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Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs.

Ahmad A, Maitah MY, Ginnebaugh KR, Li Y, Bao B, Gadgeel SM, Sarkar FH - J Hematol Oncol (2013)

Bottom Line: Ectopic up-regulation of miRNAs, especially miR-200b and let-7c, significantly diminished the erlotinib resistance of A549M cells.Inhibition of Hh signaling by GDC-0449 in EMT cells resulted in the attenuation of CSC markers and up-regulation of miR-200b and let-7c, leading to sensitization of EMT cells to drug treatment, thus, confirming a connection between Hh signaling, miRNAs and drug resistance.We demonstrate that Hh pathway, through EMT-induction, leads to reduced sensitivity to EGFR-TKIs in NSCLCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, USA. fsarkar@med.wayne.edu.

ABSTRACT

Background: Epidermal growth factor receptor- tyrosine kinase inhibitors (EGFR-TKIs) benefit Non-small cell lung cancer (NSCLC) patients, and an EGFR-TKIi erlotinib, is approved for patients with recurrent NSCLC. However, resistance to erlotinib is a major clinical problem. Earlier we have demonstrated the role of Hedgehog (Hh) signaling in Epithelial-to-Mesenchymal transition (EMT) of NSCLC cells, leading to increased proliferation and invasion. Here, we investigated the role of Hh signaling in erlotinib resistance of TGF-β1-induced NSCLC cells that are reminiscent of EMT cells.

Methods: Hh signaling was inhibited by specific siRNA and by GDC-0449, a small molecule antagonist of G protein coupled receptor smoothened in the Hh pathway. Not all NSCLC patients are likely to benefit from EGFR-TKIs and, therefore, cisplatin was used to further demonstrate a role of inhibition of Hh signaling in sensitization of resistant EMT cells. Specific pre- and anti-miRNA preparations were used to study the mechanistic involvement of miRNAs in drug resistance mechanism.

Results: siRNA-mediated inhibition as well as pharmacological inhibition of Hh signaling abrogated resistance of NSCLC cells to erlotinib and cisplatin. It also resulted in re-sensitization of TGF-β1-induced A549 (A549M) cells as well the mesenchymal phenotypic H1299 cells to erlotinib and cisplatin treatment with concomitant up-regulation of cancer stem cell (CSC) markers (Sox2, Nanog and EpCAM) and down-regulation of miR-200 and let-7 family miRNAs. Ectopic up-regulation of miRNAs, especially miR-200b and let-7c, significantly diminished the erlotinib resistance of A549M cells. Inhibition of Hh signaling by GDC-0449 in EMT cells resulted in the attenuation of CSC markers and up-regulation of miR-200b and let-7c, leading to sensitization of EMT cells to drug treatment, thus, confirming a connection between Hh signaling, miRNAs and drug resistance.

Conclusions: We demonstrate that Hh pathway, through EMT-induction, leads to reduced sensitivity to EGFR-TKIs in NSCLCs. Therefore, targeting Hh pathway may lead to the reversal of EMT phenotype and improve the therapeutic efficacy of EGFR-TKIs in NSCLC patients.

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Modulation of CSC markers and miRNAs accompanies EMT of NSCLC cells. (A) A549M cells exhibit increased expression of CSC markers Sox2, Nanog and EpCAM and GDC-0449 inhibited such TGF-β-induced expression of CSC markers. TGF-β1-induced EMT also involved changes in the expression levels of (B) miR-200 family and (C) let-7 family of miRNAs. RNU6B and RNU48 were used as miRNA controls against which the data was normalized. *p<0.05 and **p<0.01.
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Figure 4: Modulation of CSC markers and miRNAs accompanies EMT of NSCLC cells. (A) A549M cells exhibit increased expression of CSC markers Sox2, Nanog and EpCAM and GDC-0449 inhibited such TGF-β-induced expression of CSC markers. TGF-β1-induced EMT also involved changes in the expression levels of (B) miR-200 family and (C) let-7 family of miRNAs. RNU6B and RNU48 were used as miRNA controls against which the data was normalized. *p<0.05 and **p<0.01.

Mentions: In order to fully understand the mechanism(s) of drug resistance that accompany induction of EMT in NSCLC cells, we investigated CSC markers (Sox2, Nanog and EpCAM) and the expression levels of several EMT-related miRNAs in parental A549 vs. mesenchymal A549M cells. A comparison of levels of CSC markers, by western blot analysis, revealed that the protein levels of CSC markers are elevated in vehicle-treated control A549M cells (Figure 4A). Since our earlier work has established a mechanistic role of Hh signaling in EMT of these cells, we treated A549M cells with GDC-0449 to inhibit Hh signaling and found that, compared to levels in vector-treated A549M cells, GDC-0449-treated cells had significantly reduced levels of CSCs (Figure 4A). As further molecular signature of mesenchymal A549M cells, we investigated some miRNAs that have been implicated in the EMT of cancer cells. We chose two families of miRNAs, the miR-200 and let-7 families, and our data revealed that several member miRNAs of these EMT-regulating miRNA families are down-regulated in the mesenchymal cells (Figure 4B-C). In particular miR-200b and let-7c miRNAs were found to be the most significantly down-regulated miRNAs from the two respective families. These results are consistent with the documented epithelial phenotype promoting role of these two miRNA families.


Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs.

Ahmad A, Maitah MY, Ginnebaugh KR, Li Y, Bao B, Gadgeel SM, Sarkar FH - J Hematol Oncol (2013)

Modulation of CSC markers and miRNAs accompanies EMT of NSCLC cells. (A) A549M cells exhibit increased expression of CSC markers Sox2, Nanog and EpCAM and GDC-0449 inhibited such TGF-β-induced expression of CSC markers. TGF-β1-induced EMT also involved changes in the expression levels of (B) miR-200 family and (C) let-7 family of miRNAs. RNU6B and RNU48 were used as miRNA controls against which the data was normalized. *p<0.05 and **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3852827&req=5

Figure 4: Modulation of CSC markers and miRNAs accompanies EMT of NSCLC cells. (A) A549M cells exhibit increased expression of CSC markers Sox2, Nanog and EpCAM and GDC-0449 inhibited such TGF-β-induced expression of CSC markers. TGF-β1-induced EMT also involved changes in the expression levels of (B) miR-200 family and (C) let-7 family of miRNAs. RNU6B and RNU48 were used as miRNA controls against which the data was normalized. *p<0.05 and **p<0.01.
Mentions: In order to fully understand the mechanism(s) of drug resistance that accompany induction of EMT in NSCLC cells, we investigated CSC markers (Sox2, Nanog and EpCAM) and the expression levels of several EMT-related miRNAs in parental A549 vs. mesenchymal A549M cells. A comparison of levels of CSC markers, by western blot analysis, revealed that the protein levels of CSC markers are elevated in vehicle-treated control A549M cells (Figure 4A). Since our earlier work has established a mechanistic role of Hh signaling in EMT of these cells, we treated A549M cells with GDC-0449 to inhibit Hh signaling and found that, compared to levels in vector-treated A549M cells, GDC-0449-treated cells had significantly reduced levels of CSCs (Figure 4A). As further molecular signature of mesenchymal A549M cells, we investigated some miRNAs that have been implicated in the EMT of cancer cells. We chose two families of miRNAs, the miR-200 and let-7 families, and our data revealed that several member miRNAs of these EMT-regulating miRNA families are down-regulated in the mesenchymal cells (Figure 4B-C). In particular miR-200b and let-7c miRNAs were found to be the most significantly down-regulated miRNAs from the two respective families. These results are consistent with the documented epithelial phenotype promoting role of these two miRNA families.

Bottom Line: Ectopic up-regulation of miRNAs, especially miR-200b and let-7c, significantly diminished the erlotinib resistance of A549M cells.Inhibition of Hh signaling by GDC-0449 in EMT cells resulted in the attenuation of CSC markers and up-regulation of miR-200b and let-7c, leading to sensitization of EMT cells to drug treatment, thus, confirming a connection between Hh signaling, miRNAs and drug resistance.We demonstrate that Hh pathway, through EMT-induction, leads to reduced sensitivity to EGFR-TKIs in NSCLCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, USA. fsarkar@med.wayne.edu.

ABSTRACT

Background: Epidermal growth factor receptor- tyrosine kinase inhibitors (EGFR-TKIs) benefit Non-small cell lung cancer (NSCLC) patients, and an EGFR-TKIi erlotinib, is approved for patients with recurrent NSCLC. However, resistance to erlotinib is a major clinical problem. Earlier we have demonstrated the role of Hedgehog (Hh) signaling in Epithelial-to-Mesenchymal transition (EMT) of NSCLC cells, leading to increased proliferation and invasion. Here, we investigated the role of Hh signaling in erlotinib resistance of TGF-β1-induced NSCLC cells that are reminiscent of EMT cells.

Methods: Hh signaling was inhibited by specific siRNA and by GDC-0449, a small molecule antagonist of G protein coupled receptor smoothened in the Hh pathway. Not all NSCLC patients are likely to benefit from EGFR-TKIs and, therefore, cisplatin was used to further demonstrate a role of inhibition of Hh signaling in sensitization of resistant EMT cells. Specific pre- and anti-miRNA preparations were used to study the mechanistic involvement of miRNAs in drug resistance mechanism.

Results: siRNA-mediated inhibition as well as pharmacological inhibition of Hh signaling abrogated resistance of NSCLC cells to erlotinib and cisplatin. It also resulted in re-sensitization of TGF-β1-induced A549 (A549M) cells as well the mesenchymal phenotypic H1299 cells to erlotinib and cisplatin treatment with concomitant up-regulation of cancer stem cell (CSC) markers (Sox2, Nanog and EpCAM) and down-regulation of miR-200 and let-7 family miRNAs. Ectopic up-regulation of miRNAs, especially miR-200b and let-7c, significantly diminished the erlotinib resistance of A549M cells. Inhibition of Hh signaling by GDC-0449 in EMT cells resulted in the attenuation of CSC markers and up-regulation of miR-200b and let-7c, leading to sensitization of EMT cells to drug treatment, thus, confirming a connection between Hh signaling, miRNAs and drug resistance.

Conclusions: We demonstrate that Hh pathway, through EMT-induction, leads to reduced sensitivity to EGFR-TKIs in NSCLCs. Therefore, targeting Hh pathway may lead to the reversal of EMT phenotype and improve the therapeutic efficacy of EGFR-TKIs in NSCLC patients.

Show MeSH
Related in: MedlinePlus