Abstract B5: A novel high-content screening approach to identify inhibitors of lysosome anterograde trafficking and tumor invasion

Abstract

Abstract Background: Tumor cell invasion and metastasis are a key factors contributing to morbidity and mortality in cancer. Previous studies from our laboratory have demonstrated that intracellular trafficking of lysosomes plays an important role in regulating cancer cell invasion. The outward movement of lysosomes stimulated by certain growth factors and acidic extracellular tumor microenvironment is accompanied by increased protease secretion and tumor cell invasion. Troglitazone (an anti-diabetic) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), both with sodium-proton exchangers inhibitory activity, stimulate inward movement of peripheral lysosomes in prostate cancer cells forming an aggregate near the nucleus (termed juxtanuclear lysosome aggregation) resulting in reduced protease secretion and tumor cell invasion. We therefore propose that drugs that affect lysosome trafficking could potentially lower tumor invasiveness. This represents a novel approach in translational cancer research that could potentially discover new effective anti-invasion and anti-metastatic drugs. Materials and Methods: DU 145 prostate cancer cells were seeded in 96 wells plates. Compounds to be screened were applied overnight in each well, and the next day cells fixed and immunostained for lysosome associated protein-1 (LAMP-1). Acidic media and EIPA were used as negative and positive control respectively for lysosome position. A Cellomics-based biocompartmental algorithm was used to measure the relative position of lysosomes in the cells following exposure to compounds at 5 micromolar (μM). The parameters for a drug “hit” were based on Z'-factor calculated using the positive and negative controls. Results: A total of 2240 drug compounds from 4 different libraries (repositioning and phytochemical) were screened. We identified 18 “hits” and further explored Niclosamide as a drug that inhibited lysosome trafficking. This is an anti-helminth drug that affects the oxidative phosphorylation of the parasites and has been studied in colon cancer invasion and metastasis. A dose of Niclosamide of 1 μM and less was not toxic to DU145 cells, normal myofibroblasts or normal epithelial cells. Acidic extracellular pH induced lysosome redistribution was blocked by Niclosamide at a dose of 625 nanomolar (nM) and above. HGF and EGF induced lysosome redistribution was blocked by Niclosamide at a dose of 312 nM and above. A time course analysis demonstrated that Niclosamide altered lysosomal positioning beginning at 2-4 hours of exposure. Effective concentrations did not affect microtubules. At a dose of 1 μM and exposure time of 4 hours, Niclosamide exhibits no significant effect on ATP level. Conclusion: We have developed a valid high content screening approach to identify drugs that inhibit lysosome trafficking, a process that plays a key role in tumor invasion. Niclosamide exhibits an inhibitory effect on lysosome trafficking at doses as low as 312 to 615 nM and with exposure times as low as 2 hours. Further studies assessing its mechanism of action on lysosome position and effect on cancer cell invasion are ongoing. We are also beginning to explore the other 17 “hits” we discovered including a few that are currently used only for neurological indications. Finally, we are screening larger chemical diversity libraries for compounds that could be developed into tumor invasion inhibitors. Citation Format: Hazem El-Osta, Samantha Dykes, Jennifer Carroll, Floyd Galiano, James Cardelli. A novel high-content screening approach to identify inhibitors of lysosome anterograde trafficking and tumor invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B5.