Project 2 is based on preliminary data demonstrating that prostate cancer-derived exocytosed microvesicles of endosomal origin, exosomes (EX), containing ?v integrins provide a paracrine mechanism of regulation of prostate cancer cell motility. During the current funding cycle, we have investigated whether the ?v6 and ?v3 integrins known to be highly up-regulated in cancer and metastasis, are transferred among different subsets of prostate cancer cells through EX and have the ability to functional aberrations in the recipient cells. We have recently described that these ?v integrins are present in EX of several prostate cancer cells, are transferred from donor to recipient cells and are localized to the cell surface indicating they are functional. The quality of our EX preparations, obtained by differential ultracentrifugation, was tested by electron microscopyJ continuous sucrose gradient, and biochemical characterization using CD63, CD81 and Flotillin-1, as markers known to be enriched in EXJ furthermore, EX internalization was determined by confocal microscopy. The active state of ?v integrins is confirmed in cell migration assays which show that the ?v6 integrin, transferred through EX, promotes cell migration of recipient cells mediated by specific ligands. Furthermore, we show that focal adhesion kinase and src, known downstream effectors of integrins in cell motility, are localized in EX as active phosphorylated forms indicating that these microvesicles may transfer entire signaling compartments to a recipient cell. To evaluate the relevance of our findings, we purified EX from sera of TRAMP mice, which had developed prostate cancer and show that the ?v3 integrin is expressed in these EX. Furthermore, in collaboration with Core B, we show a proteomics analysis of EX (i) and modulation of EX content in response to downregulation of ?v6 (ii). These results show a unique EX protein signature compared with cell lysates and uncover an increase in GRP94 levels in EX upon downregulation of ?v6. In summary, this study shows that ?v integrins are transferred among different subsets of prostate cancer cells through EX, promote cell migration through interaction with specific ligands, and are found in EX purified from serum. Based on these data, three specific aims will investigate the role of the ?v integrins delivered by EX in intercellular communications with respect to phenotypic changes occurring upon uptake of EX by either cancer cells or myeloid-derived suppressor recipient cells (Aim 1), characterize the role of integrins, their downstream effectors and associated transmembrane proteins transferred through EX in cancer cell bioenergetics, modulation of the cellular stress response, and activation of cell motility kinases (Aim 2), and test the composition of EX and role of EX-mediated prostate cancer progression using SCID mice as well as castrated, prostate-specific Pten conditional knockout mice (Aim 3). Overall this study will bring new insights in the role of EX containing ?v integrins in prostate cancer progression.
This project is focused on the study of a paracrine mechanism of regulation of prostate cancer cell motility. The results will bring new insights in the role of containing ?v-integrins exosomes, exocytosed microvesicles of endosomal origin, in prostate cancer metastasis and will pave the way for future studies aimed to test the diagnostic and therapeutic potential of exosomes containing ?v-integrins in prostate cancer.
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