Epidermal growth factor receptor-targeted tyrosine kinase inhibitors (EGFR-TKIs) have become first-line drugs used for non-small cell lung cancer (NSCLC) treatment. However, drug resistance to EGFR-TKIs will be developed inevitably due to the repeated use of these drugs. In the present study, we isolated cancer stem cells (CSCs) from the PC9 NSCLC cell line. We then observed that the PC9 CSCs showed significant resistance to erlotinib compared with the PC9 non-CSCs. Erlotinib failed to suppress the phosphorylation of PI3K and AKT in PC9 CSCs, although the EGFR was inhibited sufficiently. Mechanically, we observed aberrant upregulation of microRNA-23a (miR-23a) and downregulation of PTEN in PC9 CSCs compared to PC9 non-CSCs. Luciferase reporter assays proved that PTEN was the target of miR-23a in PC9 CSCs. Furthermore, knockdown of miR-23a enhanced the antitumor effect of erlotinib by increasing the expression of PTEN. In addition, transfection with miR-23a inhibitors promoted the erlotinib-dependent inhibition of PI3K/AKT pathway, thus, suppressing the proliferation and inducing apoptosis in PC9 CSCs. These results propose that upregulation of miR-23a is a potential mechanism associated with resistance to EGFR-TKIs in lung cancer stem cells. Inhibition of miR-23a serves as a novel therapeutic strategy to eliminate the EGFR-TKIs resistance of lung cancer stem cells.
Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality worldwide. Overall 5-year survival rate of NSCLC is lower than 5% (
Recently, studies has demonstrated that cancer stem cells (CSCs), which are a small subgroup of cells in tumor possessing the ability of self-renewal, are capable of driving tumorigenesis. Accumulating evidence indicated that lung cancer stem cells are responsible for resistance to chemotherapeutics and EGFR-TKIs (
MicroRNAs (miRNAs) are single-stranded RNAs with a length of 21–25 nucleotides. They regulate cancer initiation and progression by regulating the expression of cancer-related genes. miRNAs usually act as negative regulators of gene expression by binding to the mRNAs at the 3untranslated region (3′UTR) (
The NSCLC cell line PC9 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and was cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). For isolating CSCs of PC9 cell line, FACSVantage (BD FACSCalibur; BD Biosciences, San Diego, CA, USA) was used to identify and collect the CD133-positive PC9 cells (CD133-FITC antibody was purchased from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). To estimate the purity of CSC population in PC9, flow cytometry was performed.
Negative control oligonucleotides (miR-NC), microRNA-23a mimics (miR-23a), miR-23a inhibitors (anti-miR-23a) and PTEN siRNA were purchased from Guangzhou RiboBio, Co., Ltd. (Guangzhou, China). For transfection, 50 pmol/ml miR-NC, miR-23a, anti-miR-23a or PTEN siRNA were packaged with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). Then, PC9 CSCs were incubated with RNAs in serum-free medium (Gibco). Six hours later, the culture medium was changed to 10% FBS RPMI-1640 medium for 24 h.
PC9 CSCs (5×103) or PC9 non-CSCs (5×103) were plated on 96-well plates with 200 µl RPMI-1640 medium. Then, cells were transfected with RNAs followed by treating with EGFR-TKIs for 48 h. After incubation, 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added into the medium for 4 h. Subsequently, we replaced the culture medium with 150 µl dimethyl sulfoxide (DMSO) for 30 min. Cell viability of PC9 CSCs and PC9 non-CSCs was evaluated according to the absorbance at 570 nm using a microplate reader. Half maximal inhibitory concentration (IC50) of EGFR-TKIs to PC9 CSCs and PC9 non-CSCs was calculated according to the results of cell viability assays.
PC9 CSCs (5×105) or PC9 non-CSCs (5×105) were plated on 6-well plates with 2 ml RPMI-1640 medium. Then, cells were transfected with RNAs followed by treating with 0.04 µM erlotinib for 48 h. After incubation, 3H- thymidine was added into the culture medium for 6 h. Then, 3H-thymidine incorporation assays were performed to determine the cell proliferation of PC9 CSCs and PC9 non-CSCs.
PC9 CSCs and PC9 non-CSCs were seeded at a density of 50 cells/cm2 (185 cells/well) in a 12-well plate format. Subsequently, cells were treated with 0.04 µM erlotinib for 10 days. After treatment, cell colonies were stained with crystal violet/ethanol mixture and then were counted under a microscope. The proportion of colonies formed is shown as a percentage of its untreated control (
Total RNA was extracted from the PC9 CSCs and PC9 non-CSCs using TRIzol reagent (Invitrogen). Expression of miR-23a, Oct4 and Sox2 was measured by One-Step RT-qPCR with SYBR Premix Ex Taq (Takara Bio Inc., Otsu, Japan) according to the manufacturers instructions. U6 snRNA was used as the internal control to calculate the relative expression of miR-23a. GAPDH was used as the internal control to calculate the relative expression of Oct4 and Sox2. 2−∆∆CT method was used to analyze the results of RT-qPCR (
Total proteins were extracted from the PC9 CSCs and PC9 non-CSCs using RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) and then quantified by bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins were then separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were then blocked in 5% skim milk for 1 h at room temperature and then incubated overnight at 4°C with the following antibodies: anti-PTEN, anti-EGFR, anti-phosphorylated EGFR, anti-PI3K, anti-phosphorylated PI3K, anti-AKT, anti-phosphorylated AKT, anti-pro-caspase-9, anti-cleaved caspase-9, anti-pro-caspase-3, anti-cleaved caspase-3 and anti-β-actin (Cell Signaling Technology). After washing the membranes with Tris-buffered saline, they were incubated with goat anti-rabbit secondary antibody (Cell Signaling Technology) for 1 h at room temperature. The blots were visualized using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA).
PC9 CSCs (5×105) or PC9 non-CSCs (5×105) were plated on 6-well plates with 2 ml RPMI-1640 medium. Then, cells were transfected with RNAs followed by treating with 0.04 µM erlotinib for 48 h. After incubation, cells were collected and were stained with PI and Annexin V (Sigma-Aldrich) for 20 min at room temperature. Subsequently, percentage of apoptotic cells was quantified using flow cyto-metry.
3′UTR of PTEN mRNA was amplified and inserted into pGL3 Luciferase reporter vectors (Promega, Madison, WI, USA) downstream of the luciferase gene. PC9 CSCs (1×105) were plated on 24-well plates with 500 µl RPMI-1640 medium. Then, cells were transfected with 50 pmol/ml miR-23a mimics, 2 µg/ml firefly luciferase reporters and 100 ng/ml
Quantitative data are presented as the mean ± SD. All of the experiments were repeated in triplicate. For comparison analysis, the statistical analysis was performed by the Students t-test using SPSS 15.0 software (SPSS; Chicago, IL, USA). A value of P<0.05 was considered to indicate a statistically significant difference.
To study the effect of EGFR-TKIs on lung cancer stem cells, we isolated the CSCs population of PC9 using CD133 antibody. Our separated PC9 CSCs expressed high level of CD133, and CD133 negative PC9 cell population was considered as the non-CSCs (
Suppression of EGFR/PI3K/AKT pathway is the mechanism of EGFR-TKIs in killing the NSCLC cells (
To investigate the mechanism by which erlotinib failed to inhibit the PI3K/AKT pathway in PC9 CSCs, we detected the miRNA expression in PC9 cell line. We found that the expression of miR-23a was significantly upregulated in PC9 CSCs rather than PC9 non-CSCs (
The preceding results demonstrated that miR-23a which was overexpressed in PC9 CSCs decreased the PTEN expression. We therefore investigated whether knockdown of miR-23a enhances the antitumor effect of erlotinib on PC9 CSCs. Results of western blot analysis illustrated that transfection with miR-23a antisense oligonucleotides (anti-miR-23a) significantly increased the expression of PTEN, whereas the PTEN siRNA abolished the effect of anti-miR-23a (
To investigate the effect of anti-miR-23a on other EGFR-TKIs, we treated the PC9 CSCs with gefitinib and afatinib after transfection with anti-miR-23a and PTEN siRNA. The results of MTT demonstrated that knockdown of miR-23a also enhanced the antitumor effect of gefitinib and afatinib by upregulating the expression of PTEN (
Approximately 25% NSCLC patients exhibit mutation for epidermal growth factor receptor (EGFR). For these NSCLC patients, the cancer cells are sensitive to EGFR-TKIs such as erlotinib, gefitinib and afatinib. As the treatment of EGFR-TKIs is the targeted molecular therapy without serious side-effects for NSCLC patients, EGFR-TKIs have become the first-line therapy for NSCLC (
MicroRNA-23a is reported to function as an oncogene in various human cancers. In NSCLC, high expression level of miR-23a shows poor prognosis (
Phosphatase and tensin homologue (PTEN) is an important regulator for suppressing tumorigenesis and cancer development. PTEN mutation or loss induced uncontrolled cell cycle (
In conclusion, we demonstrated that lung cancer stem cells express low level of PTEN. As PTEN is targeted by miR-23a, we proved that introduction with miR-23a antisense oligonucleotides resensitize lung cancer stem cells to EGFR-TKIs by suppressing PI3K/AKT pathway. Combination of miR-23a antisense oligonucleotides and EGFR-TKIs could be attractive strategy for treatment of NSCLC.
Thanks are due to all the contributors who assisted with this study.
PC9 CSCs are resistant to erlotinib. (A) CD133 antibody was used to sort the CSC population in PC9 cell line. Purity of PC9 CSCs and PC9 non-CSCs was analyzed by flow cytometry. *P<0.05 vs. unsorted PC9 cells. (B) Expression of Oct4 and Sox2 in PC9 CSCs and PC9 non-CSCs was detected by RT-qPCR. *P<0.05 vs. PC9 non-CSCs. (C) Antitumor effect of erlotinib on PC9 CSCs and PC9 non-CSCs was evaluated by MTT assays. *P<0.05. IC50 of erlotinib was compared between PC9-CSCs and PC9-non-CSCs. *P<0.05 vs. PC9 non-CSCs.
Erlotinib failed to suppress the phosphorylation of PI3K and AKT in PC9 CSCs. (A) Phosphorylation of EGFR, PI3K and AKT in PC9 non-CSCs and PC9 CSCs was detected by western blot analysis after they were treated with 0.04 µM erlotinib. (B) Cell proliferation of PC9 non-CSCs and PC9 CSCs was measured by 3H-thymidine incorporation assays after they were treated with 0.04 µM erlotinib. **P<0.01. (C) Colony formation of PC9 non-CSCs and PC9 CSCs was evaluated after they were treated with 0.04 µM erlotinib. **P<0.01. (D) Apoptosis of PC9 non-CSCs and PC9 CSCs was measured by flow cytometry after they were treated with 0.04 µM erlotinib.
PTEN is the target of miR-23a. (A) Expression of miR-23a in PC9 CSCs and PC9 non-CSCs was detected by RT-qPCR. *P<0.05 vs. PC9 non-CSCs. (B) PTEN expression in PC9 CSCs and PC9 non-CSCs was measured by western blot analysis. (C) PTEN is predicted as the target of miR-23a by TargetScan databases. (D) miR-23a decreased the activities of luciferase in pGL3-PTEN plasmid. *P<0.05 vs. miR-NC group. (E) Western blot analysis was performed to detect the expression of PTEN after the PC9 CSCs were transfected with miR-23a mimics or antisense oligonucleotides.
Anti-miR-23a resensitizes PC9 CSCs to erlotinib by upregulating the expression of PTEN. (A) Transfection efficiency of anti-miR-23a and PTEN siRNA on regulating the PTEN expression was detected by western blot analysis. (B) Effect of anti-miR-23a, PTEN siRNA and miR-23a on regulating the anti-tumor effect of erlotinib on PC9 CSCs. *P<0.05. (C) Expression of PTEN and phosphorylation of EGFR, PI3K and AKT were detected by western blot analysis after the PC9 CSCs were treated with anti-miR-23a, PTEN siRNA and erlotinib (0.04 µM). (D) 3H-thymidine incorporation assays were performed to evaluate the effect of anti-miR-23a, PTEN siRNA and erlotinib on cell proliferation in PC9 CSCs. *P<0.05 vs. erlotinib+miR-NC group. #P<0.05 vs. erlotinib+miR-NC group. (E) Flow cytometry was performed to measure apoptosis of PC9 CSCs treated with anti-miR-23a, PTEN siRNA and erlotinib. (F) Cleavage of caspase-9 and caspase-3 in PC9 CSCs was detected by western blot analysis after they were treated with anti-miR-23a, PTEN siRNA and erlotinib.
miR-23a antisense oligonucleotides enhance the antitumor effect of other EGFR-TKIs. (A) Anti-miR-23a promoted the cell death of PC9 CSCs induced by gefitinib (0.04 µM) or afatinib (0.04 µM). *P<0.05. (B) miR-23a antisense oligonucleotides decreased the IC50 of gefitinib, erlotinib and afatinib to PC9 CSCs. *P<0.05.