Masahiro Matsuki

CASY-1, an ortholog of calsyntenins/alcadeins, is essential for learning in Caenorhabditis elegans

Calsyntenins/alcadeins are type I transmembrane proteins with two extracellular cadherin domains highly expressed in mammalian brain. They form a tripartite complex with X11/X11L and APP (amyloid precursor protein) and are proteolytically processed in a similar fashion to APP. Although a genetic association of calsyntenin-2 with human memory performance has recently been reported, physiological roles and molecular functions of the protein in the nervous system are poorly understood. Here, we show that CASY-1, the Caenorhabditis elegans ortholog of calsyntenins/alcadeins, is essential for multiple types of learning. Through a genetic screen, we found that casy-1 mutants show defects in salt chemotaxis learning. casy-1 mutants also show defects in temperature learning, olfactory adaptation, and integration of two sensory signals. casy-1 is widely expressed in the nervous system. Expression of casy-1 in a single sensory neuron and at the postdevelopmental stage is sufficient for its function in salt chemotaxis learning. The fluorescent protein-tagged ectodomain of CASY-1 is released from neurons. Moreover, functional domain analyses revealed that both cytoplasmic and transmembrane domains of this protein are dispensable, whereas the ectodomain, which contains the LG/LNS-like domain, is critically required for learning. These results suggest that learning is modulated by the released ectodomain of CASY-1.

Olfactory plasticity is regulated by pheromonal signaling in Caenorhabditis elegans

Too Close for Comfort Pheromones are often used for sexual communications in animals, but they can also serve as a measure of population density. Now, Yamada et al. (p. 1647) have found that population density in the nematode worm Caenorhabditis elegans regulates plasticity of olfactory behavior, in which attraction to an odorant decreases after prolonged exposure. Using two rounds of genetic screens, a peptide named SNET-1 and a homolog of a mammalian transmembrane peptidase neprilysin were found to mediate pheromonal regulation. This regulation of olfactory behavior may serve to coordinate the behavior of individual animals in relation to the status of the whole population. A nematode odor response is regulated by population density through dauer pheromone, a neuropeptide, and neprilysin peptidase. Population density–dependent dispersal is a well-characterized strategy of animal behavior in which dispersal rate increases when population density is higher. Caenorhabditis elegans shows positive chemotaxis to a set of odorants, but the chemotaxis switches from attraction to dispersal after prolonged exposure to the odorants. We show here that this plasticity of olfactory behavior is dependent on population density and that this regulation is mediated by pheromonal signaling. We show that a peptide, suppressor of NEP-2 (SNET-1), negatively regulates olfactory plasticity and that its expression is down-regulated by the pheromone. NEP-2, a homolog of the extracellular peptidase neprilysin, antagonizes SNET-1, and this function is essential for olfactory plasticity. These results suggest that population density information is transmitted through the external pheromone and endogenous peptide signaling to modulate chemotactic behavior.

GPC-1, a G Protein γ-Subunit, Regulates Olfactory Adaptation in Caenorhabditis elegans

Caenorhabditis elegans genome carries two Gγ genes, gpc-1 and gpc-2, and two Gβ genes, gpb-1 and gpb-2. Of these, gpc-2 and gpb-1 are expressed ubiquitously and are essential for viability. Through a genetic screen, we identified gpc-1 as essential for olfactory adaptation. While wild-type animals show decreased chemotaxis to the odorant benzaldehyde after a short preexposure to the odorant, gpc-1 mutants are still attracted to the odorant after the same preexposure. Cell-specific rescue experiments show that gpc-1 acts in the AWC olfactory neurons. Coexpression of GPC-1 and GPB-1, but not GPB-2, caused enhanced adaptation, indicating that GPC-1 may act with GPB-1. On the other hand, knock down of gpc-2 by cell-targeted RNAi caused reduced chemotaxis to the odorant in unadapted animals, indicating that GPC-2 mainly act for olfactory sensation and the two Gγs have differential functions. Nonetheless, overexpression of gpc-2 in AWC neurons rescued the adaptation defects of gpc-1 mutants, suggesting partially overlapping functions of the two Gγs. We further tested genetic interaction of gpc-1 with several other genes involved in olfactory adaptation. Our analyses place goa-1 Goα and let-60 Ras in parallel to gpc-1. In contrast, a gain-of-function mutation in egl-30 Gqα was epistatic to gpc-1, suggesting the possibility that gpc-1 Gγ may act upstream of egl-30 Gqα.

Targeting of tumor growth and angiogenesis underlies the enhanced antitumor activity of lenvatinib in combination with everolimus

The combination of lenvatinib, a multiple receptor tyrosine kinase inhibitor, plus everolimus, a mammalian target of rapamycin (mTOR) inhibitor, significantly improved clinical outcomes versus everolimus monotherapy in a phase II clinical study of metastatic renal cell carcinoma (RCC). We investigated potential mechanisms underlying the antitumor activity of the combination treatment in preclinical RCC models. Lenvatinib plus everolimus showed greater antitumor activity than either monotherapy in three human RCC xenograft mouse models (A‐498, Caki‐1, and Caki‐2). In particular, the combination led to tumor regression in the A‐498 and Caki‐1 models. In the A‐498 model, everolimus showed antiproliferative activity, whereas lenvatinib showed anti‐angiogenic effects. The anti‐angiogenic activity was potentiated by the lenvatinib plus everolimus combination in Caki‐1 xenografts, in which fibroblast growth factor (FGF)‐driven angiogenesis may contribute to tumor growth. The combination showed mostly additive activity in vascular endothelial growth factor (VEGF)‐activated, and synergistic activity against FGF‐activated endothelial cells, in cell proliferation and tube formation assays, as well as strongly suppressed mTOR‐S6K‐S6 signaling. Enhanced antitumor activities of the combination versus each monotherapy were also observed in mice bearing human pancreatic KP‐1 xenografts overexpressing VEGF or FGF. Our results indicated that simultaneous targeting of tumor cell growth and angiogenesis by lenvatinib plus everolimus resulted in enhanced antitumor activity. The enhanced inhibition of both VEGF and FGF signaling pathways by the combination underlies its superior anti‐angiogenic activity in human RCC xenograft models.

Lenvatinib Suppresses Angiogenesis through the Inhibition of both the VEGFR and FGFR Signaling Pathways

Cardiovascular disease is the leading cause of death in the World [1]. As the major risk factor for cardiovascular disease, hypertension has been identified as the most important cause of disability and the leading risk factor for death globally [2]. The causes of hypertension are complex and are related to genetic factors, lifestyle, diet structure, and environmental factors, including air pollution [3]. Long-term exposure to ambient air pollution increases cardiovascular mortality rates [4,5]. Air pollution has also been associated with the incidence of nonfatal myocardial infarction and stroke [6-8]. Hypertension is one of the most important risk factors for cardiovascular disease, and has been ranked as the leading cause for death and disability worldwide [9,10].

Abstract 3264: Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models

Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of vascular endothelial growth factor (VEGF) receptors VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor (FGF) receptors FGFR1, 2, 3, and 4; the platelet-derived growth factor (PDGF) receptor PDGFRα; KIT; and RET. Lenvatinib showed antitumor activity against various tumor types mainly through its potent inhibition of angiogenesis, and is currently marketed for the treatment of patients with radioactive iodine-refractory differentiated thyroid cancer. Recently, lenvatinib in combination with everolimus has shown longer progression free survival compared to lenvatinib or everolimus alone in renal cell carcinoma in Phase 2 study. The aim of this study is to elucidate the activity of the combination of lenvatinib and everolimus in preclinical human RCC xenograft models. We examined antitumor activity in two human RCC (A-498 and Caki-1) xenograft models orally treated with lenvatinib (10 mg/kg), everolimus (30 mg/kg), and the combination of lenvatinib and everolimus for 1 or 2 weeks. The antitumor proliferation and antiangiogenic effects were evaluated by immunohistochemistry (IHC) using anti Ki67 antibody and anti CD31 antibody, respectively. The induction of apoptosis was detected by TUNEL assay. To analyze the gene expression profile of tumor samples, microarray analysis were also conducted. The antitumor activity of the combination of lenvatinib and everolimus was greater than that of either agent administered alone in A-498 and Caki-1 xenograft models. The combination caused tumor regression and had no remarkable body weight loss. IHC analysis revealed decrease of microvessel density in lenvatinib and combination groups, and also decrease in the proportion of proliferative cells in everolimus treated and combination-treated group in A-498 model. In TUNEL assay, significant induction of apoptosis was observed only in the combination-treatment group. The analysis of gene expression profile in A-498 xenograft tumors also supported these results: lenvatinib alone upregulated hypoxia-related genes and everolimus decreased proliferation-related genes. The combination of these 2 drugs induced blends of the gene expression changes caused by each single treatment. Our results indicate that treatment of lenvatinib in combination with everolimus caused significant antitumor effect by combining the potent antiangiogenic activity of lenvatinib as well as direct antitumor activity of everolimus in A-498 model. These preclinical results provide one of the mechanisms to support the significant clinical benefit observed in RCC with the combination of lenvatinib and everolimus. Citation Format: Yusuke Adachi, Masahiro Matsuki, Atsumi Yamaguchi, Yoichi Ozawa, Kiyochi Okamoto, Kaoru Mitsuhashi, Takayuki Kimura, Taisuke Hoshi, Osamu Tohyama, Kenji Tai, Makoto Ogo, Yasuhiro Funahashi, Junji Matsui. Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3264.

Lenvatinib inhibits angiogenesis and tumor fibroblast growth factor signaling pathways in human hepatocellular carcinoma models.

Unresectable hepatocellular carcinoma (uHCC) is one of the most lethal and prevalent cancers worldwide, and current systemic therapeutic options for uHCC are limited. Lenvatinib, a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs), recently demonstrated a treatment effect on overall survival by statistical confirmation of noninferiority to sorafenib in a phase 3 study of uHCC. Here, we investigated mechanisms underlying the antitumor activity of lenvatinib in preclinical HCC models. In vitro proliferation assay of nine human HCC cell lines showed that lenvatinib selectively inhibited proliferation of FGF signal‐activated HCC cells including FGF19‐expressing Hep3B2.1‐7. Lenvatinib suppressed phosphorylation of FRS2, a substrate of FGFR1–4, in these cells in a concentration‐dependent manner. Lenvatinib inhibited in vivo tumor growth in Hep3B2.1‐7 and SNU‐398 xenografts and decreased phosphorylation of FRS2 and Erk1/2 within the tumor tissues. Lenvatinib also exerted antitumor activity and potently reduced tumor microvessel density in PLC/PRF/5 xenograft model and two HCC patient‐derived xenograft models. These results suggest that lenvatinib has antitumor activity consistently across diverse HCC models, and that targeting of tumor FGF signaling pathways and anti‐angiogenic activity underlies its antitumor activity against HCC tumors.

Abstract 1805: Antitumor activity of lenvatinib mesilate in human hepatocellular carcinoma preclinical models

Hepatocellular carcinoma (HCC) is a one of the leading causes of cancer death worldwide; however systemic therapeutic option for unresectable HCC is limited. Liver carcinogenesis is a complex process and various pathways have been found to be deregulated. Of those pathways, aberrant activation of fibroblast growth factor 19 (FGF19)/FGF receptor 4 (FGFR4) axis has been hypothesized to participate in the development of HCC with poor prognosis. Lenvatinib mesilate (lenvatinib) is an orally administered multi-targeted tyrosine kinase inhibitor (TKI) that selectively inhibits vascular endothelial growth factor receptors (VEGFR)1-3, FGFR1-4, platelet-derived growth factor receptor (PDGFR)α, RET, and KIT. In this study, we investigated the activities of lenvatinib against human HCC cells with or without amplification/overexpression of FGF19 gene in vitro and in vivo. Lenvatinib inhibited in vitro proliferation of human HCC cell lines Hep 3B2.1-7 and HuH-7 with IC50 values of 0.23 and 0.42 μmol/L (0.086 and 0.16 μmol/L as unbound IC50), respectively, whereas it did not show potent antiproliferative activity against PLC/PRF/5 (IC50 >10 μmol/L). Both Hep 3B2.1-7 and HuH-7 were reported to have an amplification of FGF19 gene and their cell growth was dependent on FGF19/FGFR4 axis. Sorafenib inhibited in vitro proliferation of the 3 HCC cell lines with IC50 values of 2.2 - 5.3 μmol/L (0.59 - 1.4 μmol/L as estimated unbound IC50 based on previously reported protein binding). Next, antitumor activity was evaluated in 2 human HCC cell line (PLC/PRF/5 and Hep 3B2.1-7) xenograft models. Lenvatinib treatments at 1 - 100 mg/kg resulted in tumor growth inhibition with a maximal antitumor effect giving a minimum T/C value (minT/C) of 14% via its antiangiogenic activity in the PLC/PRF/5 model, and at 3 - 30 mg/kg with minT/C of 31% in the Hep 3B2.1-7 model. Sorafenib showed significant antitumor effect at doses ≥30 mg/kg with minT/C of 30% in the PLC/PRF/5 model, and at 10 and 30 mg/kg with minT/C of 47% in the Hep 3B2.1-7 model. Finally, we tested antitumor activity in HCC patient-derived xenograft (PDX) model (LI0050), and PDX-derived cell line (LIXC-012) xenograft model with FGF19 overexpression in mice. Lenvatinib significantly inhibited the tumor growth of LI0050 at 10 and 30 mg/kg with minT/C of 33%, and that of LIXC-012 at 3 - 30 mg/kg with minT/C of 25%. Treatment with sorafenib at 30 mg/kg was intolerable in LI0050 model, and sorafenib showed significant antitumor activity at 30 mg/kg with T/C of 55%, but not 10 mg/kg in the LIXC-012 model. In conclusion, these results indicate that lenvatinib has potent preclinical antitumor activity in human HCC xenograft models with or without amplification/overexpression of FGF19. Furthermore, the maximum antitumor effect of lenvatinib was greater than that of sorafenib in this study. Direct effect of lenvatinib on proliferation of HCC cells with an aberrant FGF signaling pathway in tumor warrants further investigation. Citation Format: Masahiro Matsuki, Yuji Yamamoto, Taisuke Hoshi, Takayuki Kimura, Yasuhiro Funahashi, Junji Matsui. Antitumor activity of lenvatinib mesilate in human hepatocellular carcinoma preclinical models [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 1805. doi:10.1158/1538-7445.AM2017-1805

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

Abstract 3262: Effects of lenvatinib mesilate in combination with everolimus on VEGF and FGF-driven angiogenesis and tumor growth

Lenvatinib mesilate (lenvatinib) is an orally available inhibitor for multiple receptor tyrosine kinases (RTKs) that selectively inhibits the kinase activities of VEGFR1, 2, and 3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1, 2, 3, and 4; PDGFRα; KIT; and RET. Recently, lenvatinib in combination with everolimus has shown longer progression free survival compared to lenvatinib or everolimus alone in renal cell carcinoma in a Phase 2 study. In this study, we evaluated the effect of lenvatinib in combination with everolimus on VEGF and bFGF-driven angiogenesis to elucidate the mechanism of combination action in preclinical models. Preclinical studies provide a plausible biologic rationale for the significant clinical benefit observed in RCC with the combination of lenvatinib and everolimus. Effects of lenvatinib, everolimus, and its combination on VEGF or bFGF activated intracellular signaling were analyzed in HUVEC by western blotting. Combination effects of lenvatinib and everolimus on VEGF and bFGF-induced proliferation or tube formation of HUVEC were examined using combination indexes (CI). Antitumor activities were tested in the KP-1/VEGF or KP-1/FGF models, where VEGF or FGF-induced tumor angiogenesis and tumor growth were enhanced in nude mice due to overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells. Lenvatinib inhibited the VEGF or bFGF-driven phosphorylation of Erk1/2 (Thr202/Tyr204), S6K (Thr389), and S6K (Thr421/Ser424), and S6 (Ser235/Ser236), indicating the inhibition of both the MAPK pathway and the mTOR-S6K-S6 pathway. Everolimus inhibited the phosphorylation of S6K (Thr389), S6K (Thr421/Ser424), and S6 (Ser235/Ser236), but not Erk1/2. The combination showed greater inhibition for the phosphorylation of S6K (Thr421/Ser424) and S6 (Ser235/Ser236) than each single agent. Inhibitory activity of the combination at several molar ratios was mostly additive for VEGF-driven proliferation (CI: 0.799-1.167) and mostly synergistic for bFGF-driven tube formation (CI: 0.469-0.741). In the KP-1/VEGF or KP-1/FGF xenogtaft models, lenvatinib, everolimus, and the combination (p.o., qd x 14) significantly inhibited tumor growth compared to vehicle. In addition the combination of lenvatinib (7.5 mg/kg) and everolimus (15 mg/kg) showed significantly greater antitumor activity than higher dose of either lenvatinib (10 mg/kg) or everolimus (30 mg/kg) monotherapy. These results demonstrated enhancement of the inhibitory activity against VEGF and FGF-induced angiogenesis by the combination of lenvatinib with everolimus, and the synergistic enhancement against bFGF-induced angiogenesis unlike other VEGFR2 TKIs. The vertical inhibition of angiogenic signaling pathways with lenvatinib (RTK) and everolimus (mTOR) may contribute to enhanced antiangiogenic activity by dual targeting of the mTOR-S6K-S6 pathway. Citation Format: Kaoru Mitsuhashi, Takayuki Kimura, Taisuke Hoshi, Osamu Tohyama, Kenji Tai, Makoto Ogo, Masahiro Matsuki, Atsumi Yamaguchi, Yoichi Ozawa, Yusuke Adachi, Kiyoshi Okamoto, Junji Matsui, Yasuhiro Funahashi. Effects of lenvatinib mesilate in combination with everolimus on VEGF and FGF-driven angiogenesis and tumor growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3262.