Double-stranded RNA-dependent protein kinase regulates the motility of breast cancer cells

Abstract

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway.

Figure 1. Activation of PKR inhibits the motility of breast cancer cells.

A: MDA-MB231 cells (5x10⁴) were placed into the upper compartments of migration chambers (transwells) in the presence of PIC (0, 5 and 10 ng/ml). The transwells were incubated at 37°C in 5% CO₂ overnight. The number of migrated MDA-MB231 cells was measured as described under the Materials and Methods. B: The number of migrated BT474 and SKBR3 cells in the presence of PIC treatment (0 or 10 ng/ml) was determined as described above. C: The expression of phosphorylated PKR and total PKR in MCF7, MB231, BT474 and SKRB3 cells were determined by immunoblotting. D: MDA-MB231 cells were exposed to PIC (0, 5 and 10 ng/ml) for 36 h and cell migration was determined by wound healing migration assay as described under the Materials and Methods. E: MDA-MB231 cells were exposed to PIC (0, 5, 10, 50 or 100 ng/ml) with/without serum for 24 and 36 h. The cell viability was determined with MTT assay. The number of viable cells after PIC treatment was presented relative to untreated controls. F: MDA-MB231 cells were treated with PIC (0 or 10 ng/ml) for indicated time courses. Cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR and eIF2α. The expression of GAPDH served as a loading control. G: The relative levels of pPKR and pelF2α were quantified as described under the Materials and Methods and normalized to the expression of PKR and elF2α, respectively. Each datum point was the mean ± SEM of three independent experiments. * denotes a statistically significant difference from untreated controls (p<0.05).

Figure 2. Effect of PKR inhibitor on PIC-induced inhibition of cell migation.

A: MDA-MB231 cells were pretreated with a selective PKR inhibitor (PKR-I, 500 nM) for 24 hours followed by PIC (0 or 10 ng/ml) exposure for 6 hours. Cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR, eIF2α, p38 MAPK and MK2. The expression of actin served as a loading control. B: The relative levels of pPKR, pelF2α, pp38 and pMK2 were quantified as described under the Materials and Methods and normalized to the expression of PKR, elF2α, p38 MAPK and MK2, respectively. * denotes a statistically significant difference from untreated groups. # denotes a significant difference from PIC-treated groups. C: MDA-MB231 cells were pretreated with PKR-I (500 nM) for 24 h then placed into the upper compartments of migration chambers in the presence of PIC (0 or 10 ng/ml). The number of MDA-MB231 cells that migrated through the transwells was measured as described under the Materials and Methods. The experiment was replicated three times. * denotes a statistically significant difference from non-PIC-treated groups. # denotes a significant difference from PIC-treated groups. & denotes a significant difference from the untreated group.

Figure 3. Activation of PKR impairs lamellipodia formation.

MDA-MB231 cells were treated with PIC (0, 1, 5 or 10 ng/ml) for 12 hours, and then equal amounts of cells were seeded on fibronectin-coated culture wells, allowing attachment for 3 hours. A: After attaching, the phase-contrast images were captured using a Zeiss Axiovert 40C photomicroscope. The images of cells treated with PIC (0 or 10 ng/ml) are presented. Scale bar  = 50 µm. B: Cells with extended leading areas (lamellipodia) were counted in ten randomly selected fields in each treatment group. The percentage of cells with lamellipodia was determined. C: MDA-MB231 cells were treated with PIC (0 or 10 ng/ml) for 12 hours. The distribution of the actin cytoskeleton was detected by fluorescent staining (Alexa Fluor 488 Phalloidin) as described under the Materials and Methods. The arrow indicates the lamellipodia (the leading edge). Scale bar  = 10 µm. D: MDA-MB231 cells were treated with PIC (0, 1, 5 or 10 ng/ml) for 12 hours, and then equal amounts of cells were seeded on fibronectin-coated culture wells, allowing attachment for 3 hours. Cell spreading areas were measured randomly for at least 25 cells for each treatment group. The experiment was replicated three times. Each datum point was the mean ± SEM of three independent experiments. * denotes a significant difference from untreated controls. # denotes a significant difference from PIC (1 ng/ml)-treated groups. § denotes a significant difference from PIC (5 ng/ml)-treated groups (p<0.05).

Figure 4. Activation of PKR inhibits cofilin.

A: MDA-MB231 cells were treated with PIC (0 or 10 ng/ml) for indicated times. There were two controls; cells received no treatment (con) or cells were treated with Lipofectamine 2000 (Lipo). Cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR, p38 MAPK, MK2, LIMK1 and cofilin. The expression of actin served as a loading control. B: The relative levels of pp38, pMK2, pLIMK and pcofilin were quantified as described under the Materials and Methods and normalized to the expression of p38 MAPK, MK2, LIMK1 and cofilin respectively. * denotes a significant difference from untreated controls. C: MDA-MB231 cells were exposed to PIC (0 or 10 ng/ml) for 6 hours, and then cells were seeded on fibronectin-coated culture wells, allowing attachment for 3 hours. The expression of cofilin and actin was detected by immunofluorescent staining as described under the Materials and Methods. Arrows indicate the lamellipodia. Scale bar  = 10 µm. These experiments were replicated three times.

Figure 5. Effect of p38 MAPK inhibitor on PIC-stimulated cell signaling.

MDA-MB231 cells were pretreated with a selective p38 MAPK inhibitor (SB203580, 5 µM) for 2 hours followed by PIC (0 or 10 ng/ml) treatments for an additional 6 hours. A: After the treatment, cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR, p38 MAPK, MK2, LIMK1 and cofilin. The expression of actin served as a loading control. B: The relative levels of pPKR, pp38, pMK2, pLIMK and pcofilin were quantified as described under the Materials and Methods and normalized to the expression of PKR, p38 MAPK, MK2, LIMK1 and cofilin respectively. * denotes a significant difference from untreated controls. C: The expression of phospho-cofilin and phospho-MK2 was visualized by immunofluorescent staining as described under the Materials and Methods. Scale bar  = 100 µm. D: MDA-MB231 cells were pretreated with SB203580 (5 µM) for 2 hours then placed into the upper compartments of migration chambers (transwells) in the presence of PIC (0 or 10 ng/ml). The number of MDA-MB231 cells that migrated through the transwells was determined as described under the Materials and Methods. The experiment was replicated three times. * denotes a statistically significant difference from controls. # denotes a significant difference from PIC-treated groups.

Figure 6. Effect of PKR siRNA on PIC-regulated cell migation.

MDA-MB231 cells were transfected with control siRNA or PKR siRNA for 48 h. A: Following transfection, cell lysates were collected and the expression PKR was determined by immunoblotting. B: Following transfection, MDA-MB231 cells were placed into the upper compartments of the migration chambers in the presence of PIC (0 or 10 ng/ml). The number of MDA-MB231 cells that migrated through the transwells was measured as described under the Materials and Methods. * denotes a statistically significant difference from controls. # denotes a significant difference from PIC-treated groups. C: Following transfection, MDA-MB231 cells were exposed to PIC (0 or 10 ng/ml) for 6 hours. Cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR, eIF2α, p38 MAPK and MK2. The expression of tubulin served as a loading control. D: The relative levels of pPKR, pelF2α, pp38 and pMK2 were quantified as described under the Materials and Methods and normalized to the expression of PKR, elF2α, p38 MAPK and MK2, respectively. The experiment was replicated three times. * denotes a statistically significant difference from controls. # denotes a significant difference from PIC-treated groups.

Figure 7. Activation of PKR inhibits dynamic cell membrane ruffling.

A: MDA-MB231 cells were pretreated with DMSO (control), PKR-I (500 nM) or SB203580 (5 µM) for indicated hours, then followed by the treatment of PIC (10 ng/ml) for 6 hours. Top panel: Phase-contrast frames were acquired every 10 seconds for 10 minutes on a time-lapse microscope using a 60X oil-immersion lens. Scale bar  = 10 µm. Bottom panel: The corresponding kymographs of the movie generated along a line transecting the cell membrane on the lamellipodia are presented. B: Relative rate of cell membrane ruffling was calculated from kymographs. The experiments were replicated five times. * denotes a statistically significant difference from all other groups (p<0.05).

Figure 8. Schematic illustration of PKR-mediated cell signaling that regulates cell migration.

The activation or high expression of PKR results in the activation of p38 MAPK/MK2 which stimulates the phosphorylation of LIMK. The activated LIMK inhibits cofilin, resulting in the suppression of cell migration.