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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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Related in: MedlinePlus

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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pone-0016068-g009: 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.

Mentions: Based on existing evidence in the literature and our current data, we propose a model where epithelial tumor cells could be chronically exposed to TGF-β1 excreted by either stromal cells, immune cells or the tumor cells within the tumor microenvironment, resulting in the up-regulation of Shh both at the mRNA and at the protein levels and consequently causes activation of Hh signaling and the acquisition of EMT phenotype, which is responsible for tumor cell aggressiveness and metastasis (Fig. 9). Therefore, the inhibition of Shh signaling could be a useful approach for reducing tumor aggressiveness in NSCLC, and as such, the reversal of EMT could also be useful for re-sensitization of drug-resistant NSCLC to conventional therapeutics, which would likely contribute to the improved survival of patients who rightfully deserve better treatment outcomes.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016068-g009: 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.
Mentions: Based on existing evidence in the literature and our current data, we propose a model where epithelial tumor cells could be chronically exposed to TGF-β1 excreted by either stromal cells, immune cells or the tumor cells within the tumor microenvironment, resulting in the up-regulation of Shh both at the mRNA and at the protein levels and consequently causes activation of Hh signaling and the acquisition of EMT phenotype, which is responsible for tumor cell aggressiveness and metastasis (Fig. 9). Therefore, the inhibition of Shh signaling could be a useful approach for reducing tumor aggressiveness in NSCLC, and as such, the reversal of EMT could also be useful for re-sensitization of drug-resistant NSCLC to conventional therapeutics, which would likely contribute to the improved survival of patients who rightfully deserve better treatment outcomes.

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