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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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The positive correlation between RUFY3 and PAK1 in gastric cancer cells and clinical gastric cancer tissue samples. (a) The protein levels of RUFY3 in gastric cancer cell lines (BGC-823, MKN45, AGS MGC-803, SGC-7901, BGC-823 and MKN1) relative to the normal gastric epithelial cell line (GES-1) were analyzed by western blot. (b) The protein level of RUFY3 is positively correlated with PAK1 in gastric cancers and matched adjacent normal gastric tissue samples. Total protein from gastric cancer samples was extracted, and the protein levels of RUFY3 and PAK1 were measured by western blot. (c) Histogram showed the relative level of RUFY3 in gastric cancer tissues. Among 40 patients with gastric cancer, 28 of 40 (70%) samples revealed >50% increase in the RUFY3 level relative to their matched non-tumor adjacent tissues (P=0.003). (d) The relative protein level of RUFY3 was plotted against that of PAK1 in gastric cancers tissue samples with Spearman's correlation statistical analysis from (b). Spearman's correlation coefficient is 0.661 (P=0.002). (e) Immunohistochemical analyses of RUFY3 and PAK1 expression in adjacent normal gastric tissues and metastatic gastric cancer. Scale bar, 50 μm
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fig6: The positive correlation between RUFY3 and PAK1 in gastric cancer cells and clinical gastric cancer tissue samples. (a) The protein levels of RUFY3 in gastric cancer cell lines (BGC-823, MKN45, AGS MGC-803, SGC-7901, BGC-823 and MKN1) relative to the normal gastric epithelial cell line (GES-1) were analyzed by western blot. (b) The protein level of RUFY3 is positively correlated with PAK1 in gastric cancers and matched adjacent normal gastric tissue samples. Total protein from gastric cancer samples was extracted, and the protein levels of RUFY3 and PAK1 were measured by western blot. (c) Histogram showed the relative level of RUFY3 in gastric cancer tissues. Among 40 patients with gastric cancer, 28 of 40 (70%) samples revealed >50% increase in the RUFY3 level relative to their matched non-tumor adjacent tissues (P=0.003). (d) The relative protein level of RUFY3 was plotted against that of PAK1 in gastric cancers tissue samples with Spearman's correlation statistical analysis from (b). Spearman's correlation coefficient is 0.661 (P=0.002). (e) Immunohistochemical analyses of RUFY3 and PAK1 expression in adjacent normal gastric tissues and metastatic gastric cancer. Scale bar, 50 μm

Mentions: To examine the role of RUFY3 and its relationship with PAK1 in gastric cancer, we first investigated the protein levels of RUFY3 and PAK1 in gastric cancer cell lines (BGC-823, MKN45, AGS, MGC-803, SGC-7901 and MKN1) relative to the normal gastric epithelial cell line (GES-1) by western blot. As shown in Figure 6a, compared with GES-1, RUFY3 and PAK1 were highly expressed in BGC-823, MKN45, AGS and MGC-803 cells. In addition, we measured the protein levels of RUFY3 and PAK1 in 40 pairs of gastric cancer tissue samples with invasive carcinoma at pathologic TNM III and TNM IV stages through western blot. Among 40 patients with gastric cancer, 28 of 40 (70%) samples revealed >50% increase in the RUFY3 level relative to their matched non-tumor adjacent tissues (Figures 6b and c, P=0.003 and Table 1), and PAK1 had high expression as well in most of the gastric cancer tissues, which was consistent with the previous reports on PAK1 expression in cancer cells and gastric cancer samples.18, 20 Moreover, RUFY3 levels in gastric cancer sample were also analyzed and plotted against the level of PAK1. The 10 representative samples of gastric cancer showed a positive correlation expression between PAK1 and RUFY3 (Figure 6b). After quantifying the protein fragments, an obvious positive correlation was observed between RUFY3 and PAK1 expression in tumor tissue samples (P=0.002; Table 2), and the Spearman's correlation coefficient was perfect (Figures 6d, R=0.661).


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)

The positive correlation between RUFY3 and PAK1 in gastric cancer cells and clinical gastric cancer tissue samples. (a) The protein levels of RUFY3 in gastric cancer cell lines (BGC-823, MKN45, AGS MGC-803, SGC-7901, BGC-823 and MKN1) relative to the normal gastric epithelial cell line (GES-1) were analyzed by western blot. (b) The protein level of RUFY3 is positively correlated with PAK1 in gastric cancers and matched adjacent normal gastric tissue samples. Total protein from gastric cancer samples was extracted, and the protein levels of RUFY3 and PAK1 were measured by western blot. (c) Histogram showed the relative level of RUFY3 in gastric cancer tissues. Among 40 patients with gastric cancer, 28 of 40 (70%) samples revealed >50% increase in the RUFY3 level relative to their matched non-tumor adjacent tissues (P=0.003). (d) The relative protein level of RUFY3 was plotted against that of PAK1 in gastric cancers tissue samples with Spearman's correlation statistical analysis from (b). Spearman's correlation coefficient is 0.661 (P=0.002). (e) Immunohistochemical analyses of RUFY3 and PAK1 expression in adjacent normal gastric tissues and metastatic gastric cancer. Scale bar, 50 μm
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Related In: Results  -  Collection

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fig6: The positive correlation between RUFY3 and PAK1 in gastric cancer cells and clinical gastric cancer tissue samples. (a) The protein levels of RUFY3 in gastric cancer cell lines (BGC-823, MKN45, AGS MGC-803, SGC-7901, BGC-823 and MKN1) relative to the normal gastric epithelial cell line (GES-1) were analyzed by western blot. (b) The protein level of RUFY3 is positively correlated with PAK1 in gastric cancers and matched adjacent normal gastric tissue samples. Total protein from gastric cancer samples was extracted, and the protein levels of RUFY3 and PAK1 were measured by western blot. (c) Histogram showed the relative level of RUFY3 in gastric cancer tissues. Among 40 patients with gastric cancer, 28 of 40 (70%) samples revealed >50% increase in the RUFY3 level relative to their matched non-tumor adjacent tissues (P=0.003). (d) The relative protein level of RUFY3 was plotted against that of PAK1 in gastric cancers tissue samples with Spearman's correlation statistical analysis from (b). Spearman's correlation coefficient is 0.661 (P=0.002). (e) Immunohistochemical analyses of RUFY3 and PAK1 expression in adjacent normal gastric tissues and metastatic gastric cancer. Scale bar, 50 μm
Mentions: To examine the role of RUFY3 and its relationship with PAK1 in gastric cancer, we first investigated the protein levels of RUFY3 and PAK1 in gastric cancer cell lines (BGC-823, MKN45, AGS, MGC-803, SGC-7901 and MKN1) relative to the normal gastric epithelial cell line (GES-1) by western blot. As shown in Figure 6a, compared with GES-1, RUFY3 and PAK1 were highly expressed in BGC-823, MKN45, AGS and MGC-803 cells. In addition, we measured the protein levels of RUFY3 and PAK1 in 40 pairs of gastric cancer tissue samples with invasive carcinoma at pathologic TNM III and TNM IV stages through western blot. Among 40 patients with gastric cancer, 28 of 40 (70%) samples revealed >50% increase in the RUFY3 level relative to their matched non-tumor adjacent tissues (Figures 6b and c, P=0.003 and Table 1), and PAK1 had high expression as well in most of the gastric cancer tissues, which was consistent with the previous reports on PAK1 expression in cancer cells and gastric cancer samples.18, 20 Moreover, RUFY3 levels in gastric cancer sample were also analyzed and plotted against the level of PAK1. The 10 representative samples of gastric cancer showed a positive correlation expression between PAK1 and RUFY3 (Figure 6b). After quantifying the protein fragments, an obvious positive correlation was observed between RUFY3 and PAK1 expression in tumor tissue samples (P=0.002; Table 2), and the Spearman's correlation coefficient was perfect (Figures 6d, R=0.661).

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