Interplay between Trx-1 and S100P promotes colorectal cancer cell epithelial-mesenchymal transition by up-regulating S100A4 through AKT activation

Abstract

We previously reported a novel positive feedback loop between thioredoxin-1 (Trx-1) and S100P, which promotes the invasion and metastasis of colorectal cancer (CRC). However, the underlying molecular mechanisms remain poorly understood. In this study, we examined the roles of Trx-1 and S100P in CRC epithelial-to-mesenchymal transition (EMT) and their underlying mechanisms. We observed that knockdown of Trx-1 or S100P in SW620 cells inhibited EMT, whereas overexpression of Trx-1 or S100P in SW480 cells promoted EMT. Importantly, S100A4 and the phosphorylation of AKT were identified as potential downstream targets of Trx-1 and S100P in CRC cells. Silencing S100A4 or inhibition of AKT phosphorylation eliminated S100P- or Trx-1-mediated CRC cell EMT, migration and invasion. Moreover, inhibition of AKT activity reversed S100P- or Trx-1-induced S100A4 expression. The expression of S100A4 was higher in human CRC tissues compared with their normal counterpart tissues and was significantly correlated with lymph node metastasis and poor survival. The overexpression of S100A4 protein was also positively correlated with S100P or Trx-1 protein overexpression in our cohort of CRC tissues. In addition, overexpression of S100P reversed the Trx-1 knockdown-induced inhibition of S100A4 expression, EMT and migration and invasion in SW620 cells. The data suggest that interplay between Trx-1 and S100P promoted CRC EMT as well as migration and invasion by up-regulating S100A4 through AKT activation, thus providing further potential therapeutic targets for suppressing the EMT in metastatic CRC.

Keywords: S100A4; S100P; colorectal cancer; epithelial-mesenchymal transition; thioredoxin-1.

Figure 1

The expression levels of S100P, Trx‐1, S100A4 and EMT‐associated proteins in SW480 and SW620 cells. A, S100P, Trx‐1, S100A4 and EMT‐associated proteins (E‐cadherin, vimentin and fibronectin) were examined by Western blotting. β‐actin was used as the loading control. B, EMT morphological changes induced by S100P or Trx‐1. Representative microscopic views of SW480 and SW620 cells were shown. Scale bar, 50 μm

Figure 2

Effects of Trx‐1 and S100P on epithelial–mesenchymal transition of colorectal carcinoma cells. (A) Western blotting revealed that overexpression of Trx‐1 resulted in a decreased expression of epithelial marker E‐cadherin and increased expressions of mesenchymal markers (vimentin and fibronectin), S100A4 and phosphorylated AKT (P‐AKT) in SW480 cells, whereas knockdown of Trx‐1 by shRNA resulted in an increased expression of E‐cadherin and decreased expressions of vimentin, fibronectin, S100A4 and P‐AKT in SW620 cells. (B) Western blotting showed that overexpression of S100P resulted in a decreased expression of E‐cadherin and increased expressions of vimentin, fibronectin, S100A4 and P‐AKT in SW480 cells, whereas knockdown of S100P by shRNA resulted in an increased expression of E‐cadherin and decreased expressions of vimentin, fibronectin, S100A4 and P‐AKT in SW620 cells. β‐Actin was used as the loading control. (C) Immunofluorescence staining of Trx‐1 overexpression down‐regulated E‐cadherin expression while up‐regulating vimentin expression in SW480 cells. (D) Knockdown of Trx‐1 by shRNA up‐regulated E‐cadherin expression and down‐regulated vimentin expression in SW620 cells. (E) S100P overexpression caused a decrease in E‐cadherin expression and an increase in the expression of vimentin in SW480 cells. (F) Knockdown of S100P by shRNA increased the expression of E‐cadherin and decreased vimentin expression in SW620 cells. Nuclei were counterstained using DAPI (blue). Merged figures show the co‐localization of E‐cadherin or vimentin (red) with DAPI. Scale bar = 20 μm

Figure 3

Inhibition of AKT activity induces the mesenchymal‐to‐epithelial transition, decreased S100A4 expression and migration and invasion of SW620 cells. (A) SW620 cells were treated with MK‐2206 (3 μg/mL and 6 μg/mL), an AKT inhibitor, for 24 hours. The expression levels of AKT, phosphorylated AKT (P‐AKT), S100A4, E‐cadherin, vimentin and fibronectin were measured by Western blotting. β‐Actin was used as the loading control. (B) Transwell assays showed that MK‐2206 suppressed SW620 cell migration and invasion. SW620 cells were treated with 3 μg/mL MK‐2206 for 24 hours before performing the Transwell assays (**P ˂ .01). Values represent relative change in migration and invasion normalized to an arbitrary value of 1 for controls

Figure 4

Silencing S100A4 by siRNA induces mesenchymal‐to‐epithelial transition and inhibits migration and invasion of SW620 cells. (A) SW620 cells were transfected with S100A4‐siRNA for 48 hours. The levels of AKT, phosphorylated AKT (P‐AKT), S100A4, E‐cadherin, vimentin and fibronectin were measured by Western blotting. β‐Actin was used as the loading control. (B) Silencing S100A4 by siRNA inhibited migration and invasion rate of SW620 cells by up to 41.2 and 70.5%, respectively. The SW620 cells were transfected with S100A4‐siRNA for 48 hours before performing the Transwell assays (**P ˂ .01)

Figure 5

Expression of S100A4 in colorectal carcinoma tissues and its prognostic significance in colorectal cancer patients. (A) Strong immunopositive staining of cancerous tissue, H score = 200; (B) moderate immunopositive staining of cancerous tissue, H score = 120; (C) negative staining of cancerous tissue, H score = 0; D, S100A4 expression of colorectal carcinoma tissue was significantly higher than that of the matched adjacent normal tissues as indicated by IHC. **P < .01. Scale bar = 20 μm. E, Significant up‐regulation of S100A4 protein expression by IHC was shown in colorectal carcinoma with lymph node metastases, relative to colorectal carcinoma without lymph node metastasis. *P < .05. F, Kaplan‐Meier survival analysis according to CRC patients with S100A4 overexpression (log‐rank test, P = .042)

Figure 6

Silencing S100A4 or inhibiting AKT activity reserves Trx‐1‐mediated epithelial–mesenchymal transition (EMT), migration and invasion. A representative Western blot (A) and the summarized data (B) showed that the level of E‐cadherin increased and the levels of the S100A4 and vimentin decreased after silencing S100A4 or blocking AKT phosphorylation with MK‐2206 in SW480‐Trx‐1 cells. SW480‐Trx‐1 cells were transfected with 50 μM S100A4‐siRNA for 48 hours or treated with AKT inhibitor MK‐2206 (6 μg/mL). β‐Actin was used as the loading control. (C,D) Transwell assays demonstrated that the enhanced migration and invasive abilities of SW480‐Trx‐1 cells were inhibited after silencing S100A4 or blocking the phosphorylation of AKT

Figure 7

Silencing S100A4 or inhibiting AKT activity reverses S100P‐mediated EMT, migration and invasion. A representative Western blot (A) and the summarized data (B) showed that the level of E‐cadherin increased and the levels of S100A4 and vimentin decreased after silencing S100A4 or blocking phosphorylation of AKT with MK‐2206 in SW480‐S100P cells. SW480‐S100P cells were transfected with 50 μM S100A4‐siRNA for 48 hours or treated with AKT inhibitor MK‐2206 (6 μg/mL). β‐Actin was used as the loading control. (C, D) Transwell assays showed that the enhanced migration and invasive abilities of SW480‐S100P cells were inhibited after silencing S100A4 or blocking phosphorylation of AKT

Figure 8

S100P partially reverses the inhibition of EMT, migration and invasion caused by Trx‐1 knockdown. A representative Western blot (A) and the summarized data (B) showed that overexpression of S100P partially reversed Trx‐1 silencing‐induced mesenchymal–epithelial transition in SW620 cells. SW620‐shTrx‐1 cells were transfected with Lenti‐S100P; the expression levels of E‐cadherin, S100A4 and vimentin were assessed by Western blotting. β‐Actin was used as the loading control. (C, D) Overexpression of S100P partly reversed Trx‐1 silencing‐induced inhibition of migration and invasion in SW620 cells. (E) Model for crosstalk between S100P and Trx‐1 that ultimately promotes colorectal cancer cell EMT, migration and invasion by up‐regulating S100A4 through AKT activation