Sayaka Tanaka

Presenilin-dependent intramembrane cleavage of ephrin-B1

BackgroundPresenilin-dependent γ-secretase cleavage of several transmembrane proteins, including amyloid-β precursor protein and Notch, mediates the intramembrane proteolysis to liberate their intracellular domains that are involved in cellular signaling. Considering γ-secretase inhibitors as therapeutics for Alzheimers disease, understanding the physiologically and biologically important substrate for γ-secretase activity in brains is emerging issue. To elucidate the molecular mechanism and physiological role of γ-secretase, we screened candidate molecules for γ-secretase substrates.ResultsWe show that ephrin-B1, that participates in cell-cell repulsive and attractive signaling together with its Eph receptor, constitutively undergoes ectodomain shedding and that the residual membrane-tethered fragment is sequentially cleaved by γ-secretase to release the intracellular domain. Furthermore, overexpression of membrane-tethered ephrin-B1 caused protrusion of numerous cellular processes consisted of F-actin, that required the preservation of the most C-terminal region of ephrin-B1. In contrast, soluble intracellular domain translocated into the nucleus and had no effect on cell morphology.ConclusionOur findings suggest that ephrin-B is a genuine substrate for γ-secretase and regulates the cytoskeletal dynamics through intramembrane proteolysis.

SPIO-PICsome: Development of a highly sensitive and stealth-capable MRI nano-agent for tumor detection using SPIO-loaded unilamellar polyion complex vesicles (PICsomes)

Size controllable polyion complex vesicles (PICsomes), composed of biocompatible poly(ethylene glycol) (PEG) and poly(amino acid)s, have an extremely prolonged lifetime in the bloodstream that enables them to accumulate effectively in tumors via the enhanced permeability and retention (EPR) effect. The purpose of this study was to use PICsomes to synthesize a highly sensitive MRI contrast agent for more precise tumor detection. We synthesized SPIO-Cy5-PICsomes (superparamagnetic iron oxide nanoparticle-loaded Cy5-cross-linked Nano-PICsomes) and characterized them using dynamic light scattering and transmission electron microscopy in vitro and evaluated their ability to detect subcutaneously grafted tumors in vivo with MRI. The transverse relaxivity (r2) of the SPIO-Cy5-PICsomes (r2=663±28mM(-1)s(-1)) was 2.54 times higher than that of bare clinically-used SPIO. In in vivo MRI experiments on mice subcutaneously grafted with colon-26 tumor cells, the tumor signal was significantly altered at 3h after SPIO-Cy5-PICsome administration and persisted for at least 24h. Small and early-stage in vivo tumors (3days after grafting, approximately 4mm(3)) were also clearly detected with MRI. SPIO-loaded PICsomes are sensitive MRI contrast agents that can act as a powerful nanocarrier to detect small tumors for early diagnosis.

Systemically Injectable Enzyme‐Loaded Polyion Complex Vesicles as In Vivo Nanoreactors Functioning in Tumors

The design and construction of nanoreactors are important for biomedical applications of enzymes, but lipid- and polymeric-vesicle-based nanoreactors have some practical limitations. We have succeeded in preparing enzyme-loaded polyion complex vesicles (PICsomes) through a facile protein-loading method. The preservation of enzyme activity was confirmed even after cross-linking of the PICsomes. The cross-linked β-galactosidase-loaded PICsomes (β-gal@PICsomes) selectively accumulated in the tumor tissue of mice. Moreover, a model prodrug, HMDER-βGal, was successfully converted into a highly fluorescent product, HMDER, at the tumor site, even 4u2005days after administration of the β-gal@PICsomes. Intravital confocal microscopy showed continuous production of HMDER and its distribution throughout the tumor tissues. Thus, enzyme-loaded PICsomes are useful for prodrug activation at the tumor site and could be a versatile platform for enzyme delivery in enzyme prodrug therapy.

Transcription Factor YY1 Contributes to Tumor Growth by Stabilizing Hypoxia Factor HIF-1α in a p53-Independent Manner

In response to hypoxic stress, hypoxia-inducible factor (HIF)-1α is a critical transcription factor regulating fundamental cellular processes, and its elevated expression level and activity are associated with poor outcomes in most malignancies. The transcription factor Yin Yang 1 (YY1) is an important negative regulator of the tumor suppressor factor p53. However, the role of YY1 under tumor hypoxic condition is poorly understood. Herein, we show that inhibition of YY1 reduced the accumulation of HIF-1α and its activity under hypoxic condition, and consequently downregulated the expression of HIF-1α target genes. Interestingly, our results revealed that the downregulation of HIF-1α by inhibiting YY1 is p53-independent. Functionally, the in vivo experiments revealed that inhibition of YY1 significantly suppressed growth of metastatic cancer cells and lung colonization and also attenuated angiogenesis in a p53-null tumor. Collectively, our findings unraveled a novel mechanism by which YY1 inhibition disrupts hypoxia-stimulated HIF-1α stabilization in a p53-independent manner. Therefore, YY1 inhibition could be considered as a potential tumor therapeutic strategy to give consistent clinical outcomes independent of p53 status.

Rapid Cancer Fluorescence Imaging Using A γ-Glutamyltranspeptidase-Specific Probe For Primary Lung Cancer

BACKGROUND: We set out to examine the activity of γ-glutamyltranspeptidase (GGT) in lung cancer and the validity of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) for intraoperative imaging of primary lung cancer. METHODS: GGT activities and mRNA expression levels of GGT1 (one of the GGT subtypes) in five human lung cancer cell lines were examined by fluorescence imaging and quantitative reverse transcription polymerase chain reaction. In vivo imaging of an orthotopic A549 xenograft model in nude mice was performed to confirm its applicability to intraoperative imaging. Furthermore, ex vivo imaging of 73 specimens from lung cancer patients were performed and analyzed to calculate the sensitivity/specificity of gGlu-HMRG for lung cancer diagnosis. RESULTS: GGT activities and mRNA expression levels of GGT1 are diverse depending on cell type; A549, H441, and H460 showed relatively high GGT activities and expression levels, whereas H82 and H226 showed lower values. In the in vivo mouse model study, tiny pleural dissemination and hilar/mediastinal lymph node metastasis (less than 1 mm in diameter) were clearly detected 15 minutes after topical application of gGlu-HMRG. In the ex vivo study of specimens from patients, the sensitivity and specificity of gGlu-HMRG were calculated to be 43.8% (32/73) and 84.9% (62/73), respectively. When limited to female patients, never smokers, and adenocarcinomas, these values were 78.9% (15/19) and 73.7% (14/19), respectively. CONCLUSIONS: Although GGT activity of lung cancer cells vary, gGlu-HMRG can serve as an intraoperative imaging tool to detect small foci of lung cancer when such cells have sufficient GGT activity.

Regulation of endothelial Fas expression as a mechanism of promotion of vascular integrity by mural cells in tumors

Angiogenesis is a multi‐step process that culminates in vascular maturation whereby nascent vessels stabilize to become functional, and mural cells play an essential role in this process. Recent studies have shown that mural cells in tumors also promote and maintain vascular integrity, with wide‐reaching clinical implications including the regulation of tumor growth, metastases, and drug delivery. Various regulatory signaling pathways have been hitherto implicated, but whether regulation of Fas‐dependent apoptotic mechanisms is involved has not yet been fully investigated. We first compared endothelial FAS staining in human pancreatic ductal adenocarcinomas and colon carcinomas and show that the latter, characterized by lower mural cell coverage of tumor vasculature, demonstrated higher expression of FAS than the former. Next, in an in vitro coculture system of MS‐1 and 10T1/2 cells as endothelial and mural cells respectively, we show that mural cells decreased endothelial Fas expression. Then, in an in vivo model in which C26 colon carcinoma cells were inoculated together with MS‐1 cells alone or with the further addition of 10T1/2 cells, we demonstrate that mural cells prevented hemorrhage. Finally, knockdown of endothelial Fas sufficiently recapitulated the protection against hemorrhage seen with the addition of mural cells. These results together suggest that regulation of endothelial Fas signaling is involved in the promotion of vascular integrity by mural cells in tumors.

Abstract 5113: Rapid Cancer Imaging By GGT-targeted Fluorescence Probe For Primary Lung Cancer

Introduction Lung cancer is the leading cause of cancer death in Japan, however, it has been difficult to detect and diagnose precisely lung cancer with a diameter less than 1cm to date. The purpose of this study is to investigate clinical application of novel GGT-targeted fluorescence probe for detecting the primary lung cancer in an intraoperative manner. Methods As a fluorescence probe for γ-glutamyltranspeptidase (GGT), γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) was used. gGlu-HMRG is non-fluorescent, but is converted to a highly fluorescent hydroxymethyl rhodamine green (HMRG) upon reaction with the enzyme, which tends to accumulate in GGT-overexpressing cancer cells. First, we examined GGT activity of lung cancer cell lines, A549, H460, H441, H82 and H226, by applying gGlu-HMRG and evaluating fluorescence intensities by confocal fluorescence microscopy. We also compared mRNA expression level of GGT1 (one of the subtypes of GGT) by qRT-PCR. Further, by transfecting siRNA targeted to GGT1, we investigated the target of gGlu-HMRG. Next we performed in vivo imaging of orthotopic A549 lung cancer xenograft model in nude mouse to confirm the validity of fluorescence imaging. Finally, we carried out ex vivo fluorescence imaging of 73 human lung cancers and normal lung tissues which were surgically resected, and the fluorescence intensities were analyzed by Receiver Operating Characteristics curve. Results A549, H460 and H441 cells with high GGT1 expression could be visualized with high fluorescence intensity after application of gGlu-HMRG within several minutes, whereas H82 and H226 cells with relatively low GGT1 expression could not. We ascertained that the target of gGlu-HMRG was GGT1 by fluorescence imaging and qRT-PCR with lung cancer cell lines transfected with siRNA for GGT1. In lung cancer xenograft model, pleural dissemination, hilar and mediastinal lymph node metastasis and the surface of lung cancer were clearly detected within 15 minutes after topical drip of gGlu-HMRG. We confirm that every fluorescent lesion was adenocarcinoma pathologically. In ex vivo human lung cancer fluorescence imaging, the sensitivity, specificity and accuracy were calculated to be 43.8% (32/73cases), 84.9% (62/73cases) and 64.4% (94/146), respectively. The adenocarcinomas, cancer of female or never smoker were more significantly detected by fluorescence imaging (p Conclusions We suggest that intraoperative application of gGlu-HMRG to detect pleural dissemination, small mediastinal lymph nodal metastasis, or other small foci of the lung cancer cells on surgical margin might be feasible when the cancer cells overexpress GGT. Intraoperative application of fluorescence probe is highly expected in near future. Citation Format: Haruaki Hino, Mitsuaki Kawashima, Tomonori Murayama, Junji Ichinose, Kentaro Kitano, Kazuhiro Nagayama, Jun-ichi Nitadori, Masaki Anraku, Tomohiro Murakawa, Kasue Mizuno, Sayaka Tanaka, Mako Kamiya, Nobuhiro Nishiyama, Kazunori Kataoka, Kohei Miyazono, Yasuteru Urano, Jun Nakajima. Rapid Cancer Imaging By GGT-targeted Fluorescence Probe For Primary Lung Cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5113. doi:10.1158/1538-7445.AM2015-5113

Inositol 1, 4, 5-trisphosphate receptor interacts with the SNARE domain of syntaxin 1B

Inositol 1, 4, 5-trisphosphate receptors (IP3Rs) are intracellular ligand-gated Ca2+ channels that mediate Ca2+ release from the endoplasmic reticulum (ER) into the cytosol and function in diverse cellular processes including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. The Ca2+ release activity of IP3Rs is tightly regulated by many factors including IP3R-binding proteins. We show that IP3Rs interact with syntaxin 1 (Syx1), a membrane trafficking protein that regulates various plasma-membrane ion channels including N-, P/Q, and L-type voltage-gated Ca2+ channels, voltage-gated potassium channels, and an epithelial sodium channel. We found that a SNARE-domain of Syx1B, one of the two Syx1 isoforms, directly interacted with the type1 IP3R (IP3R1) internal coupling domain, a known modulator for channel opening. These results indicate that Syx1B is an IP3R-interacting protein and that its interaction may play a crucial role in regulating the channel activity of IP3Rs in Syx1B-expressing cells.