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PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion.

Wang G, Zhang Q, Song Y, Wang X, Guo Q, Zhang J, Li J, Han Y, Miao Z, Li F - Cell Death Dis (2015)

Bottom Line: Importantly, we found that the inhibitory effect of cell migration and invasion is significantly enhanced by knockdown of both PAK1 and RUFY3 compared with knockdown of RUFY3 alone or PAK1 alone.Strikingly, we found significant upregulation of RUFY3 in gastric cancer samples with invasive carcinoma at pathologic TNM III and TNM IV stages, compared with their non-tumor counterparts.Therefore, these findings provide important evidence that PAK1 can positively regulate RUFY3 expression, which contribute to the metastatic potential of gastric cancer cells, maybe blocking PAK1-RUFY3 signaling would become a potential metastasis therapeutic strategy for gastric cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China.

ABSTRACT
Actin protrusion at the cell periphery is central to the formation of invadopodia during tumor cell migration and invasion. Although RUFY3 (RUN and FYVE domain containing 3)/SINGAR1 (single axon-related1)/RIPX (Rap2 interacting protein X) has an important role in neuronal development, its pathophysiologic role and relevance to cancer are still largely unknown. The purpose of this study was to elucidate the molecular mechanisms by which RUFY3 involves in gastric cancer cell migration and invasion. Here, our data show that overexpression of RUFY3 leads to the formation of F-actin-enriched protrusive structures at the cell periphery and induces gastric cancer cell migration. Furthermore, P21-activated kinase-1 (PAK1) interacts with RUFY3, and promotes RUFY3 expression and RUFY3-induced gastric cancer cell migration; inhibition of PAK1 attenuates RUFY3-induced SGC-7901 cell migration and invasion. Importantly, we found that the inhibitory effect of cell migration and invasion is significantly enhanced by knockdown of both PAK1 and RUFY3 compared with knockdown of RUFY3 alone or PAK1 alone. Strikingly, we found significant upregulation of RUFY3 in gastric cancer samples with invasive carcinoma at pathologic TNM III and TNM IV stages, compared with their non-tumor counterparts. Moreover, an obvious positive correlation was observed between the protein expression of RUFY3 and PAK1 in 40 pairs of gastric cancer samples. Therefore, these findings provide important evidence that PAK1 can positively regulate RUFY3 expression, which contribute to the metastatic potential of gastric cancer cells, maybe blocking PAK1-RUFY3 signaling would become a potential metastasis therapeutic strategy for gastric cancer.

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PAK1 regulates RUFY3-mediated cell migration and invasion. (a) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The exogenous expression of RUFY3 and PAK1 were verified by western blot. (b and c) Inhibition of PAK1 attenuates RUFY3-mediated cell migration and invasion. (b)The SGC-7901 cells transfected with GFP-RUFY3 or GFP vector, which were along with PAK1-siRNA-treated (PAK1 siRNA) or control siRNA-treated (Control siRNA), were used to perform the wound-healing assay. (Left panel) Photographs represented the cells migrating into the wounded area. Scale bars: 50.0 μm. (Middle panel) Histogram showed the percentage of wound closure. Data are the average of at least three independent experiments with similar results (*P <0.05, **P<0.01, compared with 0 h). (Right panel) The efficacy of PAK1-shRNA was determined by western blot. (c) SGC-7901 cells transfected with GFP-RUFY3 were treated with or without IPA-3 (5 μM) for 24 h, Me2SO (DMSO) as a control, and used to perform the transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The exogenous expression of RUFY3 was demonstrated by western blotting when cells were treated with or without IPA-3. (d) Knockdown of both PAK1 and RUFY3 can significantly inhibit cell invasion. The RUFY3-shRNA1 was transfected into stable expressing PAK1-shRNA lentivirus SGC-7901 cells and BGC-823 cells to perform the transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnifications) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The efficacys of PAK1-shRNA and RUFY3-siRNA were determined by western blot
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fig5: PAK1 regulates RUFY3-mediated cell migration and invasion. (a) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The exogenous expression of RUFY3 and PAK1 were verified by western blot. (b and c) Inhibition of PAK1 attenuates RUFY3-mediated cell migration and invasion. (b)The SGC-7901 cells transfected with GFP-RUFY3 or GFP vector, which were along with PAK1-siRNA-treated (PAK1 siRNA) or control siRNA-treated (Control siRNA), were used to perform the wound-healing assay. (Left panel) Photographs represented the cells migrating into the wounded area. Scale bars: 50.0 μm. (Middle panel) Histogram showed the percentage of wound closure. Data are the average of at least three independent experiments with similar results (*P <0.05, **P<0.01, compared with 0 h). (Right panel) The efficacy of PAK1-shRNA was determined by western blot. (c) SGC-7901 cells transfected with GFP-RUFY3 were treated with or without IPA-3 (5 μM) for 24 h, Me2SO (DMSO) as a control, and used to perform the transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The exogenous expression of RUFY3 was demonstrated by western blotting when cells were treated with or without IPA-3. (d) Knockdown of both PAK1 and RUFY3 can significantly inhibit cell invasion. The RUFY3-shRNA1 was transfected into stable expressing PAK1-shRNA lentivirus SGC-7901 cells and BGC-823 cells to perform the transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnifications) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The efficacys of PAK1-shRNA and RUFY3-siRNA were determined by western blot

Mentions: Next, we decided to examine whether PAK1 can also influence the RUFY3-mediated cell migration and invasion. As expected, a significant increase in wound- healing cell migration was clearly seen in SGC-7901 cells expressing RUFY3 and PAK1 compared with cells expressing RUFY3 alone (Supplementary Figure S4a, left panel), and the relative migrating distance of cells was significantly longer (Supplementary Figure S4a, right panel). In addition, a matrigel-coated Boyden chamber system was used to quantify the invasive ability of SGC-7901 cells expressing RUFY3 and PAK1. Remarkably, at the 24 h, the effect of promoting SGC-790 cell invasion induced by RUFY3 can be more significantly facilitated by overexpressing PAK1 compared with cells expressing RUFY3 alone (Figure 5a, left panel), and the relative number of invading cells was significantly increased (Figure 5a, right panel), indicating that PAK1 as an important role in RUFY3-mediated facilitating gastric cancer cell migration and invasion. These results suggest that overexpressing PAK1 facilitates RUFY3-mediated migration and invasion of gastric cancer cells.


PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion.

Wang G, Zhang Q, Song Y, Wang X, Guo Q, Zhang J, Li J, Han Y, Miao Z, Li F - Cell Death Dis (2015)

PAK1 regulates RUFY3-mediated cell migration and invasion. (a) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The exogenous expression of RUFY3 and PAK1 were verified by western blot. (b and c) Inhibition of PAK1 attenuates RUFY3-mediated cell migration and invasion. (b)The SGC-7901 cells transfected with GFP-RUFY3 or GFP vector, which were along with PAK1-siRNA-treated (PAK1 siRNA) or control siRNA-treated (Control siRNA), were used to perform the wound-healing assay. (Left panel) Photographs represented the cells migrating into the wounded area. Scale bars: 50.0 μm. (Middle panel) Histogram showed the percentage of wound closure. Data are the average of at least three independent experiments with similar results (*P <0.05, **P<0.01, compared with 0 h). (Right panel) The efficacy of PAK1-shRNA was determined by western blot. (c) SGC-7901 cells transfected with GFP-RUFY3 were treated with or without IPA-3 (5 μM) for 24 h, Me2SO (DMSO) as a control, and used to perform the transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The exogenous expression of RUFY3 was demonstrated by western blotting when cells were treated with or without IPA-3. (d) Knockdown of both PAK1 and RUFY3 can significantly inhibit cell invasion. The RUFY3-shRNA1 was transfected into stable expressing PAK1-shRNA lentivirus SGC-7901 cells and BGC-823 cells to perform the transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnifications) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The efficacys of PAK1-shRNA and RUFY3-siRNA were determined by western blot
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fig5: PAK1 regulates RUFY3-mediated cell migration and invasion. (a) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The exogenous expression of RUFY3 and PAK1 were verified by western blot. (b and c) Inhibition of PAK1 attenuates RUFY3-mediated cell migration and invasion. (b)The SGC-7901 cells transfected with GFP-RUFY3 or GFP vector, which were along with PAK1-siRNA-treated (PAK1 siRNA) or control siRNA-treated (Control siRNA), were used to perform the wound-healing assay. (Left panel) Photographs represented the cells migrating into the wounded area. Scale bars: 50.0 μm. (Middle panel) Histogram showed the percentage of wound closure. Data are the average of at least three independent experiments with similar results (*P <0.05, **P<0.01, compared with 0 h). (Right panel) The efficacy of PAK1-shRNA was determined by western blot. (c) SGC-7901 cells transfected with GFP-RUFY3 were treated with or without IPA-3 (5 μM) for 24 h, Me2SO (DMSO) as a control, and used to perform the transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The exogenous expression of RUFY3 was demonstrated by western blotting when cells were treated with or without IPA-3. (d) Knockdown of both PAK1 and RUFY3 can significantly inhibit cell invasion. The RUFY3-shRNA1 was transfected into stable expressing PAK1-shRNA lentivirus SGC-7901 cells and BGC-823 cells to perform the transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μm. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnifications) from three independent experiments (*P <0.05, **P<0.01, compared with control vector). (Right panel) The efficacys of PAK1-shRNA and RUFY3-siRNA were determined by western blot
Mentions: Next, we decided to examine whether PAK1 can also influence the RUFY3-mediated cell migration and invasion. As expected, a significant increase in wound- healing cell migration was clearly seen in SGC-7901 cells expressing RUFY3 and PAK1 compared with cells expressing RUFY3 alone (Supplementary Figure S4a, left panel), and the relative migrating distance of cells was significantly longer (Supplementary Figure S4a, right panel). In addition, a matrigel-coated Boyden chamber system was used to quantify the invasive ability of SGC-7901 cells expressing RUFY3 and PAK1. Remarkably, at the 24 h, the effect of promoting SGC-790 cell invasion induced by RUFY3 can be more significantly facilitated by overexpressing PAK1 compared with cells expressing RUFY3 alone (Figure 5a, left panel), and the relative number of invading cells was significantly increased (Figure 5a, right panel), indicating that PAK1 as an important role in RUFY3-mediated facilitating gastric cancer cell migration and invasion. These results suggest that overexpressing PAK1 facilitates RUFY3-mediated migration and invasion of gastric cancer cells.

Bottom Line: Importantly, we found that the inhibitory effect of cell migration and invasion is significantly enhanced by knockdown of both PAK1 and RUFY3 compared with knockdown of RUFY3 alone or PAK1 alone.Strikingly, we found significant upregulation of RUFY3 in gastric cancer samples with invasive carcinoma at pathologic TNM III and TNM IV stages, compared with their non-tumor counterparts.Therefore, these findings provide important evidence that PAK1 can positively regulate RUFY3 expression, which contribute to the metastatic potential of gastric cancer cells, maybe blocking PAK1-RUFY3 signaling would become a potential metastasis therapeutic strategy for gastric cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China.

ABSTRACT
Actin protrusion at the cell periphery is central to the formation of invadopodia during tumor cell migration and invasion. Although RUFY3 (RUN and FYVE domain containing 3)/SINGAR1 (single axon-related1)/RIPX (Rap2 interacting protein X) has an important role in neuronal development, its pathophysiologic role and relevance to cancer are still largely unknown. The purpose of this study was to elucidate the molecular mechanisms by which RUFY3 involves in gastric cancer cell migration and invasion. Here, our data show that overexpression of RUFY3 leads to the formation of F-actin-enriched protrusive structures at the cell periphery and induces gastric cancer cell migration. Furthermore, P21-activated kinase-1 (PAK1) interacts with RUFY3, and promotes RUFY3 expression and RUFY3-induced gastric cancer cell migration; inhibition of PAK1 attenuates RUFY3-induced SGC-7901 cell migration and invasion. Importantly, we found that the inhibitory effect of cell migration and invasion is significantly enhanced by knockdown of both PAK1 and RUFY3 compared with knockdown of RUFY3 alone or PAK1 alone. Strikingly, we found significant upregulation of RUFY3 in gastric cancer samples with invasive carcinoma at pathologic TNM III and TNM IV stages, compared with their non-tumor counterparts. Moreover, an obvious positive correlation was observed between the protein expression of RUFY3 and PAK1 in 40 pairs of gastric cancer samples. Therefore, these findings provide important evidence that PAK1 can positively regulate RUFY3 expression, which contribute to the metastatic potential of gastric cancer cells, maybe blocking PAK1-RUFY3 signaling would become a potential metastasis therapeutic strategy for gastric cancer.

Show MeSH
Related in: MedlinePlus