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

DU145 cells were purchased from ATCC (ATCC-HTB-81, Manassas, VA) and maintained in RPMI 1640 media (Mediatech, Corning, NY) supplemented with 10% Fetal Bovine Serum (FBS). HeLa cells were obtained from ATCC (ATCC-CCL-2) and maintained in DMEM media (Mediatech) supplemented with 10% FBS. Cells were grown at 37 °C in 5% CO₂ and passaged upon reaching 75% confluence.

Troglitazone, AG490, Bay11, SP600125, PD169316, and SB203580 were purchased from Cayman Chemicals (Ann Arbor, MI). Hepatocyte Growth Factor, SB202474, AG1478, U0126, and SU11274 were purchased from Calbiochem (San Diego, CA). SB239063 and LY294002 were obtained from Enzo Life Sciences (Farmingdale, NY). Epidermal Growth Factor and 5(N-Ethyl-N-isopropyl) amiloride (EIPA) were acquired from Sigma (St. Louis, MO). Antibodies recognizing total p38 MAPK and phosphorylated EGFR Y845, Met Y1234/1235, AKT S473, MAPK 44/42 T202/204, and p38 MAPK T180/Y182 were used at 1:1000 and supplied by Cell Signaling Technology (Beverly, MA). Antibodies recognizing total EGFR (1:1000), AKT1 (1:4000) and ERK 1/2 (1:4000) were obtained from Santa Cruz Biotechnology (Dallas, TX). The total c-Met (1:1000) antibody was purchased from Life Technologies (Carlsbad, CA). The α-tubulin antibody was purchased from NeoMarkers (Fremont, CA) and was used at 1:20,000. The LAMP-1 H4A3 antibody was supplied by the Developmental Studies Hybridoma Bank at the University of Iowa and was used at a 1:200 dilution for immunofluorescence. Matrigel, anti-EEA1, and anti-GM130 were obtained from BD Bioscience (San Jose, CA) and used at 1:100. DQ-collagen IV, Oregon Green or 635 Phalloidin (1:200) and mounting media containing DAPI plus SlowFade Gold reagent were obtained from Invitrogen Life Technologies (Grand Island, NY). Dylight 594 donkey anti-mouse was purchased from Jackson Immuno Research (West Grove, PA) and used at 1:200. Secondary antibodies (HRP- conjugated anti-mouse and anti-rabbit) for western blot were purchased from GE Healthcare, Pittsburgh, PA and used at 1:5000. Since a majority of the pharmacological inhibitors were solubilized in DMSO, a DMSO concentration of 0.1% was in contact with the cells and used as a control in all pharmacological inhibitory experiments.

Experiments conducted in 2-diminesional cell culture, cells were seeded at ~50% confluence on glass cover slips. Following treatment, cells were fixed with ice cold 4% paraformaldyhide (PFA) pH 7.2 for 20 min. Cells were washed twice with phosphate buffered saline (PBS) then incubated for 1 h with primary antibody diluted in 0.25% bovine serum albumin (BSA) and 0.1% Saponin in PBS (BSP). After incubation with primary antibody, cells were washed twice with PBS and incubated with fluorescently conjugated secondary antibody diluted in BSP for 1 h. To visualize the cytoskeleton, cells were incubated with phalloidin diluted in BSP for 20 min. Cells were then washed three times in PBS and mounted using DAPI with Slow Fade Gold reagent. Images were taken using an Olympus UPlanFl 40X/0.75 objective on an Olympus BX50 microscope, utilizing a Roper Scientific Sensys Camera, and MetaMorph software. Images were pseudocolored and merged using ImageJ. For 3-dimensional immunofluorescence of LAMP-1, all reagents were warmed to 37 °C. Cultures were fixed with 4% PFA for 20 min then quenched with 100 mM glycine in PBS for 10 min. Cells were then washed 2X with PBS and permeabilized/blocked for 30 min with 10% donkey serum and 1% F(ab)₂ Fragment anti-mouse (Jackson IR, West Grove, PA) diluted in BSP. Cells were washed 2X in PBS with the remainder of the protocol remaining the same as for 2-dimensional immunofluorescence. Images were taken using a HCX Plan Apo 63X/1.4–0.6 oil objective on a Leica TCS SP5 microscope utilizing Leica LAS AF software.

3D cultures supplemented with DQ-collagen IV were prepared using a modification of a previously described protocol [22]. Briefly, 120 μL ice cold Matrigel was supplemented with 25 μg/mL DQ-collagen IV, plated on coverslips, and allowed to solidify at 37 °C for 15 min. 1X10⁵ cells were diluted in media containing serum and plated on top of the solidified extracellular matrix for two days to allow for colony formation. Once multicellular colonies were visualized, the media was replaced with serum free media containing inhibitors and/or growth factor for 48 h. Colonies were then fixed for 30 min with 37 °C 4% PFA and washed twice with warm PBS. After staining and imaging, images were analyzed for extracellular DQ-collagen IV signal using Image J. Briefly, a mask was generated to include the area of the phalloidin staining. This area was subtracted from the DQ-collagen IV signal using Image Calculator. Remaining extracellular DQ-collagen IV signal was recorded as integrated density and displayed as arbitrary units.

Performed as previously described [16].

LysoTracker software was a generous gift from Meiyappan Solaiyappan at Johns Hopkins University [23]. This program was used to analyze the distance of fluorescently labeled lysosomes from the nucleus border. Twenty-five representative cells spanning three independent experiments were analyzed for each experimental condition.

50 μL of a 1:5 dilution of Matrigel in serum free RPMI was plated on Costar Transwell Permeable Support inserts with 8.0 μm pores and allowed to solidify at 37 °C for two hours. Matrigel was re-hydrated with 50 μL serum free media for an additional 30 min at 37 °C. 1X10⁴ cells including pharmacological inhibitors and/or EGF were seeded in a total volume of 100 μL on top of the insert and allowed to invade for 48 h. Growth factor and inhibitor treatments were maintained in serum free media for the duration of the experiment. Transwell membranes were then fixed with 4% PFA for 20 min and stained with crystal violet for 20 min. Transwell inserts were washed with PBS and cells remaining on the top of the insert were removed using a cotton swab. Five representative 10X fields were counted from three independent experiments.

ImageJ software was used for western blot quantification. The ratio of the intensity of each protein band to its corresponding tubulin load control was calculated and graphed.

Cell scattering is a morphological readout for in vitro motility and is often associated with tumor cell response to growth factor stimulation. Signaling through both the HGF/c-Met and EGF/EGFR is a potent inducer of cell scattering in the DU145 prostate cancer cell line [24‐27]. We used several specific inhibitors to test whether these two receptor tyrosine kinases utilized similar down-stream signaling cascades to regulate scattering. DU145 cells were treated with specific inhibitors of PI3K/AKT (LY29004) [28], MEK/ERK (U0126) [29] or p38 MAPK (SB203580) [30] then stimulated with either HGF or EGF. All inhibitors were used at 5 or 10 μM unless otherwise noted. These inhibitor concentrations have been previously shown by our lab to be pathway specific and not inhibit other signaling pathways under the conditions of this study [16]. Cells were fixed and stained with FITC-labeled phalloidin to visualize F-actin and the percent of scattered cells was analyzed for each experimental condition (Fig. 1a; quantified in Fig. 1b). Control-treated cells assumed a cobblestone morphology which lost cell-cell adhesions upon treatment with growth factors. Inhibition of PI3K/AKT or MEK/ERK inhibited HGF-mediated scattering as previously described [17]. However, only p38 inhibition, and not inhibition of PI3K/AKT or MEK/ERK, blocked EGF-mediated cell scattering. This suggests that HGF/c-Met and EGF/EGFR regulate cell motility via different downstream pathways and that p38 MAPK activity is necessary for EGF/EGFR-mediated scattering.

In addition to stimulating cell motility/scattering, HGF has been reported to redistribute lysosomes from the perinuclear region to the cell periphery and this lysosome redistribution is necessary for HGF-mediated invasion [15, 17]. We therefore asked whether EGF stimulation would similarly cause anterograde lysosome trafficking. DU145 cells were treated with EGF or HGF and then fixed and stained for lysosome-associated membrane protein-1 (LAMP-1) (red), actin (green), and DAPI (blue) (Fig. 2a; quantified in Fig. 2b). Similar to what was observed with HGF treatment, EGF stimulation resulted in anterograde trafficking of LAMP-1 positive lysosomes to actin rich cellular protrusions.

Several studies suggest that c-Met and EGFR undergo crosstalk and can transactivate each other; raising the possibility that EGF stimulation drives lysosome trafficking through c-Met transactivation [31‐33]. To test whether EGFR transactivates c-Met, DU145 cells were first pre-treated with the c-Met inhibitor SU11274 [34] or the EGFR inhibitor AG1478 [35] and then stimulated with HGF or EGF. Western blot analysis revealed that HGF specifically activated c-Met signaling, which was not reduced in the presence of the EGFR inhibitor. Additionally, EGF activated EGFR and downstream EGFR signaling was not depleted under conditions of c-Met inhibition (Fig. 2c; quantified in Additional file 1: Figure S1). Dulak et al. suggested that EGF signaling results in c-Met activation at later time points [31]. Therefore, we treated cells with EGF over a 24-h time period and probed for EGFR and c-Met activation by western blot (Fig. 2d; quantified in Additional file 1: Figure S1). No increase in c-Met phosphorylation was observed at early or late timepoints post EGF stimulation, suggesting that there was no EGFR/c-Met signaling crosstalk in our system. In order to assess whether EGF-stimulated anterograde lysosome trafficking is EGFR specific, we treated cells with the EGFR inhibitor AG1478 or the c-Met inhibitor SU11274 in the presence or absence of EGF and observed the redistribution of LAMP-1 positive vesicles (red) by immunofluorescence microscopy (Fig. 2e; quantified in 2f). EGF-mediated anterograde lysosome trafficking was blocked by the addition of the EGFR inhibitor, but not the c-Met inhibitor. Together, these data suggest that EGF/EGFR signaling stimulates anterograde lysosome trafficking and this is not due to crosstalk with or transactivation of c-Met.

To examine whether other organelles redistribute to the periphery in response to EGF, cells were stimulated with EGF for 16 h then stained for markers of early endosomes, mitochondria, or the cis-golgi (Additional file 2: Figure S2). Organelle distribution relative to the nucleus was observed using immunofluorescence microscopy. EEA1 positive early endosomes were mostly diffuse throughout the cytoplasm, and did not re-localize to the cell periphery upon stimulation with EGF. Moreover, mitochondria and the cis-Golgi remained closely localized near the nucleus in both control and EGF treated cells. Thus, of the tested organelles, only LAMP-1 positive lysosomes underwent anterograde trafficking in response to EGF stimulation.

Previous studies characterized NHEs as key regulators of anterograde lysosome trafficking in response to HGF stimulation [16, 17]. EGF stimulation is also known to activate plasma membrane NHEs [36, 37], raising the possibility that NHEs also regulate anterograde lysosome trafficking in response to EGF stimulation. To test this, we treated DU145 cells with 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA), a general NHE inhibitor, or Troglitazone (Tro), an PPARγ agonist that we previously characterized as having a potent inhibitory effect on NHE function, in the presence or absence of EGF [14]. Cells were fixed and stained for LAMP-1 (red), actin (green), and DAPI (blue) (Fig. 3a; quantified in 3b). NHE inhibition with either EIPA or Tro prevented EGF-mediated anterograde lysosome trafficking. Similarly, EIPA treatment also prevented EGF-stimulated lysosome trafficking in HeLa cells (Additional file 3: Figure S3).

We next investigated whether juxtanuclear lysosome aggregation would prevent EGF-stimulated invasion or cell motility. Cells were stimulated with EGF in the presence or absence of EIPA and allowed to invade through a Matrigel-coated Boyden chamber. Cells were fixed and stained with crystal violet and the number of invasive cells were counted (Fig. 3c). Under conditions where lysosomes were clustered in perinuclear region as a result of EIPA treatment, EGF-stimulated invasion was reduced to levels comparable to that of control cells. Conversely, when cells under these same treatment conditions were assayed for cell motility using a scratch wound healing assay (Fig. 3d; quantified in Fig. 3e), NHE inhibition and prevention of lysosomal anterograde trafficking did not reduce overall cell motility. Therefore, the reduction of invasion upon EIPA treatment was not due to a reduction in overall cell motility. These results suggest that NHE inhibition and anterograde lysosome trafficking are necessary for EGF-mediated lysosome trafficking and invasion, but have no effect on overall cell motility.

We identified p38 MAPK as a key regulator of EGF-mediated cell scattering (Fig. 1), and questioned whether p38 MAPK activity also controlled EGF-mediated anterograde lysosome trafficking. To identify which signaling pathways were activated in response to EGF treatment in our system, DU145 cells were stimulated with EGF over time and assayed for levels of total or phosphorylated EGFR, ERK, AKT, and p38 MAPK by western blot (Fig. 4a; quantified in Additional file 4: Figure S4). EGF/EGFR activation results in the phosphorylation and activation of all tested downstream signaling proteins to varying degrees. To assess whether any of these downstream signaling components regulated EGF-induced lysosome trafficking, cells were pretreated with specific inhibitors of MEK/ERK (U0126), PI3K/AKT (LY294002) or p38α/β (SB203580) followed by stimulation with EGF. Cells were fixed and stained for LAMP-1 (red), actin (green), and DAPI (blue). Immunofluorescence microscopy revealed that p38 inhibition, but not inhibition of PI3K/AKT or MEK/ERK blocked EGF-mediated anterograde lysosome trafficking (Fig. 4b; quantified in Fig. 4c). Inhibition of p38 MAPK also blocked EGF-driven anterograde lysosome trafficking in HeLa cells (Additional file 3: Figure S3). To further confirm the involvement of p38 MAPK in the process of EGF-mediated anterograde lysosome trafficking, we used two additional p38 inhibitors, PD169316 and SB239063. SB202474 is an inactive analog of SB203580 and functions as a negative control. DU145 PCa cells were treated with the various p38 inhibitors in the presence or absence of EGF and lysosome positioning was assessed by immunofluorescence of LAMP-1 (red), actin (green), and DAPI (blue) (Additional file 5: Figure S5A). Treatment with either PD169316 or SB239063 prevented EGF-mediated anterograde lysosome trafficking. However, treatment with the inactive analog SB202474 failed to inhibit EGF-mediated lysosome trafficking, and LAMP-1 positive vesicles (red) were found out near the cell periphery (arrows) similar to what was seen with EGF treatment alone. In order to assess whether these p38 inhibitors were working, cells were pre-treated with each p38 inhibitor and then stimulated with EGF. Parallel western blot analysis revealed that all p38 inhibitors blocked EGF-mediated phosphorylation of p38, while the inactive analog (SB202474) did not (Additional file 5: Figure S5B). We also tested whether other downstream signaling pathways were involved in EGF-mediated anterograde lysosome trafficking. Cells were treated with inhibitors of Janus kinase-2 (JAK2, AG490), c-Jun N-terminal kinase (JNK, SP600125), or nuclear factor-κB (NFκB, Bay11) in the presence or absence of EGF and position of LAMP-1 positive vesicles (red) was analyzed by immunofluorescence (Additional file 5: Figure S5C). Inhibition of JAK, JNK, or NFκB did not prevent EGF-mediated anterograde lysosome trafficking. Collectively, these data indicated that p38 MAPK activity is necessary for EGF-mediated lysosome redistribution.

Previous reports suggest that EGFR does not effectively internalize or signal in the absence of p38 MAPK activity [38‐41]. If EGFR is not signaling properly, this may be one explanation for the inhibition of cell scattering and anterograde lysosome trafficking seen upon p38 inhibition. To assess EGFR signaling, cells were treated with 100 ng/mL EGF in the presence or absence of the p38 inhibitor SB203580 or with decreasing concentrations of EGF and assessed by western blot (Fig. 5a; quantified in Additional file 6: Figure S6). Treatment with SB203580 blocked EGF-mediated p38 activity, but had no effect on levels of phosphorylated EGFR, ERK, or AKT (lane 2, Fig. 5a). This suggests that the PI3K/AKT and MEK/ERK signaling pathways are not suppressed as a result of off target effects of SB203580. Downstream signaling was maintained at comparable levels across a range of EGF concentrations (100 ng/mL- 3 ng/mL) (lane 4–10, Fig. 5a), even though receptor activation was reduced at the lower concentrations. We applied the same treatment conditions to a scattering assay (Fig. 5b) and found that DU145 cells still scattered with treatment of EGF as low as 1.56 ng/mL. Cells were then treated with vehicle, SB203580, SB203580 plus EGF, or varying concentrations of EGF and stained for actin (green), LAMP-1 (red) and DAPI (blue). Lysosome redistribution to the periphery still occurred in cells treated with 3 ng/mL EGF (Fig. 5c; quantified in Fig. 5d). Collectively these data support the idea that p38 inhibition does not significantly alter EGFR signaling in our system and that DU145 cells still undergo downstream signaling, scattering, and anterograde lysosome trafficking in response to very low levels of EGFR activation. Therefore, the loss of p38 activity results in the inhibition of EGF-driven anterograde lysosome movement, and this is not due to a reduction in overall EGFR signaling.

Cell culture on a 2D plastic or glass surface does not accurately represent the 3-dimensional (3D) architecture of a solid tumor. Recent advances in 3D culture suggest that cell phenotypes vary greatly between cells cultured in 2D vs. 3D environments [42]. We observed that EGF stimulation resulted in anterograde lysosome trafficking in 2D culture (Fig. 2), and queried whether this same phenotype was maintained in cells grown in 3D culture. To address this, we cultured DU145 cells on Matrigel in the presence or absence of EGF. Cells were fixed and stained for DAPI (blue) actin (red), and LAMP-1 (green) and images were collected using confocal microscopy (Fig. 6a). Control-treated DU145 cells formed spheroid-like colonies, indicative of non-invasive cells. In contrast, EGF-treated cells formed irregular colonies and many cells had a mesenchymal morphology, suggesting that EGF stimulates an invasive phenotype in 3D culture. Additionally, LAMP-1 positive vesicles were localized to actin rich cellular protrusions along the leading edge of EGF-treated cells.

Our lab has previously characterized anterograde lysosome trafficking events as being necessary for acidic extracellular pH and HGF-mediated invasion and cathepsin B secretion in 2D [14‐17]. However, the role of EGF-mediated anterograde lysosome trafficking in 3D invasion and protease secretion was never investigated. To test this, we performed 3D–Matrigel invasion assays in the presence of DQ-collagen IV, a dye-quenched collagen that fluoresces upon proteolytic cleavage [22, 43]. DU145 cells were grown on a matrix of DQ-collagen IV and Matrigel and incubated with the p38 inhibitor SB203580, the NHE inhibitor EIPA, or vehicle control in the presence or absence of EGF. Cells were fixed and stained for actin (red) and imaged using confocal microscopy. Green represents cleaved DQ-collagen IV as a readout for protease activity (Fig. 6b; quantified in Fig. 6c). Cells grown in the absence of EGF form spheroid-like colonies with minimal cleaved DQ-collagen IV fluorescence. Cells treated with EGF exhibit a more invasive phenotype characterized by the loss of spheroid colony morphology and the appearance of cellular protrusions. This invasive morphology was accompanied by increased DQ-collagen IV fluorescence (green) indicating increased protease secretion and activity. EGF-driven invasive morphology and protease activity was reduced in the presence of SB203580 and EIPA. Collectively, these results indicate that anterograde lysosome trafficking occurs in a more physiologically relevant culture model and that lysosome trafficking contributes to the invasive and proteolytic phenotype of EGF-stimulated cells grown in 3D culture.