CD2AP inhibits metastasis in gastric cancer by promoting cellular adhesion and cytoskeleton assembly

Abstract

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that the alter of the cellular cytoskeleton and intercellular adhesion. CD2-associated protein (CD2AP) is one of the critical proteins regulating cytoskeleton assembly and intercellular adhesion. However, no study has investigated the expression and biological significance of CD2AP in gastric cancer (GC) to date. Therefore, the aim of our study was to explore if the expression of CD2AP is associated with any clinical features of GC and to elucidate the underlying mechanism. Immunohistochemistry of 620 patient tissue samples indicated that the expression of CD2AP is downregulated in DGC. Moreover, a low CD2AP level was indicative of poor patient prognosis. In vitro, forced expression of CD2AP caused a significant decrease in the migration and invasion of GC cells, whereas depletion of CD2AP had the opposite effect. Immunofluorescence analysis indicated that CD2AP promoted cellular adhesion and influenced cell cytoskeleton assembly via interaction with the F-actin capping protein CAPZA1. Overall, the upregulation of CD2AP could attenuate GC metastasis, suggesting CD2AP as a novel biomarker for the prognosis and treatment of patients with GC.

Keywords: CD2AP; cytoskeleton; gastric cancer; intercellular adhesion; invasion; migration.

Figure 1

The expression of CD2AP is significantly lower in diffuse gastric cancer. A, Immunohistochemical expression of CD2AP in IGC, DGC, paired normal tissue, normal gastric lamina propria, and normal gastric epithelium. B, Select the gland region by IPP, analyze the IOD of the CD2AP positive region and the total area of the gland, and calculate the CD2AP score. C, In the paired specimens, the expression of CD2AP in DGC tissues (n = 15) was significantly lower than that in the matched normal gastric mucosa (***P < .001), but not in IGC (n = 37) and mixed gastric cancer (n = 4) (P > .05). D, The expression of CD2AP in DGC is significantly lower than that in IGC, mixed type and normal gastric mucosa (***P < .001). E, Violin picture show CD2AP expression in DGC, IGC, and normal gastric tissue. For each Violin picture, median and ranges are indicated (*P < .05, ***P < .001). CD2AP, CD2‐associated protein; DGC, Diffuse gastric cancer; IGC, intestinal gastric cancer; IOD, integrated optical density, IPP, Image Pro Plus 6 [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

GC patients with a low expression level of CD2AP have a poor prognosis. A and B, Low CD2AP expression in 564 gastric cancer predicts poor OS times and DFS times. C and D, Low CD2AP protein expression in 187 DGC predicts poor OS times and DFS times. E‐H, Low CD2AP protein expression in GED data sets (GSE14208, GSE15459, GSE57303, GSE62254) predicts poor OS times. I, Low CD2AP protein expression in 375 TCGA gastric cancer data sets predicts poor OS times. CD2AP, CD2‐associated protein; DGC, diffuse gastric cancer; DFS, disease‐free survival; GC, gastric cancer; OS, overall survival; TCGA, The Cancer Genome Atlas [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Depletion of CD2AP expression enhanced gastric cancer cell migration and invasion. A and B, The specificity of the two different CD2AP siRNAs in downregulating CD2AP gene expression. BGC‐823, MGC‐803 transfected with empty (siNC) or CD2AP siRNAs (si‐CD2AP‐1, si‐CD2AP‐2) were analyzed by immunoblotting with an antibody to CD2AP. An antibody to GAPDH was used as an equal loading control. The effect of CD2AP depletion on in vitro migration and invasion ability of BGC‐823, MGC‐803 cells. C, The migration rate and invasion rates of CD2AP‐overexpressing BGC‐823, MGC‐803 cell lines were markedly increased compared with that of the control cells (***P < .001). D, The proliferation rates of the CD2AP‐depleting BGC‐823 cells were not significantly different from those of control cells. CD2AP, CD2‐associated protein; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; siRNA, small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Overexpression of CD2AP inhibition gastric cancer cell migration and invasion. A‐C, BGC‐823, MGC‐803 transfected with pcDNA3.1 and pcDNA3.1‐CD2AP‐HA upregulated CD2AP gene expression, MGC‐803‐CD2AP induction CD2AP expression by 1 µg/mL DOX, and were analyzed by immunoblotting with an antibody to CD2AP. An antibody to GAPDH was used as an equal loading control. The effect of CD2AP‐overexpressing depletion on in vitro migration and invasion ability of BGC‐823, MGC‐803 cell lines. D and E, The migration rate and invasion rates of CD2AP‐depleting BGC‐823, MGC‐803, MGC‐803‐CD2AP cell lines were markedly increased compared with that of the control cells. (***P < .001). F, The proliferation rates of the CD2AP‐overexpressing BGC‐823 cells were not significantly different from those of control cells. CD2AP, CD2‐associated protein; DOX, doxycycline; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Overexpression of CD2AP promoted cellular adhesion and cytoskeleton assembly in gastric cancer cells. A, MGC‐803‐CD2AP cell induced express by 1 µg/mL DOX for 48 hours, the cytoskeleton was labeled with Phalloidin. B, The average area of the cytoskeleton is significantly enhanced in CD2AP overexpression MGC‐803‐CD2AP cell (***P < .001). C, MGC‐803‐CD2AP cell induced express by 1 µg/mL DOX for 48 hours, cell adhesion spots were labeled with zyxin antibody. D, The number of adhesion spots in cell is significantly increased in CD2AP overexpression MGC‐803‐CD2AP cell (***P < .001). E and F, MGC‐803‐CD2AP cell induced express by 1 µg/mL DOX for 48 hours, overexpress CD2AP significantly increase the adhesion ability of GC cells (***P < .001). CD2AP, CD2‐associated protein; DOX, doxycycline; GC, gastric cancer [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

CD2AP interact with CAPZA1 mediated cellular adhesion and cytoskeleton assembly. A, Transfected MGC‐803 with pcDNA3.1‐CD2AP plasmid, empty plasmid transfected as control, Using Anti‐HA Magnetic Beads binding to HA‐ta, Western blot analysis show CD2AP interact with CAPZA1. B, Extracted MGC‐803 cell protein and incubated with anti‐capza1 antibody to form antigen‐antibody mixture, immunoprecipitation with Protein A/G Magnetic Beads, Western blot analysis show CD2AP interact with CAPZA1. C, MGC‐803‐CD2AP transfected with siNC or si‐CAPZA1 were analyzed by Western blot analysis with an antibody to CAPZA1. An antibody to GAPDH was used as an equal loading control. D and E, MGC‐803‐CD2AP cell transfected with si‐CAPZA1 and induced CD2AP expression by DOX. Depletion of CAPZA1 significantly rescues the ability of CD2AP to inhibit migration and invasion of GC cell (***P < .001). F‐H, MGC‐803‐CD2AP cell transfected with si‐CAPZA1 and induced CD2AP expression by DOX, Cell adhesion spots were labeled with zyxin antibody, the cytoskeleton were labeled with Phalloidin, depletion of CAPZA1 significantly rescue the ability of CD2AP which enhanced the assembly of cytoskeleton F‐actin and the number of focal adhesion spots (***P < .001). I and J, MGC‐803‐CD2AP cell transfected with si‐CAPZA1 and induced CD2AP expression by DOX. Depletion of CAPZA1 significantly rescues the ability of CD2AP to inhibit cell adhesion of GC cell (***P < .001). CD2AP, CD2‐associated protein; DOX, doxycycline; GC, gastric cancer [Color figure can be viewed at wileyonlinelibrary.com]