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

Maitah MY, Ali S, Ahmad A, Gadgeel S, Sarkar FH - PLoS ONE (2011)

Bottom Line: Induction of EMT was found to be consistent with aggressive characteristics such as increased clonogenic growth, cell motility and invasion.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, United States of America.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3020967&req=5

pone-0016068-g004: 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.

Mentions: In order to assess the mechanism by which chronic TGF-β1 treatment induced EMT and tumor cell aggressiveness, we focused our investigation on Hh signaling because it has been implicated in EMT induction, metastasis and invasion [20]–[24], [26], [34], [34]–[37]. Interestingly, we found a dramatic increase in the expression of Hh pathway ligand Shh both at the mRNA and protein levels in A549-M cells whereas the parental A549 cells showed undetectable levels of Shh mRNA (Fig. 3A and Fig. 3B), which is consistent with published data showing that A549 parental cells contains undetectable levels of Shh expression [35]. In order to further confirm our findings documenting up-regulation of Shh by TGF-β1 treatment, and the induction of EMT in A549 NSCLC cell lines, we treated another NSCLC cell line (H2030 cells) with TGF-β1 for two weeks, and we found a significant increase in the expression of Shh mRNA, which was consistent with the induction of EMT marker ZEB1 and down-regulation of epithelial marker E-cadherin (Fig. 4A). These results suggest that TGF-β1 induced EMT is mediated by the transcriptional activation of Shh, which is the first such report in the literature.


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

Maitah MY, Ali S, Ahmad A, Gadgeel S, Sarkar FH - PLoS ONE (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020967&req=5

pone-0016068-g004: 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.
Mentions: In order to assess the mechanism by which chronic TGF-β1 treatment induced EMT and tumor cell aggressiveness, we focused our investigation on Hh signaling because it has been implicated in EMT induction, metastasis and invasion [20]–[24], [26], [34], [34]–[37]. Interestingly, we found a dramatic increase in the expression of Hh pathway ligand Shh both at the mRNA and protein levels in A549-M cells whereas the parental A549 cells showed undetectable levels of Shh mRNA (Fig. 3A and Fig. 3B), which is consistent with published data showing that A549 parental cells contains undetectable levels of Shh expression [35]. In order to further confirm our findings documenting up-regulation of Shh by TGF-β1 treatment, and the induction of EMT in A549 NSCLC cell lines, we treated another NSCLC cell line (H2030 cells) with TGF-β1 for two weeks, and we found a significant increase in the expression of Shh mRNA, which was consistent with the induction of EMT marker ZEB1 and down-regulation of epithelial marker E-cadherin (Fig. 4A). These results suggest that TGF-β1 induced EMT is mediated by the transcriptional activation of Shh, which is the first such report in the literature.

Bottom Line: Induction of EMT was found to be consistent with aggressive characteristics such as increased clonogenic growth, cell motility and invasion.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, United States of America.

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.

Show MeSH
Related in: MedlinePlus