The histone deacetylase inhibitor cambinol prevents acidic pHe-induced anterograde lysosome trafficking independently of sirtuin activity

AbstractCommon features of the solid tumor microenvironment, such as acidic extracellular pH and growth factors, are known to induce the redistribution of lysosomes from a perinuclear region to a position near the plasma membrane. Lysosome/plasma membrane juxtaposition facilitates invasion by allowing for the release of lysosomal proteases, including cathepsin B, which contribute to matrix degradation. In this study we identified the sirtuin 1/sirtuin 2 (SIRT1/2) inhibitor cambinol acts as a drug that inhibits lysosome redistribution and tumor invasion. Treatment of cells with cambinol resulted in a juxtanuclear lysosome aggregation (JLA) similar to that seen upon treatment with the PPARγ agonist, troglitazone (Tro). Like Tro, cambinol required the activity of ERK1/2 in order to induce this lysosome clustering phenotype. However, cambinol did not require the activity of Rab7, suggesting that this drug causes JLA by a mechanism different from what is known for Tro. Additionally, cambinol-induced JLA was not a result of autophagy induction. Further investigation revealed that cambinol triggered JLA independently of its activity as a SIRT1/2 inhibitor, suggesting that this drug could have effects in addition to SIRT1/2 inhibition that could be developed into a novel anti-cancer therapy

Lysosome trafficking is necessary for EGF-driven invasion and is regulated by p38 MAPK and Na+/H+ exchangers

Abstract Background Tumor invasion through a basement membrane is one of the earliest steps in metastasis, and growth factors, such as Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF), stimulate this process in a majority of solid tumors. Basement membrane breakdown is one of the hallmarks of invasion; therefore, tumor cells secrete a variety of proteases to aid in this process, including lysosomal proteases. Previous studies demonstrated that peripheral lysosome distribution coincides with the release of lysosomal cathepsins. Methods Immunofluorescence microscopy, western blot, and 2D and 3D cell culture techniques were performed to evaluate the effects of EGF on lysosome trafficking and cell motility and invasion. Results EGF-mediated lysosome trafficking, protease secretion, and invasion is regulated by the activity of p38 mitogen activated protein kinase (MAPK) and sodium hydrogen exchangers (NHEs). Interestingly, EGF stimulates anterograde lysosome trafficking through a different mechanism than previously reported for HGF, suggesting that there are redundant signaling pathways that control lysosome positioning and trafficking in tumor cells. Conclusions These data suggest that EGF stimulation induces peripheral (anterograde) lysosome trafficking, which is critical for EGF-mediated invasion and protease release, through the activation of p38 MAPK and NHEs. Taken together, this report demonstrates that anterograde lysosome trafficking is necessary for EGF-mediated tumor invasion and begins to characterize the molecular mechanisms required for EGF-stimulated lysosome trafficking

A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

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

The Arf-like GTPase Arl8b is essential for three-dimensional invasive growth of prostate cancer in vitro and xenograft formation and growth in vivo

Cancer is a multistep process that requires cells to respond appropriately to the tumor microenvironment, both in early proliferative stages and in later invasive disease. Arl8b is a lysosome localized Arf-like GTPase that controls the spatial distribution of lysosomes via recruitment of kinesin motors. Common features of the tumor microenvironment such as acidic extracellular pH and various growthfactors stimulate lysosome trafficking to the cell periphery (anterograde), which is critical for tumor invasion by facilitating the release of lysosomal proteases to promote matrix remodeling. Herein we report for the first time that Arl8b regulates anterograde lysosome trafficking in response to hepatocyte growth factor, epidermal growth factor, and acidic extracellular pH. Depletion of Arl8b results in juxtanuclear lysosome aggregation, and this effect corresponds with both diminished invasive growth and proteolytic extracellular matrix degradation in a three-dimensional model of prostate cancer. Strikingly, we found that depletion of Arl8b abolishes the ability of prostate cancer cells to establish subcutaneous xenografts in mice. We present evidence that Arl8b facilitates lipid hydrolysis to maintain efficient metabolism for a proliferative capacity in low nutrient environments, suggesting a likely explanation for the complete inability of Arl8b-depleted tumor cells to grow in vivo. In conclusion, we have identified two mechanisms by which Arl8b regulates cancer progression: 1) through lysosome positioning and protease release leading to an invasive phenotype and 2) through control of lipid metabolism to support cellular proliferation. These novel roles highlight that Arl8b is a potential target for the development of novel anti-cancer therapeutics

Niclosamide inhibits lysosome trafficking, motility, and invasion in glioma A172 cells.

<p><b>(A)</b> A172 cells were treated for 8 hours with DMSO, 25 μM EIPA, or varying concentrations of niclosamide diluted in low pH media (pH 6.4). Next, cells were fixed and stained for LAMP-1 (red), actin (green) and DAPI (blue). Lysosomes in DMSO control have a peripheral location whereas in niclosamide or EIPA-treated cells they are located around the nucleus (as indicated by arrows). <u>Scale bars: 10</u> μ<u>m</u>. <b>(B)</b> A172 cells were plated in collagen-coated 96 well plates and allowed to form a confluent monolayer prior to wounding. Next, 20% matrigel was added to the wells for which invasion is to be studied. Cells were allowed to migrate or invade in the presence of DMSO, 33 ng/mL HGF or 100 ng/mL EGF in the presence or absence of 0.3 μM niclosamide. Motility and invasion were calculated by IncuCyte Imager and the relative wound density percentage at 24 hours post wound is shown. Error bars represent the SD from at least 3 independent experiments. * p<0.05 treatment versus serum free or DMSO, ** p<0.01 treatment versus serum free or DMSO.</p

Bafilomycin A1, a specific inhibitor of vacuolar-type proton pump, induced perinuclear distribution of lysosomes.

<p><b>(A)</b> DU145 cells were treated for 16 hours with DMSO, 25 μM EIPA, 0.1 μM bafilomycin, 1 μM niclosamide or 50 μM chloroquine. Then, pH was dropped to 6.4 in all conditions for an additional 2 hours. Cells were then fixed and immunostained for LAMP1 (red), DAPI (blue) and actin (green). <u>Scale bars: 10</u> μ<u>m</u>. <b>(B)</b> DU145 cells were treated for 16 hours with DMSO, bafilomycin A1 (Baf), niclosamide (Nic), or EIPA in serum free (SF) and complete media (S). Lysosome distribution was calculated “mean ring spot count channel 3” using the Cellomics Imager. Error bars represent the SD from at least 3 independent experiments. * denotes statistical significance (p<0.05) relative to treatment with DMSO.</p

Lysosomes in niclosamide-treated cells are not leaky and are not autophagosomes.

<p><b>(A)</b> LC3-mCherry-GFP transfected DU145 cells were treated with 1 μM niclosamide for 4 hours or 24 hours and cells were fixed and stained for LAMP-1. Dashed circles represent nuclei. Arrows indicate increased colocalization of LC3-GFP with LAMP-1. <u>Scale bars: 10</u> μ<u>m</u>. <b>(B)</b> DU145 cells were incubated with Acridine Orange, washed, and treated with DMSO, niclosamide or chloroquine for 16 hours. Red represents acridine orange accumulation in the acidic lysosomes, as seen in DMSO condition. In cells treated with chloroquine or different concentrations of niclosamide, the leaked acridine orange in cytosol is no more concentrated in the acidic lysosomal compartment and therefore its color turns into green. <u>Scale bars: 10</u> μ<u>m</u>. <b>(C)</b> DU145 cells were loaded with dextran 40kDa (green) and then treated overnight with DMSO, 10 μM mefloquine, or 0.6 μM niclosamide. Cells were fixed and stained for DAPI (blue). In control cells lysosomes have a peripheral distribution (bold arrows), whereas in niclosamide treated cells lysosomes are intact and found near the nucleus (arrowheads). Mefloquine induces lysosomal membrane permeabilization, LMP, as manifested by green cytosolic haziness (thin arrows) and is the positive control. <u>Scale bars: 10</u> μ<u>m</u></p

Niclosamide blocks growth factor-induced motility and invasion of DU145 prostate cancer cells.

<p><b>(A)</b> DU145 cells were seeded in collagen-coated 96 well plates and allowed to form a confluent monolayer prior to wounding with a 96 well scratcher. For invasion assays, matrigel was added on top of the cells after wounding. Cells were allowed to migrate or invade in the presence of 33 ng/mL HGF or 100 ng/mL EGF in the presence or absence of 0.3 μM niclosamide. Wound closure was measured in real time using the IncuCyte Imager. Dotted lines indicate wound size at time 0 and 24 hours. <b>(B)</b> Motility and invasion were calculated by the IncuCyte platform as relative wound density percentage at 24 hours. Error bars represent the SD from at least 3 independent experiments. * denotes statistical significance (p<0.01) of niclosamide versus respective control.</p