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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

Knock-down of Shh sensitizes A549M cells to standard therapies. Cell proliferation of A549M cells was significantly reduced after treatment with erlotinib (A) and cisplatin (B) following Shh knock-down. Cells were first treated with vehicle (A549M-control) or with specific si-RNA against Shh (A549M-siShh) for 48 hours and then with indicated concentrations of erlotinib/cisplatin for 24 hours. Parental A549 cells were included as a control to verify the induced resistance of A549M cells to erlotinib/cisplatin. All the plotted values are relative to vehicle-treated A549 cells. *p<0.05 and **p<0.01.
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Figure 2: Knock-down of Shh sensitizes A549M cells to standard therapies. Cell proliferation of A549M cells was significantly reduced after treatment with erlotinib (A) and cisplatin (B) following Shh knock-down. Cells were first treated with vehicle (A549M-control) or with specific si-RNA against Shh (A549M-siShh) for 48 hours and then with indicated concentrations of erlotinib/cisplatin for 24 hours. Parental A549 cells were included as a control to verify the induced resistance of A549M cells to erlotinib/cisplatin. All the plotted values are relative to vehicle-treated A549 cells. *p<0.05 and **p<0.01.

Mentions: Next, we evaluated whether Hedgehog (Hh) inhibition can sensitize A549M cells to erlotinib or cisplatin. We first used siRNA approach and inhibited Shh, a ligand of the Hh pathway to test whether the knock-down of Shh sensitizes A549M cells to erlotinib and cisplatin. A549M cells were transfected with Shh-specific siRNA, control cells were transfected with scrambled siRNA and the cells were treated with erlotinib or cisplatin. Additionally, parental A549 cells were included in the experiment to confirm comparatively increased resistance of A549M cells to erlotinib and cisplatin. As previously shown [3], siRNA against Shh was found to significantly down-regulate the expression of Shh. A549M cells with Shh knock-down showed significant reduction in cell proliferation (p<0.05) when treated with erlotinib (Figure 2A) and cisplatin (Figure 2B). To confirm the impact of inhibition of Hh signaling on drug resistance, we treated A549M cells with pharmacological inhibitor GDC-0449 for 72 h, followed by treatment with erlotinib or cisplatin, and the cell viability was assessed after 72 h of treatment. A549M cells were more resistant to erlotinib and cisplatin, compared to parental A549 cells, and A549M cells treated with GDC-0449 showed reduced cell proliferation (Table 1), as evidenced by lower IC50 of both the drugs in the cells pre-treated with GDC-0449. This suggests that Hh inhibitor GDC-0449 sensitizes mesenchymal phenotypic cells to standard therapy. The results of GDC-0449 sensitization of A549M cells, at a few selected doses of erlotinib (Figure 3A) and cisplatin (Figure 3B) clearly show that the A549M cells pre-treated with GDC-0449 are more sensitive to the drugs. It is interesting to note that GDC-0449 was not able to sensitize the parental A549 cells (Figure 3A-B),which may be due to the fact that the parental cells do not express appreciable levels of Shh.


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)

Knock-down of Shh sensitizes A549M cells to standard therapies. Cell proliferation of A549M cells was significantly reduced after treatment with erlotinib (A) and cisplatin (B) following Shh knock-down. Cells were first treated with vehicle (A549M-control) or with specific si-RNA against Shh (A549M-siShh) for 48 hours and then with indicated concentrations of erlotinib/cisplatin for 24 hours. Parental A549 cells were included as a control to verify the induced resistance of A549M cells to erlotinib/cisplatin. All the plotted values are relative to vehicle-treated A549 cells. *p<0.05 and **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Knock-down of Shh sensitizes A549M cells to standard therapies. Cell proliferation of A549M cells was significantly reduced after treatment with erlotinib (A) and cisplatin (B) following Shh knock-down. Cells were first treated with vehicle (A549M-control) or with specific si-RNA against Shh (A549M-siShh) for 48 hours and then with indicated concentrations of erlotinib/cisplatin for 24 hours. Parental A549 cells were included as a control to verify the induced resistance of A549M cells to erlotinib/cisplatin. All the plotted values are relative to vehicle-treated A549 cells. *p<0.05 and **p<0.01.
Mentions: Next, we evaluated whether Hedgehog (Hh) inhibition can sensitize A549M cells to erlotinib or cisplatin. We first used siRNA approach and inhibited Shh, a ligand of the Hh pathway to test whether the knock-down of Shh sensitizes A549M cells to erlotinib and cisplatin. A549M cells were transfected with Shh-specific siRNA, control cells were transfected with scrambled siRNA and the cells were treated with erlotinib or cisplatin. Additionally, parental A549 cells were included in the experiment to confirm comparatively increased resistance of A549M cells to erlotinib and cisplatin. As previously shown [3], siRNA against Shh was found to significantly down-regulate the expression of Shh. A549M cells with Shh knock-down showed significant reduction in cell proliferation (p<0.05) when treated with erlotinib (Figure 2A) and cisplatin (Figure 2B). To confirm the impact of inhibition of Hh signaling on drug resistance, we treated A549M cells with pharmacological inhibitor GDC-0449 for 72 h, followed by treatment with erlotinib or cisplatin, and the cell viability was assessed after 72 h of treatment. A549M cells were more resistant to erlotinib and cisplatin, compared to parental A549 cells, and A549M cells treated with GDC-0449 showed reduced cell proliferation (Table 1), as evidenced by lower IC50 of both the drugs in the cells pre-treated with GDC-0449. This suggests that Hh inhibitor GDC-0449 sensitizes mesenchymal phenotypic cells to standard therapy. The results of GDC-0449 sensitization of A549M cells, at a few selected doses of erlotinib (Figure 3A) and cisplatin (Figure 3B) clearly show that the A549M cells pre-treated with GDC-0449 are more sensitive to the drugs. It is interesting to note that GDC-0449 was not able to sensitize the parental A549 cells (Figure 3A-B),which may be due to the fact that the parental cells do not express appreciable levels of Shh.

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