Snail regulates Nanog status during the epithelial-mesenchymal transition via the Smad1/Akt/GSK3β signaling pathway in non-small-cell lung cancer

Abstract

The epithelial-mesenchymal transition (EMT), a crucial step in cancer metastasis, is important in transformed cancer cells with stem cell-like properties. In this study, we established a Snail-overexpressing cell model for non-small-cell lung cancer (NSCLC) and investigated its underlying mechanism. We also identified the downstream molecular signaling pathway that contributes to the role of Snail in regulating Nanog expression. Our data shows that high levels of Snail expression correlate with metastasis and high levels of Nanog expression in NSCLC. NSCLC cells expressing Snail are characterized by active EMT characteristics and exhibit an increased ability to migrate, chemoresistance, sphere formation, and stem cell-like properties. We also investigated the signals required for Snail-mediated Nanog expression. Our data demonstrate that LY294002, SB431542, LDN193189, and Noggin pretreatment inhibit Snail-induced Nanog expression during EMT. This study shows a significant correlation between Snail expression and phosphorylation of Smad1, Akt, and GSK3β. In addition, pretreatment with SB431542, LDN193189, or Noggin prevented Snail-induced Smad1 and Akt hyperactivation and reactivated GSK3β. Moreover, LY294002 pretreatment prevented Akt hyperactivation and reactivated GSK3β without altering Smad1 activation. These findings provide a novel mechanistic insight into the important role of Snail in NSCLC during EMT and indicate potentially useful therapeutic targets for NSCLC.

Figure 1. Snail upregulation is correlated with the malignancy of human non-small-cell lung cancer (NSCLC) tissues

(A) Representative images of immunohistochemical staining of Snail in specimens from 55 NSCLC patients. In different tumor types (normal tissue, adenocarcinoma, squamous cell carcinoma, and adenosquamous carcinoma), the expression level of Snail was obvious in high-grade but not low-grade NSCLC tumors. (B) The expression level of Snail was analyzed and quantified by an experienced pathologist; **p < 0.01 and ***p < 0.001 indicate statistical significance as compared to the control (normal tissue).

Figure 2. Snail overexpression induces the epithelial–mesenchymal transition (EMT) in A549 and CL1-5 cells

(A) Phase-contrast images present the morphology of A549 cells expressing empty vector (A549-vector cells), A549 cells overexpressing Snail (A549-Snail cells), CL1-0 cells, and CL1-5 cells. Immunofluorescent staining of E-cadherin and vimentin (green fluorescence) show the changes in EMT biomarkers. Nuclei were stained with Hoechst 33342 (blue fluorescence). (B) Western blots showing the expression of epithelial markers and mesenchymal markers in A549-vector cells, A549-Snail cells, CL1-0 cells, and CL1-5 cells. In Snail-expressing cells, expression of mesenchymal markers increased, but expression of epithelial markers decreased. (C) The number of migrating cells was significantly increased in Snail-expressing cells; ***p < 0.001 indicates statistical significance as compared to the control. (D) Chemoresistance as evaluated by the MTT assay. The LC₅₀ for cisplatin in A549-vector and A549-Snail cells was 134.6 nM and 170.3 nM, respectively. The LC₅₀ for cisplatin in CL1-0 and CL1-5 cells was 148.4 nM and 287.6 nM, respectively; CL1-5 is more resistant to cisplatin than CL1-0.

Figure 3. Overexpression of Snail enhances in vivo metastatic and tumorigenic abilities in A549 cells

(A) The in vivo pulmonary metastatic colonies assay was performed as described in the Methods section. Both the images and the analyzed data (N = 5) demonstrate the aggressive metastatic capacity of A549 cells overexpressing Snail (A549-Snail cells) as compared to A549 cells expressing empty vector (A549-vector cells); ***p < 0.001 indicates statistical significance as compared to the A549-vector cells. (B) A549-vector cells or A549-Snail cells (1 × 10⁴ cells) were injected into the subrenal space in NOD/SCID mice. The growth curves of xenograft tumors in NOD/SCID mice show that transplanted A549-Snail cells are capable of tumorigenesis. Data are shown as mean ± standard deviation (N = 5).

Figure 4. Snail overexpression induces stem cell-like signatures during the epithelial–mesenchymal transition

(A/E) The mRNA expression of stemness genes (Oct4, Sox2, Nanog) was higher in A549 cells overexpressing Snail (A549-Snail cells) than in A549 cells expressing empty vector (A549-vector cells). However, only Nanog expression was higher in CL1-5 cells than in CL1-0 cells. (B/F) The number of spheroid-like bodies formed was significantly higher in Snail-expressing cells; ***p < 0.001 indicates statistical significance as compared to the control. (C/D/G/H) Cell-surface markers (CD24, CD44, and CD133) were analyzed by flow cytometry as described in the Methods section. Increases in the CD44^high/CD24^low subpopulation (C) and the surface expression of CD133 (D) were found in A549-Snail cells as compared to the A549-vector cells.

Figure 5. The expression of Snail and Nanog are highly correlated in lung cancer tissues

(A) Representative specimens of low-grade and high-grade lung tumors immunostained using antibodies specific to Snail and Nanog. (B) Positive correlation between levels of Snail and Nanog in lung tumors, and the level of Nanog as a novel prognostic marker for lung cancer patients.

Figure 6. PI-3 kinase/Akt activation and GSK3β inactivation are required for Snail-induced Nanog expression

(A) Remarkable activation of Akt (Ser-473 phosphorylation) and inactivation of GSK3β (Ser-9 phosphorylation) are found in A549 cells overexpressing Snail (A549-Snail cells) as compared to A549 cells expressing empty vector (A549-vector cells). Both Akt and GSK3β phosphorylation could be blocked by LY294002 pretreatment (10 μM; 1 h). In addition, Snail-induced Nanog expression was blocked by LY294002 pretreatment. (B/C) Data were quantified by densitometric analysis and expressed as the mean ± standard deviation from at least 3 independent experiments. (D) Remarkable activation of Smad1 and inactivation of Smad2 were found in the A549-Snail cells as compared to the A549-vector cells. Neither Smad1 phosphorylation nor Smad2 dephosphorylation was affected by LY294002 pretreatment. (E) Data were quantified by densitometric analysis and expressed as the mean ± standard deviation from at least 3 independent experiments; *p < 0.05, **p< 0.01, and ***p < 0.001 indicate statistical significance as compared to the A549-vector cells; ##p < 0.01 and ###p < 0.001 indicate statistical significance as compared to the A549-Snail cells.

Figure 7. Snail-induced Nanog expression is regulated by Smad1/Akt/GSK3β pathway activation

Snail-induced Akt and Smad1 activation and GSK3β inactivation was inhibited by SB431542 (A), LDN193189 (B), and Noggin (C) pretreatment. In addition, Snail-induced Nanog expression was prevented by SB431542, LDN193189, and Noggin pretreatment. (D/E/F) Data were quantified by densitometric analysis and expressed as the mean ± standard deviation from at least 3 independent experiments; ***p < 0.001 indicates statistical significance as compared to the A549-vector cells; ##p < 0.01 and ###p < 0.001 indicate statistical significance as compared to the A549-Snail cells.

Figure 8. The Smad1/Akt/GSK3β pathway is consistently activated in CL1-5 cells but is downregulated in Snail-silenced CL1-5 cells

(A) CL1-5 cells, which endogenously express Snail, exhibited greater activation of the Smad1/Akt/GSK3β pathway and upregulated Nanog expression in comparison to CL1-0 cells. Both SB431542 and LDN193189 decreased activation of the Smad1/Akt/GSK3β pathway and downregulated Nanog expression in CL1-5 cells. Unlike Akt and GSK3β phosphorylation, which was completely inhibited by LY294002, Smad1 phosphorylation did not respond to LY294002. (B/C) Scramble and Smad1 siRNA were expressed in CL1-5 cells for 40 h. The Smad1 siRNA fully suppressed the Smad1/Akt/GSK3β pathway as well as Nanog expression. In addition, the expression of mesenchymal markers decreased, but the expression of epithelial markers increased in Smad-silenced CL1-5 cells as compared to CL1-0 cells. (D) The endogenously expressed Snail was silenced in CL1-5 cells. An increase in E-cadherin and a decrease in vimentin expression were found in Snail-silenced CL1-5 cells. (E) In addition, a reduction in the number of migrating cells was observed in Snail-silenced CL1-5 cells as compared to the control cells (transfected with Scramble siRNA); ***p < 0.001 indicates statistical significance as compared to the control. (F) Either a decrease in the level of activation of the Smad1/Akt/GSK3β pathway or a reduction in Nanog expression was found in Snail-silenced CL1-5 cells.

Figure 9. Diagram showing the induction of stem cell-like properties by Snail via the activation of the Smad1/Akt/GSK3β pathway and subsequent upregulation of Nanog expression