Mamoru Yanagimachi

Circulating miRNA biomarkers for Alzheimer's disease.

A minimally invasive diagnostic assay for early detection of Alzheimers disease (AD) is required to select optimal patient groups in clinical trials, monitor disease progression and response to treatment, and to better plan patient clinical care. Blood is an attractive source for biomarkers due to minimal discomfort to the patient, encouraging greater compliance in clinical trials and frequent testing. MiRNAs belong to the class of non-coding regulatory RNA molecules of ∼22 nt length and are now recognized to regulate ∼60% of all known genes through post-transcriptional gene silencing (RNAi). They have potential as useful biomarkers for clinical use because of their stability and ease of detection in many tissues, especially blood. Circulating profiles of miRNAs have been shown to discriminate different tumor types, indicate staging and progression of the disease and to be useful as prognostic markers. Recently their role in neurodegenerative diseases, both as diagnostic biomarkers as well as explaining basic disease etiology has come into focus. Here we report the discovery and validation of a unique circulating 7-miRNA signature (hsa-let-7d-5p, hsa-let-7g-5p, hsa-miR-15b-5p, hsa-miR-142-3p, hsa-miR-191-5p, hsa-miR-301a-3p and hsa-miR-545-3p) in plasma, which could distinguish AD patients from normal controls (NC) with >95% accuracy (AUC of 0.953). There was a >2 fold difference for all signature miRNAs between the AD and NC samples, with p-values<0.05. Pathway analysis, taking into account enriched target mRNAs for these signature miRNAs was also carried out, suggesting that the disturbance of multiple enzymatic pathways including lipid metabolism could play a role in AD etiology.

Antibody to Thrombin Receptor Inhibits Neointimal Smooth Muscle Cell Accumulation Without Causing Inhibition of Platelet Aggregation or Altering Hemostatic Parameters After Angioplasty in Rat

An antibody was raised in rabbits against SFFLRNPSEDTFEQF peptide, which is an NH2-terminal peptide of the thrombin-cleaved rat thrombin receptor. In vitro, the antibody inhibited rat smooth muscle cell proliferation but had no effect on rat platelet aggregation or clotting time. These data indicate that the antibody is a specific blocker of the thrombin receptor-signaling pathway in rat smooth muscle cells but does not work as a blocker in rat platelets, suggesting the existence of a second thrombin receptor in the platelets. Using an in vivo balloon catheter-induced injury model in rats, we examined the effect of the anti-rat thrombin receptor IgG on intimal smooth muscle cell accumulation 2 weeks after angioplasty. Analysis of the ratio of intimal to medial cross-sectional areas showed that injection of immune IgG resulted in 43.7% and 53.1% reduction (P<0.01) of neointimal smooth muscle cell accumulation compared with saline and nonimmune IgG treatment, respectively. Moreover, the injection of immune IgG caused a significant decrease of thrombin receptor mRNA expression and also 40.5% and 43.0% decreases (P<0.01) of the proliferating cell nuclear antigen (PCNA) index in the intima compared with the PCNA index after saline and nonimmune IgG treatment, respectively. The suppression of the PCNA index was also observed in the immune IgG-treated group at an early stage after angioplasty. These results suggest that thrombin receptor activation is involved in the proliferation and accumulation of neointimal smooth muscle cells induced by balloon injury.

Squalene synthase inhibitors reduce plasma triglyceride through a low-density lipoprotein receptor-independent mechanism

Inhibitors of squalene synthase are considered to be candidate drugs to reduce both plasma cholesterol and triglyceride. However, little is known about the mechanism of squalene synthase inhibitor-specific effect on plasma triglyceride. In this study, we confirmed the triglyceride-lowering effect of ER-27856, a potent squalene synthase inhibitor prodrug, in rhesus monkeys. To determine the role of low-density lipoprotein (LDL) receptor in the triglyceride-lowering effect of squalene synthase inhibitors, we intravenously administered ER-28448, the active form of ER-27856, to Watanabe heritable hyperlipidemic (WHHL) rabbits for 4 days. In heterozygotes, ER-28448 reduced plasma cholesterol and triglyceride by 52% and 37%, respectively. In homozygous rabbits, in contrast, ER-28448 lowered plasma triglyceride by 40% but did not lower plasma cholesterol. Orally administered ER-27856 reduced plasma triglyceride in homozygous animals but atorvastatin and bezafibrate did not. In hepatocytes isolated from homozygous WHHL rabbits, squalene synthase inhibitors but not atorvastatin reduced triglyceride biosynthesis. These data demonstrate that squalene synthase inhibitors reduced plasma triglyceride through an LDL receptor-independent mechanism, which was distinct from that of the triglyceride-lowering action of atorvastatin or bezafibrate. The reduction of hepatic triglyceride biosynthesis may play an important role in the hypotrigyceridemic action of squalene synthase inhibitors.

Abstract 4502: Eribulin alters vascular function in human triple-negative (TN) breast MX-1 and MDA-MB-231 tumor xenograft models as measured by DCE-MRI.

Objective: Eribulin mesylate (ERI) is a simplified synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. Literature reports have increasingly shown tubulin binding agents have vascular disruptive activity. In this study, we evaluated the effect of eribulin on tumor vasculature using DCE-MRI of TN human breast MX-1 xenografts in nude rats. Methods: The effects of ERI on human TN breast MX-1 and MDA-MB-231 tumor are examined in nude rats and nude mice. ERI was administered at MTD dose (0.3 mg/kg at Q4D4 in nude rats and 3.0 mg/kg at D1 in nude mice). In nude rats, DCE-MRI was conducted on a Bruker 4.7 T scanner with Magnevist contrast. Imaging was conducted on day -1, 6 hrs, day 2, day 5-6 and day 10. In nude mice, morphology of tumor vasculature was analyzed with immunohistochemistry (IHC) staining with anti-mouse endothelial marker CD31 antibody. Microvessel density (MVD) and vessel perimeter were analyzed using Aperio Image Scope. Results and discussion: ERI inhibited tumor growth of MX-1 (day 5; 290 +/- 169 vs 1195 +/- 36 mm3, N=5, p Conclusions: ERI altered tumor vasculature acutely in the tumor rim and increase vascular perfusion in the tumor core 5 days after treatment. In addition to regulation of mitosis, ERI may have induced normalization of tumor vasculature in preclinical TN breast cancer cell models. Further analysis of the mechanism of ERI on vascular normalization will be warranted. Citation Format: Paul J. McCracken, Ken Ito, Mamoru Yanagimachi, Xavier Tizon, Peggy Provent, Shanqin Xu, Namita Kumar, Denice Welsh, Tyler J. Teceno, Galina Kuznetsov, Yasuhiro Funahashi. Eribulin alters vascular function in human triple-negative (TN) breast MX-1 and MDA-MB-231 tumor xenograft models as measured by DCE-MRI. [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 4502. doi:10.1158/1538-7445.AM2013-4502

Abstract 1522: Gene expression profiling (GEP) reveals Epithelial Mesenchymal Transition (EMT) genes selectively differentiating eribulin sensitive breast cancer cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Objectives: Eribulin mesylate (ERI) is a simplified synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. In this study, we investigated whether selective signal pathways were associated with ERI activity compared to paclitaxel (PTX), which stabilize microtubules, based on GEP using 3 cancer cell line panels in vitro. Methods: In vitro anti-proliferation activity (IC50) was determined in 27 breast, 19 endometrial and 21 ovarian cancer cell lines treated with ERI and PTX. We performed GEP for the same cancer panels treated for 24 hours at conc. of 10 x IC50 using Affimetrix Human Genome U133 Plus 2.0 arrays. We applied paired t-test (p 1.5) to identify genes significantly altered between treatments. Pathway enrichment was performed using Metacore and Ingenuity Pathway Analysis software. Cell lines were clustered by hierarchical clustering based on Pearson correlation. Results: We determined the sets of genes differentially altered between ERI and PTX treatments in 3 cancer panels. Regardless of cancer panels, several tubulins (TUBA1B, TUBA1C, TUBA3D, TUBA4A, TUBB2C, TUBBG1, TUBB3, TUBB2A, TUBB2C, TUBB and TUBB6) had significantly lower expression in cell lines treated with ERI, compared to PTX. Our analysis revealed that expression profiles of the gene sets correlated with drug sensitivity in cancer panels (p=0.004 for ERI in breast cancer, p=0.06 for PTX in endometrial cancer, p=0.05 for ERI in ovarian cancer). Pathway enrichment analysis of the gene revealed that the common pathways altered between 2 treatments in 3 cancer panels were related to cytoskeleton remodeling (p<0.002) and role of NEK in cell cycle regulation (p<0.0005). We also identified pathways specific to cancer types. EMT was enriched in genes with significantly altered expression between the two drugs for breast (p=3.4e-07) and endometrial cancer (p=0.001), but not for ovarian cancer. Expression of genes from EMT pathway correlated with breast cancer ERI sensitivity (p=0.1) and endometrial cancer PTX sensitivity (p=0.07). In breast cancer most genes from the EMT signature were up-regulated in ERI resistant cluster, allowing us differentiate cell line sensitivity based on expression. In addition, we found selective signaling pathways were associated to drug sensitivity in breast cancer panels among clustered 394 pathways based on GEP (13 and 4 pathways for ERI and PTX respectively, p<0.01). Conclusion: GEP analysis showed that gene sets altered with ERI and PTX were correlated to drug sensitivity in vitro anti-proliferative activity among 3 cancer panels. Breast cancer is the best target to differentiate ERI sensitivity based on GEP compared to PTX and EMT genes are selectively related to ERI sensitivity. Citation Format: Zoltan Dezso, Judith Oestreicher, Amy Weaver, Tadashi Kadowaki, Mamoru Yanagimachi, Junji Matsui, Sergei I. Agoulnik, Yasuhiro Funahashi. Gene expression profiling (GEP) reveals Epithelial Mesenchymal Transition (EMT) genes selectively differentiating eribulin sensitive breast cancer cell lines. [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 1522. doi:10.1158/1538-7445.AM2013-1522

Abstract 1089: Effect of lenvatinib (E7080) on site specific phosphorylation in cancer cells with activated FGFR2 signaling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective: Somatic mutations in FGFR2 contribute to development of malignant tumors. It has been reported that 10-12% of endometrial carcinomas have mutations in the FGFR2 gene and FGFR2 is thought to be a target for novel anti-cancer agents. Lenvatinib is an inhibitor of multiple receptor tyrosine kinases, such as VEGFR, FGFR, PDGFR, RET and Kit. Lenvatinib inhibited angiogenesis driven by both VEGF and FGF in preclinical models. The purpose of this study is to determine the effect of lenvatinib on site specific phosphorylation of FGFR2 expressed in tumor cells, which are addicted to FGF signaling by either amplification or active mutations of FGFR2. Methods: The gastric cancer cell line, SNU-16, and endometrial cancer cell lines, AN3CA and MFE-296, were examined for phosphorylation status of FGFR2. Phosphorylation of FGFR2 was detected by western blotting after immunoprecipitation (IP-WB) with anti-FGFR2 antibody and the site specific phosphorylation status was assessed by liquid chromatography mass spectrometry (LC-MS). Inhibitory activity of lenvatinib and sorafenib against the phosphorylation of FGFR2 was examined followed by FGF-2 stimulation. Results: Phosphorylation of FGFR2 was promoted in SNU-16 because of gene amplification. Inhibitory activity of lenvatinib and sorafenib (0.001-10 uM) was examined against the phosphorylation of FGFR2. IP-WB analysis showed that lenvatinib inhibited the phosphorylation of FGFR2 at 1 uM and 10 uM, while sorafenib inhibited only at 10 uM. Next, we performed LC-MS analysis to determine whether lenvatinib and sorafenib selectively affect site specific phosphorylation of FGFR2. LC-MS analysis showed three different statuses of phosphorylation in FGFR2, single phosphorylation of either Y656 or Y657 and dual phosphorylation of Y656 and Y657, and both agents inhibited phosphorylation at 10 uM despite the phosphorylation status. IP-WB analysis also showed a constitutive activation of FGFR2 in AN3CA and MFE-296 (mutation in FGFR2 kinase domain) compared to other endometrial cancer cells without mutation. Lenvatinib, but not by sorafenib, decreased the phosphorylation of FGFR2 in AN3CA and MFE-296 at 1 uM, consistent with the observation in SNU-16. LC-MS analysis in AN3CA demonstrated the phosphorylation on Y657 was clearly inhibited by both agents, although the level of phosphorylation on Y656 was increased by sorafenib, but not by lenvatinib. Conclusions: IP-WB analysis showed that lenvatinib inhibited the phosphorylation of FGFR2 at lower concentration in comparison with sorafenib. LC-MS analysis indicated that inhibitory activity of both agents was different among phosphorylation sites of FGFR2 and also types of FGFR2-activated cancer cells. These results suggested that lenvatinib may exert an unique anti-tumor activity through the inhibition of FGFR2 signaling, in addition to the anti-angiogenic activity via inhibition of VEGFR2 kinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1089. doi:1538-7445.AM2012-1089