CIP4 targeted to recruit GTP-Cdc42 involving in invadopodia formation via NF-κB signaling pathway promotes invasion and metastasis of CRC

Abstract

Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family, which plays an important role in regulating cell membrane and actin, has been reported to interact with Cdc42 and be closely associated with tumor invadopodia formation. In this study, we found that CIP4 expression was significantly higher in human CRC tissues and correlated with the CRC infiltrating depth and metastasis, as well as the lower survival rate in patients. In cultured CRC cells, knockdown of CIP4 inhibited cell migration and invasion ability in vitro and tumor metastasis in vivo, while the overexpression of CIP4 promoted invadopodia formation and matrix degradation ability. We then identified GTP-Cdc42 as a directly interactive protein of CIP4, which was upregulated and recruited by CIP4. Furthermore, activated NF-κB signaling pathway was found in CIP4 overexpression of CRC cells contributing to invadopodia formation, while the inhibition of either CIP4 or Cdc42 led to the suppression of the NF-κB pathway and resulted in a decreased quantity of invadopodia. Our findings suggested that CIP4 targets to recruit GTP-Cdc42 and directly combines with it to accelerate invadopodia formation and function by activating NF-κB signaling pathway, thus promoting CRC infiltration and metastasis.

Keywords: CIP4; Cdc42; NF-κB; colorectal cancer; invadopodia; invasion; metastasis.

Graphical abstract

Figure 1

CIP4 expression in CRC tissues correlates with tumor development, invasiveness, and patient survival rate (A) CIP4 protein expression in 14 pairs of human CRC tissues (T) and adjacent normal tissues (N) was detected by western blot. The quantification of protein levels was normalized to GAPDH. ∗∗∗p < 0.0001. (B) CIP4 protein expression in 107 paraffin-embedded normal human colorectal tissues (normal) and CRC tissues (adjacent, tumor 1, tumor 2) was detected by immunohistochemical staining (scale, 200μm, 100μm) and analyzed by scores. The error bars represent the mean ± SD; ∗∗∗p < 0.0001. (C) Comparison of CIP4 expression in the invasion front (a) and tumor central area (b) of CRC. (D) Bioinformatics analysis shows the relationship between CIP4 expression and patient survival times; p = 0.0353.

Figure 2

CIP4 promotes CRC cell migration and invasion in vitro and tumor metastasis in vivo (A) The establishment of CIP4 stable knockdown (Lovo and HT29) and overexpression (HCT116 and DLD1) cell lines. CIP4 protein expression was detected by western blotting. (B) A wound healing assay was performed to evaluate the ability of cells to migrate (scale, 200 μm), and their migration ability was identified by wound healing percentage. The error bars represent the mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01. (C) The ability of cells to migrate in CRC cells was detected by the transwell migration assay (scale, 100 μm). The error bars represent the mean ± SD; ∗∗p < 0.01, ∗∗∗p < 0.001. (D) The invasion ability in CRC cells was detected by the Matrigel-coated Boyden chamber invasion assay (scale, 100 μm). The error bars represent the mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001. (E) The effect of CIP4 on tumor metastasis was assessed by an orthotopic xenograft CRC mouse model  (scale, 100 μm). The number of liver metastatic nodules in individual mice was counted under the microscope and analyzed (∗p < 0.05, n = 3).

Figure 3

CIP4 is sufficient for invadopodia formation and function in CRC cells (A) Quantification of cells with invadopodia was analyzed by immunofluorescence. The white arrowhead indicates the invadopodia. The error bars represent the mean ± SD (n = 200 cells). ∗∗p < 0.01, ∗∗∗p < 0.001. Magnification: 60×. (B) SEM showed the cell morphology in the indicated cells. Magnification: 1,000×, 2,000×, and 4,000×. (C) Matrix degradation assay was performed to analyze the invasion ability in cells. The quantification of FITC-gelatin degradation was detected by immunofluorescence. The error bars represent the mean ± SD (n = 100 cells). ∗p < 0.05, ∗∗p < 0.01. Magnification: 120×. (D) SEM showed the morphology of matrix degradation. Magnification: 150×, 2,000×.

Figure 4

CIP4 promotes the expression and activation of Cdc42 (A) Cdc42 protein expression in 14 pairs of human CRC tissues (T) and adjacent normal tissues (N) was detected by western blot. The quantification of protein levels was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ∗∗∗p < 0.001. (B) Cdc42 expression was detected by immunohistochemistry staining in 58 paraffin-embedded normal human colorectal tissues and CRC tissues and analyzed by scores. The error bars represent the mean ± SD; ∗∗p < 0.01. Representative photographs of CIP4 and Cdc42 IHC staining (scale, 200 μm, 100 μm) of normal tissue (Normal) and CRC tissue (tumor 1, tumor 2), as indicated. Spearman’s correlation analysis showed the relationship between the CIP4 and Cdc42 expression levels in 58 human CRC tissues (r = 0.297, p = 0.023). (C) We used western blot to analyze the expression of CIP4, Cdc42, and GTP-Cdc42 in CIP4 knockdown Lovo and HT29 cells and CIP4 overexpressing HCT116 and DLD1 cells. Grayscale values were normalized to GAPDH. (D) The expression of CIP4 and Cdc42 in indicated cells was observed by immunofluorescence. Magnification: 60×.

Figure 5

CIP4 directly interacts with activated Cdc42 to accelerate invadopodia function (A) Co-localization of CIP4 (green) and Cdc42 (red) in Lovo and HT29 cells was observed by immunofluorescence. Magnification: 180×. The Pearson’s correlation and overlap coefficient were shown in bar graph format. The error bars represent the mean ± SD (n = 5). (B) GST pull down experiments on CIP4 protein with mutants of activated Cdc42. GST and HIS were analyzed by western blot. (C) The localization of CIP4 and invadopdia was observed in Lovo and HT29 cells by confocal laser scanning. The white arrowhead indicates the co-localization of CIP4 and invadopodia. Magnification: 180×. (D) The localization of FLAG-tagged activated Cdc42 and invadopdia was observed in CIP4 overexpression or low-expression cells by confocal laser scanning. The white arrowhead indicates the co-localization of FLAG-tagged activated Cdc42 and invadopodia. Magnification: 180×.

Figure 6

The NF-κB signaling pathway is involved in accelerating invadopodia formation regulated by CIP4 through GTP-Cdc42 (A and B) We used western blot to analyze the expression of CIP4, Cdc42, GTP-Cdc42, p65, p-p65, and p65 in the nucleus in Lovo (the CIP4-knockdown cells were treated with LPS, 10 μg/mL for 0.5 h) and HCT116 (the CIP4-overexpressed cells were treated with ML141, 20μM for 36 h, or QNZ, 10μM for 8 h) cells, as indicated. Grayscale values were normalized to GAPDH and histone H3 in the nucleus. (C and D) The quantification of cells with invadopodia was analyzed by immunofluorescence. Lovo CIP4-knockdown cells were treated with LPS, 10 μg/mL for 0.5 h, HCT116 CIP4-overexpression cells were treated with ML141, 20μM for 36 h, or QNZ, 10μM for 8 h. The white arrowhead indicates the invadopodia. The error bars represent the mean ± SD (Lovo: p = 0.0002, HCT116: p < 0.0001, n = 200 cells). Magnification: 60×.