Everolimus inhibits the proliferation and migration of epidermal growth factor receptor-resistant lung cancer cells A549 via regulating the microRNA-4328/phosphatase and tensin homolog signaling pathway.

Lung cancer is the most common cancer type worldwide, and investigating novel therapeutics methods for the treatment of chemoresistant lung cancer are of notable clinical significance. Reverse transcription-quantitative polymerase chain reaction and western blotting assays were performed to analyze the expression levels of phosphatase and tensin homolog (PTEN) and microRNA-4328 (miR-4328), and Cell Counting Kit-8 (CCK-8) and Transwell migration assays were conducted to evaluate the proliferation and migration of A549 cells, respectively. Everolimus was observed to upregulate the expression of PTEN and inhibit the proliferation and migration of A549 cells in a dose-dependent manner. The knockdown of PTEN abolished the effects of everolimus on the proliferation and migration of A549 cells, and everolimus was demonstrated to upregulate PTEN, and inhibit the proliferation and migration of A549 cells via downregulating miR-4328. Collectively, the results of the present study indicate that everolimus inhibited the proliferation and migration of EGFR-resistant A549 lung cancer cells via regulating the miR-4328/PTEN signaling pathway. Copyright: © Xiang et al.

Introduction

Lung cancer is one of the most common malignant cancer types, and was the leading cause of cancer-associated mortality globally in 2016 (1,2). Despite the prevalence of the disease, the prognosis of patients with lung cancer remains poor, with a 5-year survival rate of ~6–14% in males and 7–18% in females (3). Therefore, it is imperative to understand the mechanism of lung carcinogenesis and identify any candidate driver genes, which may be targeted by cancer therapy.

Epidermal growth factor receptor (EGFR) is a well-known oncogene in non-small cell lung cancer (NSCLC). EGFR-tyrosine kinase inhibitors (EGFR-TKIs) are an effective therapeutic method for the treatment of patients with NSCLC who harbor EGFR-activating mutations (4). However, the majority of patients with NSCLC develop drug resistance ~10 months following chemotherapy (5). Therefore, sensitizing EGFR-resistant lung cancer cells is important to cancer therapy.

Everolimus is an oral mechanistic target of rapamycin (mTOR) inhibitor derived from rapamycin that inhibits the tumorigenic phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mTOR driver pathway, and everolimus is currently approved for the treatment of metastatic (hormone-receptor positive, human EGFR2-negative) breast cancer, well-differentiated pancreatic neuroendocrine tumors, gastrointestinal or lung cancer and tyrosine-kinase-inhibitor-resistant renal cell carcinoma (6–8). The mTOR signaling pathway serves a central role in tumor cell proliferation in vitro and tumor growth in vivo (9). Numerous clinical trials using everolimus for a number of cancer types, including metastatic triple negative breast cancer and recurrent adult low-grade gliomas, are currently underway (10–12).

The phosphatase and tensin homolog (PTEN) is a crucial tumor suppressor gene located on chromosome 10q23.31 (13). Since PTEN is frequently inactivated in numerous human cancer types, including colorectal and breast cancer, through point mutations as well as deletions, it is an enticing therapeutic target for activation (14). PTEN-deficient cancer cells are hypothesized to be principal targets of mTOR inhibitors due to the loss of PTEN resulting in the activation of AKT and the subsequent upregulation of mTOR activity (14). MicroRNAs (miRs/miRNAs) are important in the regulation of PTEN, and it has been demonstrated that a number of miRNAs, including miR-92a and miR-215, have the potential to regulate the expression of PTEN (15,16). miR-4328 is a novel miR, and has been identified to be significantly downregulated in mucinous cystadenocarcinoma, compared with mucinous cystadenoma (17), and serves a critical role in the tension force-induced bone formation (18). Additionally, a previous study used the miRTarBase database to predict that miR-4328 could target PTEN (19). Recently, Seront et al (20) revealed that the loss of PTEN was associated with the resistance to the mTOR inhibitor everolimus in patients with advanced bladder cancer. However, whether PTEN can also serve an important role in EGFR-resistant lung cancer cells remains largely unknown.

To the best of our knowledge, the present study is the first to determine whether everolimus influences the proliferation and migration of EGFR-resistant A549 lung cancer cells, which are naturally resistant to the EGFR-TKI geritinib (21,22). Furthermore, the present study also preliminarily investigated the regulatory mechanism of everolimus on EGFR-resistant lung cancer cells.

Materials and methods

Reagents

Everolimus was purchased from Novartis Pharma AG (Basel, Switzerland), and anti-PTEN (cat. no. 9188; 1:1,000) and anti-β-actin (cat. no. 3700; 1:5,000) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Prior to addition to cell cultures, everolimus was prepared in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM.

Cell lines and everolimus treatment

A549 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 50 U/ml penicillin and 50 µg/ml streptomycin. All culture supplements were by Invitrogen (Thermo Fisher Scientific, Inc.) and cells were maintained at 37°C with 5% CO2.

Cells were treated at 37°C for 72 h with everolimus at 50 or 100 nM or a vehicle(0.01% DMSO) as a negative control.

Cell transfection

A549 cells were plated at a density of 1×105 cells/well, and allowed to adhere overnight in a 6-well dish, and the cells were transfected with PTEN small interfering RNA (siRNA), miR-4328 mimics or non-specific negative control, which were synthesized by GenePharma (GenePharma, Shanghai, China), using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The sequences were as follows: PTEN siRNA, 5′-GGCUAAGUGAAGAUGACAATT-3′; miR-4328 mimics, 5′-CCAGUUUUCCCAGGAUU-3′; and non-specific negative control, 5′-UUCUCCGAACGUGUCACGUTT-3′. At 24 h following transfection, everolimus was used to treat the transfected A549 cells, as aforementioned.

Cell proliferation assay

Cellular proliferation ability of A549 cells was measured by Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's protocol. At 24 h following transfection, cells were seeded into 96-well plates at a density of 8×103 cells/well with 100 µl cell culture medium (with 10% FBS), and at the indicated time points (days 1, 2, 3, 4 and 5), 10 µl CCK-8 was added to each well. The absorbance at 450 nm was detected by a plate reader following incubation for 1 h at 37°C. The experiments were repeated three times.

Cell migration assay

The migration abilities of A549 cells were analyzed with a Transwell assay, as described previously (23). Briefly, the cells were digested and seeded into the upper chamber (Transwell inserts) at a density of 3×105 cells/ml with cell culture medium containing 10% FBS. Cell culture medium supplemented with 20% FBS was added to the lower chamber, and incubated at 37°C for 36 h. Following this incubation, the membrane was stained with 0.1% crystal violet for 30 min at room temperature. These stained cells were counted using a light microscope following washing with PBS at ×20 magnification. The experiments were performed three times.

Luciferase reporter assay

pmiR-PTEN 3′untranslated region (UTR) wild-type or pmiR-PTEN 3′UTR mutant were transfected into A549 cells with pRL-TK vectors (Promega Corporation, Madison, WI, USA) with Lipofectamine 2000 for 24 h. Subsequently, 100 nM everolimus was added to treat the transfected A549 cells for 48 h at 37°C. Using a Dual Luciferase Reporter assay system (Promega Corporation), the luciferase activity was measured with a microplate luminometer (Infinite F200; Tecan, Männedorf, Switzerland) and the relative luciferase activity was normalized to firefly luciferase activity. The experiments were repeated three times.

Reverse transcription-quantitative polymerase chain reaction analysis (RT-qPCR)

Total RNA was isolated from the A549 cells using the RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol, and used for RT-qPCR. To detect the relative expression levels of PTEN, RT-qPCR was performed. The total PCR volume was 20 µl and consisted of 10 µl 2X Power SYBR® Green PCR Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 2 µl cDNA (5 ng/µl) and 1 µl primer mix (10 µM each). Using the LightCycler 480 II (Roche Applied Science, Penzberg, Germany) to perform PCR amplification and the procedure for PCR amplification was as follows: Denaturation at 95°C for 10 min, 40 cycles of 95°C for 15 sec, and then 60°C for 1 min. The comparative Cq method was used to calculate the relative gene expression level (24), and the expression levels of detected gene were normalized to an endogenous reference GAPDH. Additionally, those relative to the calibrator were provided by the formula 2−ΔΔCq (25). With the use of the Hairpin-it™ miR-4328 qRT-PCR Primer Set (Shanghai GenePharma, Co., Ltd., Shanghai, China), the relative quantity of miR-4328 was measured. The primers were as follows: PTEN forward, 5′-TGGATTCGACTTAGACTTGACCT-3′ and reverse, 5′-GGTGGGTTATGGTCTTCAAAAGG-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′. The experiments were performed three times.

Western blot analysis

Cultured A549 cells were harvested and lysed with radioimmunoprecipitation assay buffer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) was subsequently added, and cells were incubated on ice for protein extraction for 30 min. The protein concentration was measured by the bicinchoninic acid method (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Equal amounts of proteins (10 µg per sample) were separated by SDS-PAGE (12% gels) and transferred onto polyvinylidene fluoride membranes, which were blocked in 10% skimmed milk in PBS containing 0.1% Tween-20 for 2 h at room temperature. The membranes were probed overnight at 4°C with the aforementioned anti-PTEN and anti-β-actin primary monoclonal antibodies and in horseradish peroxidase (HRP)-conjugated secondary antibodies (rabbit anti-mouse and mouse anti-rabbit IgG; cat. nos. 58802 and 93702, respectively; 1:2,000; Cell Signaling Technology) for 1 h at 25°C. Using an enhanced chemiluminescence reagent (Sigma-Aldrich; Merck KGaA) to detect the target, β-actin was used as the normalization control. ImageJ software version 1.41 (National Institutes of Health, Bethesda, MD, USA) was used for the densitometry analysis. The experiments were performed three times.

Statistical analyses

Data was analyzed by GraphPad Prism version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) and presented as mean ± standard deviation. For multiple comparison analysis, one-way analysis of variance followed by Tukey's multiple comparison post-test was performed. P<0.05 was considered to indicate a statistically significant difference.

Results

Everolimus upregulates the expression of PTEN and inhibits the proliferation and migration of A549 cell in a dose-dependent manner

The present study treated A549 cells using everolimus at concentrations of 50 and 100 nM, and the PTEN expression was observed to be significantly enhanced (P<0.01), and the expression of the 100 nM group was increased, compared with the 50 nM group (Fig. 1A and B). The effects of everolimus on the cell proliferation and migration of lung cancer cells were also detected, and everolimus was demonstrated to significantly inhibit the proliferation and migration of A549 cells at 50 and 100 nM (P<0.05; Fig. 1C-E). The everolimus treatment also significantly upregulated the mRNA levels of PTEN at 50 and 100 nM (P<0.001; Fig. 1F).

Figure 1.

Everolimus upregulates the expression of PTEN and inhibits the proliferation and migration of A549 cells in a dose-dependent manner. (A) Everolimus increased the PTEN protein expression as detected by western blotting. (B) Quantification with statistical analysis of the western blotting results. (C) Effect of everolimus on the proliferation of A549 cells as analyzed with a Cell Counting Kit-8 assay. (D) Effect of everolimus on the migration of A549 cells. The migration of cells was measured with Transwell migration assays (×200 magnification). (E) Quantification and statistical analysis of the migration assays. (F) Everolimus increased the PTEN mRNA expression as detected by reverse transcription-quantitative polymerase chain reaction analysis. **P<0.01, ***P<0.001 vs. 0 nM everolimus. PTEN, phosphatase and tensin homolog; OD, optical density.

Knockdown of PTEN abolishes the effects of everolimus on the proliferation and migration of A549 cells

In order to investigate the roles of PTEN in the therapeutic process of everolimus on the A549 cells, transfection experiments were performed to knockdown the expression of PTEN and examine the effects of everolimus treatment. PTEN siRNA significantly reduced the expression of PTEN following everolimus treatment (P<0.001; Fig. 2A and B). Notably, it was identified that the knockdown of PTEN significantly enhanced the proliferation of A549 cells in the everolimus-treated samples (P<0.05; Fig. 2C). The present study also identified that silencing of PTEN reduces the effects of everolimus treatment on the migration of A549 cells (Fig. 2D and E).

Figure 2.

Knockdown of PTEN abolishes the effects of everolimus on the proliferation and migration of A549 cells. (A) Knockdown of PTEN was analyzed by western blotting. (B) Quantification and statistical analysis of the western blotting results. (C) Effect of knockdown of PTEN on the condition of everolimus on the proliferation of A549 cells analyzed with a Cell Counting Kit-8 assay. (D) Effect of knockdown of PTEN following treatment with everolimus on the migration of A549 cells. The migration of cells was measured with Transwell migration assays (×200 magnification). (E) Quantification and statistical analysis of the migration assay results. ***P<0.001 vs. 0 nM everolimus; ###P<0.001 vs. 100 nM everolimus without PTEN siRNA. PTEN, phosphatase and tensin homolog; OD, optical density; siRNA, small interfering RNA.

Everolimus upregulates PTEN and inhibits the proliferation and migration of A549 cells via downregulating miR-4328

The present study further demonstrated that everolimus significantly downregulated the expression of miR-4328, which has previously been predicted as the candidate upstream regulatory molecule for PTEN (19) (Fig. 3A). To confirm the association between miR-4328 and PTEN, a luciferase reporter assay was performed and treatment with everolimus significantly decreased the reporter activity in A549 cells following transfection with a wild-type vector (P<0.001; Fig. 3B). Additionally, when transfection with a mutant vector was performed, the reporter activity was not significantly altered between the everolimus treatment and negative control groups (Fig. 3B). The everolimus-treated A549 cells were additionally transfected with miR-4328 mimics, and the upregulation of PTEN induced by everolimus was inhibited at the mRNA and protein levels (Fig. 3C and D). In order to confirm the role miR-4328 in the treatment of everolimus, miR-4328 mimics were transfected into cells treated with everolimus, and the results demonstrated that overexpression of miR-4328 significantly reduced the effects of everolimus on the proliferation and migration of A549 cells (Fig. 3E-G).

Figure 3.

Everolimus upregulates PTEN and inhibits the proliferation and migration of A549 cells via downregulating miR-4328. (A) Everolimus decreased the expression of miR-4328 expression as detected by reverse transcription-quantitative polymerase chain reaction assay. (B) A dual-luciferase reporter assay was performed to detect the binding of miR-4328 and PTEN 3′untranslated region. miR-4328 mimics determined the effect of everolimus on the PTEN (C) mRNA and (D) protein expression. miR-4328 mimics demonstrated the effect of everolimus on the proliferation and migration of A549 cells detected with (E) Cell Counting Kit-8 and (F) a Transwell assay (×200 magnification). (G) Quantification and statistical analysis of the result of the Transwell assay. **P<0.01, ***P<0.001 vs. 0 nM everolimus treatment. ##P<0.01; ###P<0.001 vs. 100 nM everolimus without miR mimics. PTEN, phosphatase and tensin homolog; OD, optical density; NC, negative control; miR, microRNA.

Discussion

Everolimus, an oral mTOR inhibitor derived from rapamycin, inhibits the kinase activity of the raptor/mTOR complex directly by binding to FK506-binding protein 12, thus forming an inhibitory complex with mTOR (26). This type of mTOR inhibitor is well characterized for its anti-neoplastic properties and regulates cell proliferation via integrating signals from growth factors, nutrients, cytokines, hormones and cellular stress (9,27).

Everolimus is currently approved by the U.S. Food and Drug Administration for use with cases of advanced renal cell carcinoma and is routinely used in oncology practice as a substitute for traditional cytotoxic chemotherapy (28,29). Additionally, everolimus was recommended by the European Neuroendocrine Tumor Society consensus guidelines as a first-line treatment for advanced, progressive lung cancer (30,31). Lane et al (32) reported that everolimus inhibited vascular endothelial growth factor (VEGF)-stimulated proliferation of human endothelial cells and impaired the release of VEGF from tumor cells. Kuwahara et al (33) further demonstrated that the volumes of oral squamous cell carcinoma SAS cell- and radioresistant SAS (SAS-R)-derived tumors, as well as radio-sensitized SAS-R tumors, in xenograft tumor models were reduced following treatment with everolimus, and the influence of everolimus on tumor growth control was derived from promoting thrombosis in the SAS-R tumors rather than inhibiting VEGF expression in tumor cells. Previous studies also reported that everolimus has been approved as first-line and second-line therapy for the treatment of advanced renal cell carcinoma (29,34). In a study investigating human carcinoid lung neuroendocrine tumors (NET) xenografts, Johnbeck et al (35) demonstrated that everolimus notably inhibited almost 50% of tumor growth, compared with the placebo-treated tumors, indicating that everolimus has an anti-proliferative effect on H727 NET. Based on the currently available literature, US and European health authorities have approved the use of everolimus in the treatment of advanced pancreatic NET (35). These studies indicated that everolimus serves an important role in the abolition of tumors. This is consistent with the data of the present study demonstrating that everolimus significantly inhibited the proliferation and migration of EGFR-resistant A549 lung cancer cells in a dose-dependent manner.

It has been reported that tumor cells with the loss of PTEN function were sensitive to everolimus (36). PTEN is the second most frequently compromised tumor suppressor in human malignancies (37), and complete deficiency of PTEN protein expression is significantly associated with advanced cancer stage and poor prognosis (38,39). Loss of PTEN is associated with cancer progression, and 26% of primary breast cancer cases had low PTEN levels (38,40–42). PTEN is involved in the regulation of the cell cycle and apoptosis by blocking the PI3K/AKT signaling pathway (43,44). Furthermore, PTEN may directly bind to tumor protein 53 (p53) and increase p53 protein level (45).

miRs are expressed in a tissue-specific manner, serve a pivotal role in tumorigenesis and are dysregulated in a variety of cancer types, including ovarian cancer, lung cancer and colorectal cancer (46–48). In the present study, everolimus was revealed to upregulate the expression of PTEN in a dose-dependent manner. Additionally, silencing of PTEN reduced the effects of everolimus on the proliferation and migration of A549 cells. Notably, the present study also demonstrated that everolimus upregulated PTEN and inhibited the proliferation and migration of A549 cells via downregulating miR-4328.

The data of the present study revealed that everolimus inhibited the proliferation of EGFR-resistant A549 lung cancer cells via regulating the miR-4328/PTEN signaling pathway, and indicated that everolimus has a therapeutic effect on EGFR-resistant lung cancer cells. A limitation of the present study is that the effect of everolimus on the miR-4328/PTEN signaling pathway was only detected in a single cell line; therefore, these findings should be confirmed in animal models in the future.