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Abstract
The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and DIAPH3 silencing evokes a transition to an amoeboid tumor phenotype in multiple cell backgrounds. This amoeboid transformation is accompanied by increased tumor cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the formation of atypically large (>1 μm) membrane blebs that can be shed as extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of nano-sized EV (e.g., exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused EV shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of EV production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells. DU145 EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV produced as a result of DIAPH3 loss or growth factor stimulation may condition the tumor microenvironment through multiple mechanisms, including the proliferation of cancer cells and suppression of tumor-infiltrating immune cells.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank Dr Anthony Hill of the Boston Children’s Hospital IDDRC Imaging Core for technical assistance with confocal imaging. This study was supported by NIH 1R01CA143777, 1R01CA112303, and US Department of Defense PC093459 (to M.R.F), NIH 5R00CA131472 (to D.D.V.), a Ladies Auxiliary VFW Cancer Research Postdoctoral Fellowship (to S.M.) and the Fishbein Family IC Research Foundation/Interstitial Cystitis Association (ICA), Pilot Research Program/ICA, New York Academy of Medicine, and Boston Children’s Hospital Faculty Development (to J.K.). The research described was also supported by NIH/National Center for Advancing Translational Science (NCATS) UCLA CTSI Grant Number UL1TR000124. J.K. is an American Urological Association Foundation Research Scholar and a Harvard Medical School Eleanor and Miles Shore Scholar. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contributions
J.K. and M.R.F. conceived of the study, designed experiments, evaluated data, and wrote the paper. J.K., S.M., M.L., and D.B. performed experiments. D.T.U. provided expertise and supervised data interpretation. S.M. and D.D.V. contributed conceptually and intellectually and to the writing of the manuscript.