Hideyuki Yokote

AZD2171 Shows Potent Antitumor Activity Against Gastric Cancer Over-Expressing Fibroblast Growth Factor Receptor 2/Keratinocyte Growth Factor Receptor

Purpose: AZD2171 is an oral, highly potent, and selective vascular endothelial growth factor signaling inhibitor that inhibits all vascular endothelial growth factor receptor tyrosine kinases. The purpose of this study was to investigate the activity of AZD2171 in gastric cancer. Experimental Design: We examined the antitumor effect of AZD2171 on the eight gastric cancer cell lines in vitro and in vivo. Results: AZD2171 directly inhibited the growth of two gastric cancer cell lines (KATO-III and OCUM2M), with an IC50 of 0.15 and 0.37 μmol/L, respectively, more potently than the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib. Reverse transcription-PCR experiments and immunoblotting revealed that sensitive cell lines dominantly expressed COOH terminus–truncated fibroblast growth factor receptor 2 (FGFR2) splicing variants that were constitutively phosphorylated and spontaneously dimerized. AZD2171 completely inhibited the phosphorylation of FGFR2 and downstream signaling proteins (FRS2, AKT, and mitogen-activated protein kinase) in sensitive cell lines at a 10-fold lower concentration (0.1 μmol/L) than in the other cell lines. An in vitro kinase assay showed that AZD2171 inhibited kinase activity of immunoprecipitated FGFR2 with submicromolar Ki values (∼0.05 μmol/L). Finally, we assessed the antitumor activity of AZD2171 in human gastric tumor xenograft models in mice. Oral administration of AZD2171 (1.5 or 6 mg/kg/d) significantly and dose-dependently inhibited tumor growth in mice bearing KATO-III and OCUM2M tumor xenografts. Conclusions: AZD2171 exerted potent antitumor activity against gastric cancer xenografts overexpressing FGFR2. The results of these preclinical studies indicate that AZD2171 may provide clinical benefit in patients with certain types of gastric cancer.

EphA4 promotes cell proliferation and migration through a novel EphA4-FGFR1 signaling pathway in the human glioma U251 cell line

The Eph receptor tyrosine kinases and their ephrin ligands form a unique cell-cell contact-mediated bidirectional signaling mechanism for regulating cell localization and organization. High expression of Eph receptors in a wide variety of human tumors indicates some roles in tumor progression, which makes these proteins potential targets for anticancer therapy. For this purpose, we did gene expression profiling for 47 surgical specimens of brain tumors including 32 high-grade glioma using a microarray technique. The analysis, focused on the receptor tyrosine kinases, showed that EphA4 mRNA in the tumors was 4-fold higher than in normal brain tissue. To investigate the biological significance of EphA4 overexpression in these tumors, we analyzed EphA4-induced phenotypic changes and the signaling mechanisms using human glioma U251 cells. EphA4 promoted fibroblast growth factor 2-mediated cell proliferation and migration accompanied with enhancement of fibroblast growth factor 2-triggered mitogen-activated protein kinase and Akt phosphorylation. In addition, active forms of Rac1 and Cdc42 increased in the EphA4-overexpressing cells. Furthermore, we found that EphA4 formed a heteroreceptor complex with fibroblast growth factor receptor 1 (FGFR1) in the cells and that the EphA4-FGFR1 complex potentiated FGFR-mediated downstream signaling. Thus, our results indicate that EphA4 plays an important role in malignant phenotypes of glioblastoma by enhancing cell proliferation and migration through accelerating a canonical FGFR signaling pathway. [Mol Cancer Ther 2008;7(9):2768–78]

Pertuzumab, a novel HER dimerization inhibitor, inhibits the growth of human lung cancer cells mediated by the HER3 signaling pathway

A humanized anti‐HER2 monoclonal antibody pertuzumab (Omnitarg, 2C4), binding to a different HER2 epitope than trastuzumab, is known as an inhibitor of heterodimerization of the HER receptors. Potent antitumor activity against HER2‐expressing breast and prostate cancer cell lines has been clarified, but this potential is not clear against lung cancers. The authors investigated the in vitro anti‐tumor activity of pertuzumab against eight non‐small cell lung cancer cells expressing various members of the HER receptors. A lung cancer 11_18 cell line expressed a large amount of HER2 and HER3, and its cell growth was stimulated by an HER3 ligand, heregulin (HRG)‐α. Pertuzumab significantly inhibited the HRG‐α‐stimulated cellular growth of the 11_18 cells. Pertuzumab blocked HRG‐α‐stimulated phosphorylation of HER3, mitogen‐activated protein kinase (MAPK), and Akt. In contrast, pertuzumab failed to block epidermal growth factor (EGF)‐stimulated phosphorylation of EGF receptor (EGFR) and MAPK. Immunoprecipitation showed that pertuzumab inhibited HRG‐α‐stimulated HER2/HER3 heterodimer formation. HRG‐α‐stimulated HER3 phosphorylation was also observed in the PC‐9 cells co‐overexpressing EGFR, HER2, and HER3, but the cell growth was neither stimulated by HRG‐α nor inhibited by pertuzumab. The present results suggest that pertuzumab is effective against HRG‐α‐dependent cell growth in lung cancer cells through inhibition of HRG‐α‐stimulated HER2/HER3 signaling. (Cancer Sci 2007; 98: 1498–1503)

SRPX2 is overexpressed in gastric cancer and promotes cellular migration and adhesion.

SRPX2 (Sushi repeat containing protein, X‐linked 2) was first identified as a downstream molecule of the E2A‐HLF fusion gene in t(17;19)‐positive leukemia cells and the biological function of this gene remains unknown. We found that SRPX2 is overexpressed in gastric cancer and the expression and clinical features showed that high mRNA expression levels were observed in patients with unfavorable outcomes using real‐time RT‐PCR. The cellular distribution of SRPX2 protein showed the secretion of SRPX2 into extracellular regions and its localization in the cytoplasm. The introduction of the SRPX2 gene into HEK293 cells did not modulate the cellular proliferative activity but did enhance the cellular migration activity, as shown using migration and scratch assays. The conditioned‐medium obtained from SRPX2‐overexpressing cells increased the cellular migration activity of a gastric cancer cell line, SNU‐16. In addition, SRPX2 protein remarkably enhanced the cellular adhesion of SNU‐16 and HSC‐39 and increased the phosphorylation levels of focal adhesion kinase (FAK), as shown using western blotting, suggesting that SRPX2 enhances cellular migration and adhesion through FAK signaling. In conclusion, the overexpression of SRPX2 enhances cellular migration and adhesion in gastric cancer cells. Here, we report that the biological functions of SRPX2 include cellular migration and adhesion to cancer cells.

Preoperative embolization of meningiomas fed by ophthalmic branch arteries

BACKGROUND The efficacy of preoperative embolization for hypervascular meningiomas mainly fed by the branches of the ophthalmic arteries was examined. CASES AND METHODS Five hypervascular meningiomas mainly fed by the branches of the ophthalmic arteries, four posterior ethmoidal arteries, one anterior falx artery, and one recurrent middle meningeal artery were embolized with Gel-foam powder, polyvinyl alcohol (PVA) particles, and/or microcoils as a preoperative treatment using a microcatheter. RESULTS Catheterization of the ophthalmic and tumor feeding artery was possible and feeding arteries and lesion embolization were effective to reduce the bleeding during surgery in all cases. In three cases, visual acuity and visual field were preserved. However, in one case, visual acuity and visual field defect appeared due to the migration of Gelfoam powder (Upjohn, Kalamazoo, MI) into the retinal artery. In another case, the retinal artery was embolized with the feeding arteries since the patient was already blind. CONCLUSION Embolization of hypervascular meningioma feeding vessels arising from the ophthalmic artery is possible and effective with preservation of vision, if embolic agents are injected gently enough not to reflux into the central retinal artery.

N-Glycan fucosylation of epidermal growth factor receptor modulates receptor activity and sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor.

The glycosylation of cell surface proteins is important for cancer biology processes such as cellular proliferation or metastasis. α1,6‐Fucosyltransferase (FUT8) transfers a fucose residue to n‐linked oligosaccharides on glycoproteins. Herein, we study the effect of fucosylation on epidermal growth factor receptor (EGFR) activity and sensitivity to an EGFR‐specific tyrosine kinase inhibitor (EGFR‐TKI). The increased fucosylation of EGFR significantly promoted EGF‐mediated cellular growth, and the decreased fucosylation by stable FUT8 knockdown weakened the growth response in HEK293 cells. The overexpression of FUT8 cells were more sensitive than the control cells to the EGFR‐TKI gefitinib, and FUT8 knockdown decreased the sensitivity to gefitinib. Finally, to examine the effects in a human cancer cell line, we constructed stable FUT8 knockdown A549 cells, and found that these cells also decreased EGF‐mediated cellular growth and were less sensitive than the control cells to gefitinib. In conclusion, we demonstrated that the modification of EGFR fucosylation affected EGF‐mediated cellular growth and sensitivity to gefitinib. Our results provide a novel insight into how the glycosylation status of a receptor may affect the sensitivity of the cell to molecular target agents. (Cancer Sci 2008; 99: 1611–1617)

Enhancement of cisplatin sensitivity in high mobility group 2 cDNA-transfected human lung cancer cells.

To elucidate the role of high mobility group 2 protein (HMG2) in cis‐diamminedichloroplatinum (II) (cisplatin, CDDP) sensitivity, we constructed a human HMG2‐transfected human non‐small cell lung cancer cell line, PC‐14/HMG2. The HMG2 mRNA expression level was approximately twice those of parental PC‐14 and mock‐transfected PC‐14/CMV. Gel mobility shift assay revealed a CDDP‐treated DNA‐protein complex in the nuclear extract of PC‐14/HMG2, which was not found in the extracts of PC‐14 and PC‐14/CMV. This complex formation was subject to competition by CDDP‐treated non‐specific salmon sperm DNA, indicating that ectopic HMG2 recognizes CDDP‐damaged DNA. PC‐14/HMG2 showed more than 3‐fold higher sensitivity to CDDP than PC‐14 and PC‐14/CMV. The intracellular platinum content of PC‐14/HMG2 after exposure to 300 μM CDDP was 1.1 and 1.5 times that of PC‐14 and PC‐14/CMV, respectively. Cellular glutathione levels were not different in these cell lines. Repair of DNA interstrand cross‐links determined by alkaline elution assay was decreased in PC‐14/HMG2. These results suggest that HMG2 may enhance the CDDP sensitivity of cells by inhibiting repair of the DNA lesion induced by CDDP.

Identification of Predictive Biomarkers for Response to Trastuzumab Using Plasma FUCA Activity and N-Glycan Identified by MALDI-TOF-MS

The aim of this study was to identify glycobiological biomarkers that indicate sensitivity to trastuzumab, a humanized monoclonal antibody against HER2 in plasma samples from breast cancer patients. Plasma samples were obtained from 24 breast cancer patients treated with trastuzumab monotherapy. The catalytic activities of plasma alpha1-6, fucosyltransferase (FUT8) and alpha-L fucosidase (FUCA) were analyzed using high-performance liquid chromatography (HPLC) and spectrophotometer, respectively. The plasma N-glycan profiles were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Plasma FUT8 activity was not significantly correlated with either the clinical response or progression-free survival (PFS). On the other hand, plasma FUCA activity was significantly correlated with PFS (p < 0.05). The MALDI-TOF-MS analysis of the plasma N-glycan profile revealed that the expression of 2534 m/z N-glycan was lower in patients with progressive disease (PD) and was correlated with PFS. Low expression of 2534 m/z N-glycan discriminated between PD and non-PD with 75% sensitivity and 82% specificity. We demonstrated that the plasma FUCA activity and 2534 m/z N-glycan may be predictive biomarkers of sensitivity to trastuzumab. Our results suggest that glycosylation analysis may provide useful information for determining clinical cancer therapy and provide novel insight into biomarker studies using glycobiological tools in the field of breast cancer.

p16INK4 expression is associated with the increased sensitivity of human non-small cell lung cancer cells to DNA topoisomerase I inhibitors.

Inactivation of p16INK4, an inhibitor of cyclin‐dependent kinases 4 (CDK4) and 6 (CDK6), may be essential for ontogenesis in non‐small cell lung cancer (NSCLC). We examined the sensitivity of two clones of P16INK4‐transfected NSCLC cell line with homozygous deletion of p16INK4, A549/pl6‐l and 2, to DNA topoisomerase I (topo I) inhibitors. A549/pl6‐l and ‐2 showed 7.7‐ and 9.1‐fold increases in sensitivity to CPT‐11 (11,7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]carbonyloxycamptothecin), respectively, compared with A549 cells. Ectopic p16INK4‐expressing cells also showed ∼4.0‐fold increase in sensitivity to SN‐38 (7‐ethyl‐10‐hydroxycamptothecin), the active metabolite of CPT‐11, compared to the parent cells. The topo I‐mediated DNA relaxation activities of ectopic p16INK4‐expressing cells were approximately 5 times higher than those of the parent cells. Northern and western blot analyses indicate that these increased topo I activities of ectopic p16INK4‐expressing cells were due to an elevated topo I mRNA level and an increase in topo I protein. The chemosensitivity to topo I inhibitors, topo I mRNA level, protein content and activity of a pl6INK4 revertant, lacking functional p16INK4, tended to be restored toward those of the parental phenotype to some extent. These results suggest that p161NK4 expression is closely associated with the increased sensitivity of ectopic pl6INK4‐expressing NSCLC cells to topo I inhibitors. The up‐regulation of topo I mRNA level, protein content and activity may he responsible for this hypersensitivity.

Tissue plasminogen activator thrombolysis and transluminal angioplasty in the treatment of basilar artery thrombosis: Case report

A 61-year-old man with basilar artery thrombotic occlusion was successfully treated by a combination of local infusion of tissue plasminogen activator (t-PA) followed by percutaneous transluminal angioplasty (PTA). t-PA superselective infusion combined with the mechanical destruction of the clot by the guide wire was effective for recanalization. A Stealth dilation catheter, with a 2.5-mm balloon diameter and 10-mm length, was used for PTA. A balloon inflation time of 60 seconds with 4 atmospheres of pressure was delivered for successful dilation of the basilar artery. It was confirmed to be widely patent on follow-up angiography 40 days after the combined procedure with no further ischemic attacks apparent on clinical examination at 6 months.