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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.

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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).
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pone-0016068-g007: 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).

Mentions: In order to further investigate whether the inhibition of Shh autocrine signaling leads to the reduction in cell migration, invasion, and tumorigenesis in other NSCLC cell lines that expresses Shh, we chose H1299 and H1650 cell lines, both of which were derived from lung metastasis of NSCLC patients. Both cell lines have been shown to be resistant to chemotherapy and targeted therapy (e.g. Erlotinib) [17], [38], [39]. Our results confirmed that both the cell lines expressed Shh as documented by qRT-PCR and Western blot analysis (Fig. 7A). Treatment of H1650 cells with Shh inhibitors GDC-0449 showed decreased cell migration, invasion and tumorigenic characteristics (Fig. 7B–C and Fig. S1), which clearly provide strong experimental support in favor of the role Shh in EMT phenotype. It is important to note that the treatment of H1650 cells with GDC-0449 led to the partial reversal of the EMT phenotype as documented by the reduced expression of fibronectin and ZEB1, and the increased expression of E-cadherin (Fig. 7D and E), which is consistent with the data in A549-M cells as presented under Fig. 5D and E. Moreover, these results are also consistent with the knock-down of Shh by Shh-specific siRNA in H1650 cells with robust transfection efficiency (Fig. 8A), and resulting in a significant reduction in cell migration and invasion (Fig. 8B and Fig. 8C). In addition, the treatment of H1299 cells with GDC-0449 or cyclopamine led to a significant reduction in invasion and tumorigenic behavior as assessed by clonogenic growth (Fig. S2A-B and Fig. S1). The treatment of H1299 cells with GDC-0449 also led to the partial reversal of the EMT phenotype as documented by reduced expression of fibronectin and ZEB1, and the increased expression of E-cadherin (Fig. S2C-D).


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016068-g007: 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).
Mentions: In order to further investigate whether the inhibition of Shh autocrine signaling leads to the reduction in cell migration, invasion, and tumorigenesis in other NSCLC cell lines that expresses Shh, we chose H1299 and H1650 cell lines, both of which were derived from lung metastasis of NSCLC patients. Both cell lines have been shown to be resistant to chemotherapy and targeted therapy (e.g. Erlotinib) [17], [38], [39]. Our results confirmed that both the cell lines expressed Shh as documented by qRT-PCR and Western blot analysis (Fig. 7A). Treatment of H1650 cells with Shh inhibitors GDC-0449 showed decreased cell migration, invasion and tumorigenic characteristics (Fig. 7B–C and Fig. S1), which clearly provide strong experimental support in favor of the role Shh in EMT phenotype. It is important to note that the treatment of H1650 cells with GDC-0449 led to the partial reversal of the EMT phenotype as documented by the reduced expression of fibronectin and ZEB1, and the increased expression of E-cadherin (Fig. 7D and E), which is consistent with the data in A549-M cells as presented under Fig. 5D and E. Moreover, these results are also consistent with the knock-down of Shh by Shh-specific siRNA in H1650 cells with robust transfection efficiency (Fig. 8A), and resulting in a significant reduction in cell migration and invasion (Fig. 8B and Fig. 8C). In addition, the treatment of H1299 cells with GDC-0449 or cyclopamine led to a significant reduction in invasion and tumorigenic behavior as assessed by clonogenic growth (Fig. S2A-B and Fig. S1). The treatment of H1299 cells with GDC-0449 also led to the partial reversal of the EMT phenotype as documented by reduced expression of fibronectin and ZEB1, and the increased expression of E-cadherin (Fig. S2C-D).

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