The Arf-like GTPase Arl8b is essential for three-dimensional invasive growth of prostate cancer in vitro and xenograft formation and growth in vivo - PubMed (original) (raw)
The Arf-like GTPase Arl8b is essential for three-dimensional invasive growth of prostate cancer in vitro and xenograft formation and growth in vivo
Samantha S Dykes et al. Oncotarget. 2016.
Abstract
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 growth factors 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.
Keywords: Arl8b; invasion; lipid metabolism; lysosome; xenograft.
Conflict of interest statement
The authors declare no conflicts of interest.
Figures
Figure 1. Arl8b knockdown prevents low pH-, EGF-, and HGF-induced lysosome trafficking to the cell membrane
A. DU145 and PPC1 cells were transduced with lentiviral-delivered shRNA sequences targeted to Arl8b (KD) or non-targeted (NT) shRNA. Immunoblots confirm knockdown. B. DU145 and PPC1 cells were treated with low pH for 2 hours, or 33 ng/mL HGF or 100 ng/mL EGF for 18 hours then fixed, stained for LAMP-1 (Red), phalloidin (Green), and DAPI (Blue). C. Quantitated lysosome distribution from 25 cells shown as mean ± SEM; *=p<0.001 compared to NT control, #=p<0.05 compared to NT control.
Figure 2. Arl8b knockdown prevents the formation of invasive outgrowths and matrix degradation
A. DU145 and C. PPC1 cells were grown in Matrigel and DQ-Collagen IV for 72 hours under the indicated experimental conditions then fixed and stained with phalloidin (red). Green represents DQ-collagen IV cleavage as a readout for protease secretion. Images are representative of three independent experiments and are quantified in B. and D.. Data are shown as mean ± SEM;*=p<0.05 compared to NT.
Figure 3. Arl8b knockdown does not affect HGF- or EGF-induced signaling or cell scattering
A. DU145 NT and Arl8b KD cells were serum starved 30 minutes prior to treating with 33 ng/mL HGF or 100 ng/mL EGF for 20 minutes. Cell lysates were analyzed by immunoblot for the indicated proteins. B. Ratio of normalized densitometry from three independent immunoblots. Data are shown as mean ± SEM. C. DU145 NT and Arl8b KD cells were treated with 33 ng/mL HGF or 100 ng/mL EGF overnight in serum-free media. Cells were fixed and stained for actin. Representative images from 10X and 63X fields are shown, N=3. D. Quantitative analysis of cell scattering from three independent experiments. Data are shown as mean ± SEM; *=p<0.001 compared to NT control and #=p<0.001 compared to KD control.
Figure 4. Defective Arl8b KD motility is not rescued by co-culture with NT cells
A. DU145 or B. PPC1 cells were grown on top of transwell inserts in serum-free media and allowed to migrate toward serum-containing media for 48 hours. Cells were fixed and stained with crystal violet. Images represent 10X fields of cells that have migrated to the underside of the insert, N=3. Graphs represent the average number of motile cells from three independent experiments; *=p<0.001 compared to NT.
Figure 5. Arl8b depletion prevents tumor growth in vivo
A. PPC1 NT or Arl8b KD cells were grown subcutaneously in SCID/bg mice and tumor volume was measured over time. Data are shown as mean ± SEM; *=p<0.001 compared to Arl8b shRNA. B. Immediately following subcutaneous injections, remaining cell suspensions were directly seeded onto 10 cm dishes through the same needle and syringe used for injections and allowed to settle for 24 hours in complete media. C. PPC1 NT or Arl8b KD cells were grown subcutaneously in SCID/bg mice and tumor volume was measured over time. Data are shown as mean ± SEM; *=p<0.001 compared to Arl8b shRNA. D. Tumor weight was measured at time of harvest. Data are shown as mean ± SEM; *=p<0.001 compared to NT shRNA. E. Representative mice from each experimental group are shown.
Figure 6. Arl8b depletion imposes an aberrant lipogenic phenotype to prostate cancer cells and impairs proliferation under limited nutrient conditions
A. PPC1 NT and Arl8b KD cells were seeded to approximately 15% confluency prior to proliferation analysis in complete growth media (10% serum, 100% glucose). Data are shown as mean ± SEM; *=p<0.05 compared to NT serum-free and #=p<0.01 compared to cells grown in complete media, or B. serum-free media at 100%, 30%, 10%, or 3% of normal glucose concentration. Relative change in confluency over 4 days was determined using the IncuCyte ZOOM imaging system and analysis software. Conditions were tested in quadruplicate for three independent experiments. Data are shown as mean ± SEM; *=p<0.01 compared to NT and $=p<0.05 compared to NT. C. Biotinylated cell surface protein from DU145 or PPC1 NT and Arl8b KD cells growing in complete growth media was isolated and immunoblotted for Glut1. Densitometric analysis of Glut1 expression relative to Tubulin is shown. D. DU145 or PPC1 NT and Arl8b KD cells serum starved for twelve hours were fixed and stained for neutral lipid inclusion bodies. Representative images from 63x-objective confocal microscopy are shown and quantitation from 40x-objective confocal fields (five fields, three independent experiments) is graphed. Data are shown as mean ± SEM; *=p<0.01 compared to NT. E. RNA from DU145 or PPC1 NT and Arl8b KD cells grown in complete media was analyzed by qPCR for acetyl CoA carboxylase (ACACA) and ATP citrate lysase (ACLY). Data are shown as mean ± SEM; *=p<0.01.
Figure 7. Proposed model for Arl8b regulation of protease-dependent tumor invasion and activation of the lipogenic phenotype
A. Arl8b regulates lysosome trafficking to the periphery, which facilitates release of lysosomal proteases and ECM remodeling. P: protease. B. Arl8b regulates hydrolysis of internalized and/or stored lipids preventing unnecessary, metabolically inefficient de novo lipogenesis. This mechanism would maintain adequate ATP production supporting a proliferative capacity in a low nutrient environment. LP: lysosomal proteases, TAG: triaclyglycerides, CE: cholesterol esters, LAL: lysosomal acid lipase.
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