Abstract 4065: A novel imaging-based high-throughput assay identifies Niclosamide as inhibitor of lysosome anterograde trafficking and tumor invasion

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Tumor cell invasion and metastasis are key factors contributing to morbidity and mortality in cancer. Previous studies from our laboratory have demonstrated that the outward movement of lysosomes stimulated by certain growth factors or acidic media is accompanied by increased cathepsin B secretion and consequently tumor cell invasion. Troglitazone and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) stimulate inward movement of lysosomes in prostate cancer cells resulting in reduced cathepsin B secretion and tumor cell invasion. We propose to screen for repurposed drugs that block lysosome trafficking and lower tumor invasiveness. Materials and Methods: DU 145 prostate cancer cells seeded in 96-wells plates were treated with the compounds overnight, then immunostained for lysosomal-associated membrane protein-1 (LAMP-1). Acidic media and EIPA were used as negative and positive control respectively. An imaging-based automated system was used to measure the position of lysosomes relatively to the nucleus. A variety of biochemical and cell biological approaches were used to further characterize the properties of the lead compound. Effect on lysosomal protease secretion was conducted using a fluorometric-based cathepsin B assay. Motility and Invasion studies were conducted using Incucyte system acquiring real-time pictures of cells covered with matrigel. Results: A total of 2240 repurposed drugs were screened. We identified 18 “hits” and further explored Niclosamide, a drug that inhibits lysosome trafficking. A dose of Niclosamide below 1 microM was not toxic on DU145 cells, normal myofibroblasts or epithelial cells. Niclosamide blocked acidic-induced lysosome redistribution at a dose of 625 nanomolar (nM) and above, and blocked HGF and EGF-induced lysosome redistribution at a dose of 312 nM and above. Niclosamide altered lysosomal positioning beginning at 2-4 hours of exposure. At a dose of 1 microM and exposure time of 4 hours or below, Niclosamide exhibited no significant effect on ATP level, microtubules or actin. The effect of Niclosamide on lysosome positioning was not affected by disruption of actin, or microtubules, neither by inhibition of PI3K or MAPK pathways. Cathepsin B secretion induced by extracellular acidic pH was blocked by Niclosamide. HGF and EGF-induced cancer cell invasion and motility were both reduced by Niclosamide. A decrease in extracellular matrix degradation was noticed when cells grown on DQ collagen gel were treated with Niclosamide. Conclusion: We have developed a valid high-content screening approach to identify drugs that inhibit lysosome trafficking and reduce tumor invasion. Further characterization of the other “hit”, including drugs that are used for neurological indications, are ongoing on glioma cells. Our findings represent a novel approach in cancer research that could potentially bring effective drugs that inhibits tumor invasion. Citation Format: Hazem E. El-Osta, Samantha Dykes, Magdalena Circu, Jennifer Carroll, Kinsey Kelly, Floyd Galiano, Glenn Mills, James Cardelli. A novel imaging-based high-throughput assay identifies Niclosamide as inhibitor of lysosome anterograde trafficking and tumor invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4065. doi:10.1158/1538-7445.AM2014-4065