HSP27 modulates epithelial to mesenchymal transition of lung cancer cells in a Smad-independent manner

Abstract

Epithelial to mesenchymal transition (EMT) is induced by transforming growth factor-β1 (TGF-β1) and is a crucial event for cancer cells to acquire invasive and metastatic phenotypes. However, the signals that induce EMT in cancer cells have yet to be adequately defined. In this study, a proteomic investigation was performed to understand the signaling pathway of the EMT of lung cancer using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. The protein expression profiles of A549 were compared to those of A549 cells treated with TGF-β1. Of more than 2,000 protein spots shown by 2D-DIGE, 53 were found to be up- or down-regulated upon induction with TGF-β1. In the 53 protein spots, the protein level of heat shock protein (HSP) 27 was found to increase significantly. HSP27 protein was higher in two different lung cancer cell lines, demonstrating the EMT phenomenon with TGF-β1. Notably, the silencing of HSP27 enhanced spindle integration, resulting in an additive effect with TGF-β1-induced EMT. Furthermore, the TGF-β1-induced HSP27 increase was not affected by the suppression of Smad2 and Smad3 in A549 cells. These results suggest that HSP27 was involved in TGF-β1-induced EMT in a Smad-independent manner in lung cancer cells and may provide an effective clinical strategy in lung cancer patients whose tumors are dependent on TGF-β1-induced EMT.

Figure 1

(A) TGF-β1-stimulated EMT of A549 lung cancer cells. A549 cells were treated with 5 ng/ml of TGF-β1 for 72 h and then designated as A549/TGF-β1 cells. The cells were observed under a light microscope. A549 cells exhibited a classic epithelial morphology. By contrast, A549/TGF-β1 appeared to be less uniformly epithelial. (B) A549/TGF-β1 cells were subjected to 2D-DIGE. (C) Expression levels of HSP27 following TGF-β1 treatment were analyzed in four lung cancer cell lines using 2D-DIGE. Among the four cell lines, EMT was observed in A549, RERF-LC-KJ and LC2-ad cells, but not PC9, following TGF-β1 stimulation. Data are the mean ± SD from three independent experiments. ^*p<0.05 when compared to the respective parent cells. (D) HSP27 expression was analyzed by Western blot analysis. Compared to the A549 cells, A549/TGF-β1 revealed an increased level of HSP27.

Figure 1

(A) TGF-β1-stimulated EMT of A549 lung cancer cells. A549 cells were treated with 5 ng/ml of TGF-β1 for 72 h and then designated as A549/TGF-β1 cells. The cells were observed under a light microscope. A549 cells exhibited a classic epithelial morphology. By contrast, A549/TGF-β1 appeared to be less uniformly epithelial. (B) A549/TGF-β1 cells were subjected to 2D-DIGE. (C) Expression levels of HSP27 following TGF-β1 treatment were analyzed in four lung cancer cell lines using 2D-DIGE. Among the four cell lines, EMT was observed in A549, RERF-LC-KJ and LC2-ad cells, but not PC9, following TGF-β1 stimulation. Data are the mean ± SD from three independent experiments. ^*p<0.05 when compared to the respective parent cells. (D) HSP27 expression was analyzed by Western blot analysis. Compared to the A549 cells, A549/TGF-β1 revealed an increased level of HSP27.

Figure 2

(A) Morphologic change of A549 cells treated with HSP27 siRNA. A549 and A549/TGF-β1 cells were treated with control siRNA or HSP27 siRNA. A549/TGF-β1 cells treated with HSP27 siRNA showed morphological evidence of EMT. (B) Protein expression of EMT markers in A549 and A549/TGF-β1 cells treated with HSP27 siRNA. Loss of E-cadherin expression occurred in A549/TGF-β1 cells treated with HSP27 siRNA, whereas a gain in N-cadherin expression was noted in A549/TGF-β1 cells with control siRNA.

Figure 3

(A) Morphologic change of A549 cells treated with siRNA of Smad2 or Smad3. A549 and A549/TGF-β1 cells were treated with control siRNA or siRNA of Smad2 or Smad3. A549/TGF-β1 cells treated with Smad2 or Smad3 suppressed EMT morphologically. (B) Protein expression of EMT markers in A549 or A549/TGF-β1 cells treated with specific siRNA of Smad2 and Smad3. A549/TGF-β1 cells treated with Smad2- or Smad3-specific siRNA still expressed E-cadherin and suppressed N-cadherin expression. (C) Protein expression of HSP27 following the transfection of Smad2/3 double siRNA. HSP27 expression was unchanged by the transfection of Smad2- plus Smad3-specific siRNAs in A549/TGF-β1 cells. (D) Protein expression of Smad and p-Smad of A549 cells treated with siRNA of HSP27. The expression of Smad2, Smad3, p-Smad2 and p-Smad3 was unchanged by the HSP27-specific siRNA transduction.

Figure 3

(A) Morphologic change of A549 cells treated with siRNA of Smad2 or Smad3. A549 and A549/TGF-β1 cells were treated with control siRNA or siRNA of Smad2 or Smad3. A549/TGF-β1 cells treated with Smad2 or Smad3 suppressed EMT morphologically. (B) Protein expression of EMT markers in A549 or A549/TGF-β1 cells treated with specific siRNA of Smad2 and Smad3. A549/TGF-β1 cells treated with Smad2- or Smad3-specific siRNA still expressed E-cadherin and suppressed N-cadherin expression. (C) Protein expression of HSP27 following the transfection of Smad2/3 double siRNA. HSP27 expression was unchanged by the transfection of Smad2- plus Smad3-specific siRNAs in A549/TGF-β1 cells. (D) Protein expression of Smad and p-Smad of A549 cells treated with siRNA of HSP27. The expression of Smad2, Smad3, p-Smad2 and p-Smad3 was unchanged by the HSP27-specific siRNA transduction.