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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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Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. (a) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μm) are magnified in the right images ( × 600; scale bars, 24 μm). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (**P<0.01, compared with GFP vector). Protein expression was confirmed by western blotting assays using GFP-tagged antibody when equal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference protein. (b and c) RUFY3 colocalizes with F-actin at the cell periphery. SGC-7901 cells were transiently transfected with pEGFP-C1 or pEGFP-RUFY3. Rhodamine-conjugated phalloidin was used to detect F-actin. After 24 h transfection, cells were fixed and permeabilized. (b) Images were captured using a scanning confocal fluorescence microscope and one confocal section is shown in each image. Scale bars, 10 μm. (c) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with F-actin at the cell periphery. The data show mean±S.E.M. (**P<0.01, compared with GFP vector), in which ~40 transfected cells were observed. (d) Colocalization of GFP-RIPX and myosinIIb at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of myosinIIb (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 10 μm. (e) Colocalization of GFP-RIPX and integrin β5 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of integrin β5 (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 20 μm. (f) Colocalization of GFP-RIPX and integrin α3β1 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. Scale bars, 20 μm
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fig1: Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. (a) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μm) are magnified in the right images ( × 600; scale bars, 24 μm). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (**P<0.01, compared with GFP vector). Protein expression was confirmed by western blotting assays using GFP-tagged antibody when equal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference protein. (b and c) RUFY3 colocalizes with F-actin at the cell periphery. SGC-7901 cells were transiently transfected with pEGFP-C1 or pEGFP-RUFY3. Rhodamine-conjugated phalloidin was used to detect F-actin. After 24 h transfection, cells were fixed and permeabilized. (b) Images were captured using a scanning confocal fluorescence microscope and one confocal section is shown in each image. Scale bars, 10 μm. (c) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with F-actin at the cell periphery. The data show mean±S.E.M. (**P<0.01, compared with GFP vector), in which ~40 transfected cells were observed. (d) Colocalization of GFP-RIPX and myosinIIb at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of myosinIIb (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 10 μm. (e) Colocalization of GFP-RIPX and integrin β5 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of integrin β5 (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 20 μm. (f) Colocalization of GFP-RIPX and integrin α3β1 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. Scale bars, 20 μm

Mentions: Previous studies suggested that RUFY3 was localized in growth cones in nerve cells.17, 18 Here, we detect the localization of RUFY3 in gastric cancer cell lines. The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3, and GFP vector was used as a control. Meanwhile, to further indicate the localization of GFP-RUFY3 at the migrating edge, we showed the localization of a cell undergoing a scratch wound assay and found that most of the SGC-7901 cells expressing GFP-RUFY3 formed the protrusion at the cell periphery by sequential scanning, and rarely detected in GFP-expressing cells (Figure 1a). We then analyzed whether RUFY3 colocalized with F-actin by confocal microscopy. As shown in Figure 1b, the GFP-RUFY3 was significantly colocalized with F-actin at the cell periphery in the majority of cells (Figure 1c). In addition, we measured the quantitation of colocalization with color scatterplots, and the analysis results showed that RUFY3 highly colocalized with F-actin at the cell periphery (Supplementary Figure S5a).


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)

Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. (a) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μm) are magnified in the right images ( × 600; scale bars, 24 μm). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (**P<0.01, compared with GFP vector). Protein expression was confirmed by western blotting assays using GFP-tagged antibody when equal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference protein. (b and c) RUFY3 colocalizes with F-actin at the cell periphery. SGC-7901 cells were transiently transfected with pEGFP-C1 or pEGFP-RUFY3. Rhodamine-conjugated phalloidin was used to detect F-actin. After 24 h transfection, cells were fixed and permeabilized. (b) Images were captured using a scanning confocal fluorescence microscope and one confocal section is shown in each image. Scale bars, 10 μm. (c) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with F-actin at the cell periphery. The data show mean±S.E.M. (**P<0.01, compared with GFP vector), in which ~40 transfected cells were observed. (d) Colocalization of GFP-RIPX and myosinIIb at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of myosinIIb (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 10 μm. (e) Colocalization of GFP-RIPX and integrin β5 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of integrin β5 (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 20 μm. (f) Colocalization of GFP-RIPX and integrin α3β1 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. Scale bars, 20 μm
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fig1: Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. (a) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μm) are magnified in the right images ( × 600; scale bars, 24 μm). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (**P<0.01, compared with GFP vector). Protein expression was confirmed by western blotting assays using GFP-tagged antibody when equal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference protein. (b and c) RUFY3 colocalizes with F-actin at the cell periphery. SGC-7901 cells were transiently transfected with pEGFP-C1 or pEGFP-RUFY3. Rhodamine-conjugated phalloidin was used to detect F-actin. After 24 h transfection, cells were fixed and permeabilized. (b) Images were captured using a scanning confocal fluorescence microscope and one confocal section is shown in each image. Scale bars, 10 μm. (c) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with F-actin at the cell periphery. The data show mean±S.E.M. (**P<0.01, compared with GFP vector), in which ~40 transfected cells were observed. (d) Colocalization of GFP-RIPX and myosinIIb at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of myosinIIb (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 10 μm. (e) Colocalization of GFP-RIPX and integrin β5 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of integrin β5 (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 20 μm. (f) Colocalization of GFP-RIPX and integrin α3β1 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. Scale bars, 20 μm
Mentions: Previous studies suggested that RUFY3 was localized in growth cones in nerve cells.17, 18 Here, we detect the localization of RUFY3 in gastric cancer cell lines. The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3, and GFP vector was used as a control. Meanwhile, to further indicate the localization of GFP-RUFY3 at the migrating edge, we showed the localization of a cell undergoing a scratch wound assay and found that most of the SGC-7901 cells expressing GFP-RUFY3 formed the protrusion at the cell periphery by sequential scanning, and rarely detected in GFP-expressing cells (Figure 1a). We then analyzed whether RUFY3 colocalized with F-actin by confocal microscopy. As shown in Figure 1b, the GFP-RUFY3 was significantly colocalized with F-actin at the cell periphery in the majority of cells (Figure 1c). In addition, we measured the quantitation of colocalization with color scatterplots, and the analysis results showed that RUFY3 highly colocalized with F-actin at the cell periphery (Supplementary Figure S5a).

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