Abstract 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 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.

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.

We conducted an expression analysis of prostate stem cell antigen (PSCA) in normal urogenital tissues, benign prostatic hyperplasia (n = 21), prostatic intraepithelial neoplasia (n = 33), and primary, (n = 137) and metastatic (n = 42) prostate adenocarcinoma, using isotopic in situ hybridization on tissue microarrays. In normal prostate, we observe PSCA expression in the terminally differentiated, secretory epithelium; strong expression was also seen in normal urothelium. Forty-eight percent of primary and 64% of metastatic prostatic adenocarcinomas expressed PSCA RNA. Our studies did not confirm a positive correlation between level of PSCA RNA expression and high Gleason grade. We characterized monoclonal anti-PSCA antibodies that recognize PSCA expressed on the surface of live cells, are efficiently internalized after antigen recognition, and kill tumor cells in vitro in an antigen-specific fashion upon conjugation with maytansinoid. Unconjugated anti-PSCA antibodies demonstrated efficacy against PSCA-positive tumors by delaying progressive tumor growth in vivo. Maytansinoid-conjugated antibodies caused complete regression of established tumors in a large proportion of animals. Our results strongly suggest that maytansinoid-conjugated anti-PSCA monoclonal antibodies should be evaluated as a therapeutic modality for patients with advanced prostate cancer.

To stimulate the dorsolateral frontal cortex, 12 healthy, adult, human females played contract bridge for 1.5 h between initial and final blood sample collections. Flow cytometric analyses of samples, performed in triplicate, showed a significant increase in CD4-positive T lymphocytes. The dorsolateral frontal cortical thickness is significantly and bilaterally reduced in immune-incompetent female, nude mice. Thymic transplants reverse the deficient cortical thickness and CD4-positive cell numbers.

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-gamma in several tissues and elevated serum TNF-alpha 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.