokuzo Arao, Hiroyuki Ito, Masahiro Fukuoka, Kazuto Nishio, Kazuhiko Nakagawa
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  ABSTRACT: Somatic mutations in the epidermal growth factor receptor (EGFR) gene are a predictor of response to treatment with EGFR tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). However, mechanisms of de novo resistance to these drugs in patients harboring EGFR mutations have remained unclear. We examined whether the mutational status of KRAS might be associated with primary resistance to EGFR-TKIs in EGFR mutation-positive patients with NSCLC. Forty patients with NSCLC with EGFR mutations who were treated with gefitinib or erlotinib and had archival tissue specimens available were enrolled in the study. KRAS mutations were analyzed by direct sequencing. Three (7.5%) of the 40 patients had progressive disease, and two (67%) of these three individuals had both KRAS and EGFR mutations. Our results suggest that KRAS mutation is a negative predictor of response to EGFR-TKIs in EGFR mutation-positive patients with NSCLC.
  Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 03/2010; 5(3):399-400. · 4.55 Impact Factor
• Article: Bortezomib potentially inhibits cellular growth of vascular endothelial cells through suppression of G2/M transition.

  Daisuke Tamura, Tokuzo Arao, Kaoru Tanaka, Hiroyasu Kaneda, Kazuko Matsumoto, Kanae Kudo, Keiichi Aomatsu, Yoshihiko Fujita, Takashi Watanabe, Nagahiro Saijo, Yoshikazu Kotani, Yoshihiro Nishimura, Kazuto Nishio
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  ABSTRACT: Bortezomib, a selective 26S proteasome inhibitor, has shown clinical benefits against refractory multiple myeloma. The indirect anti-angiogenic activity of bortezomib has been widely recognized; however, the growth-inhibitory mechanism of bortezomib on vascular endothelial cells remains unclear, especially on the cell cycle. Here, we showed that bortezomib (2 nM of the IC(50) value) potently inhibited the cellular growth of human umbilical vascular endothelial cells (HUVECs) via a vascular endothelial growth factor receptor (VEGFR)-independent mechanism resulting in the induction of apoptosis. Bortezomib significantly increased the vascular permeability of HUVECs, whereas a VEGFR-2 tyrosine kinase inhibitor decreased it. Interestingly, a cell cycle analysis using flow cytometry, the immunostaining of phospho-histone H3, and Giemsa staining revealed that bortezomib suppressed the G2/M transition of HUVECs, whereas the mitotic inhibitor paclitaxel induced M-phase accumulation. A further analysis of cell cycle-related proteins revealed that bortezomib increased the expression levels of cyclin B1, the cdc2/cyclin B complex, and the phosphorylation of all T14, Y15, and T161 residues on cdc2. Bortezomib also increased the ubiquitination of cyclin B1 and wee1, but inhibited the kinase activity of the cdc2/cyclin B complex. These protein modifications support the concept that bortezomib suppresses the G2/M transition, rather than causing M-phase arrest. In conclusion, we demonstrated that bortezomib potently inhibits cell growth by suppressing the G2/M transition, modifying G2/M-phase-related cycle regulators, and increasing the vascular permeability of vascular endothelial cells. Our findings reveal a cell cycle-related mode of action and strongly suggest that bortezomib exerts an additional unique vascular disrupting effect as a vascular targeting drug.
  Cancer Science 03/2010; 101(6):1403-8. · 3.33 Impact Factor
• Article: FOXQ1 is overexpressed in colorectal cancer and enhances tumorigenicity and tumor growth.

  Hiroyasu Kaneda, Tokuzo Arao, Kaoru Tanaka, Daisuke Tamura, Keiichi Aomatsu, Kanae Kudo, Kazuko Sakai, Marco A De Velasco, Kazuko Matsumoto, Yoshihiko Fujita, Yasuhide Yamada, Junji Tsurutani, Isamu Okamoto, Kazuhiko Nakagawa, Kazuto Nishio
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  ABSTRACT: Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21(CIP1/WAF1) expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 +/- 301 versus 1735 +/- 769 mm3, P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family.
  Cancer Research 02/2010; 70(5):2053-63. · 7.86 Impact Factor
• Article: mTOR signal and hypoxia-inducible factor-1 alpha regulate CD133 expression in cancer cells.

  Kazuko Matsumoto, Tokuzo Arao, Kaoru Tanaka, Hiroyasu Kaneda, Kanae Kudo, Yoshihiko Fujita, Daisuke Tamura, Keiichi Aomatsu, Tomohide Tamura, Yasuhide Yamada, Nagahiro Saijo, Kazuto Nishio
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  ABSTRACT: The underlying mechanism regulating the expression of the cancer stem cell/tumor-initiating cell marker CD133/prominin-1 in cancer cells remains largely unclear, although knowledge of this mechanism would likely provide important biological information regarding cancer stem cells. Here, we found that the inhibition of mTOR signaling up-regulated CD133 expression at both the mRNA and protein levels in a CD133-overexpressing cancer cell line. This effect was canceled by a rapamycin-competitor, tacrolimus, and was not modified by conventional cytotoxic drugs. We hypothesized that hypoxia-inducible factor-1 alpha (HIF-1 alpha), a downstream molecule in the mTOR signaling pathway, might regulate CD133 expression; we therefore investigated the relation between CD133 and HIF-1 alpha. Hypoxic conditions up-regulated HIF-1 alpha expression and inversely down-regulated CD133 expression at both the mRNA and protein levels. Similarly, the HIF-1 alpha activator deferoxamine mesylate dose-dependently down-regulated CD133 expression, consistent with the effects of hypoxic conditions. Finally, the correlations between CD133 and the expressions of HIF-1 alpha and HIF-1 beta were examined using clinical gastric cancer samples. A strong inverse correlation (r = -0.68) was observed between CD133 and HIF-1 alpha, but not between CD133 and HIF-1 beta. In conclusion, these results indicate that HIF-1 alpha down-regulates CD133 expression and suggest that mTOR signaling is involved in the expression of CD133 in cancer cells. Our findings provide a novel insight into the regulatory mechanisms of CD133 expression via mTOR signaling and HIF-1 alpha in cancer cells and might lead to insights into the involvement of the mTOR signal and oxygen-sensitive intracellular pathways in the maintenance of stemness in cancer stem cells.
  Cancer Research 10/2009; 69(18):7160-4. · 7.86 Impact Factor
• Article: [Molecular diagnostic methods designed for clinical approach to cancer of unknown origin].

  Yoshihiko Fujita, Kazuto Nishio
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  ABSTRACT: Several studies have shown that patterns of gene expression remain consistent with the tissue of origin in cancer samples. Gene expression profiling may therefore offer a promising new technology to build a site of origin classifier with the ultimate aim to determine the origin of cancer of unknown primary(CUP). A single cDNA microarray platform was used to profile 229 tumors of known origin(14 tumor types). This data set was subsequently used for training and validation of a support vector machine(SVM)classifier, demonstrating 89% accuracy to predict a site of origin(13 types). Applying this microarray SVM classifier to 13 cases of CUP, a high confidence prediction was made in 11 of 13 cases. These predictions were supported by comprehensive review of the patients' clinical histories. Thus, data generated using both microarray and quantitative PCR can be used to train and validate a cross-platform SVM model with high prediction accuracy.
  Gan to kagaku ryoho. Cancer & chemotherapy 07/2009; 36(6):923-6.