High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

Abstract

Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.

Boosting the biogenesis and secretion of mesenchymal stem cell-derived exosomes

“…GW4869 and manumycin-A were the first and few molecules known to inhibit exosome secretion [6,20]. Recently, prostate cancer cells were screened with small molecules to test their effects on exosome production [5,6], but the molecules identified in that screen (tipifarnib, nitrefazole, pentetrazol, etc.) have not been tested or shown to work in other cell types [5], despite evidence of cell type-specific effects of small molecules on exosome secretion [6].…”

“…A recent screening approach in prostate cancer cells identified 23 significant exosome stimulators and inhibitors [5], with fenoterol, norepinephrine, forskolin, methyldopamine, and mephenesin the top five stimulators. However, the screen was limited to a single prostate cancer cell type, and the identified molecules have yet to be tested or shown to work in other cell types [5,6].…”

“…A recent screening approach in prostate cancer cells identified 23 significant exosome stimulators and inhibitors [5], with fenoterol, norepinephrine, forskolin, methyldopamine, and mephenesin the top five stimulators. However, the screen was limited to a single prostate cancer cell type, and the identified molecules have yet to be tested or shown to work in other cell types [5,6]. Given that manumycin-A, a microbial metabolite, inhibits exosome secretion in C4-2B castration-resistant prostate cancer cells but not in normal RWPE-1 cells, it is possible that small molecule modulators have different effects on different cell types [6].…”

Tumor‐derived exosomes (TDEs): How to avoid the sting in the tail

“…Mounting evidence indicates that changes in cancer cells and the tumor microenvironment can regulate the biogenesis, composition, and function of TDEs, however relative little is known about many of these processes, which could offer new avenues for tumor interventions. Indeed, at least one recent study has utilized a high‐throughput screening approach to identify the potential of repurposed drugs to act as selective inhibitors of exosome biogenesis . This approach represents a tempting means to limit the negative local and systemic effects of TDEs on tumor development and metastasis, including their effects on the antitumor immune response.…”

“…Indeed, at least one recent study has utilized a high-throughput screening approach to identify the potential of repurposed drugs to act as selective inhibitors of exosome biogenesis. 188 This approach represents a tempting means to limit the negative local and systemic effects of TDEs on tumor development and metastasis, including their effects on the antitumor immune response. However, since all cells appear to secrete exosomes, this type of intervention appears likely to cause significant side-effects unless drugs can be identified that target tumorselective mechanisms regulating exosome biogenesis.…”