Loss of the 14-3-3σ is essential for LASP1-mediated colorectal cancer progression via activating PI3K/AKT signaling pathway

Abstract

LIM and SH3 protein 1 (LASP1) can promote colorectal cancer (CRC) progression and metastasis, but the direct evidence that elucidates the molecular mechanism remains unclear. Here, our proteomic data showed that LASP1 interacted with 14-3-3σ and decreased the expression of 14-3-3σ in CRC. Deletion of 14-3-3σ was required for LASP1-mediated CRC cell aggressiveness. In vitro gain- and loss-of-function assays showed that 14-3-3σ suppressed the ability of cell migration and decreased the phosphorylation of AKT in CRC cells. We further observed clearly co-localization between AKT and 14-3-3σ in CRC cells. Treatment of PI3K inhibitor LY294002 markedly prevented phosphorylation of AKT and subsequently counteract aggressive phenotype mediated by siRNA of 14-3-3σ. Clinically, 14-3-3σ is frequently down-regulated in CRC tissues. Down-regulation of 14-3-3σ is associated with tumor progression and poor prognosis of patients with CRC. Multivariate analysis confirmed low expression of 14-3-3σ as an independent prognostic factor for CRC. A combination of low 14-3-3σ and high LASP1 expression shows a worse trend with overall survival of CRC patients. Our research paves the path to future investigation of the LASP1-14-3-3σ axis as a target for novel anticancer therapies of advanced CRC.

Figure 1. LASP1 negatively regulates 14-3-3σ expression by protein interaction.

(A) DIGE was performed to screen the differentially expressed proteins in SW480/LASP-1 cells or SW620 cells that were transfected with LASP-1 siRNA and control cells. The enlarged images of two differentially expressed protein spots in DIGE analysis were shown. The protein spots are indicated (white arrows). (B) Western blot was performed to detect the expression of 14-3-3σ and LASP1 protein in indicated cells. The immunosignal was quantified using densitometric scanning software, and relative protein abundance was determined by normalisation with levels of β-actin. Each bar represented the mean ± SD. The results were reproduced in three independent experiments. The asterisk (*) indicates P < 0.05. The asterisk (**) indicates P < 0.01. (C) Endogenous interaction between 14-3-3σ and LASP1 in CRC cells. Cells were lysed and purified by anti-LASP1 or anti-14-3-3σ affinity gel; protein pellets were analyzed by western blot with anti-LASP1 or anti-14-3-3σ. (D) The subcellular localization of 14-3-3σ and LASP1 in indicated cells was assessed by immunofluorescence staining. The full-length blots/gels are presented in Supplementary Fig. S3.

Figure 2. 14–3–3σ suppresses cell migration via inhibiting phosphorylation of AKT.

(A) Western blot analysis for the expression of 14-3-3σ in CRC cell lines. Immunosignals were quantified by densitometric scanning. 14-3-3σ expression in the individual cells was calculated as 14-3-3σ expression relative to GAPDH expression. Data are means + SD from three independent experiments. (B) The representative figures and data of transwell assay for SW620 cells were transfected with 14-3-3σ vector. Each bar represented the mean ± SD. The results were reproduced in three independent experiments. (C) The representative figures and data of transwell assay for SW480 and HCT116 cells co-infected with 14-3-3σ siRNA and PI3K inhibitor LY294002. (D) Western blot analysis of phosphorylated AKT in indicated cells transfected with 14-3-3σ vector or co-infected with 14-3-3σ siRNA and PI3K inhibitor LY294002. The immunosignal was quantified using densitometric scanning software, and relative protein abundance was determined by normalisation with total levels of AKT. Each bar represented the mean ± SD. The results were reproduced in three independent experiments. The asterisk (*) indicates P < 0.05. The asterisk (**) indicates P < 0.01. (E) The subcellular localization of 14-3-3σ and AKT in indicated cells was assessed by immunofluorescence staining. (F) Endogenous interaction between 14-3-3σ and AKT in CRC cells. Cells were lysed and purified by anti-AKT or anti-14-3-3σ affinity gel; protein pellets were analyzed by western blot with anti-AKT or anti-14-3-3σ. Representative figures were shown. The results were reproduced in 3 independent experiments. The full-length blots/gels are presented in Supplementary Fig. S6.

Figure 3. Loss of 14-3-3σ is essential for LASP1-mediated cell aggressiveness.

(A) Representative figures and data of transwell assay for indicated cells. (B) Western blot analysis of phosphorylated AKT in indicated cells co-infected with transfected with 14-3-3σ and LASP1 vector or siRNA. The immunosignal was quantified using densitometric scanning software, and relative protein abundance was determined by normalisation with total levels of AKT. Each bar represented the mean ± SD. The results were reproduced in three independent experiments. The asterisk (*) indicates P < 0.05. The asterisk (**) indicates P < 0.01. The full-length blots/gels are presented in Supplementary Fig. S7.

Figure 4. 14–3–3σ is frequently down-regulated in CRC tissues.

(A) IHC analysis of 14-3-3σ protein expression in CRC tissues (T) and adjacent non-tumor tissues (N). (B) IHC analysis of 14-3-3σ protein expression in the junction of benign and malignant colorectal lesions. In the bottom row, right picture (f) corresponded with high magnification of left picture (e). Scale bars were shown in the lower right corner of each picture. N, adjacent non-tumor tissues. T, CRC tissues. (C) Graphical illustration of statistical 14-3-3σ distribution in 116 cases of CRC tissues. 14-3-3σ is significantly higher in CRC than adjacent non-tumorous tissue. The high expression of 14-3-3σ is more frequently found in CRC with lymph node metastasis (N1 + N2) than in CRC without lymph node metastasis (N0). (D) Kaplan-Meier survival curves and univariate analyses (log-rank) for CRC patients with positive expression versus negative expression or low expression versus high expression of 14-3-3σ in 116 cases of CRC tissues.

Figure 5. 14–3–3σ expression is inversely correlated with LASP1 expression in CRC tissues.

(A) Western blot analysis of LASP1 and 14-3-3σ in CRC tissues (T) and adjacent non-tumor tissues (N). (B) The expression of LASP1 and 14-3-3σ in the paired CRC tissues and matched normal tissues. The immunosignal was quantified using densitometric scanning software, and the relative protein abundance was determined by normalization with β-actin. (C) A statistically significant inverse correlation between 14-3-3σ and LASP1 protein was observed in CRC specimens. The full-length blots/gels are presented in Supplementary Fig. S8.

Figure 6. LASP1 and 14-3-3σ corporately contribute to CRC aggressiveness.

(A) Paraffin-embedded CRC sections were stained with anti-14-3-3σ or anti-LASP1 antibodies. Visualizations of two representative cases were shown. Lower panels are corresponding high magnification of upper panels. The low expression of 14-3-3σ is more frequently found in CRC cases with LASP1 overexpression. (B) Prognostic significance of a combination of 14-3-3σ and LASP1 assessed using Kaplan-Meier survival and univariate analyses (log-rank) for CRC patients in 116 cases. (C) A hypothetical model illustrating that loss of the 14-3-3σ is essential for LASP1-mediated colorectal cancer progression via activating PI3K/AKT signaling pathway.