Abstract
Rationale: Non–small cell lung cancers carrying epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), but patients ultimately develop drug resistance and relapse. Although epithelial-mesenchymal transition (EMT) can predict resistance to EGFR TKIs, the molecular mechanisms are still unknown.
Objectives: To examine the role of EMT regulators in resistance to gefitinib.
Methods: The expression level of EMT regulators in gefitinib-sensitive cells (PC9) and gefitinib-resistant cells (PC9/gef) was determined using quantitative real-time reverse transcription–polymerase chain reaction and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of EMT regulators on gefitinib resistance in vitro, and a xenograft mouse model was used for in vivo confirmation. In addition, cancer cells from 44 patients with malignant pleural effusions of lung adenocarcinoma were collected for analysis of EMT regulator mRNA by quantitative real-time reverse transcription–polymerase chain reaction.
Measurements and Main Results: Slug expression, but not that of snail, twist, or zeb-1, was significantly increased in PC9/gef compared with PC9 cells. Slug knockdown in PC9/gef cells reversed resistance to gefitinib, and overexpression of Slug in PC9 cells protected cells from gefitinib-induced apoptosis. Silencing of Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis primarily through Bim up-regulation and activation of caspase-9. Slug enhanced tumor growth in a xenograft mouse model, even with gefitinib treatment. In clinical samples, Slug expression was significantly higher in cancer cells with resistance to EGFR TKIs than in treatment-naive cancer cells.
Conclusions: Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.
Molecular-targeting therapeutics, such as EGFR tyrosine kinase inhibitors, have become an important part of lung cancer treatment strategies. One challenge confronted by targeted cancer therapies is the potential for drug resistance development.
Silencing Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis, a process that was mediated mainly through restoration of Bim expression and caspase-9 activation.
Altered expression of the epidermal growth factor receptor (EGFR) has been identified in a variety of human tumors, including lung, breast, head and neck, and ovarian cancers (3, 4). Activated EGFR has been reported to promote cell survival, proliferation, invasion, and metastasis through activation of JAK/STAT (Janus kinase/signal transducers and activators of transcription), PI3K (phosphoinositol 3-kinase)/Akt, and MAPK (mitogen-activated protein kinase) pathways (4, 5). These observations have established EGFR as a target for cancer therapy and have led to the development of the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Recent research has indicated that patients with non–small cell lung cancer (NSCLC) with EGFR-activating mutations exhibit a dramatic clinical response to EGFR TKIs (6, 7). In clinical practice, Asians, females, nonsmokers, and patients with adenocarcinoma respond preferentially to EGFR TKIs (8). These are also the patient groups with a high rate of EGFR mutations. Despite the dramatic initial responses to such inhibitors, most patients ultimately develop drug resistance and relapse.
Most activating mutations in the EGFR gene result in a single substitution of arginine for leucine at position 858 (L858R) and 15-nucleotide deletion (del_E746-A750) in exon 19 (9). Several clinical studies have shown that a second-site point mutation at position 790 (T790M) is responsible for approximately half of all cases of patients with lung adenocarcinoma who develop resistance to EGFR TKIs (8, 10). Amplification of the protooncogene MET (encoding the hepatocyte growth factor receptor) also contributes to EGFR TKI resistance and is detected in about 20% of these patients (11). The possibility that other mechanisms may also be involved in EGFR TKI resistance cannot be excluded.
Epithelial–mesenchymal transition (EMT), a process by which epithelial cells lose cell polarity and convert to a mesenchymal phenotype, is essential for embryonic development, cancer progression, and chemotherapy resistance (12–14). EMT plays an important role in resistance to EGFR TKIs, during which cancer cells lose epithelial markers, such as E-cadherin (15–17). In contrast, strong expression of E-cadherin enhances gefitinib sensitivity in NSCLC cells with a mesenchymal phenotype (17). Although EMT can predict resistance to gefitinib or erlotinib (15, 16, 18), the molecular mechanisms are still unknown.
Transcriptional reprogramming via the transcription factors Slug, snail, zeb-1, and twist can lead to EMT. These EMT regulators can repress the expression of E-cadherin and have been reported to affect sensitivity to chemotherapeutic drugs (19, 20). However, the mechanism of action of EMT regulators with respect to resistance to anticancer drugs, especially EGFR TKIs, is not well defined at present. Here, we found that the EMT regulator Slug contributes to the development of resistance to gefitinib in lung adenocarcinomas containing EGFR-activating mutations. Therefore, inhibition of Slug may be an effective strategy for more successful treatment of gefitinib-resistant lung cancers. Some of the results of these studies have been reported in the form of an abstract (21).
The human lung adenocarcinoma cell line PC9 and derivative PC9/gef clones were gifts from Dr. C. H. Yang (Graduate Institute of Oncology, Cancer Research Center, National Taiwan University). HCC827 was purchased from the American Type Culture Collection (Manassas, VA). Slug was silenced in PC9/gef cells using small interfering RNA (siRNA) duplexes targeting human Slug (SNAI2), synthesized by Ambion (Austin, TX). Slug overexpression in PC9 cells was accomplished by infecting cells with lentiviruses containing the entire human Slug coding region, prepared using the ViraPower Lentiviral Expression System (Invitrogen, Carlsbad, CA), as described by the manufacturer. A stock solution of gefitinib, a gift from AstraZeneca (London, UK), was prepared in dimethyl sulfoxide and stored at −20°C.
EGFR mutations were detected by MALDI-TOF MS according to the user manual guidelines for the MassARRAY system (SEQUENOM, San Diego, CA) (22).
The colorimetric MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit; Promega, Madison, WI) was used to determine the number of viable cells. The absorbance at 490 nm was recorded using a VICTOR3 multilabel reader (PerkinElmer, Waltham, MA).
Cells grown on chamber slides (BD Bioscience, San Diego, CA) were fixed in 4% paraformaldehyde for 15 minutes. Fixed cells were incubated in blocking solution (0.1% saponin and 0.2% BSA in phosphate-buffered saline [PBS]) to prevent nonspecific staining. The cells were then incubated with primary antibodies for 1 hour and then incubated with fluorescence-conjugated secondary antibodies for 30 minutes. After immunostaining, the chamber slides were stained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, Louis, MO).
Apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit (BD Bioscience). Cells were trypsinized and washed twice with ice-cold PBS. The cell pellet was resuspended in Annexin V binding buffer. Fluorescein isothiocyanate–conjugated Annexin V (1 μg/ml) and propidium iodide (50 μg/ml) were added to the cells and incubated for 15 minutes at room temperature. Cells were analyzed using a Cytomic FC500 flow cytometer (Beckman Coulter, Brea, CA). The activity of caspase-9 was measured with a luminescence kit (caspase-Glo 9 assays; Promega).
Animal experiments were approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taiwan. Four- to 6-week-old nu/nu athymic male mice were obtained from the Laboratory Animal Center of National Applied Research Laboratories (Taipei, Taiwan). Cells were injected subcutaneously into the lower rear flank of the mice. When tumor volumes reached approximately 200 mm3, as measured by calipers, mice were randomly allocated into groups of six animals to receive gefitinib (10 mg/kg/d) or vehicle by oral gavage. All mice were sacrificed on Day 20 after their tumors had been measured.
This study was approved by the Institutional review board (IRB) of National Taiwan University Hospital. Between July 2009 and March 2010, we consecutively collected the pleural effusions from patients who received thoracentesis in the chest ultrasonography examination room of the National Taiwan University Hospital. The pleural fluids of patients were acquired aseptically in vacuum bottles by thoracentesis. The red blood cells in the specimen were hemolyzed using RBC lysis buffer. The remaining cells were washed twice with PBS and cultured in complete RPMI 1640 media, as described previously (23). Media were replaced every 2 to 3 days and cells were harvested after 10 days. Total RNA was extracted from cultured cells using the TRIzol reagent (Invitrogen), and the expression of Slug mRNA was determined using quantitative reverse transcriptase–polymerase chain reaction (RT-PCR), as previously described (24).
Student t test was used to compare the means of two groups. Two-sided P values less than 0.05 were considered significant. All analyses were performed using SPSS software (version 15.0 for Windows; SPSS Inc.) Additional details of measurement techniques are available in an online supplement.
PC9 cells expressing a mutant EGFR with a deletion in exon 19 are a gefitinib-sensitive NSCLC cell line (25). PC9/gef cells were selected from parental PC9 cells that had been continuously exposed to increasing concentrations of gefitinib (Dr. C. H. Yang, personal communication). PC9 and PC9/gef cells were exposed to gefitinib, and cell viability was evaluated using an MTS assay. The viability of PC9/gef cells was unaffected by increasing concentrations of gefitinib up to 5 μM (IC50 > 5 μM), whereas PC9 cells were very sensitive to gefitinib, with an IC50 value of 0.041 μM (Figure 1A). A T790M mutant of EGFR and MET amplification are the known mechanisms of acquired gefitinib resistance in lung cancer. We analyzed EGFR for the DNA substitution corresponding to the T790M mutation by MALDI-TOF MS genotypic analysis and direct sequencing. As shown in Figure 1B and Figure E1 in the online supplement, we found no evidence for this mutation in parental PC9 cells, PC9/gef cells, or PC9/gef subclones (PC9/gef B4, PC9/gef C2, PC9/gef C4, PC9/gef C7). Quantification of MET gene copy number by real-time PCR showed that no MET amplification occurred in gefitinib-resistant PC9/gef or subclones (Figure 1C). These results suggest that other mechanisms are responsible for resistance to gefitinib in PC9/gef cells.
Figure 1. PC9/gef was resistant to gefitinib, but there was no T790M mutation or MET amplification. (A) PC9 and PC9/gef cells were exposed to various doses of gefitinib for 5 days and assayed for survival by MTS assay. PC9 was sensitive to gefitinib treatment. PC9/gef was resistant to gefitinib. For each dose, six repeat wells were treated. The curves are the mean survival data for three independent experiments. Error bars show the standard deviations (***P < 0.001). (B) Epidermal growth factor receptor (EGFR) T790M (C to T alteration in 2,369th nucleotide) detection by high sensitive MALDI-TOF nucleotide mass spectrometry. Shifted signals due to incorporated nucleotides (C or T) of unextended detection probes (U) after single nucleotide extension reaction can be identified in the spectrum. There was no T790M mutation (T) detected in PC9, PC9/gef, and its subclones (PC9/gef B4, C2, C4, and C7). H1975, lung adenocarcinoma cell line harboring mutant EGFR L858R and T790M, is a positive control. H1650, lung adenocarcinoma cell line harboring exon 19 deletion, is a negative control. The results shown are representative of three independent experiments. (C) The copy number of the MET gene was evaluated by real-time quantitative polymerase chain reaction of genomic DNA. No MET amplifications were noted in PC9/gef and its subclones. The value represents the mean of triplicate for each subclone. The experiment was repeated three times with similar results.
[More] [Minimize]To determine whether EMT contributes to gefitinib resistance in mutant EGFR-expressing lung cancer cells, we analyzed the expression of the epithelial marker E-cadherin and the mesenchymal marker vimentin by Western blotting and immunofluorescence staining. As shown in Figure 2A, the expression of E-cadherin was decreased in PC9/gef cells compared with that in PC9 cells; in contrast, vimentin was increased in PC9/gef cells. Furthermore, the invasivity of PC9/gef cells was increased relative to that of PC9 cells (Figure 2B). These results indicate that gefitinib-resistant PC9/gef cancer cells had undergone EMT. The transcription factors Slug, snail (SNAI1), zeb-1, and twist can induce EMT (26, 27). To verify that resistance to gefitinib was associated with EMT-related transcription factors, we explored the expression levels of snail, Slug, twist, and zeb-1 by Western blotting and quantitative real-time RT-PCR. As shown in Figure 2C, the protein expression of Slug, but not that of snail, twist, or zeb-1, was markedly increased in gefitinib-resistant PC9/gef cells. The expression of Slug mRNA in PC9/gef clones was clearly higher than in PC9 cells, and the expression levels of snail, twist, and zeb-1 were not increased in gefitinib-resistance subclones (Figure 2D). Taken together, these data indicate that Slug expression may play a crucial role in gefitinib resistance in this cell model.
Figure 2. The epithelial-mesenchymal transition (EMT) regulator Slug played a role in gefitinib resistance. (A) The expression of E-cadherin, vimentin, and α-tubulin was evaluated by Western blot (left). Immunofluorescence staining of vimentin, E-cadherin (green), and propidium iodide (blue) was analyzed by confocal microscopy (right). The results shown are representative of three independent experiments. (B) Matrigel invasion assay of PC9 and PC9/gef cells. Columns, number of cells invaded across the membrane. Data represent mean ± SD of three independent experiments. (**P < 0.01). (C) The expression of EMT-related regulators was evaluated by Western blot. (D) The expression of mRNA of Slug, snail, twist, and zeb-1 was evaluated by quantitative real-time RT-PCR. Data represent mean ± SD of three independent experiments (***P < 0.001).
[More] [Minimize]We evaluated the effect of Slug on gefitinib resistance in PC9/gef cells by siRNA-mediated knockdown of Slug. First, we established which siRNA most efficiently knocked down Slug expression in CL1–5 cells, which highly express Slug (Figure 3A), selecting Slug siRNA 3 (si-Slug 3) over other Slug siRNAs (si-Slug 1, si-Slug 2). Treatment of PC9/gef cells with 50 nM si-Slug 3 (control) for 48 hours reduced the levels of Slug and vimentin compared with that in cells treated with scrambled siRNA (si-scramble) but increased E-cadherin levels (Figures 3B and 3C). Transfection of PC9/gef cells with si-Slug 3 enhanced gefitinib-induced apoptosis, increasing the percentage of apoptotic cells from 12 to 30% (Figure 3D). Knockdown of Slug expression in PC9/gef by a commercial pool of Slug siRNA (pool si-Slug) also increased the percentage of apoptotic cells after gefitinib treatment (Figure E2). These results suggest that knockdown of Slug expression reverses gefitinib resistance in PC9/gef cells. Moreover, even in the absence of gefitinib treatment, transfection of si-Slug 3 resulted in about 10% of apoptotic cells compared with that of si-scramble, indicating that Slug depletion may induce apoptosis of PC9/gef cells independent of gefitinib treatment. PC9/gef cells transfected with si-Slug 3 or si-scramble and treated with 0.05 μM gefitinib were then assayed for caspase-9 activity by Western blotting and luminescent assays. As shown in Figure 3F and 3G, gefitinib induced a marked increase in the activity of caspase-9 in PC9/gef cells after knockdown of Slug.
Figure 3. Knockdown of Slug expression reversed gefitinib resistance in PC9/gef cells. (A) CL1–5 cells were transfected with different Slug small interfering RNAs (siRNAs) (si-Slug 1, si-Slug 2, si-Slug 3) or scramble siRNA (si-scramble). The expression of Slug was evaluated by real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Data represent mean ± SD of three independent experiments (**P < 0.01). (B) PC9/gef cells were transfected with si-Slug3 or si-scramble. The expression of E-cadherin mRNA was evaluated by real-time RT-PCR. Data represent mean ± SD of three independent experiments (*P < 0.05). (C) The expressions of Slug, E-cadherin, and vimentin was determined by Western blot analysis after transfection of PC9/gef cells with si-Slug3 or si-scramble. Lamin B and α-tubulin were as loading control. The results shown are representative of three independent experiments. (D) Cells were incubated with 0.05 μM gefitinib for 40 hours after transfection of si-scramble or si-Slug3. Cells were then stained with annexin V–fluorescein isothiocyanate and propidium iodide, and analyzed by flow cytometry. The results shown are representative of three independent experiments. (E) The percentage of apoptotic cells was expressed as the sum of the bottom right (early state of apoptosis) and top right quadrants (late stage of apoptosis). The percentage of apoptotic cells from three independent experiments is shown. Error bars show the mean ± standard deviation. Data represent mean ± SD of three independent experiments (***P < 0.001). (F and G) Caspase 9 activity of these cells was analyzed by luminescent assay (F) and Western blot (G). Data represent mean ± SD of three independent experiments. (***P < 0.001).
[More] [Minimize]To investigate if Slug plays a protective role against gefitinib-induced apoptosis, we established Slug-overexpressing clones from gefitinib-sensitive PC9 and HCC827 cells, both of which express EGFR mutants containing deletions in exon 19. As shown in Figure 4A and Figure E4A, Slug-transfected clones (PC9-Slug and HCC827-Slug) expressed higher levels of Slug protein than the control clones (PC9-mock and HCC827-mock). Moreover, after gefitinib treatment, the percentage of apoptotic cells in cultures of PC9-Slug and HCC827-Slug decreased compared with that in control clones (Figures 4B and 4C). Similar results were obtained after overexpression of Slug in the SK-MES-1 lung cancer cell line, which is sensitive to gefitinib by virtue of expression of wild-type EGFR (17). In these cells, gefitinib treatment of cells transiently transfected with Slug reduced the percentage of cell death compared with that in mock-transfected controls (Figure E3). To investigate whether Slug overexpression affected caspase-9 activities after gefitinib treatment, we used Western blot analysis and caspase activity assays. As shown in Figures 4C and 4D, after treatment with gefitinib for 48 hours, caspase-9 activity was attenuated in Slug-overexpressing cells. These results indicate that high levels of Slug may provide protection against gefitinib-induced apoptosis. Next, to determine whether overexpression of Slug protected PC9 cells against gefitinib treatment in vivo, we inoculated athymic nude mice with PC9-Slug or PC9-mock cells and then administered 10 mg/kg/d gefitinib or vehicle control. As shown in Figure 4E, the administration of gefitinib induced significant regression of PC9-mock tumor xenografts, but not of PC9-Slug xenografts, compared with vehicle groups (P = 0.003).
Figure 4. Overexpression of Slug protects PC9 cells from gefitinib-induced apoptosis. (A) The expression of Slug was evaluated by Western blot in PC9-mock and PC9-Slug. (B) Cells was exposed to 0.5 μM gefitinib for 24 hours and assayed for apoptosis using flow cytometry. The columns are the mean for three independent experiments. Error bars show the standard deviations (***P < 0.001). (C and D) PC9-Slug and PC9-mock cells were incubated with 0.5 μM gefitinib for 16 hours and then caspase 9 activity of these cells was measured by Western blot (C) and luminescent assay (D). The results shown are representative of three independent experiments (**P < 0.01). (E) PC9-mock or PC9-Slug cells were injected into athymic nude mice subcutaneously. The mice were treated with gefitinib (10 mg/kg/d) or vehicle for 20 days and their tumor growths were measured every 5 days (n = 9 mice per group). Bars represent standard error of the mean. Asterisks indicate statistically significant difference between PC9-mock with gefitinib treatment and PC9-mock with vehicle treatment groups (**P < 0.01).
[More] [Minimize]In previous reports, Slug has been shown to protect cells through regulation of antiapoptotic (28) and BH3-only proteins (29). Our results showed that gefitinib-induced apoptosis in Slug-silenced PC9/gef cells was associated with the intrinsic apoptotic pathway (Figures 3D and 3E). This pathway involves Bcl-2 family member proteins, which can be divided into antiapoptotic, proapoptotic, and BH3-only proteins (30). To determine if the Bcl-2 family plays a role in gefitinib-induced apoptosis in Slug-knockdown PC9/gef cells, we examined the expression of Bcl-2, BcL-xL, Puma, Bad, and Bim by Western blotting. Neither BH3-only proteins (Bad, Puma) nor antiapoptosis proteins (Bcl-2, Bcl-xL) were significantly altered after gefitinib treatment of Slug-knockdown PC9/gef cells (Figure 5A). In contrast, the expression of three isoforms of Bim—Bim short (Bims), Bim long (BimL), and Bim extra long (BimEL)—were greatly increased in si-Slug 3–transfected PC9/gef cells treated with gefitinib (Figure 5A). To explore the functional relationship between Slug and Bim, we transfected PC9/gef cells with both Slug siRNA and Bim siRNA, or with si-scramble, si-Slug 3, or Bim siRNA alone, and then treated cells with gefitinib. Co-knockdown of Slug and Bim significantly reduced the percentage of apoptotic cells after gefitinib treatment compared with knockdown of Slug alone (Figure 5B). Collectively, these results suggest that Slug suppresses the expression of proapoptotic Bim in gefitinib-treated cells. To confirm this, we treated Slug-overexpressing PC9-Slug cells with gefitinib and analyzed for the expression of Bim. As shown in Figures 5C and 5D, Bim expression after gefitinib treatment was decreased in PC9-Slug cells compared with PC9-mock cells; thus, gefitinib-induced Bim up-regulation was abolished by Slug. These results indicate that the depletion of Slug reversed the gefitinib resistance of PC9/gef through up-regulation of Bim.
Figure 5. Role of Bim in gefitinib induced apoptosis of Slug-depleted PC9/gef cells. (A) Cells were incubated with 0.05 μM gefitinib for 40 hours after transfection of si-scramble or si-Slug3. Bim, bad, bcl-2, bcl-xl, puma expression of these cells were analyzed by Western blot. The results shown are representative of three independent experiments. (B) PC9/gef cells were transfected individually with si-scramble, si-Slug3, si-Bim, or combination of si-Slug3 and si-Bim and analyzed by Western blot. After transfection of siRNAs, PC9/gef cells were treated with 0.05 μM gefitinib for 40 hours and analyzed for apoptotic cells using flow cytometry. The data are presented as the average percentage of cells staining annexin V–fluorescein isothiocyanate and propidium iodide from three independent experiments (***P < 0.001). (C and D) BimEL expression of PC9, PC9-mock, and PC9-Slug cells were analyzed by Western blot (C) and real-time reverse transcriptase–polymerase chain reaction (D) after treatment with gefitinib. The results shown are representative of three independent experiments (**P < 0.01).
[More] [Minimize]To confirm the role of Slug in EGFR TKI resistance, we collected cancer cells from 44 malignant pleural effusions of lung adenocarcinomas (24 sampled at the time of diagnosis of lung cancer and 20 sampled after the development of acquired resistance to EGFR TKI) for determination of Slug expression (31). The clinical characteristics of the 44 patients are listed in Table E2. There were no differences in the clinical characteristics between the treatment-naive patients and patients with tumors with acquired resistance to EGFR TKI.
Real-time quantitative RT-PCR was used to determine the amount of Slug mRNA. As shown in Figure 6A, Slug mRNA expression in lung adenocarcinomas after the development of acquired EGFR TKI resistance (1.43 ± 1.72, n = 20) was significantly higher than that before treatment (0.55 ± 0.56, n = 24; P = 0.039, Student t test).
Figure 6. The expression of Slug increased after development of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in patients with lung cancer. (A) Box plot of Slug mRNA expression in lung adenocarcinoma patients by real-time reverse transcriptase–polymerase chain reaction. Box shows median and interquartile range ± 95% confidence interval. Patient samples were tested in triplicate (*P < 0.05). (B) The expression of Slug mRNA of lung cancer cells in three paired samples at initial diagnosis and after acquired EGFR TKI resistance.
[More] [Minimize]In addition, we collected three paired patient samples (treatment-naive and after development of acquired resistance to EGFR TKI) for analysis of Slug mRNA expression. These samples revealed a trend toward increased Slug expression after the development of acquired EGFR TKI resistance (Figure 6B). The EGFR mutation status and the duration of EGFR TKI use are listed in Table E3.
There has been substantial recent development in molecular-targeted therapies, an available option in addition to conventional cancer treatments. Molecular-targeted therapy drugs can interfere with and block specific molecular pathways involved in cancer cell growth and progression (1, 3). One such target that has been extensively studied is activated EGFR, which can be effectively blocked by EGFR TKIs. However, the development of resistance to EGFR TKIs continues to critically limit the long-term control of cancer using this strategy. In this study, we found that EMT correlates with resistance to gefitinib in lung cancer. Using gain- and loss-of-function approaches, we also showed that expression of the EMT regulator Slug is required for gefitinib resistance. Silencing Slug in cells with acquired gefitinib resistance restored gefitinib-induced apoptosis, a process that is mediated mainly through increased activation of Bim and caspase-9.
Resistance to EGFR TKIs in many cancers harboring wild-type EGFR is associated with loss of the epithelial marker E-cadherin (15–17), suggesting involvement of EMT in the resistance mechanism. Consistent with this, ectopic expression of E-cadherin in NSCLC cells, which possess a mesenchymal phenotype, enhances gefitinib sensitivity (18). Yauch and colleagues also reported that E-cadherin expression is a novel biomarker capable of predicting the clinical efficacy of erlotinib in patients with NSCLC (17). Cells of the A549 NSCLC cell line, which expresses wild-type EGFR and exhibits resistance to gefitinib, become more aggressive and more resistant to gefitinib on loss of biomarkers associated with epithelial status and gain of biomarkers associated with mesenchymal status (32). These observations suggest that EMT contributes to primary resistance to gefitinib, although it should be noted that these studies do not completely mimic clinical reality because wild-type lung cancer cells are intrinsically resistant to gefitinib. Using two types of mutant EGFR-expressing cell lines—gefitinib-sensitive parental cells and derivative clones that had developed gefitinib resistance—we found that expression of E-cadherin was decreased and expression of vimentin and Slug were increased in cells with acquired resistance to gefitinib. Our results thus suggest that EMT contributes to acquired resistance to EGFR TKIs in EGFR-mutant, gefitinib-sensitive cells.
Expression of the EMT regulators Slug, snail, twist, and zeb-1, which cause EMT induction through transcriptional reprogramming, may differentially influence EGFR TKI resistance. Recent reports have shown that zeb-1 mRNA levels are inversely related to gefitinib sensitivity in NSCLCs that express wild-type EGFR (15, 18). Snail has also been shown to down-regulate the transcription of E-cadherin and ErbB3, which contribute to gefitinib sensitivity (33, 34). However, zeb-1 and snail expression were not significantly different between gefitinib-sensitive PC9 cells and gefitinib-resistant PC9/gef cells, suggesting that zeb-1 and snail are not involved in the acquired resistance to gefitinib in lung cancer cells, although they may be involved in intrinsic resistance.
Slug, a member of the snail superfamily of zinc finger transcription factors, is the key EMT regulator responsible for conferring acquired resistance to EGFR TKIs in NSCLC cells. Our previous studies showed that the expression of Slug promotes the invasivity of tumor cells through increased activity of metalloproteinase-2 and suppression of E-cadherin (24). The mechanism of Slug-induced invasiveness depends on p53 status; notably, mutant p53 can stabilize Slug protein (35). A recent reported suggested that reducing the expression of Slug enhanced the sensitivity of neuroblastoma cell lines to imatinib mesylate, a soluble small-molecule TKI, by attenuating Bcl-2 expression (28). Slug can also antagonize p53-mediated apoptosis in hematopoietic progenitors by repressing Puma. Thus, Slug could regulate cancer cell survival via direct or indirect transcriptional regulation of proapoptotic and antiapoptotic genes (29, 36, 37), although further study will be required to resolve the molecular details. Therefore, in addition to EGFR T790M and MET amplification, resistance to gefitinib may be mediated through Slug-induced EMT, which confers resistance by interfering with the balance between apoptosis and antiapoptosis.
We screened for changes in Bcl-2 family members after knockdown of Slug and treatment with gefitinib. We found that only Bim was altered by depletion of Slug in the acquired gefitinib-resistance model after treatment with gefitinib, suggesting that Slug affected gefitinib sensitivity in cancer cells through changes in the expression of Bim. Specifically, treatment of Slug-knockdown PC9/gef cells with gefitinib resulted in increased expression of Bim, suggesting that Slug may suppress Bim expression in response to gefitinib. Consistent with this, we also found that both Bim protein and mRNA were decreased in Slug-overexpressing PC9-Slug cells after gefitinib treatment. Our results are in accord with previous reports, which have shown that Bim serves as an executioner of EGFR TKI-induced apoptosis in lung cancer cell lines containing activating EGFR mutants (38), and siRNA-mediated depletion of Bim causes insensitivity to gefitinib and erlotinib (39).
To the best of our knowledge, the role of Slug in cancer cells from patients treated with EGFR TKIs has not been explored. The present study showed that the mean mRNA levels of Slug expression were significantly higher in cancer cells from EGFR TKI-treated patients after the development of acquired resistance to EGFR TKIs than in treatment-naive cancer cells. Although the difference is not so dramatic, it is consistent with the results of our in vitro cell line model, in which Slug mRNA levels were increased in cells with acquired resistance to gefitinib (PC9/gef) compared with parental cells (PC9). An increase in Slug expression was also noted in the three patients for whom paired samples were available for the analysis of Slug expression before and after EGFR TKI treatment. In addition to the presence of the T790M mutation, changes in Slug may prove to be useful for monitoring drug resistance in patients who have received EGFR TKI treatment, but because of the heterogeneity in the mechanisms of acquired resistance additional clinical studies are necessary to confirm this hypothesis.
An appropriate combination of gefitinib administration and Slug depletion might be a potential approach to lung cancer therapy. First, Slug suppression alone led to a degree of spontaneous apoptosis in PC9/gef cells. Second, Slug depletion appeared to markedly sensitize PC9/gef cells to gefitinib-induced apoptosis. Third, Slug supported EGFR-mutant tumor growth in an animal xenograft model, even with gefitinib treatment. Taken together, our data suggest that the development of Slug-targeted drugs is a promising strategy for enhancing the efficacy of molecular-targeted drugs and improving the effectiveness of lung cancer therapy.
The authors thank the NTU Microarray Core Facility of National Research Program for Genomic Medicine of Taiwan for technical assistance and the Department of Medical Research in National Taiwan University Hospital for facility support.
| 1. | Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. Non-small cell lung cancer: Epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 2008;83:584–594. |
| 2. | Wheeler DL, Dunn EF, Harari PM. Understanding resistance to EGFR inhibitors—impact on future treatment strategies. Nat Rev Clin Oncol 2010;7:493–507. |
| 3. | Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003;21:2787–2799. |
| 4. | Ono M, Kuwano M. Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs. Clin Cancer Res 2006;12:7242–7251. |
| 5. | Gazdar AF. Personalized medicine and inhibition of EGFR signaling in lung cancer. N Engl J Med 2009;361:1018–1020. |
| 6. | Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–2139. |
| 7. | Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–1500. |
| 8. | Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG, Halmos B. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 2005;352:786–792. |
| 9. | Gazdar AF, Minna JD. Inhibition of EGFR signaling: All mutations are not created equal. PLoS Med 2005;2:e377. |
| 10. | Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG, Varmus H. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005;2:e73. |
| 11. | Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, Lindeman N, Gale CM, Zhao X, Christensen J, et al. Met amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 2007;316:1039–1043. |
| 12. | Kajiyama H, Shibata K, Terauchi M, Yamashita M, Ino K, Nawa A, Kikkawa F. Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells. Int J Oncol 2007;31:277–283. |
| 13. | Wang X, Ling MT, Guan XY, Tsao SW, Cheung HW, Lee DT, Wong YC. Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells. Oncogene 2004;23:474–482. |
| 14. | Yang AD, Fan F, Camp ER, van Buren G, Liu W, Somcio R, Gray MJ, Cheng H, Hoff PM, Ellis LM. Chronic oxaliplatin resistance induces epithelial-to-mesenchymal transition in colorectal cancer cell lines. Clin Cancer Res 2006;12:4147–4153. |
| 15. | Frederick BA, Helfrich BA, Coldren CD, Zheng D, Chan D, Bunn PA Jr, Raben D. Epithelial to mesenchymal transition predicts gefitinib resistance in cell lines of head and neck squamous cell carcinoma and non-small cell lung carcinoma. Mol Cancer Ther 2007;6:1683–1691. |
| 16. | Thomson S, Buck E, Petti F, Griffin G, Brown E, Ramnarine N, Iwata KK, Gibson N, Haley JD. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 2005;65:9455–9462. |
| 17. | Yauch RL, Januario T, Eberhard DA, Cavet G, Zhu W, Fu L, Pham TQ, Soriano R, Stinson J, Seshagiri S, et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 2005;11:8686–8698. |
| 18. | Witta SE, Gemmill RM, Hirsch FR, Coldren CD, Hedman K, Ravdel L, Helfrich B, Dziadziuszko R, Chan DC, Sugita M, et al. Restoring e-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res 2006;66:944–950. |
| 19. | Arumugam T, Ramachandran V, Fournier KF, Wang H, Marquis L, Abbruzzese JL, Gallick GE, Logsdon CD, McConkey DJ, Choi W. Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer. Cancer Res 2009;69:5820–5828. |
| 20. | Voulgari A, Pintzas A. Epithelial-mesenchymal transition in cancer metastasis: mechanisms, markers and strategies to overcome drug resistance in the clinic. Biochim Biophys Acta 2009;1796:75–90. |
| 21. | Chang TH, Shih JY, Tsai MF, Yang CH, Yang PC. Transcription factor slug conferring resistance to EGFR tyrosine kinase inhibitor gefitinib [abstract]. Proceedings of the 101st Annual Meeting of the American Association for Cancer Research 2010:A625. |
| 22. | Thomas RK, Baker AC, Debiasi RM, Winckler W, Laframboise T, Lin WM, Wang M, Feng W, Zander T, MacConaill L, et al. High-throughput oncogene mutation profiling in human cancer. Nat Genet 2007;39:347–351. |
| 23. | Basak SK, Veena MS, Oh S, Huang G, Srivatsan E, Huang M, Sharma S, Batra RK. The malignant pleural effusion as a model to investigate intratumoral heterogeneity in lung cancer. PLoS ONE 2009;4:e5884. |
| 24. | Shih JY, Tsai MF, Chang TH, Chang YL, Yuan A, Yu CJ, Lin SB, Liou GY, Lee ML, Chen JJ, et al. Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma. Clin Cancer Res 2005;11:8070–8078. |
| 25. | Koizumi F, Shimoyama T, Taguchi F, Saijo N, Nishio K. Establishment of a human non-small cell lung cancer cell line resistant to gefitinib. Int J Cancer 2005;116:36–44. |
| 26. | Moreno-Bueno G, Cubillo E, Sarrio D, Peinado H, Rodriguez-Pinilla SM, Villa S, Bolos V, Jorda M, Fabra A, Portillo F, et al. Genetic profiling of epithelial cells expressing e-cadherin repressors reveals a distinct role for snail, slug, and e47 factors in epithelial-mesenchymal transition. Cancer Res 2006;66:9543–9556. |
| 27. | Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2007;7:415–428. |
| 28. | Vitali R, Mancini C, Cesi V, Tanno B, Mancuso M, Bossi G, Zhang Y, Martinez RV, Calabretta B, Dominici C, et al. Slug (snai2) down-regulation by RNA interference facilitates apoptosis and inhibits invasive growth in neuroblastoma preclinical models. Clin Cancer Res 2008;14:4622–4630. |
| 29. | Wu WS, Heinrichs S, Xu D, Garrison SP, Zambetti GP, Adams JM, Look AT. Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell 2005;123:641–653. |
| 30. | Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell 2004;116:205–219. |
| 31. | Jackman D, Pao W, Riely GJ, Engelman JA, Kris MG, Janne PA, Lynch T, Johnson BE, Miller VA. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J Clin Oncol 2010;28:357–360. |
| 32. | Rho JK, Choi YJ, Lee JK, Ryoo BY, Na II, Yang SH, Kim CH, Lee JC. Epithelial to mesenchymal transition derived from repeated exposure to gefitinib determines the sensitivity to EGFR inhibitors in A549, a non-small cell lung cancer cell line. Lung Cancer 2009;63:219–226. |
| 33. | De Craene B, Gilbert B, Stove C, Bruyneel E, van Roy F, Berx G. The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Res 2005;65:6237–6244. |
| 34. | Rosell R, Taron M, Reguart N, Isla D, Moran T. Epidermal growth factor receptor activation: how exon 19 and 21 mutations changed our understanding of the pathway. Clin Cancer Res 2006;12:7222–7231. |
| 35. | Wang SP, Wang WL, Chang YL, Wu CT, Chao YC, Kao SH, Yuan A, Lin CW, Yang SC, Chan WK, et al. P53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug. Nat Cell Biol 2009;11:694–704. |
| 36. | Haupt S, Alsheich-Bartok O, Haupt Y. Clues from worms: a slug at puma promotes the survival of blood progenitors. Cell Death Differ 2006;13:913–915. |
| 37. | Zilfou JT, Spector MS, Lowe SW. Slugging it out: fine tuning the p53-PUMA death connection. Cell 2005;123:545–548. |
| 38. | Cragg MS, Kuroda J, Puthalakath H, Huang DC, Strasser A. Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics. PLoS Med 2007;4:1681–1689; discussion 1690. |
| 39. | Costa DB, Halmos B, Kumar A, Schumer ST, Huberman MS, Boggon TJ, Tenen DG, Kobayashi S. BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations. PLoS Med 2007;4:1669–1679; discussion 1680. |
