Abstract
Background/Aim: We evaluated the radiosensitizing effect of the combination treatment of trametinib, a MEK inhibitor, and temsirolimus, an mTOR inhibitor, on non-small-cell lung carcinoma (NSCLC) cells. Materials and Methods: The effects of combining trametinib and temsirolimus with radiation in NSCLC cell lines were evaluated using clonogenic survival and apoptosis assays. DNA double-strand breaks and cell cycle distribution were analyzed using flow cytometry. Tumor volume was measured to determine the radiosensitivity in lung cancer xenograft models. Results: Exposure of lung cancer cells to a combination of trametinib and temsirolimus reduced clonogenic survival and promoted radiation-induced apoptosis. Combined inhibition of MEK and mTOR induced prolonged expression of γH2AX after irradiation and resulted in prolonged G2/M cell cycle arrest after irradiation in A549 cells. In vivo studies revealed that co-administration of the drugs sensitizes lung cancer xenografts to radiotherapy. Conclusion: The combination of trametinib and temsirolimus can enhance lung cancer radiosensitivity in vitro and in vivo.
- Lung cancer
- MEK inhibitor
- mTOR inhibitor
- radiation
- trametinib
- temsirolimus
Lung cancer is the leading cause of cancer-related deaths worldwide (1). Approximately 70% of patients are diagnosed with advanced disease; outcomes remain poor, with a median survival time of 8-10 months and a 5-year survival rate of 20-25% (2, 3). Radiotherapy is an important treatment option for lung cancer. It has been reported that approximately 50% of all cancer patients and 65-76% of lung cancer patients receive radiation treatment (4, 5). Since radiotherapy generally destroys radiosensitive cells, many patients experience tumor recurrence and metastasis due to radioresistant cancer cells. Therefore, radioresistance remains a major obstacle for the treatment of cancer by radiotherapy. Although the mechanisms of radioresistance are largely unknown, recent studies have shown that radiation sensitivity of tumor cells is associated with several signal transduction pathways (6, 7). In particular, the MAPK and PI3K/AKT/mTOR pathways, which are key signaling cascades in regulating cell growth, survival, and proliferation during cancer progression, play a central role in the development of radioresistance in non-small cell lung cancer (NSCLC) and other tumors (7-16). Hyperactivation of either of these pathways is one of the factors responsible for the development of cancer cells with increased resistance to radiotherapy.
Therefore, dual targeting of these cascades may be an attractive strategy to sensitize tumor cells to radiation. Trametinib, approved for BRAF-driven NSCLC and melanoma, is a potent and highly selective allosteric inhibitor of MEK1/2 (17, 18). Temsirolimus, a rapamycin analog, is one of the most widely used mTOR inhibitors against metastatic renal cell carcinoma (19).
In this study, we evaluated the radiosensitizing effect of the combination therapy of trametinib and temsirolimus, thereby targeting two signaling pathways, on NSCLC by investigating cell apoptosis, DNA damage, and cell cycle arrest.
Materials and Methods
Cells. The NSCLC cell lines A549 and NCI-H460 were purchased from the American Type Culture Collection (Manassas, VA, USA). Both cell lines were cultured at 37°C and 5% CO2 in RPMI1640 medium (Hyclone Laboratories, Waltham, MA, USA) containing 10% fetal bovine serum.
Reagents. Trametinib (a MEK inhibitor) and temsirolimus (an mTOR inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA). Crystal violet, propidium iodide (PI), RNase A, and paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Annexin V–fluorescein isothiocyanate (FITC) Apoptosis Detection Kit was purchased from BD Biosciences (San Jose, CA, USA). A protein assay kit, for quantification of proteins, was purchased from Bio-Rad (Richmond, CA, USA). Antibodies against phospho-histone H2AX, phospho-chk2 (Thr68), phospho-p70S6K, phospho-p90RSK, cleaved caspase3, cleaved caspase8, and cleaved poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p53, phospho-p53 (Ser15), p27, and GAPDH as well as horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (CA, USA). FITC-labeled secondary antibody was purchased from Invitrogen (Eugene, OR, USA). An enhanced chemiluminescence (ECL) western blotting detection kit was supplied by Dozen (Seoul, Republic of Korea).
Drug treatment and irradiation. The drugs were stored as frozen aliquots in dimethyl sulfoxide (DMSO) at –70°C and diluted in culture medium at the time of use. Trametinib (100 nM) and temsirolimus (50 nM) were added 1 h before irradiation and the drug treatment was continued post-irradiation. A Biobeam 8000 (Gamma-Service Medical GmbH, Leipzig, Germany) Cesium-137 gamma-ray irradiator was used to irradiate the cells at a dose rate of 2.6 Gy/min.
Clonogenic survival assay. Cells were plated in duplicate at single-cell density. After 18 h, cells were treated with trametinib, temsirolimus, or both for 1 h prior to irradiation with 0, 2, 4, 6, and 8 Gy. Following irradiation, the cells were subjected to 24 h post-irradiation treatment with each drug. After 14 days of incubation, cells were fixed with 70% methanol and stained with 0.5% crystal violet. Survival was calculated relative to that of unirradiated cells [survival=(plating efficiency of treated cells)/(plating efficiency of control cells) where plating efficiency=(number of colonies formed by treated cells)/(number of colonies formed by untreated cells)] (20).
Apoptosis assay. Apoptosis assay was performed at 48 and 72 h after irradiation. Apoptosis induced by each treatment was detected using the Annexin V–FITC Apoptosis Detection Kit in accordance with the manufacturer’s protocol and was quantified by FACS analysis.
Western blot analysis. The cultured cells were harvested and sonicated in lysis buffer (1% SDS, 5% glycerol, 1 mM EDTA, 30 mM Tris-HCl, pH 6.8), followed by centrifugation and quantification. Western blotting was performed by SDS-PAGE using a 10-12% gel. After electrophoresis, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane and then washed with PBS–Tween 20 buffer containing 5% non-fat skim milk. The membranes were incubated with primary antibodies at 4°C overnight and then with secondary antibodies at a dilution of 1:2,000 at room temperature for 1 h. After successive washes, the membranes were developed using the ECL kit and analyzed using the ImageQuant™LAS4000 image analysis system (GE Healthcare, Uppsala, Sweden).
Detection of histone γH2AX by flow cytometry. Detection of histone γH2AX was performed at 1, 24, and 48 h after irradiation (8 Gy). For analysis, at least 10,000 cells were trypsinized, washed with phosphate-buffered saline (PBS), and fixed in 2% paraformaldehyde for 30 min at room temperature. After washing with PBS, the cells were permeabilized with 90% methanol for 30 min on ice. The histone γH2AX antibody was added at a dilution of 1:400 in 1% bovine serum albumin (BSA) and incubated for 1 h at room temperature. The cells were again washed twice with PBS and then incubated in the dark with FITC-labeled secondary antibody at a dilution of 1:100 in 1% BSA for 30 min at room temperature. Fluorescence-activated cell sorting (FACS) analysis was performed using the FACSCanto™ II flow cytometer (BD Biosciences, San Jose, CA, USA).
Cell cycle analysis. Cell cycle analysis was conducted at 24, 48, and 72 h after irradiation. Cells were trypsinized, washed with ice-cold PBS, and fixed with 70% ethanol. Fixed cells were washed with PBS and stained with propidium iodide (PI, 50 μg/ml) and RNase A (50 μg/ml) in the dark for 15 min at room temperature. The DNA content in the nucleus of each cell was determined by FACS analysis.
Xenograft tumor models. All animal experiments were approved by the institutional animal care and use committee. Six-week-old female BALB/c nude mice were purchased from Orient Bio (Seongnam, Gyeonggi, Republic of Korea). The animals were housed at 24°C ± 1°C with a 12-h light:dark cycle and had free access to a standard diet and distilled water for 1 week prior to the experiment. A549 cells were subcutaneously injected (3 × 106 cells) with 50% Matrigel (BD Biosciences, Bedford, MA, USA) into the hind leg of each mouse. When the average tumor volume reached approximately 100 mm3, the mice were randomly assigned to the following eight treatment groups of six animals each: control, trametinib, temsirolimus, trametinib + temsirolimus, control + radiation, trametinib + radiation, temsirolimus + radiation, and trametinib + temsirolimus + radiation. Trametinib was administered via oral gavage at a dose of 0.3 mg/kg/day for 5 days per week. Temsirolimus was administered intraperitoneally (i.p.) at a dose of 5 mg/kg/day for 5 days per week. The mice received 2 Gy once daily for 4 consecutive days. Tumor volume was determined from caliper measurements of tumor length (L) and width (W) according to the formula LW2/2. Tumor size was measured 5 times per week for 3 weeks.
Statistical analysis. Statistical significance was assessed by t-test (two-sample assuming unequal variances) and expressed as the mean±standard error. Differences were considered significant at p<0.05.
Results
Combined inhibition of MEK and mTOR reduces clonogenic survival after radiation treatment in A549 and NCI-H460 lung cancer cells. First, the cytotoxic potential of MEK and mTOR inhibitors in A549 and NCI-H460 NSCLC cell lines was examined over a range of concentrations from 0 to 10 μM. Cell viability was quantified by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) after 24 h drug exposure (data not shown). The optimal drug concentrations were determined when the extent of cell viability reached 80-90% in these cell lines, and accordingly, 100 nM of trametinib and 50 nM of temsirolimus were used for subsequent experiments.
To examine the radiosensitizing effects of MEK and mTOR inhibitors in both cell lines, the clonogenic survival assay was performed (Figure 1A and B). The cells were treated with trametinib, temsirolimus, or both for 1 h prior to irradiation with 0, 2, 4, 6, and 8 Gy. Following irradiation, the cells were subjected to 24 h post-irradiation treatment with each drug. After 14 days, we observed a distinct decrease in the survival rate when trametinib and temsirolimus were combined with radiation compared with either treatment alone.
Combination treatment of trametinib and temsirolimus enhances radiation-induced apoptosis in A549 cells. To evaluate radiation-induced apoptosis, flow cytometry was performed by Annexin V staining at 48 and 72 h after irradiation with 8 Gy in A549 cells. Cells were pretreated with trametinib (100 nM) and/or temsirolimus (50 nM) for 1 h prior to irradiation. The combination treatment of trametinib and temsirolimus in irradiated cells led to a significant increase in apoptosis (Figure 2A and B). Western blot analysis showed increased levels of proapoptotic molecules, such as FAS, cleaved caspase 8/caspase 3, and cleaved PARP when A549 cells were treated with both drugs and radiotherapy compared with either treatment alone (Figure 2C).
Combination treatment of trametinib and temsirolimus induces prolonged expression of γH2AX after irradiation. When DNA double-strand breaks (DSBs) occur after irradiation, H2AX is phosphorylated at a very early time, forming γH2AX foci. The γH2AX foci disappear once the DNA damage is repaired. However, γH2AX persists after exposure to radiation upon treatment with radiosensitizing agents. Therefore, γH2AX is a sensitive marker to examine DNA damage induced either by ionizing radiation or DNA-damaging agents in tumor cells.
We assessed DNA DSB by detecting γH2AX foci by flow cytometry. A549 lung cancer cells were treated with trametinib (100 nM) and/or temsirolimus (50 nM) for 1 h prior to irradiation with 8 Gy. The expression of histone γH2AX was evaluated at 1, 24, 48, and 72 h after irradiation by flow cytometry and western blotting (Figure 3A and B). The expression of p70S6K, a serine/threonine kinase phosphorylated by mTOR, and p90RSK, a serine/threonine kinase phosphorylated by ERK, was evaluated as a marker of each drug effect. The expression of histone γH2AX was increased immediately after irradiation but decreased in a time-dependent manner in drug-free control cells. However, interestingly, the expression of radiation-induced histone γH2AX was maintained in cells treated with the combination of drugs. Reduction in radiation-induced DNA damage occurred within 24 h after irradiation, and co-administration of MEK and mTOR inhibitors prolonged DNA damage due to the radiosensitizing effects of these drugs.
The combination treatment of trametinib and temsirolimus causes prolonged G2/M cell cycle arrest after irradiation in A549 cells. We evaluated changes in cell cycle phase distribution after treatment of A549 cells with mTOR and MEK inhibitors as well as irradiation. For this analysis, cells were pretreated with trametinib (100 nM) and/or temsirolimus (50 nM) for 1 h before irradiation with 8 Gy. The cells were further incubated with the drugs for 24, 48, and 72 h after irradiation. Then, the cells were stained with propidium iodide, and cell cycle phase distribution was analyzed by flow cytometry.
This experiment showed that radiation-induced G2/M arrest decreased in a time-dependent manner in drug-free control cells, while cells treated with trametinib alone or a combination of trametinib and temsirolimus exhibited prolonged radiation-induced G2/M arrest (Figure 4A).
We next examined the difference in the expression of G2/M cell cycle-related molecules (Figure 4B). We observed that the levels of phospho-chk2 (T68), p53, phospho-p53, and p27 were increased in cells treated with the combination of trametinib and temsirolimus as well as with irradiation compared to drug-free or single-agent treatment. Therefore, the combination treatment of trametinib and temsirolimus with radiotherapy prolonged the G2/M phase.
The combination treatment of trametinib and temsirolimus sensitizes lung cancer xenografts to radiotherapy. To validate our findings from in vitro experiments, we carried out in vivo experiments using a xenograft nude mouse tumor model with subcutaneously implanted A549 cells. When the average volume of xenografted tumors reached approximately 100 mm3, the mice were randomly assigned to the treatment groups. Combination treatment of trametinib and temsirolimus with radiation significantly decreased the tumor size compared to the other treatment groups (Figure 5A and B). Our results showed that the combination of MEK and mTOR inhibitors induces significant radiosensitization in lung tumor models.
Discussion
Radiation therapy is a type of cancer treatment that uses beams of intense energy to kill cancer cells and shrink tumors. The biological effect of radiation can be mainly attributed to DNA damage. DNA double-strand breaks are the most lethal lesions and are recognized as the primary mechanism of radiation-induced tumor cell death through the formation of chromosomal aberrations, cell cycle arrest, and apoptosis (21, 22).
Radiation therapy is an important treatment modality for lung cancer; however, resistance of cancer cells to radiation remains a major concern. Several signal transduction pathways, such as the MAPK and PI3K/AKT/mTOR pathways, are involved in radiotherapy resistance of many solid tumors, including NSCLC (7-16). Constitutive activation of both the MAPK as well as the PI3K/AKT/mTOR pathways leads to cancer cell proliferation, differentiation, and survival.
Many preclinical studies have demonstrated the benefit of MEK 1/2 inhibition as a radiation sensitizer in various tumors (8-10, 14). The MEK inhibitor selumetinib shows increased effects in combination with radiotherapy in lung cancer xenografts, compared with single therapy, through inhibition of the tumor hypoxia response (14). Another study reported that selumetinib enhances radiation response in vitro and in vivo by prolonging G2 arrest and increasing mitotic catastrophe after irradiation in human cancer cell lines (9). Trametinib, a potent and selective MEK1/2 inhibitor, causes radiosensitization of KRAS-driven pancreatic adenocarcinoma cells by suppressing major DNA double-strand break repair pathways. These data provide support for the combination of MEK1/2 inhibition and radiation in the treatment of malignant tumors (10).
The involvement of PI3K/AKT/mTOR activity in radioresistance has been reported in various solid tumors, including NSCLC, breast cancer, cervical cancer, glioma, and head and neck cancer (6, 11-13, 15, 16). Akt signaling promotes radiotherapy resistance in lung cancer cells and cervical cancer (15, 23). Chen et al. reported that the mTOR inhibitor everolimus enhances the radiosensitivity of NSCLC cells by suppressing epithelial-mesenchymal transition in vitro (11). A previous phase I trial of the combination of everolimus and radiation for untreated NSCLC demonstrated 41% partial response and 7% stable disease (24).
Our study shows that the combination of MEK and mTOR inhibitors with radiation results in a significant increase in NSCLC cell death and a reduction in overall tumor volume. Combination treatment of trametinib and temsirolimus 1) enhanced radiation-induced apoptosis, 2) induced prolonged expression of γH2AX and DNA DSB-related proteins after irradiation, 3) prolonged G2/M cell cycle arrest after irradiation in A549 cells, and 4) sensitized lung cancer xenografts to radiotherapy. To our knowledge, this is the first study to show that dual inhibition of MEK and mTOR provides an effective strategy to improve the radiation sensitivity of NSCLC.
Several studies have investigated the effects of co-targeting MAPK and PI3K signaling with concurrent radiotherapy as a strategy for cancer treatment. Toulany et al. showed that co-administration of MEK inhibitor (PD98059) and PI3K inhibitor (PI-103) blocked Akt reactivation, impaired double-strand break repair through non-homologous end joining, and enhanced the radiation response of K-ras-mutated NSCLC (25). These findings are consistent with the observations of our study. However, there were some differences with respect to the drugs and the cancer cell lines that were used. We used trametinib, a MEK inhibitor, and temsirolimus, an mTOR inhibitor. Both trametinib and temsirolimus are currently available and are commonly used drugs for the treatment of solid tumors. They also reported that targeting PI3K alone was not suitable for radiosensitizing K-ras-mutated NSCLC cells due to the crosstalk between the PI3K/Akt/mTOR and MEK/ERK pathways. If both pathways are not blocked, the inhibition of one pathway can lead to negative feedback and compensatory activation of the other due to the crosstalk between the two pathways. Williams et al. reported that dual targeting of MAPK and PI3K signaling with concurrent radiation therapy for the treatment of pancreatic cancer improved radiosensitization through cancer cell growth arrest and apoptosis (26).
In summary, our data show that the combination of trametinib (a highly specific MEK inhibitor) and temsirolimus (an mTOR inhibitor) sensitizes human lung cancer cells to radiation in vitro and in vivo. This effect is correlated with increased apoptosis and DNA damage and prolonged G2/M arrest in lung cancer cells. These findings suggest that dual targeting of MEK and mTOR is an efficient approach to improve the effect of radiotherapy in NSCLC.
Acknowledgements
This study was supported by a grant from the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by the Ministry of Science and ICT (MSIT), Republic of Korea (No. 50550-2020).
Footnotes
Authors’ Contributions
Cheol Hyeon Kim and Seo Yun Kim designed the experiments. Eun-Hui Jeong and Tae-Gul Lee conducted the experimental analyses. Seo Yun Kim, Eun-Hui Jeong, Hye-Ryoun Kim, and Cheol Hyeon Kim analyzed and interpreted the data. Seo Yun Kim and Cheol Hyeon Kim wrote the manuscript. All the Authors discussed the results and commented on and approved the manuscript.
Conflicts of Interest
The Authors declare that they have no conflicts of interest regarding this study.
- Received March 23, 2021.
- Revision received May 11, 2021.
- Accepted May 12, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.