Abstract
Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 “hits” were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.
Highlights
Lysosomes are multifunctional intracellular organelles containing hydrolytic enzymes that degrade macromolecules and cellular components [1]
Cells were fixed and lysosomes were identified with a lysosome associated membrane protein-1 (LAMP1) antibody, an established lysosomal marker, while the nuclei were visualized with DAPI
The Sloane group found that altered lysosomal locations correlated with increased grade of invasive brain cancer, and later showed that cathepsin B released from these more peripheral lysosomes played a role in tumor invasion [9,40,48]
Summary
Lysosomes are multifunctional intracellular organelles containing hydrolytic enzymes that degrade macromolecules and cellular components [1]. Lysosomes were classically thought to only function in cellular housekeeping, but recent evidence suggests that these organelles contribute to the pathology of many clinically relevant diseases, including malignancies. Lysosomes are involved in tumorigenesis through many different mechanisms, including dysregulated autophagy, aberrant lysosomal trafficking and exocytosis, and increased lysosome membrane permeabilization (LMP) [2,3]. Due to the wide variety of lysosome-mediated functions that play a role in tumor survival, lysosomes have recently been gaining attention as an attractive target for cancer therapeutics. Our previous studies demonstrate that lysosome trafficking plays an important role in regulating cancer cell invasion, whereby tumor cells with lysosomes located close to the plasma membrane secrete more proteases and are more invasive than cells with lysosomes clustered in the perinuclear region [4,5,6,7]. We have shown that several common features of the solid tumor microenvironment, hepatic growth factor (HGF), and acidic extracellular (pHe) trigger lysosome outward movement, accompanied by increased cathepsin B secretion and tumor cell invasion
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