Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs
© Ahmad et al.; licensee BioMed Central Ltd. 2013
Received: 17 July 2013
Accepted: 30 September 2013
Published: 7 October 2013
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.
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.
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.
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.
KeywordsNSCLC Erlotinib resistance Hh signaling miRNAs EMT GDC-0449
Lung cancer is the most common cause of cancer related mortality in the United States . The primary reason for this poor outcome in non-small cell lung cancer (NSCLC) patients is the presence of systemic metastases at diagnosis in a high proportion patients . Recent studies have shown that the cellular program of epithelial-to-mesencymal transition (EMT) phenotypic cells, involved in embryogenesis, is a critical step in the development of metastases. EMT is characterized by a switch from an epithelial phenotype of polarized cells with expression of epithelial markers such as E-cadherin to a mesenchymal phenotype of cells that lack polarity, are motile and have down regulation of E-cadherin. Another important characteristic of EMT cancer cells is resistance to existing cytotoxic and targeted agents, including EGFR-TKI, erlotinib. Recent data suggests that cancer cells with EMT phenotype also demonstrate stem cell like features. Pre-clinical studies suggest that measures to reverse EMT can enhance the therapeutic efficacy of erlotinib and other drugs.
The hedgehog (Hh) signaling pathway is a crucial mediator of normal organ development during embryogenesis and tissue repair during wound healing, specifically in the lung tissue. Hh pathway regulates these processes through the induction of EMT. Re-activation of the Hh pathway with induction of EMT is increasingly being implicated in carcinogenesis of many cancers. In addition, pre-clinical studies show that the inhibition of Hh pathway can reverse EMT, which in turn is associated with enhanced tumor sensitivity to cytotoxic agents. Several investigators have shown that the Hh pathway is activated in many NSCLCs. We have earlier shown that chronic exposure to TGF-β induces EMT in a NSCLC cell line A549 leading to A549 cells with greater mesenchymal features (A549M cells) . Induction of EMT in these cells was associated with activation of the Hh pathway. With the knowledge that EMT is connected to drug resistance and our own observation that Hh signaling is involved in the regulation of EMT, we questioned whether inhibition of Hh signaling can reverse the drug resistance of NSCLC cells. In our current investigation, we investigated the effect of silencing of Hh signaling, using siRNA as well as pharmacological inhibitor GDC-0449, on drug sensitivity of NSCLC cells. GDC-0449 (vismodegib) is a Hh pathway inhibitor which was approved recently for the use in patients with basal cell carcinoma of the skin, a tumor type that has activating mutations in the Hh pathway. Here we report a novel role of Hh signaling in drug resistance phenotype of NSCLC cells which mechanistically involves the regulation of EMT-related microRNAs (miRNAs).
Materials and methods
Cell lines and reagents
The human lung adenocarcinoma cell lines A549 and H1299 were purchased from the American Type Culture Collection (Manassas, VA) and maintained according to the American Type Culture Collection’s instructions. All cells were cultured in 5% CO2–humidified atmosphere at 37°C. The cell lines have been tested and authenticated through the core facility (Applied Genomics Technology Center at Wayne State University) by short tandem repeat profiling using the PowerPlex 16 System from Promega. A549 cells were treated with TGF-β1 (5 ng/ml) for 21 days to generate A549M cells (EMT phenotypic cells). Cells were treated with 20 nM GDC-0449 (Genentech) for 72 hours, before conducting individual assays. Antibodies were purchased from following sources – Sox2, Nanog, EpCAM (Cell Signaling Technology, Beverly, MA) and β-actin (Sigma-Aldrich, St Louis, MO).
Small interfering RNA (siRNA) transfection
Small interfering RNA (siRNA) specific for Hh (Shh; SHH Stealth RNAiTM siRNA) was purchased from Invitrogen. As a nonspecific control siRNA, scrambled siRNA duplex (Invitrogen) was used. Transfection was done using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) following the manufacturer’s instruction. Shh was silenced by siRNA for 48 hours prior to assay or treatment. Experiment was repeated, at least, three times independently and representative data is reported.
Cells were seeded at 2.5 × 105 cells per well in six well plates and transfected with appropriate anti-miRs/pre-miRs or miRNA-negative controls at a final concentration of 200 nM for each individual miRNA (Ambion) using DharmaFECT1 transfection reagent (Dharmacon). After 2 days of transfection, cells were split and transfected twice again before the use of these cells for specified experiments.
Cell growth inhibition studies by MTT assay
Cells were seeded at 5 × 103 cells per 100 μl of culture medium per well in 96-well plates. The number of viable cells was assessed in six wells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma). Cells were treated with Hh inhibitor (GDC-0449) for 72 hours, or with siRNA specific for Shh for 48 hours. Next, cells were treated with either Cisplatin or Erlotinib, at the specified concentrations. Control cells received equivalent amount of vehicle (0.1% DMSO) in culture medium. After treatment, cells were incubated with MTT reagent (0.5mg/ml; Sigma) at 37°C for 2 h and then with isopropanol at room temperature for 1 h. Spectrophotometric absorbance of the samples was determined by an Ultra-26 Multifunctional Microplate Reader (Tecan, Durham, NC). Cell proliferation index was calculated by assigning the vehicle-treated control cells a numerical value of 100. All the treatments, in individual experiments, therefore, represent fraction of viable proliferating cells, relative to vehicle-treated respective controls. Results were plotted as means ± SD of three separate experiments having six determinations per experiment for each experimental condition.
For miRNA analysis, total RNA was isolated using the mirVana miRNA isolation kit (Ambion). The levels of miRNAs were determined using miRNA-specific Taqman MGB probes from the Taqman MicroRNA Assay (Applied Biosystems), as described previously . The relative amounts of miRNA were normalized to internal miRNA controls RNU6B and RNU48.
Western blot analysis
For Western blot analysis, cells were lysed in RIPA buffer containing complete mini EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich, St. Louis, MO) . After resolution on 12% polyacrylamide gels under denaturing conditions, proteins were transferred to nitrocellulose membranes, incubated with appropriate primary / horseradish peroxidase-conjugated secondary antibodies and visualized using chemiluminescence detection system (Pierce, Rockford, IL).
The experimental results presented in the figures are representative of three or more independent observations. The data are presented as the mean values ± SE. Values of p < 0.05 and lower were considered to be statistically significant.
Cells with mesenchymal phenotype (A549M) are more resistant to EGFR-TKI erlotinib and cisplatin, compared to parental A549 cells
Inhibition of hedgehog signaling sensitizes mesenchymal A549M cells to erlotinib and cisplatin
GDC-0449 lowers the IC-50 of erlotinib/cisplatin in A549M / H1299 cells
% Decrease in IC50
GDC-0449 sensitizes H1299 cells to erlotinib/cisplatin
Erlotinib (A)10 μM
GDC (B)20 nM
Erlotinib + GDC [Expected (A+B)]
Erlotinib + GDC [Observed]
48.00 ± 1.8
12.81 ± 0.7
60.81 ± 1.9
68.60 ± 1.1
Cisplatin (C)9.9 μM
GDC (B)20 nM
Cisplatin + GDC [Expected (C+B)]
Cisplatin + GDC [Observed]
46.14 ± 3.1
12.81 ± 0.7
58.95 ± 2.8
71.93 ± 2.4
TGF-β1-induced EMT of NSCLC cells involves modulation of Cancer Stem Cells (CSCs) and miRNAs
Re-expression of selected miRNAs can reverse TGF-β1-induced drug resistance
miRNAs that reverse TGF-β1-induced drug resistance also play a role in GDC-0449’s inhibition of erlotinib resistance
The major findings of our study are – a) TGF-β1-induced EMT of NSCLC cells leads to increased resistance to both erlotinib and cisplatin; b) Hh signaling seems to play a role in such EMT-induced drug resistance because siRNA-mediated as well as pharmacological down-regulation of Hh signaling inhibits resistance to both the drugs and c) EMT regulating miRNAs such as miR-200b and let-7c are mechanistically involved in Hh signaling- and EMT-mediated resistance of NSCLC cells to TKI erlotinib.
Resistance to currently available anti-cancer drugs such as erlotinib represents a major clinical challenge . A number of investigational tools such as gene expression, xenograft assays and proteomic profiling techniques have been used to find biomarkers associated with sensitivity to erlotinib in a panel of sensitive and insensitive NSCLC cell lines [9, 13, 14]. These studies reported that sensitive cell lines express well-established epithelial markers E-cadherin and catenin as well as exhibit the typical cobblestone epithelial morphology and tight cell–cell junctions of epithelial cells. On the other hand, insensitive cell lines express characteristics of mesenchymal cells, including the expression of vimentin, fibronectin, and ZEB1, consistent with more fibroblastic scattered morphology. In summary, these results are suggestive of the process of EMT as an indicator of sensitivity to EGFR inhibitors. Moreover, a study by Prudkin et al. has indicated an association with these EMT markers in the sequential pathogenesis of squamous cell carcinoma , suggesting that the combination of EGFR-TKI with the inhibitor of EMT-inducing-molecules could become a novel approach toward the treatment of lung cancer, especially for NSCLC. The hedgehog (Hh) signaling pathway is involved in embryogenesis particularly in the development of the lungs. This pathway is not active in adult tissues but it can be activated in many cancers including NSCLC [16–19]. In addition, blocking Hh signaling inhibits the growth, invasion and metastasis of cancer cells, which is associated with the down-regulation of Snail and up-regulation of E-cadherin. Also, over-expression of GLI1, the effector molecule of the Hh signaling pathway, in epithelial cells, leads to an aggressive phenotype with down-regulation of E-cadherin [20, 21]. All of this evidence suggests a connection between Hh signaling and EMT which can potentially be exploited for therapy.
Based on the available literature discussed above, there seems to be a correlation between EMT, drug resistance and Hh signaling but the mechanistic details of this inter-relationship is not clearly understood. We have previously shown that there is a transcriptional up-regulation of Shh by TGF-β1 as a key step during the induction of EMT in NSCLC cell line . As the next step, we now provide evidence in support of the role for Hh signaling pathway in drug resistance phenotype of NSCLC cells that accompanies the processes of EMT. Our results show an increase in resistance to drugs when EMT is induced in NSCLC cells that are chronically exposed to TGF-β1. Resistance was enhanced to both cisplatin and erlotinib. A similar response of EMT cells to these two different drugs suggests a broader role of EMT in drug resistance that might not be confined to any particular class of anti-cancer drugs.
With the increased resistance of EMT cells to drugs, reversal of EMT for the re-sensitization of such cells is very intuitive. The challenge, however, lies in the elucidation of the regulation of EMT which can potentially help identify novel targets for therapy and reversal of EMT. Taking a cue from our previous work, we investigated Hh signaling in relation to EMT-induced drug resistance. As a proof-of-principle, we inhibited Shh by siRNA in NSCLC cells that had undergone EMT, and this resulted in re-sensitization of NSCLC cells to erlotinib and cisplatin. To make our results clinically relevant, we used a pharmacological inhibitor of Hh signaling, GDC-0449, and obtained very similar results. These results clearly demonstrate the relevance of inhibition of Hh signaling for reversal of EMT and overcoming drug resistance. In addition to the TGF-β1-induced EMT as a model, we confirmed our results in H1299 cells that have a dominant mesenchymal phenotype and also exhibit elevated levels of Shh. Re-sensitization of H1299 cells to erlotinib and cisplatin was observed after treatment with GDC-0449 further supports our hypothesis that reversal of EMT through down-regulation of Hh signaling is an effective strategy to overcome drug resistant phenotype.
Since acquired resistance to conventional therapies is a major clinical concern, re-sensitization of tumors offers a viable alternative in the absence of novel therapeutic options. Different ‘sensitizing’ agents have been investigated for their ability to reverse drug resistance [22–25]. Of interest, re-sensitization to erlotinib [26–28] as well as cisplatin [24, 29] has been demonstrated. In a recent study , miR-98 has been shown to sensitize cisplatin-resistant human lung adenocarcinoma cells. The miR-98 belongs to let-7 family of miRNAs and was down-regulated in resistant cells. These results are in agreement with our own observations where we found reduced levels of let-7 family members in erlotinib and cisplatin resistant cells. In a very recent report , the role of let-7c in determining docetaxel resistance in lung cancer model has been described. This further provides evidence in support of the role of miRNAs, particularly let-7c in a broader drug resistance phenotype with functional implications, and these results are consistent with our findings using a different class of drugs. In addition to let-7 family, we observed down-regulation of miR-200 family and, collectively, this underlines a role of EMT-regulating miRNAs in erlotinib/cisplatin resistance. In experiments involving combination of agents/drugs, a distinction between additive vs. sensitization effects is always a concern. The combined effects of Hh inhibition and erlotinib/cisplatin were found to be significantly more than the individual or simple additive effects, which is reminiscent of sensitization. Furthermore, pre-treatment of resistant A549M cells with GDC-0449 significantly lowered the IC50 values of erlotinib and cisplatin, almost to the levels of sensitive parental A549 cells, which further confirms re-sensitization.
We observed increased expression of CSC markers and modulation of miRNAs (miR-200s and let7s) in NSCLC cells with TGF-β1-induced EMT. The role of CSCs in drug resistance of lung cancer cells has been demonstrated [31, 32]. Our results showed a significant down-regulation of CSC markers Sox2, Nanog and EpCAM upon inhibition of Hh signaling in A549-M cells by GDC-0449, which provided direct evidence in support of the connection between Hh signaling and CSCs in a model system with induced EMT. Further, miR-200 and let-7 families of miRNAs are well known inhibitors of EMT [4, 33, 34] and the data on growth, invasion and metastasis of lung cancer cells [10, 35–37] fully supports their established biological activity. As expected, we observed down-regulation of these miRNAs in TGF-β1-treated cells (A549M cells). Re-expression of these miRNAs, especially re-expression of the most down-regulated miRNAs, miR-200b and let-7c, inhibited the TGF-β1-mediated resistance of NSCLC cells to erlotinib. Interestingly, we observed a direct induction of these two-miRNAs by Hh inhibitor GDC-0449 treatment. Additionally, re-expression of these two miRNAs significantly reversed EMT markers. This could explain the observed inhibition of TGF-β1-induced effects by GDC-0449. It appears that TGF-β1 mediated induction of EMT is in part mediated by down-regulation of miR-200 and let-7 family miRNAs and contributes to drug resistance. The ability of GDC-0449 to maintain the levels, through direct up-regulation of these miRNAs, abrogates the TGF-β1-induced EMT, resulting in drug resistance. It is also interesting to note that the modulation of multiple members of the same miRNA family, either miR-200 family or the let-7 family, did influence the TGF-β1/GDC-0449 effects but not to the same extent as the combination of miR-200b and let-7c. This can probably be explained by the fact that multiple members of the same miRNA family have overlapping target genes and concurrently targeting miRNAs from different families can be more effective through their combined effects on wide range of mutually exclusive targets.
In summary, our present studies established a mechanistic role of Hh signaling in EMT-associated drug resistance phenotype of NSCLC cells which is mediated through novel regulation of CSCs and the EMT. Therefore, the inhibition of Hh signaling could be a useful approach for reducing tumor aggressiveness in NSCLC, and as such, the reversal of EMT, particularly through modulation of miRNAs, could also be useful for re-sensitization of drug-resistant NSCLC cells to conventional therapeutics, which would likely contribute to improved survival of patients who rightfully deserve better treatment outcomes.
Cancer stem cells
Epidermal growth factor receptor
Non-small cell lung cancer
Tyrosine kinase inhibitor
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