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Almost all cancers show intrinsic and/or evasive resistance to vascular endothelial growth factor (VEGF) inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. From these clinical observations, the Ang2 and Tie2 (its receptor) axis has been focused on as a promising target. Here, we show a novel strategy to circumvent the resistance by combining multi-tyrosine kinase inhibitors lenvatinib (VEGF receptor, fibroblast growth factor receptor, and RET inhibitor) and golvatinib (E7050; c-Met, Tie2, and EphB4 inhibitor). Tie2 identifies a highly pro-angiogenic macrophage subset, Tie2-expressing macrophages (TEM). Angi-Tie2 and EphB4-EphrinB2 signaling plays critical roles in pericyte-mediated vessel stabilization. In vitro analyses suggested that golvatinib combined with lenvatinib inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib and lenvatinib inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable, and no macroscopic change was observed. These preclinical studies suggest that modulation of the tumor microenvironment by a strategic and well-tolerated combination of multi-targeting tyrosine kinase inhibitors may sensitize cancer to VEGF inhibitors.

We synthesized new bidentate N-heterocyclic carbene Ag and Rh complexes with xanthene skeleton. The structural of the complexes were analyzed by NMR spectroscopy and X-ray crystallography. The complexes adopted cis or trans configuration, depending on the structure of the ligand.

Despite remarkable recent progress in our chemical and structural understanding of the mechanisms of peptide bond formation by the ribosome, only very limited information is available about whether amino acid side chains affect the rate of peptide bond formation. Here, we generated a series of peptidyl-tRNAs that end with different tRNA-attached amino acids in the P-site of the Escherichia coli ribosome and compared their reactivity with puromycin, a rapidly A-site-accessing analog of aminoacyl-tRNAs. Among the 20 amino acids examined, proline was found to receive exceptionally slow peptidyl transfer to puromycin. These results raise a possibility that the peptidyl transferase activity of the ribosome may have some specificity with regard to the P-site amino acids.

We synthesized a series of new bidentate NHC (N-heterocyclic carbene) precursors with anthracene and xanthene skeletons. The corresponding NHC–Pd complexes were prepared from these precursors. The catalytic activity of these Pd complexes was examined.

The arrest sequence, FXXXXWIXXXXGIRAGP, of E. coli SecM interacts with the ribosomal exit tunnel, thereby interfering with translation elongation. Here, we studied this elongation arrest in vitro using purified translation components. While a simplest scenario would be that elongation is arrested beyond Pro166, the last arrest-essential amino acid, and that the Pro166 codon is positioned at the P site of the ribosomal peptidyl transferase center (PTC), our toeprint analyses revealed that the ribosome actually stalls when the Pro166 codon is positioned at the A site. Northern hybridization identification of the polypeptide bound tRNA and mass determination showed that the last amino acid of the arrested peptidyl-tRNA is Gly165, which is only inefficiently transferred to Pro166. Also, puromycin does not effectively release the arrested peptidyl-tRNA under the conditions of A site occupancy by Pro166-tRNA. These results reveal that secM-encoded Pro166-tRNA functions as a nonpolypeptide element in fulfilling SecM's role as a secretion monitor.

We synthesized a series of new bidentate NHC (N-heterocyclic carbene) precursors with anthracene and xanthene skeletons. The corresponding NHC–Pd complexes were prepared from these precursors. The catalytic activity of these Pd complexes was examined.

Although vascular endothelial growth factor (VEGF) inhibitors provide significant clinical benefit, they often require dose reductions or even withdrawals due to their severe toxicities. Furthermore, almost all cancers show intrinsic and/or evasive resistance to VEGF inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. Response to lenvatinib (E7080; VEGFR1-3 inhibitor) is also reported to correlate with low Ang2 level in differentiated thyroid cancer and endometrial cancer. From these clinical observations, Ang2 and its receptor Tie2 has been focused as promising targets. Here, we demonstrated mechanisms of resistance induced by Ang2 and a novel strategy to circumvent the resistance by combination of multi-tyrosine kinase inhibitors (TKIs), lenvatinib and golvatinib (E7050; c-Met, Tie2, EphB4 inhibitor). Tie2 defines a highly pro-angiogenic macrophage subset, Tie2-expressing macrophage (TEM). Ang-Tie2 and EphB4-EphrinB2 signaling play critical roles in pericyte-mediated vessel stabilization. Ectopic expression of Ang2 in thyroid cancer conferred resistance to lenvatinib and enhanced pericyte-associated endothelial network development and TEM infiltration. In vitro analyses suggested that golvatinib/lenvatinib combination inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib/lenvatinib combination inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable in mice, and no macroscopic change was observed. These results suggest that modulation of tumor microenvironment by strategic and well-tolerated combination of multi-targeting TKIs sensitizes cancer to VEGF inhibitors, which warrants further clinical investigation to determine the clinical benefit of anti-angiogenesis cancer therapy. Citation Format: Youya Nakazawa, Satoshi Kawano, Junji Matsui, Yasuhiro Funahashi, Osamu Tohyama, Hiroki Muto, Takayuki Nakagawa, Tomohiro Matsushima. Maximizing the efficacy of anti-angiogenesis cancer therapy: A multi-targeting strategy by tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2014-2980

Objectives: Eribulin mesylate (ERI) is a simplified synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. We examined effects of ERI and Paclitaxel (PTX) on blood vessel cells by gene expression profiling (GEP) in mono- and co-culture assays of pericytes and endothelial cells. Activity of ERI and PTX against cell growth inhibition and pericyte-driven in vitro angiogenesis was also studied. Methods: We first assessed IC50s of ERI and PTX in 4-day growth inhibition assay in isolated primary human brain vascular pericytes (HBVP) and human umbilical vein endothelial cells (HUVEC). Based on identified IC50s, cells were treated with 10X IC50s of ERI or PTX and GEP was analyzed at 24 h using either Affimetrix Human Genome U133 Plus 2.0 arrays or custom TaqMan Low Density Cards (TLDA) designed with 92 genes related to angiogenesis, metastasis/ Epithelial Mesenchymal Transition (EMT) and cell differentiation signal pathways. Inhibitory activity of drugs on the length of capillary-like networks was analyzed in co-culture of HUVEC with HBVP. Results: ERI and PTX inhibited cell growth of HBVP at IC50s of 1.2 and 3.1nM and HUVEC was more sensitive than HBVP by 1.9 and 3.3 fold in cell growth assay, respectively. In HUVEC, most genes were down-regulated by both ERI and PTX treatments, while in HBVP about equal number of genes were up- or down-regulated with microarray analysis. Interestingly, 63% affected genes in HUVECs overlapped for both treatments. In HBVPs, altered gene signatures were drug-dependent and an overlap was limited by 16%. ERI specifically affected genes in HIF1 and caveolar-mediated signaling pathways while PTX regulated genes in HER2, PI3K/AKT and HGF signaling pathways among others. We confirmed obtained altered GEP using TLDA and identified 42 significant genes differentially regulated by ERI and PTX in HBVP. To examine effects on pericyte-driven in vitro angiogenesis, we compared length of capillary-like networks in co-culture of HUVEC with HBVP. ERI disrupted capillary-like networks starting at about 2 nM, while PTX showed limited inhibitory activity by less than 50% even at 100-1000 nM for 4 days treatments. In co-culture assay, TLDA data showed decreased expression levels of angiogenesis-related genes DLL4 (14% compare to control), PDGFRB (70%) and metastasis/EMT-related genes ZEB1 (53%), TGFB3 (54 %), VIM (62 %) after treatment with 10X IC50s of ERI (p Conclusions: Endothelial cells and pericytes responded differently to ERI and PTX treatments and effects of ERI on GEP of pericytes were distinct from PTX. ERI inhibited pericyte-driven in vitro angiogenesis at sub nM using co-culture assay of HUVEC with HBVP. Further analysis of the role of ERI on GEP of pericytes and pericyte-driven angiogenesis in anti-tumor activity will be warranted. Citation Format: Sergei I. Agoulnik, Judith L. Oestreicher, Noel H. Taylor, Mai Uesugi, Hiroki Muto, Satoshi Kawano, Kentaro Takahashi, Kentaro Matsuura, Ken Aoshima, Junji Matsui, Yasuhiro Funahashi. Eribulin and Paclitaxel differentially affect gene expression profiling of blood vessel cells and in vitro angiogenesis in co-cultures of human endothelial cells with pericytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3830. doi:10.1158/1538-7445.AM2013-3830

Objective: Eribulin mesylate (ERI) is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. The objective of this study was to examine the effect of ERI on tumor vasculature with immunohistchemical (IHC) analysis and gene expression profiling (GEP) in normal host cells, such as endothelial cells and vascular mural cells within tumor microenvironments in human BCC xenograft models Methods: Anti-tumor activity of ERI was examined at doses of 1.5 and 3.0 mg/kg, i.v. at day 1, in human BCC MX-1, MDA-MB-231 and MDA-MB-453 sc xenografts in nude mice. For IHC and GEP analysis, tumor tissues were collected at day 4 and day 8. IHC analysis was performed using mouse CD31 antibody to stain endothelial cells. Microvessel density (MVD) and vessel perimeter were determined by using Aperio Image Scope. GEP analysis for mouse host and human tumor cells within tumor tissues was done by using mouse and human TaqMan Low Density Arrays (TLDAs) consisting of a set of 92 genes related to angiogenesis, metastasis/EMT and cell differentiation signal pathways. Results shows % of non-treatment group (NT). Results: ERI showed significant anti-tumor activity against all three human BCC xenografts in a dose dependent manner. IHC analysis showed that ERI altered morphology of tumor vasculature day 8 after treatments and increased number of vessels with small size of perimeter (300um) in both MX-1 and MDA-MB-231 xenograft models (p Conclusions: ERI induced re-modeling of tumor vasculature in human BCC xenograft models. GEP related to angiogenesis and EMT/metastasis pathway was significantly affected with ERI treatment in host cells under tumor microenvironments. ERI might cause remodeling of tumor vasculature by regulating GEP in host cells. Further investigation may be warranted to examine if the activity of ERI against host cells in tumor tissues contributed to anti-tumor activity of ERI. Citation Format: Junji Matsui, Osamu Toyama, Mitsuhiro Ino, Taro Semba, Mai Uesugi, Hiroki Muto, Judith L. Oestreicher, Kentaro Takahashi, Kentaro Matsuura, Yoshiaki Sato, Taisuke Uehara, Takayuki Kimura, Hideki Watanabe, Yoichi Ozawa, Makoto Asano, Yusuke Adachi, Ken Aoshima, Yasuhiro Funahashi. Eribulin caused re-modeling of tumor vasculature altering gene expression profiling in angiogenesis and Epithelial Mesenchymal Transition (EMT) signaling pathway of host cells within human breast cancer cell (BCC) xenografts in nude mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2013-1413