Peter Schow

Apo2L/TRAIL-Dependent Recruitment of Endogenous FADD and Caspase-8 to Death Receptors 4 and 5

Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP. The TNF homolog Apo2L/TRAIL triggers apoptosis through two distinct death receptors, DR4 and DR5; however, receptor over-expression studies have yielded conflicting results on the ligands signaling mechanism. Apo2L/TRAIL induced homomeric and heteromeric complexes of DR4 and DR5 and stimulated recruitment of FADD and caspase-8 and caspase-8 activation in nontransfected cells. TRADD and RIP, which bound TNFR1, did not bind DR4 and DR5. Thus, Apo2L/TRAIL and FasL initiate apoptosis through similar mechanisms, and FADD may be a universal adaptor for death receptors.

Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions.

Our findings not only provide a novel insight into the pathogenesis of the transplant-related atherosclerosis, but also point to a new therapeutic strategy that involves targeting of homing, differentiation and proliferation of putative smooth-muscle progenitor cells derived from the recipient. This is the first report demonstrating that circulating progenitor cells contribute to the development of proliferative diseases. AKIO SAIURA, MASATAKA SATA, YASUNOBU HIRATA, RYOZO NAGAI MASATOSHI MAKUUCHI Department of Surgery, University of Tokyo, Graduate School of Medicine, Tokyo, Japan, Department of Cardiovascular Medicine University of Tokyo, Graduate School of Medicine, Tokyo, Japan A.S. and M.S. supervised this study equally as senior authors Email: sata-2im@h.u-tokyo.ac.jp 1. McKay, R. Stem cells-hype and hope. Nature 406, 361–364 (2000). 2. Asahara, T. et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964–967 (1997). 3. Yamashita, J. et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92–96 (2000). 4. Carmeliet, P. One cell, two fates. Nature 408, 43–45 (2000). 5. Clarke, D.L. et al. Generalized potential of adult neural stem cells. Science 288, 1660–1663 (2000).

Tumor-cell resistance to death receptor-induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor–mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Bax−/− cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair–deficient tumors can cause resistance to death receptor–targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.

Forced Expression of Murine IL-17E Induces Growth Retardation, Jaundice, a Th2-Biased Response, and Multiorgan Inflammation in Mice

IL-17 is a proinflammatory cytokine, and its in vivo expression induces neutrophilia in mice. IL-17E is a recently described member of an emerging family of IL-17-related cytokines. IL-17E has been shown to bind IL-17Rh1, a protein distantly related to the IL-17R, suggesting that IL-17E probably possesses unique biological functions. In this study, we have identified the murine ortholog of IL-17E and developed transgenic mice to characterize its actions in vivo. Biological consequences of overexpression of murine (m)IL-17E, both unique to IL-17E and similar to IL-17, were revealed. Exposure to mIL-17E resulted in a Th2-biased response, characterized by eosinophilia, increased serum IgE and IgG1, and a Th2 cytokine profile including elevated serum levels of IL-13 and IL-5 and elevated gene expression of IL-4, IL-5, IL-10, and IL-13 was observed in many tissues. Increased gene expression of IFN-γ in several tissues and elevated serum TNF-α were also noted. In addition, IL-17E induces G-CSF production in vitro and mIL-17E-transgenic mice had increased serum G-CSF and exhibit neutrophilia, a property shared by IL-17. Moreover, exposure to mIL-17E elicited pathological changes in multiple tissues, particularly liver, heart, and lungs, characterized by mixed inflammatory cell infiltration, epithelial hyperplasia, and hypertrophy. Taken together, these findings suggest that IL-17E is a unique pleiotropic cytokine and may be an important mediator of inflammatory and immune responses.

Characterization of novel UCP5/BMCP1 isoforms and differential regulation of UCP4 and UCP5 expression through dietary or temperature manipulation

Mitochondrial uncoupling proteins have been implicated in the maintenance of metabolic rate and adaptational thermoregulation. We recently reported the identification of a brain‐specific mitochondrial uncoupling protein homologue, UCP4. Here we characterized another newly described member of the uncoupling protein family, termed UCP5 (also called BMCP1). UCP5 transcripts are present in multiple human and mouse tissues, with an especially high abundance in the brain and testis. Expression of UCP5 in mammalian cells reduces the mitochondrial membrane potential. Multiple isoforms of UCP5 were identified and exhibited tissue‐specific distribution and different potency in reduction of membrane potential. Furthermore, the mRNA abundance of both UCP4 and UCP5 is modulated by nutritional status or temperature in a tissue‐specific manner in mice. Brain UCP4 and UCP5 mRNA transcripts rose by 1.5‐ and 1.7‐fold, respectively, and liver UCP5 expression increased by 1.8‐fold in response to acute cold exposure. A high‐fat diet increased UCP5 mRNA in liver by 1.6‐fold selectively in the obesity‐resistant A/J but not in the obesity‐prone C57BL/6J mouse strain. Liver UCP5 expression decreased significantly with a 24 h fast and was restored to the normal level after refeeding. In contrast, brain transcripts for both genes were not significantly altered by fasting or high‐fat diet. These findings are consistent with the notion that UCP4 and UCP5 may be involved in tissue‐specific thermoregulation and metabolic changes associated with nutritional status.–Yu, X. X., Mao, W., Zhong, A., Schow, P., Brush, J., Sherwood, S. W., Adams, S. H., Pan, G. Characterization of novel UCP5/BMCP1 isoforms and differential regulation of UCP4 and UCP5 expression through dietary or temperature manipulation. FASEB J. 14, 1611–1618 (2000)

Vascular Endothelial-Junctional Adhesion Molecule (VE-JAM)/JAM 2 Interacts with T, NK, and Dendritic Cells Through JAM 3

Screening expressed sequence tag databases for endothelial-specific homologs to human junctional adhesion molecule (JAM) and A33-Ag, we identified a protein of 298 aa that represents the recently described vascular endothelial-JAM (VE-JAM)/JAM 2. We confirmed VE-JAM/JAM 2 expression to be restricted to the high endothelial venule of tonsil and lymph nodes, and we further expanded the localization to the endothelium of arterioles in and around inflammatory and tumor foci. In our functional characterizations of VE-JAM/JAM 2, we discovered that it can function as an adhesive ligand for the T cell line J45 and can interact with GM-CSF/IL-4-derived peripheral blood dendritic cells, circulating CD56+ NK cells, circulating CD56+CD3+ NK/T cells, and circulating CD56+CD3+CD8+ cytolytic T cells. In the course of our studies, we also isolated and characterized the functional VE-JAM/JAM 2 receptor, which, upon cloning, turned out to be a submitted sequence representing JAM 3 (accession number NP 113658). With these understandings, we have characterized a protein-interacting pair that can be important in the role of T, NK, and dendritic cell trafficking and inflammation.

Large Oncosomes in Human Prostate Cancer Tissues and in the Circulation of Mice with Metastatic Disease

Oncosomes are tumor-derived microvesicles that transmit signaling complexes between cell and tissue compartments. Herein, we show that amoeboid tumor cells export large (1- to 10-μm diameter) vesicles, derived from bulky cellular protrusions, that contain metalloproteinases, RNA, caveolin-1, and the GTPase ADP-ribosylation factor 6, and are biologically active toward tumor cells, endothelial cells, and fibroblasts. We describe methods by which large oncosomes can be selectively sorted by flow cytometry and analyzed independently of vesicles <1 μm. Structures resembling large oncosomes were identified in the circulation of different mouse models of prostate cancer, and their abundance correlated with tumor progression. Similar large vesicles were also identified in human tumor tissues, but they were not detected in the benign compartment. They were more abundant in metastases. Our results suggest that tumor microvesicles substantially larger than exosome-sized particles can be visualized and quantified in tissues and in the circulation, and isolated and characterized using clinically adaptable methods. These findings also suggest a mechanism by which migrating tumor cells condition the tumor microenvironment and distant sites, thereby potentiating advanced disease.

Development of a frozen cell array as a high-throughput approach for cell-based analysis.

Recent advances in molecular biology, human genetics, and functional genomics tremendously increase the number of molecular targets available for potential therapeutic and diagnostic use. To complement DNA array data, cost-efficient high-throughput technologies providing reliable information at the protein level need to be developed. Here we describe the generation of a frozen cell array that required the use of single cell suspensions and could serve various applications such as the analysis of specific antibody or ligand binding to a large panel of different cell types. As an example, binding of an anti-human epithelial cell adhesion molecule monoclonal antibody to 24 different cell lines has been analyzed using the cell array and compared to the data generated by fluorescence-activated cell sorting. The reliability and flexibility of our frozen cell array technology is compatible with the needs of high-throughput screening for drug discovery and target validation.

Abstract 430: MiRNA profiling of prostate cancer cell-derived large oncosomes identifies a signature of invasion and metastasis

Introduction: We recently demonstrated that amoeboid prostate cancer (PCa) cells shed large (1-10µm diameter), bioactive membrane particles, representing a new class of tumor microvesicles termed large oncosomes (LO). LO can transfer oncogenic signals horizontally within the tumor microenvironment, as demonstrated by activation of phospho-tyrosines and Akt in recipient cells. Methods: We used differential ultracentrifugation and immuno-flow cytometry with size beads to isolate oncosomes in the 1-10µm diameter range. The method was applied to culture medium as well as to the plasma of mice with prostate cancer. In order to identify oncosome miRNAs relevant to metastasis, we performed a GeneChip® miRNA 2.0 Array screen of oncosomes shed by tumorigenic RWPE-2 PCa cells. Non-tumorigenic RWPE-1 cells were also analyzed. Results: LO contained protease and significantly stimulated migration of tumor and endothelial cells. LO also contained Caveolin-1, the GTPase ARF6 and other proteins that we used as markers to identify them in the circulation of mice with PCa, and in murine and human tissues with metastatic disease. Oncosomes also contained RNA. The miRNA profile of the oncosomes was compared to the miRNA profile of the cells of origin. While the miRNA profile of RWPE-2 almost completely matched the profile of RWPE-1 cells, the profiles differed significantly between LO isolated from the two cell lines. In particular, RWPE-2-derived LO contained miR-1228*, miR-150*, miR-373*, miR-135a*, miR-34b and miR-491-3p, which have been implicated in cancer cell proliferation and migration. In particular, the miRNAs miR-135a* and miR-373* target and inhibit the focal adhesion kinase (FAK), which plays a role in promoting a mesenchymal to amoeboid transition. miR-135a* and miR-373* have also been demonstrated to stimulate cell invasion, and are up-regulated in metastatic prostate and breast cancer. Target prediction analysis identified members of pathways involved in metabolic deregulation, and in oncogenic intercellular signaling and cell-cell interaction specifically in the LO purified from the tumorigenic cell line. Conclusions: Collectively, our results indicate that tumor cells can use oncosome formation to actively export pro-oncogenic miRNAs and that oncosomes therefore serve as a potential source of biomarkers of tumor progression. 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 430. doi:1538-7445.AM2012-430

Abstract LB-242: Oncosomes: Large bioactive particles secreted by prostate cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose of the study: Human prostate tumor cells were recently shown by our group to secrete large (0.5-10 m diameter) bioactive microvesicles capable of activating signal transduction and inducing proliferation and migration in cells in the tumor microenvironment (Di Vizio, et al. Cancer Res. 2009 69: 5601-9). These secreted particles, termed oncosomes, were shown to be vehicles for numerous signaling proteins involved in cell growth and motility. They also contain the membrane protein caveolin-1 (Cav-1), a mediator of prostate cancer (PCa) metastasis and a circulating PCa biomarker. The purpose of this study was to develop the tools to identify, isolate, sort and characterize PCa-derived oncosomes. Experimental procedures: Secreted particles from PCa cells were ultracentrifuged at 100,000xg and the pelleted material processed on a Moflo High-Speed Cell Sorter (Beckman-Coulter) and analyzed using Summit V4.3 software (Dako Colorado, Inc.). 1, 3 and 10 m bead standards (Spherotech Inc.) were used to set size gates. To avoid capturing aggregated material, forward scatter and pulse width were set at a linear scale and only single events were gated and analyzed. Sorted particles were visualized by confocal and electron microscopy. Results: Using the above procedure, we were able to retrieve intact particles of 1-10 m in diameter secreted by LNCaP cells overexpressing the oncoprotein MyrAkt1. The nontumorigenic prostate stromal cell line WPMY-1 secreted > 10-fold fewer particles in this size range. We confirmed that these were membrane-derived particles by showing that they contained MyrAkt1, which localizes to the plasma membrane. Particles in a similar size range were also sorted after purification from LNCaP cells stably expressing a Cav-1/GFP fusion protein. Sorted particles visualized microscopically appeared to be surrounded by a lipid bilayer and to have intrinsic structure that made them resistant to collapse. Particles isolated by these methods evoked degradation of FITC-labeled collagen and contained active MMP9 and MMP2, two key proteases involved in tumor cell invasion. Conclusions: The specific type of large bioactive particle we have isolated has not been previously described in any tumor system. Studies are ongoing to detect these particles in animal models of cancer and in human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-242.