Taro Semba

Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage

BackgroundLenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study.MethodWe evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors.ResultLenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score.ConclusionThese results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

Eribulin mesylate reduces tumor microenvironment abnormality by vascular remodeling in preclinical human breast cancer models

Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. Some tubulin‐binding drugs are known to have antivascular (antiangiogenesis or vascular‐disrupting) activities that can target abnormal tumor vessels. Using dynamic contrast‐enhanced MRI analyses, here we show that eribulin induces remodeling of tumor vasculature through a novel antivascular activity in MX‐1 and MDA‐MB‐231 human breast cancer xenograft models. Vascular remodeling associated with improved perfusion was shown by Hoechst 33342 staining and by increased microvessel density together with decreased mean vascular areas and fewer branched vessels in tumor tissues, as determined by immunohistochemical staining for endothelial marker CD31. Quantitative RT‐PCR analysis of normal host cells in the stroma of xenograft tumors showed that eribulin altered the expression of mouse (host) genes in angiogenesis signaling pathways controlling endothelial cell–pericyte interactions, and in the epithelial–mesenchymal transition pathway in the context of the tumor microenvironment. Eribulin also decreased hypoxia‐associated protein expression of mouse (host) vascular endothelial growth factor by ELISA and human CA9 by immunohistochemical analysis. Prior treatment with eribulin enhanced the anti‐tumor activity of capecitabine in the MDA‐MB‐231 xenograft model. These findings suggest that eribulin‐induced remodeling of abnormal tumor vasculature leads to a more functional microenvironment that may reduce the aggressiveness of tumors due to elimination of inner tumor hypoxia. Because abnormal tumor microenvironments enhance both drug resistance and metastasis, the apparent ability of eribulin to reverse these aggressive characteristics may contribute to its clinical benefits.

An Angiogenesis Inhibitor E7820 Shows Broad-Spectrum Tumor Growth Inhibition in a Xenograft Model: Possible Value of Integrin α2 on Platelets as a Biological Marker

We reported previously that an angiogenesis inhibitor, E7820, inhibits in vitro tube formation of human umbilical vein endothelial cell through the suppression of integrin α2 expression. Here we describe the antiangiogenic and antitumor effects of E7820 in mice and discuss the feasibility of using platelet integrin α2 expression on platelets as a biological marker of the efficacy of E7820. Oral administration of E7820 significantly inhibited basic fibroblast growth factor-induced angiogenesis in Matrigel implants and human colon WiDr tumor-induced angiogenesis in a dorsal air sac model. Twice-daily treatment with E7820 clearly inhibited the s.c. tumor growth of seven tumor cell lines derived from human colon, breast, pancreas, and kidney, and completely suppressed the growth of human pancreatic KP-1 and human colon LoVo cell lines. Moreover, E7820 significantly inhibited the growth of KP-1 and human colon tumor Colo320DM cells orthotopically implanted in the pancreas and cecum, respectively. The efficacy of E7820 was comparable in the s.c. and orthotopic transplantation models. Immunohistochemical analyses using anti-CD31 antibody showed that E7820 significantly reduced microvessel density in orthotopically implanted KP-1 tumor. E7820 reduced integrin α2 expression on a megakaryocytic cell line, Dami cells, induced by phorbol 12-myristate 13-acetate treatment. It also decreased the expression level of integrin α2 on platelets withdrawn from mice bearing s.c. KP-1 tumor at a dosage close to that affording antitumor activity. These data demonstrate that E7820 showed a broad-spectrum antitumor effect in mice through inhibition of angiogenesis and indicate that the decrease of integrin α2 on platelets might serve as a biological marker for the antitumor efficacy of E7820.

Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway‐induced resistance to vascular endothelial growth factor receptor inhibitor

Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF‐induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

Enhanced anti-angiogenic effect of E7820 in combination with erlotinib in epidermal growth factor receptor-tyrosine kinase inhibitor-resistant non-small-cell lung cancer xenograft models.

Most non‐small‐cell lung cancers (NSCLCs) harboring activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (EGFR‐TKIs); however, they invariably develop resistance to these drugs. E7820 is an angiogenesis inhibitor that decreases integrin‐α2 expression and is currently undergoing clinical trials. We investigated whether E7820 in combination with erlotinib, an EGFR‐TKI, could overcome EGFR‐TKI‐resistance in the NSCLC cell lines A549 (KRAS; G12S), H1975 (EGFR; L858R/T790M), and H1650 (PTEN; loss, EGFR; exon 19 deletion), which are resistant to erlotinib. Immunohistochemical analysis was carried out in xenografted tumors to investigate anti‐angiogenesis activity and endothelial cell apoptosis levels by endothelial cell marker CD31 and TUNEL staining, respectively. Treatment with E7820 (50 mg/kg) with erlotinib (60 mg/kg) showed a synergistic antitumor effect in three xenograft models. Immunohistochemical analysis indicated that combined treatment with E7820 and erlotinib significantly decreased microvessel density and increased apoptosis of tumor‐associated endothelial cells compared with use of only one of the agents. This combination increased apoptosis in HUVECs through activation of both intrinsic and extrinsic apoptosis pathways in vitro. The combination of E7820 with erlotinib is an alternative strategy to overcome erlotinib resistance in NSCLC by enhancement of the anti‐angiogenic activity of E7820.

Abstract 1413: 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.

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

Abstract 5177: E7386 : First-in-class orally active CBP/beta-catenin modulator as an anticancer agent

Carcinogenesis is often accelerated by the aberrant activation of components molecules of Wnt signaling pathway, especially, APC and beta-catenin are frequently reported to be mutated in various cancers. Therefore, Wnt signal pathway is thought to be one of the attractive therapeutic targets. PRI-724 generated by PRISM Pharma is a small molecule inhibitor of beta-catenin and its transcriptional coactivator CREB binding protein (CBP) thereby specific modulating Wnt/beta-catenin signaling pathway by intravenous continuous infusion. Here we firstly generated orally active small molecular inhibitor, E7386. E7386 disrupted the interaction between beta-catenin and CBP in co-immunoprecipitation assay. E7386 inhibited canonical Wnt signaling pathway /TCF reporter gene activity in LiCl-stimulated HEK-293 and MDA-MB-231 in a dose dependent manner with IC50 values of 55 nmol/L and 73 nmol/L, respectively. E7386 modulated the expression of Wnt signaling pathway related genes including AXIN2 and other genes, which were down-regulated by artificial knockdown of beta-catenin. These results indicate that E7386 controls the expression of Wnt target genes through modulation of beta-catenin/CBP interaction. Next we investigated anti-polyposis effect in ApcMin/+ mice as an in vivo proof of mechanism model. ApcMin/+ mice develops polyps in the intestinal tract caused by the aberrant activation of Wnt/beta-catenin signaling pathway. Oral administration of E7386 significantly suppressed the number of polyposis in a dose dependent manner at the dose range from 8.5 to 50 mg/kg. In addition, E7386 significantly changed the expressions of Wnt related genes in whisker follicle of ApcMin/+mice model. Finally, we investigated anti-tumor activity of E7386 in vitro tumor proliferation panel against 28 human tumor cell lines. E7386 showed relatively potent anti-proliferative activity against cancer cell lines harboring exclusively mutated Wnt signaling pathway molecules such as APC or beta-catenin. E7386 also showed significant antitumor activity in a dose dependent manner on human tumor cell line xenograft harboring APC mutation. Taken together, E7386 is a first in class orally active CBP/beta-catenin modulator and showed potent inhibitory activity against aberrant activation of Wnt/beta-catenin signaling pathway. Citation Format: Kazuhiko Yamada, Yusaku Hori, Atsumi Yamaguchi, Masahiro Matsuki, Shuntaro Tsukamoto, Akira Yokoi, Taro Semba, Yoichi Ozawa, Satoshi Inoue, Yuji Yamamoto, Kentaro Iso, Kazutaka Nakamoto, Hitoshi Harada, Naoki Yoneda, Atsushi Takemura, Masayuki Matsukura,, Kenji Kubara, Takenao Odagami, Masao Iwata, Akihiko Tsuruoka, Toshimitsu Uenaka, Junji Matsui, Tomohiro Matsushima, Kenich Nomoto, Hiroyuki Kouji, Takashi Owa. E7386 : First-in-class orally active CBP/beta-catenin modulator as an anticancer agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2017-5177