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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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Related in: MedlinePlus

TGF-β1-induced EMT results in drug resistance phenotype. Dose–response curves shows that A549M cells exhibit increased cell viability, after treatment with erlotinib (A) and cisplatin (B), compared to A549 cells. Cells were treated with indicated concentrations of erlotinib/cisplatin for 72 hours and then subjected to MTT assay. The IC50 and IC90 values for different conditions are provided in the table within the individual figures. ND: IC90 could not be determined. *p<0.05.
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Figure 1: TGF-β1-induced EMT results in drug resistance phenotype. Dose–response curves shows that A549M cells exhibit increased cell viability, after treatment with erlotinib (A) and cisplatin (B), compared to A549 cells. Cells were treated with indicated concentrations of erlotinib/cisplatin for 72 hours and then subjected to MTT assay. The IC50 and IC90 values for different conditions are provided in the table within the individual figures. ND: IC90 could not be determined. *p<0.05.

Mentions: EMT phenotypic cancer cells have been shown to acquire drug resistance [5-8]. Our earlier data established that A549 cells with mesenchymal phenotype (A549M cells) acquire invasiveness in vitro as well as in vivo[3], and, therefore, we started our current investigation with the hypothesis that A549M cells should be more resistant to therapeutic drugs because of their mesenchymal phenotype. To test this hypothesis, we treated A549 and A549M cells with increasing doses of erlotinib and cisplatin for 72 h, and measured cell viability. We found significantly higher number of proliferating A549M cells than A549 cells (p<0.05) at all the tested doses of erlotinib (Figure 1A) as well as cisplatin (Figure 1B), suggesting that A549M cells are indeed more resistant to erlotinib or cisplatin, consistent with the EMT phenotype. The IC50 values as well as the IC90 values for A549M cells were significantly higher for erlotinib (Figure 1A) and cisplatin (Figure 1B), further confirming their drug resistance characteristics.


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)

TGF-β1-induced EMT results in drug resistance phenotype. Dose–response curves shows that A549M cells exhibit increased cell viability, after treatment with erlotinib (A) and cisplatin (B), compared to A549 cells. Cells were treated with indicated concentrations of erlotinib/cisplatin for 72 hours and then subjected to MTT assay. The IC50 and IC90 values for different conditions are provided in the table within the individual figures. ND: IC90 could not be determined. *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852827&req=5

Figure 1: TGF-β1-induced EMT results in drug resistance phenotype. Dose–response curves shows that A549M cells exhibit increased cell viability, after treatment with erlotinib (A) and cisplatin (B), compared to A549 cells. Cells were treated with indicated concentrations of erlotinib/cisplatin for 72 hours and then subjected to MTT assay. The IC50 and IC90 values for different conditions are provided in the table within the individual figures. ND: IC90 could not be determined. *p<0.05.
Mentions: EMT phenotypic cancer cells have been shown to acquire drug resistance [5-8]. Our earlier data established that A549 cells with mesenchymal phenotype (A549M cells) acquire invasiveness in vitro as well as in vivo[3], and, therefore, we started our current investigation with the hypothesis that A549M cells should be more resistant to therapeutic drugs because of their mesenchymal phenotype. To test this hypothesis, we treated A549 and A549M cells with increasing doses of erlotinib and cisplatin for 72 h, and measured cell viability. We found significantly higher number of proliferating A549M cells than A549 cells (p<0.05) at all the tested doses of erlotinib (Figure 1A) as well as cisplatin (Figure 1B), suggesting that A549M cells are indeed more resistant to erlotinib or cisplatin, consistent with the EMT phenotype. The IC50 values as well as the IC90 values for A549M cells were significantly higher for erlotinib (Figure 1A) and cisplatin (Figure 1B), further confirming their drug resistance characteristics.

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