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. 2011 Jan 13;6(1):e16068.
doi: 10.1371/journal.pone.0016068.

Up-regulation of sonic hedgehog contributes to TGF-β1-induced epithelial to mesenchymal transition in NSCLC cells

Ma'in Y Maitah  1 , Shadan Ali, Aamir Ahmad, Shirish Gadgeel, Fazlul H Sarkar
Affiliations

Up-regulation of sonic hedgehog contributes to TGF-β1-induced epithelial to mesenchymal transition in NSCLC cells

Ma'in Y Maitah et al. PLoS One. .

Retraction in

Abstract

Background: Lung cancer, especially non-small cell lung cancer (NSCLC) is the major cause of cancer-related deaths in the United States. The aggressiveness of NSCLC has been shown to be associated with the acquisition of epithelial-to-mesenchymal transition (EMT). The acquisition of EMT phenotype induced by TGF-β1in several cancer cells has been implicated in tumor aggressiveness and resistance to conventional therapeutics; however, the molecular mechanism of EMT and tumor aggressiveness in NSCLC remains unknown.

Methodology/principal findings: In this study we found for the first time that the induction of EMT by chronic exposure of A549 NSCLC cells to TGF-β1 (A549-M cells) led to the up-regulation of sonic hedgehog (Shh) both at the mRNA and protein levels causing activation of hedgehog signaling. These results were also reproduced in another NSCLC cell line (H2030). Induction of EMT was found to be consistent with aggressive characteristics such as increased clonogenic growth, cell motility and invasion. The aggressiveness of these cells was attenuated by the treatment of A549-M cells with pharmacological inhibitors of Hh signaling in addition to Shh knock-down by siRNA. The inhibition of Hh signaling by pharmacological inhibitors led to the reversal of EMT phenotype as confirmed by the reduction of mesenchymal markers such as ZEB1 and Fibronectin, and induction of epithelial marker E-cadherin. In addition, knock-down of Shh by siRNA significantly attenuated EMT induction by TGF-β1.

Conclusions/significance: Our results show for the first time the transcriptional up-regulation of Shh by TGF-β1, which is mechanistically associated with TGF-β1 induced EMT phenotype and aggressive behavior of NSCLC cells. Thus the inhibitors of Shh signaling could be useful for the reversal of EMT phenotype, which would inhibit the metastatic potential of NSCLC cells and also make these tumors more sensitive to conventional therapeutics.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Induction of epithelial to mesenchymal transition (EMT) in A549 cells by chronic exposure to TGF-β1:
TGF-β1 was added to A549 cells in culture media and maintained for 21 days with changing medium every third day with freshly added TGF-β1. A) Phase contrast objective microscopic pictures at 10× magnification. A549 cells morphology changed to mesenchymal phenotype (A549-M cells). Cell shape appears elongated and non-polarized. B) qRT-PCR of A549 and A549-M cells. A549-M cells showed a lower E-cadherin “epithelial marker,” and a higher ZEB1 “EMT marker”, at the mRNA levels. Delta-delta-CT was calculated, considering GAPDH as internal control and A549 parental as reference control. C) Western blot analysis where A549-M cells showed up-regulation of fibronectin “mesenchymal” marker compared to A549 parental cells.
Figure 2
Figure 2. A549-M cells shows significant increase in migration, invasive, and tumorigenic characteristics compared to A549 parental cells:
TGF-β1-induced EMT phenotypic cells (A549-M cells) were generated as discussed under “Materials and Methods” section. A: showed wound healing assay results with its quantitative analysis. A549-M cells showed much higher motility compared to A549 parental cells. B and C showing the results of matrigel-coated membrane, and colony formation assays, respectively with its quantitative analysis. Significant increase was observed in the invasion and clonogenicity of A549-M cells compared to parental A549 cells. (*  =  p<0.05).
Figure 3
Figure 3. A549-M cells showed up-regulation of sonic hedgehog (Shh) and GLI expression both at the mRNA and protein levels:
A and B showing qRT-PCR and Western blot results, respectively for the expression of Shh whereas C and D represent the expression status of GLI at the mRNA and protein levels, respectively in A549-M cells compared to parental A549 cells. E represent Western blot data of GLI1 expression in NIH-3T3 cell after culturing with A549-M-derived conditioned media showing higher levels of GLI1 expression. (*  =  p<0.05).
Figure 4
Figure 4. Shh up-regulation is concomitant with TGF-β1-induced EMT in NSCLC cell lines.
The up-regulation of Shh contributes to the EMT induction through TGF-β1. (A) H2030 cell line was treated with TGF-β1 (5 ng/ml) for two weeks, and the media was changed every three days. The qRT-PCR data showed induced expression of EMT marker ZEB1 mRNA, and reduced expression of epithelial marker E-cadherin mRNA, which was consistent with up-regulation of Shh mRNA similar to those observed in A549 cells exposed to TGF-β1. (B, C and D) A549 cells was transfected with Shh siRNA (A549-siShh) or scrambled siRNA (A549-si-ve) for 24 hrs prior to treatment with TGF-β1 (5 ng/ml) for 48 hrs, then the cells where collected for assays or re-transfected for the second time with siRNA or scrambled siRNA for 24 hrs (total 6days after siShh transfection) prior to the second time treatment with TGF-β1 (5 ng/ml) for another 48 hrs (total 5days of TGF-β1 treatment). (B) Upper panel shows transfection efficiency, and lower panel shows cellular morphology following treatments. A549-siShh maintained epithelial morphology after treatment with TGF-β1 at both time points as shown in left and right panels, respectively. (C) qRT-PCR expression of Shh mRNA showing significant down-regulation following Shh siRNA transfection (D) qRT-PCR expression of ZEB1 and E-cadherin mRNA. A549-si-ve cells showed down-regulation of epithelial marker, E-cadherin consistent with significant induction in the expression of ZEB1 as expected whereas TGF-β1 failed to show any effect on these markers in A549-siShh cells.
Figure 5
Figure 5. Up-regulation of Shh in A549-M cells contributes to increased tumor cells migration and metastatic characteristics:
A549-M cells were treated with Shh inhibitors such as Cyclopamine (2 µM) and GDC-0449 (20 nM) and assayed for wound healing (A), invasion (B) and clonogenic growth (C), and performed quantitative analysis showing attenuation of invasion by the treatment with Shh inhibitors. Western blot of A549-M cells before and after treatment with GDC-0449 (20 nM) for the expression of fibronectin (D). qRT-PCR for the expression of E-cadherin and ZEB1 mRNA in A549-M cells after treatment with GDC-0449 (20 nM) showing reversal of EMT phenotype compared to untreated A549-M cells (E). (*  =  p<0.05).
Figure 6
Figure 6. Reduction in A549-M cells motility, invasiveness, and tumorigenesis by specific knock-down of Shh using Shh-specific siRNA:
A549-M cells were transfected with Shh-specific siRNA (A): Transfection efficiency as assessed by GFP. The effect of knock-down of Shh was assessed by cell motility (wound healing) (B), invasion (C) and clonogenic growth (D) and further quantitated as detailed under “Materials and Methods” section, showing significant inhibition by Shh specific siRNA. (*  =  p<0.05).
Figure 7
Figure 7. Down-regulation of Shh autocrine signaling in NSCLC cell lines led to the reduction in tumor cell migration, invasion, and tumorigenesis:
A; both H1650 and H1299 cells expresses high levels of Shh mRNA compared to NHBE cells, and both cell lines have high Shh protein expression. B and C shows reduction in cell-invasion and the colony-forming ability of H1650 cells following treatment with Shh inhibitors such as GDC-0449 (20 nM). (D) Western blot of H1650 cells before and after treatment with GDC-0449 (20 nM) for the expression of fibronectin. (E) qRT-PCR for the expression of E-cadherin and ZEB1 mRNA in H1650 cells after treatment with GDC-0449 (20 nM) showing reversal of EMT compared to untreated H1650 cells. (*  =  p<0.05).
Figure 8
Figure 8. Down-regulation of Shh signaling in NSCLC cells lines (H1650 cells) leads to reduced cell motility and invasion.
(A): Transfection efficiency was assessed by GFP. (B) Matrigel-Coated membrane assay where cells were labeled with DiIC12 fluorescent dye. (C) Matrigel-Coated membrane assay where cells were labeled with immune-staining kit (Quik staining kit). (B and C right panel) also show quantitative data analysis. (*  =  p<0.05).
Figure 9
Figure 9. Schematic diagram showing activation of TGF-β receptor by TGF-β1which leads to the up-regulation of Shh expression.
The secreted Shh protein then activates Hh signaling pathway by inhibition of Patched (smoothened suppressor), which will repress smoothened, resulting in the activation of GLI1 and its translocation to the nucleus. GLI1 as a Hh transcription factor then could activate Hh target genes, which leads to the acquisition of EMT phenotype, and contributing to increased invasion, metastasis and drug resistance.
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