Targeting the mitogen‑activated protein kinase kinase and protein kinase A pathways overcomes acquired resistance to Selumetinib in low‑grade glioma cells
- Authors:
- Published online on: November 25, 2020 https://doi.org/10.3892/or.2020.7867
- Pages: 752-763
Abstract
Introduction
Brain tumors are the leading cause of cancer-related mortality in children, despite their lower incidence (1.12-5.14 cases per 100,000 individuals) compared with that of hematological malignancies in this age group (1,2). Pediatric low-grade gliomas (PLGGs) comprise a histologically heterogeneous group of World Health Organization grade I and II tumors (3), and they are the most common type of central nervous system tumor in children (4).
The prognosis and treatment of PLGGs depend on patient age, tumor location, dimensions and histopathological characteristics (5). When complete resection is possible, patients usually do not require any adjuvant therapy, whereas partial resection is generally associated with tumor recurrence and the patients may need additional treatment, such as chemotherapy or radiotherapy (6,7). Unfortunately, the use of ionizing radiation and conventional chemotherapies in pediatric patients may damage the developing brain, often leading to significant permanent neurocognitive and systemic complications including loss of vision and/or hearing, endocrinopathies and mood or behavior disorders (7–9). In addition, while the clinical symptoms of PLGG may take months to years to progress, its management by cytotoxic chemotherapy has been demonstrated ineffective due to the slow growth rate of the tumor.
Tumors exploit various pathways that are indispensable for the regulation of cancer cell biology (10). The extracellular signal-regulated kinase (ERK) pathway has been the subject of extensive research in recent years due to its key role in the regulation of cancer cell proliferation, survival and metastasis (11). The mitogen-activated protein kinase (MAPK)-ERK signaling pathway has been demonstrated to be fundamental for the tumorigenesis of PLGGs, and pharmacological targeting of this pathway with small molecule kinase inhibitors may represent a viable therapeutic approach (11–13). Mitogen-activated protein kinase kinase (MEK)1 and MEK2 are dual-specificity protein kinases that function in the MAPK cascade, controlling cell proliferation and differentiation; MEK1/2 activate ERK 1/2, which have wide substrate specificity, resulting in the activation of a multitude of cellular responses involved in the control of cell proliferation, differentiation and apoptosis (14–16).
Selumetinib (AZD6244; ARRY-142886) is a selective, ATP-uncompetitive inhibitor of MEK1/2 (17). Selumetinib specifically binds to MEK1/2 and induces several conformational changes in the unphosphorylated MEK1/2 enzymes by inhibiting their catalytic activity, which results in the inhibition of ERK activation and the blockade of the signal transduction pathways (17,18). This small molecule was previously investigated in clinical trials for the treatment of melanoma (NCT01974752), biliary (NCT00553332), colorectal (NCT01116271), pancreatic (NCT00372944) and advanced non-small cell lung (NCT01783197) cancer as well as a variety of other malignancies (18–21).
The antitumor effect of Selumetinib on PLGGs has yet to be fully characterized. However, as activation of the MEK-ERK pathway inhibits apoptosis and promotes cell cycle progression through increasing cyclin D expression (22), inhibition of the MAPK pathway through specific targeted therapies appears to be a promising strategy for the treatment of PLGGs.
The aim of the present study was to investigate the antitumor activity of Selumetinib in vitro in two PLGG cell lines and to determine the effects of Selumetinib treatment on the key intracellular downstream signaling pathways. In addition, a cellular model of acquired resistance to Selumetinib was established in order to investigate whether the addition of a selective protein kinase A (PKA) inhibitor cAMP analogue to Selumetinib may represent a possible synergistic therapeutic strategy for reversing acquired resistance to this MEK inhibitor.
Materials and methods
Reagents
Selumetinib (AZD6244) was purchased from AdooQ Bioscience, dissolved to 67.3 mM in DMSO and stored at −20°C. 8-Chloroadenosine 3′,5′-cyclic-monophosphate (8-Cl-cAMP) was obtained from Sigma-Aldrich; Merck KGaA, dissolved in a solution of 0.1 M NH4OH to 27.49 mM and stored at −20°C.
Cell lines
The pediatric glioma cell lines Res186 and Res259 were kindly provided by Professor Chris Jones (Glioma Team, Centre for Evolution and Cancer Divisions of Molecular Pathology and Cancer Therapeutics, The Institute of Cancer Research, London, UK). Res186 and Res259 cells were derived from a 3-year-old female patient with pilocytic astrocytoma and a 4-year-old female patient with diffuse astrocytoma, respectively (23). The PLGG cell lines were cultured at 37°C with 5% CO2 in DMEM F-12 medium (Corning, Inc.) supplemented with 10% fetal bovine serum (FBS; PAN-Biotech GmbH), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin.
The Selumetinib-resistant cell line, referred to as Res259/R, was generated by continuous exposure (6 months) of the parental cell line Res259 to gradually increasing Selumetinib concentrations (1-25 µM) and maintained in a medium containing 25 µM Selumetinib. The half maximal inhibitory concentration (IC50) values in Selumetinib-sensitive and resistant Res259 cell lines was 1 and 25 µM, respectively, with a resistance index (RI) of ~25-fold. Experiments were performed using cells cultured in a Selumetinib-free medium for ≥15 days.
Cell viability assay
Res259, Res186 and Res259/R cells were seeded in 6-well plates overnight in medium supplemented with 10% FBS at 37°C. Each cell line was optimized for seeding cell number to ensure a similar degree of confluence at the end of the experiment in the untreated (control) wells (Res259, 4×104; Res186, 5×104; Res259/R, 3.5×104 cells/well). After 24 h, the cells were starved in DMEM F-12 medium with 0.5% FBS. Vehicle (DMSO) or serial concentrations (0.01, 0.05, 0.25, 0.5, 1, 2.5, 10 and 25 µM) of Selumetinib were added to the medium, and the cells were cultured for 72 h at 37°C. Cell number and viability, as well as the number of dead cells were assessed by an automated cell counter (NucleoCounter 100®; Chemometec), and expressed as a percentage of treated cells relative to the untreated control cells. All experiments were repeated at least five times in duplicate. The IC50 was calculated using GraphPad Prism version 6.0 (GraphPad Software, Inc.).
BRAF mutational analysis
DNA extraction from Res186 and Res259 cells was performed using the QIAamp DNA Mini kit (Qiagen GmbH). Exons 11 and 15 of the BRAF gene were amplified using the following primers: Exon 11 forward, 5′-TTATTGATGCGAACAGTGAATAT-3′ and reverse, 5′-TTACAGTGGGACAAAGAATTG-3′; exon 15 forward, 5′-TCATAATGCTTGCTCTGATAGGA-3′ and reverse, 5′-GGCCAAAAATTTAATCAGTGGA-3′. Briefly, DNA (100-200 ng) was amplified in a mixture containing 1X PCR buffer (20 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl2), dNTPs (200 mM each), primers (20 pM each) and 0.5 unit GoTaq polymerase (Promega Corporation) in a final volume of 25 µl. PCR conditions were as follows: Initial denaturation at 95°C for 8 min, followed by 35 cycles of 95°C for 40 sec, 55°C for 40 sec and 72°C for 40 sec. After visualization on agarose gels, the PCR products were treated with ExoSAP-IT (USB Corporation) according to the manufacturer's protocol, amplified for 25 cycles of 95°C for 10 sec, 50°C for 5 sec and 60°C for 4 min with a BigDye Terminator cycle sequencing kit (version 3.1; Applied Biosystems; Thermo Fisher Scientific, Inc.) using forward and reverse primers, and sequenced with an ABI PRISM 3100-Avant Genetic Analyzer with Sequencing Analysis Software™ v.3.6.2 (Applied Biosystems; Thermo Fisher Scientific, Inc).
Cell cycle analysis by flow cytometry
Res186, Res 259 and Res 259/R cells were exposed to Selumetinib (1 and 2.5 µM for Res186; 0.5 and 1 µM for Res259; 5 and 25 µM for Res259/R) and collected after 24, 48 or 72 h. At the end of each incubation period at 37°C, adherent cells were trypsinized, harvested and washed with cold PBS. The cells were then counted by an automated cell counter, gently fixed in 70% v/v cold ethanol and incubated at −20°C for ≤7 days. Fixed cells were stained with a PBS solution containing RNase (5 µl RNase solution, 10 mg/ml per 2×105 cells) and 50 µg/ml propidium iodide (PI) and stored at 4°C overnight. Flow cytometric DNA ploidy analysis was performed by acquiring a minimum of 3×104 cells at a low flow rate for each sample using a six-parameter (two scatter and four fluorescence signals) EPICS-XL flow cytometer (Beckman Coulter, Inc.). Analysis of cell cycle distribution was performed using FCS Express 7 Research Edition (De Novo Software). All experiments were repeated at least three times in duplicate.
Western blotting
Res186, Res259 and Res259/R cells treated with Selumetinib were lysed in lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin and 1 mM phenylmethylsulfonyl fluoride), and protein concentration was determined by a Dc protein assay (Bio-Rad Laboratories, Inc.). Total lysates (25 µg/lane) were subjected to electrophoresis on 8-12% polyacrylamide gels, transferred to a Hybond nitrocellulose membrane (Amersham; Cytiva) and probed with appropriate dilutions of the following primary antibodies: Anti-AKT, anti-phosphorylated (p)-AKT (Ser473), anti-MAPK, anti-p-MAPK (Thr202/Tyr204), anti-p-MEK1/2, anti-MEK1/2, anti-p27, anti-p21, anti-cyclin D1, anti-poly(ADP-ribose) polymerase (PARP), anti-caspase 9, anti-caspase 3, anti-cytochrome c, anti-p85, anti-neurofibromin (NF1), anti-Bax and anti-β-actin (Table I) overnight at 4°C. The membranes were washed with 1X TBS and incubated with secondary anti-rabbit or anti-mouse antibodies conjugated with horseradish peroxidase for 2 h at room temperature. The membranes were developed using an ECL detection system (Amersham; Cytiva), and chemiluminescence signals were captured using the ChemiDoc XRS system (Bio-Rad Laboratories, Inc.). For phosphorylation analysis in Res186 and Res259 cells, the membranes were initially probed with antibodies for the p-epitopes, then stripped by incubation in 62.5 mM Tris-HCl pH 6.8, 2% SDS and 100 mM β-mercaptoethanol at 50°C for 30 min, washed, blocked and reprobed with antibodies recognizing total AKT, MEK or MAPK.
Co-treatment experiments
Before testing the combination treatment in the in vitro glioma models, PLGG cell lines were treated with 0.5-30 µM 8-Cl-cAMP alone at 37°C for 72 h, and cell viability was assessed by an automated cell counter. For the co-treatment experiment, Res259 (4×104 cells/well), Res186 (5×104 cells/well) and Res259/R (3.5×104 cells/well) were exposed to 0.01-5 µM Selumetinib and 0.5-40 µM 8-Cl-cAMP at a fixed drug ratio at 37°C for 72 h. For each cell line, the drug ratio was selected based on the respective IC30 values of the two drugs as follows: Drug ratio=IC30 (Selumetinib)/IC30 (8-Cl-cAMP). Cell viability was assessed by an automated cell counter, and proliferation inhibition (%) was determined using the following equation: Proliferation inhibition (%)=1-(mean treated cell number)/(mean control cell number) ×100. The results of the combination treatments were analyzed using the Chou-Talalay method (24), and the combination index (CI) was calculated using CalcuSyn software (Biosoft, version 1.0). CI values were interpreted as follows: <1, synergism; 1, additivity; >1, antagonism.
Statistical analysis
The data are presented as the mean ± standard deviation. GraphPad Prism software version 5.0 (GraphPad Software, Inc.) was used for statistical analyses. When comparing two groups, a two-tailed unpaired Student's t-test was used to determine statistical significance. When comparing more than two groups, one-way or two-way ANOVA was performed according to the design of the experiment. Dunnett's multiple comparison test was used to compare the treatment and control groups. P<0.05 was considered to indicate a statistically significant difference.
Results
Effects of Selumetinib on PLGG cell proliferation and viability
To determine the effects of Selumetinib on Res186 and Res259 cells, cell proliferation and viability following treatment with 0.01-25 µM Selumetinib were determined by an automated cell counter after 3-day treatment. As presented in Fig. 1A, Selumetinib reduced the proliferation of the Res186 and Res259 cells, with higher concentrations of Selumetinib achieving more effective inhibition. In Res259, the IC50 value was observed to be 1.0 µM, whereas in Res186, the IC50 value was 2.5 µM. Based on these results, Res259 cells were classified as sensitive (S), whereas Res186 cells were classed as resistant (R), with Selumetinib IC50 at 72 h of ≤1 and >1 µM, respectively. This cut-off value was selected on the basis of a previous phase I trial reported by Adjei et al (25), where 1 µM was the mean plasma drug concentration achieved in patients at the maximum tolerated dose.
BRAF mutation analysis
The sequencing analysis of BRAF mutations in the Res186 and Res259 cell lines demonstrated that both cell lines had a wild-type BRAF sequence (data not shown).
Effects of Selumetinib on the cell cycle in PLGG cell lines
Res186 and Res259 cells were exposed to the respective IC30 and IC50 of Selumetinib for 24, 48 and 72 h, and the effects on the cell distribution in different cell cycle phases was evaluated by flow cytometry. Res259 and Res186 cells were observed to accumulate at the G0/G1 phase of the cell cycle after 24- and 48-h treatment. In both cell lines, this effect was observed to be dose-dependent and was accompanied by a reduction of cells in the S phase. At 72 h, the percentages of cells in the G0/G1 phase were comparable between the treated and control cells (Fig. 2B), suggesting that the disruption of the cell cycle progression through the G0/G1 and S phases occurred within 48 h of drug exposure and was transient.
Short-term effects of Selumetinib on intracellular signaling in PLGG cell lines
To evaluate the effects of Selumetinib treatment on the key intracellular downstream signaling pathways, modulation of MEK1/2, MAPK and AKT was assessed by western blot experiments. Both cell lines were treated for various times with Selumetinib at their respective IC30 and IC50. The results demonstrated that Selumetinib treatment promoted the phosphorylation of AKT and MEK1/2 after 30 min in the two cell lines at IC30 and IC50, although a slightly lower pattern of activation was observed in the sensitive Res259 cell line. By contrast, Selumetinib completely suppressed MAPK phosphorylation in Res186 and Res259 cells. The levels of total AKT, MEK and MAPK did not change during the experiment (Fig. 3A).
The main mechanism underlying the effects of Selumetinib on the PLGG cell lines was further examined. Both PLGG cell lines exhibited an increase in the p27 protein expression levels after 24 h drug exposure at IC30 and IC50, whereas the phosphorylation of p21 was observed only at 24 h in Res186 cells. In addition, p85 was activated after 15-min treatment in Res186 cells; by contrast, in Res259 cells, the phosphorylation of p85 was high at 24 h. Selumetinib did not induce the cleavage of PARP in either cell line within 24 h of treatment (Fig. 3B).
Establishment of a Selumetinib-resistant PLGG cell line and mechanisms associated with resistance development
To establish a PLGG cell line with an acquired resistance to Selumetinib, Res259 cells were cultured long-term (6 months) and gradually exposed to increasing concentrations of the drug. Continuous and prolonged treatment resulted in a loss of sensitivity to Selumetinib and the establishment of the Res259/R cell line. As demonstrated in Fig. 4A, the IC50 value at 72 h was 25 µM, which was 25-fold higher compared with the sensitive Res259 cell line (RI, ~25).
To determine the potential molecular biomarkers for Selumetinib resistance in the newly established cell line, the expression levels of the principal intracellular proteins involved in the response to Selumetinib were first determined. In Res259/R cells, higher levels of p-MEK and p-AKT were observed compared with those in the sensitive parental glioma cell line. Furthermore, the resistant cells appeared to express a slightly higher level of p-MAPK compared with their sensitive counterparts, as illustrated in Fig. 4B. Since the resistant and sensitive cell lines appeared to exhibit different degrees of AKT, MEK and MAPK phosphorylation, the basal expression of NF1, the upstream negative regulator of MEK and AKT signaling, was next analyzed (26,27). The results demonstrated that Res259/R cells appeared to exhibit lower basal NF1 levels compared with those in the sensitive cell line Res259, suggesting that in Res259/R cells, low NF1 expression may lead to uncontrolled activation of its downstream effectors.
The effects of Selumetinib on the cell cycle progression in resistant cells were determined. Notably, it was observed that the distribution of Res259/R cells in the various phases of the cell cycle differed slightly from that in the parental Res259 cell line, as higher numbers of Res259/R cells were observed in the proliferative phases of the cell cycle (S-G2/M) compared with those of Res259 cells (≥50 and <30%, respectively) at 24 h of culture. In Res259/R cells, Selumetinib triggered cell cycle arrest at the G0/G1 phase of the cell cycle after 24 and 48 h (Fig. 4C). Following 72-h drug exposure, no marked accumulation of cells was observed in the G0/G1 phase, similar to the parental Res259 cells (Fig. 2B).
Finally, after 24-h treatment, Selumetinib promoted AKT activation in sensitive Res259 cells, whereas the high levels of p-AKT observed in the Res259/R cell line were suppressed by Selumetinib treatment. Notably, in both glioma models of resistance and sensitivity to Selumetinib, MAPK phosphorylation was inhibited following drug exposure (Fig. 4D).
Long-term effects of Selumetinib treatment on PLGG cell lines
In order to explore the long-term effects of Selumetinib treatment on the in vitro PLGG models, Res259, Res259/R and Res186 cells were treated with Selumetinib for 3 and 7 days at their respective IC30, and the expression levels of a range of proteins involved in the regulation of the cell cycle and apoptosis were assessed by western blot analysis. The modulation of the main molecular key mediators of the cell cycle following Selumetinib treatment were first examined in the resistant and sensitive glioma cell models. As presented in Fig. 5, MEK inhibition by Selumetinib induced an apparent reduction in cyclin D1 expression in Res186 and Res259 cells at different treatment times, whereas a slight decrease of this cell cycle regulator was only observed in Res259/R cells after 7 days of drug exposure. In the intrinsically resistant glioma cell line Res186, the decrease in cyclin D1 expression levels appeared to be accompanied by an increase in the levels of p21 after 3 days of drug exposure. By contrast, in Res259/R cells, a moderate reduction in cyclin D1 expression was observed after 7-day drug exposure, which was associated with upregulation of p27 rather than p21. Similarly, in the Res259 cells, a decrease in the protein expression levels of cyclin D1 was observed to be associated with a slight enhancement of p27 levels after 7 days of drug exposure. Notably, in Res259/R cells, the 6-month treatment with Selumetinib appeared to induce a reduction in the basal levels of the checkpoint proteins of the G1-S transition (cyclin D1 and p21) compared with those in the parental cell line Res259. These results may indicate a more aggressive pattern of proliferation in the cellular model with acquired resistance Selumetinib compared with that in the sensitive cell line. It was then investigated whether the apoptosis machinery was activated by Selumetinib treatment. The results demonstrated that in Res259/R cells, Selumetinib appeared to slightly enhance the expression of cleaved caspase-9 following 7-day drug exposure, which was in accordance with caspase-3 activation at the same time. Furthermore, after 7 days of treatment, the 89-kDa cleaved PARP fragment was detected, and the levels of the pro-apoptotic factor Bax appeared to be increased. The resistant cell line Res186 exhibited different modulation of apoptosis regulators compared with those in Res259/R, suggesting that the underlying mechanisms of apoptosis and cell cycle arrest may vary between the two models of intrinsic and acquired Selumetinib resistance; in Res186 cells, Selumetinib induced a reduction of total PARP levels, but no caspase activation was noted even after 7 days of treatment.
Effects of the co-treatment with 8-Cl-cAMP and Selumetinib in PLGG cell lines
A number of studies have reported the importance of cAMP signaling in glioma (28–31) and the antiproliferative effects of cAMP analogs on tumor cells (32–34). Thus, 8-Cl-cAMP was tested alone and in combination with Selumetinib to determine whether this cAMP analogue may overcome resistance to MEK inhibitors or interact with Selumetinib.
Before assessing the effects of the combination therapy, the antiproliferative effect of 8-Cl-cAMP on the PLGG cell lines was determined. Res186, Res 259 and Res259/R cells were treated with increasing concentrations of 8-Cl-cAMP for 72 h. As presented in Fig. 6A, a modest inhibition of cell viability was observed in the glioma cell lines only at high 8-Cl-cAMP concentrations (30 µM). The Selumetinib-resistant cell lines Res186 and Res259/R appeared to exhibit the same degree of sensitivity to 8-Cl-cAMP with an IC30 value of 20 µM, whereas the IC30 of Res259 was 10 µM.
For the combination experiments, PLGG cells were exposed to a range of concentrations of Selumetinib and 8-Cl-cAMP for 72 h at a fixed drug ratio for each cell line. Notably, the two resistant cell lines responded differently to the combination treatment; in the Res259/R cells, which exhibited acquired resistance to Selumetinib, co-treatment with Selumetinib and 8-Cl-cAMP induced a synergistic effect on cell viability, with the CI ranging between 0.44 and 0.68 (Fig. 6B and D). The addition of 8-Cl-cAMP did not sensitize the intrinsically resistant cell line Res186 to Selumetinib (data not shown). Similarly, in sensitive Res259 cells, combination treatment did not result in a synergistic effect (data not shown). Taken together, these results suggested that the addition of 8-Cl-cAMP to the single agent Selumetinib may overcome acquired, but not intrinsic resistance to this MEK inhibitor.
Western blot analysis was next performed on Res259/R cells to assess the activation of the apoptosis machinery and the principal effectors of the MAPK and AKT cascade following co-treatment with Selumetinib and 8-Cl-cAMP. Notably, the addition of 8-Cl-cAMP to Selumetinib appeared to reduce MEK phosphorylation induced by the Selumetinib alone within a shorter time period, but this effect was not maintained with longer exposure to the two drugs (Fig. 6C). In addition, the drug combination did not promote the expression of the main pro-apoptotic factors and did not affect p-AKT levels (Fig. 6C), thus suggesting that the synergistic effect on cell viability inhibition may involve other biochemical mechanisms. In addition, the protein expression levels of total AKT, MEK and MAPK did not change during the experiment.
Discussion
Over the last decade, gene expression profiling has provided new insights into the oncogenesis of PLGGs and has enabled the development of targeted therapies for these tumors (5). Uncontrolled activation of the MEK-MAPK signaling pathway is a well-known mechanism involved in the survival and proliferation of cancer cells in several types of malignancy, including glioma (12,13). The highly selective small molecule MEK inhibitor Selumetinib has been demonstrated to be effective in clinical trials for adult cancer as a single agent (16,17,25) or as part of combination treatments (18–21), and is currently under evaluation for patients with refractory or recurrent PLGGs (clinical trial no. NCT01089101) (35,36). Despite MEK inhibition representing a promising chemotherapeutic approach, resistance to MEK inhibitors has been reported in the context of several types of cancer, such as melanoma and acute myelod leukemia (37–42). In the present study, the different sensitivity of two in vitro models of PLGG to single-agent Selumetinib treatment was demonstrated; Res259 was classified as a sensitive cell line, and Res186 as an intrinsically resistant cellular model. Notably, despite the different responses to the treatment, neither cell line harbored BRAF mutations (data not shown). BRAF mutations, particularly BRAFV600E, have been extensively reported to be associated with sensitivity to MEK inhibitors in various types of cancer (43–47), including melanoma, colorectal carcinoma and gliomas. Consistently with the results of the present study, previous studies by Ranzani et al (48) and Ming et al (49) have also demonstrated MEK inhibition sensitivity in a subset of BRAF wild-type cancer cell lines, which indicates that BRAF mutations per se may not predict sensitivity to Selumetinib. However, since pediatric patients with BRAF-mutated PLGGs may represent the best candidates for receiving Selumetinib as an alternative treatment to standard chemotherapy (50), a potential limitation of the present study was the lack of experiments using BRAF-mutated PLGG cells, which may have been more representative of the aforementioned group of patients.
The present study next evaluated whether inhibition of MAPK may be a potential biomarker for distinguishing between sensitive and intrinsically resistant cells. Previous studies on leukemia and colorectal cancer cells have revealed a substantial reduction of phosphorylated MAPK, regardless of sensitivity to the MEK inhibitor (51–53). Similar to these studies, Selumetinib suppressed MAPK phosphorylation in Res186 and Res259 cells, suggesting that, in the PLGG cell lines used in the present study, modulation of the intracellular effector MAPK may not be the only driver of cell proliferation inhibition following exposure to Selumetinib. When Res186 and Res259 cells were exposed to their respective IC30 for an extended period, decreases in the levels of cyclin D1 in both cell lines were observed. However, in Res186 cells, cyclin D1 reduction appeared to be associated with high levels of the cyclin-dependent kinase inhibitor p21, whereas in the sensitive cells, a decrease in cyclin D1 expression appeared to be associated with the p27 rather than the p21 pathway. In order to further elucidate the mechanisms underlying Selumetinib resistance, a cellular model with acquired resistance to Selumetinib, referred to as Res259/R, was established. With the aim of identifying molecular markers of acquired resistance to Selumetinib in PLGG cells, the basal activity of the main intracellular effectors, which were previously observed to be modulated by Selumetinib, was first determined. Hyperactivation of the AKT pathway is a mechanism of resistance to MEK inhibitors in lung cancer, melanoma and colorectal cancer cells (54–56). In addition to high expression levels of AKT, previous studies have demonstrated that resistance to Selumetinib may be either mediated by a strong coincident activation of the PI3K/AKT and ERK1/2 pathways or may be partly attributed to the reactivation of the MEK-ERK signaling pathway despite ongoing treatment with Selumetinib (57,58). In line with these studies, the results of the present study suggested that exposure to Selumetinib for extended periods may lead to positive selection of resistant cells characterized by high levels of the key intracellular regulators for cell proliferation and survival (AKT and MEK). Furthermore, our in vitro model of acquired resistance to Selumetinib was observed to express lower levels of the checkpoint proteins for the G1-S transition of the cell cycle cyclin D1 and p21 compared with those in Res259 cells, which may indicate a more aggressive pattern of proliferation of Res259/R cells compared with that of the parental cell line.
Overactivation of the Ras/Raf/MEK/ERK and PI3K/AKT pathways is also involved in gliomagenesis, and their regulation is dependent on the upstream tumor suppressor protein NF1 (26). NF1 is a Ras GTPase-activating protein, the interaction of which with the Ras proto-oncogene triggers its conversion from the active GTP-bound Ras to the inactive GDP-bound form (26,27). Loss of NF1 or impaired NF1 function leads to uncontrolled activation of Ras, which in turns results in high levels of MEK/MAPK and PI3K/AKT activity (27). As higher levels of phosphorylation of AKT and MEK were observed in Res259/R cells compared with those in the Res259 cell line, NF1 expression levels were next assessed in these cellular models. The results demonstrated that the resistant cell line Res259/R appeared to exhibit lower basal NF1 levels compared with those in the sensitive Res259 cell line, suggesting that low expression of the tumor suppressor protein NF1 may lead to the uncontrolled activation of its downstream effectors in cells, characterized by acquired resistance to Selumetinib. Consistently, NF1 expression levels appeared higher in the sensitive Res259 cells, which may explain the lower AKT and MEK phosphorylation compared with that in the resistant cellular model. Contrasting results have been published regarding a possible association between the sensitivity to MEK inhibitors and NF1 expression in several types of cancer cell lines. For example, NF1-deficient acute myeloid leukemia cells and NF1-deficient glioblastoma cells have been reported to be sensitive to MEK inhibitors (59,60) and Nissan et al (61) have reported NF1 loss as a biomarker of sensitivity to the MEK inhibitor in melanoma cells. By contrast, Whittaker et al (62) have demonstrated that the loss of NF1 was a mediator of resistance to Selumetinib in melanoma cell lines. Although a complete loss of NF1 expression was not observed in Res259/R cells, it may be hypothesized that there is a possible association between low NF1 levels and Selumetinib resistance in these in vitro PLGG models.
In recent years, combination treatments have been proposed for overcoming resistance to MEK inhibitors (63). Since the PI3K/AKT pathway has been demonstrated to be hyperactivated in cancers with acquired resistance (56,64,65), the addition of PI3K, AKT or mTOR inhibitors to MEK inhibitors is currently considered one of the most promising combination strategies for the treatment of MEK inhibitor-resistant tumors (66–69). In addition, a recent study by Arnold et al (70) has demonstrated the synergistic effect on PLGG cell proliferation inhibition by combining the mTOR inhibitor TAK228 and the MEK inhibitor Trametinib. In the present study, a cAMP analogue 8-Cl-cAMP was added to Selumetinib in order to evaluate whether this combination treatment may overcome acquired resistance to Selumetinib in the in vitro PLGG models. Although the mode of action of 8-Cl-cAMP has not been completely elucidated, this site-specific cAMP analogue is likely to selectively downregulate the PKA-I isoform, a signaling protein directly involved in cell proliferation, differentiation and neoplastic transformation (34). Since the cAMP/PKA pathway serves a fundamental role in the regulation of several cancer cell functions, drugs targeting this signaling pathway such as 8-Cl-cAMP have been reported to exert antitumor effects in various types of cancer (33,34,71), including gliomas (72–74). In the present study, 8-Cl-cAMP alone induced modestly inhibited the viability of the Selumetinib-resistant and sensitive cell lines. However, the addition of this cAMP analogue to Selumetinib in Res259/R cells exerted a synergist effect on cell viability inhibition (CI<1), suggesting that 8-Cl-cAMP may help reverse acquired Selumetinib resistance in the PLGG cellular model used in the present study.
In conclusion, the present study investigated three PLGG cell lines, including Res259, Res259/R and Res186, which were characterized by sensitivity, acquired resistance and intrinsic resistance to Selumetinib, respectively. In all the evaluated in vitro models, Selumetinib exerted a cytostatic effect by inducing G0/G1 phase arrest and suppressed MAPK phosphorylation, although at different concentrations. The identification of predictive biomarkers of Selumetinib sensitivity is paramount to selecting the appropriate treatment for patients with PLGG. In this context, the results of the present study demonstrated that the acquired resistance of Res259/R cells to Selumetinib was associated with a higher basal level of p-AKT and lower levels of NF1 compared with those in the parental sensitive cell model. Although further investigations are required, co-treatment with 8-Cl-cAMP and Selumetinib may be an effective combination strategy to overcome acquired resistance to Selumetinib in PLGGs.
Acknowledgements
Not applicable.
Funding
This work was supported by Fondazione per l'Oncologia Pediatrica.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
LM and DM conceived and designed the study, collected, analyzed and interpreted the data and wrote the manuscript. ABa, AR, MM and GS collected, analyzed and interpreted the data. ABu collected the data. RR conceived and designed the study and provided financial support. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
|
Ostrom QT, Gittleman H, Fulop J, Liu M, Blanda R, Kromer C, Wolinsky Y, Kruchko C and Barnholtz-Sloan JS: CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2008-2012. Neuro Oncol. 17 (Suppl 4):iv1–iv62. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Johnson KJ, Cullen J, Barnholtz-Sloan JS, Ostrom QT, Langer CE, Turner MC, McKean-Cowdin R, Fisher JL, Lupo PJ, Partap S, et al: Childhood brain tumor epidemiology: A brain tumor epidemiology consortium review. Cancer Epidemiol Biomarkers Prev. 23:2716–2736. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P and Ellison DW: The 2016 world health organization classification of tumors of the central nervous system: A summary. Acta Neuropathol. 131:803–82. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Sievert AJ and Fisher MJ: Pediatric low-grade gliomas. J Child Neurol. 24:1397–1408. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Bergthold G, Bandopadhayay P, Bi WL, Ramkissoon L, Stiles C, Segal RA, Beroukhim R, Ligon KL, Grill J and Kieran MW: Pediatric low-grade gliomas: How modern biology reshapes the clinical field. Biochim Biophys Acta. 1845:294–307. 2014.PubMed/NCBI | |
|
Chalil A and Ramaswamy V: Low grade gliomas in children. J Child Neurol. 31:517–522. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Dasgupta T, Olow AK, Yang X, Hashizume R, Nicolaides TP, Tom M, Aoki Y, Berger MS, Weiss WA, Stalpers LJ, et al: Survival advantage combining a BRAF inhibitor and radiation in BRAF V600E-mutant glioma. Neurooncol. 126:385–393. 2016. View Article : Google Scholar | |
|
Ater Jl, Zhou T, Holmes E, Mazewski CM, Booth TN, Freyer DR, Lazarus KH, Packer RJ, Prados M, Sposto R, et al: Randomized study of two chemotherapy regimens for treatment of low-grade glioma in young children: A report from the children's oncology group. J Clin Oncol. 30:2641–2647. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Bandopadhayay P, Bergthold G, London WB, Goumnerova LC, Morales La Madrid A, Marcus KJ, Guo D, Ullrich NJ, Robison NJ, Chi SN, et al: Long-term outcome of 4,040 children diagnosed with pediatric low-grade gliomas: An analysis of the surveillance epidemiology and end results (SEER) database. Pediatr Blood Cancer. 61:1173–1179. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Weuken JW and Wesseling P: MAPK pathway activation through BRAF gene fusion in pilocytic astrocytomas; a novel oncogenic fusion gene with diagnostic, prognostic, and therapeutic potential. J Pathol. 222:324–328. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Guo YJ, Pan WW, Liu SB, Shen ZF, Xu Y and Hu LL: ERK/MAPK signalling pathway and tumorigenesis. Exp Ther Med. 19:1997–2007. 2019. | |
|
Pfister S, Janzarik WG, Remke M, Ernst A, Werft W, Becker N, Toedt G, Wittmann A, Kratz C, Olbrich H, et al: BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas. J Clin Invest. 118:1739–1749. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Tateishi K, Nakamura T and Yamamoto T: Molecular genetics and therapeutic targets of pediatric low-grade gliomas. Brain Tumor Pathol. 36:74–83. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Roskoski R Jr: MEK1/2 dual-specificity protein kinases: Structure and regulation. Biochem Biophys Res Commun. 417:5–10. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Roskoski R Jr: ERK1/2 MAP kinases: Structure, function, and regulation. Pharmacol Res. 66:105–143. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Roskoski R Jr: Allosteric MEK1/2 inhibitors including cobimetanib and trametinib in the treatment of cutaneous melanomas. Pharmacol Res. 117:20–31. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Yeh TC, Marsh V, Bernat BA, Ballard J, Colwell H, Evans RJ, Parry J, Smith D, Brandhuber BJ, Gross S, et al: Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res. 13:1576–1583. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Davies BR, Logie A, McKay JS, Martin P, Steele S, Jenkins R, Cockerill M, Cartlidge S and Smith PD: AZD6244 (ARRY- 142886), a potent inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 kinases: Mechanism of action in vivo, pharmacokinetic/pharmacodynamics relationship, and potential for combination in preclinical models. Mol Cancer Ther. 6:2209–2219. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Deming DA, Cavalcante LL, Lubner SJ, Mulkerin DL, Conte L, Eickhoff JC, Kolesar JM, Fioravanti S, Greten TF, Compton K, et al: A phase I study of selumetinib (AZD6244/ARRY-142866), a MEK1/2 inhibitor, in combination with cetuximab in refractory solid tumors and KRAS mutant colorectal cancer. Invest New Drugs. 34:168–175. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Greystoke A, Steele N, Arkenau HT, Blackhall F, Haris N, Lindsay CR, Califano R, Voskoboynik M, Summers Y, So K, et al: SELECT-3: A phase I study of selumetinib in combination with platinum-doublet chemotherapy for advanced NSCLC in the first-line setting. Br J Cancer. 117:938–946. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Melosky B, Bradbury P, Tu D, Florescu M, Reiman A, Nicholas G, Basappa N, Rothenstein J, Goffin JR, Laurie SA, et al: Selumetinib in patients receiving standard pemetrexed and platinum-based chemotherapy for advanced or metastatic KRAS wildtype or unknown non-squamous non-small cell lung cancer: A randomized, multicenter, phase II study. Canadian cancer trials group (CCTG) IND.219. Lung Cancer. 133:48–55. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Torii S, Yamamoto T, Tsuchiya Y and Nishida E: ERK MAP kinase in G cell cycle progression and cancer. Cancer Sci. 97:697–702. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Bax DA, Little SE, Gaspar N, Perryman L, Marshall L, Viana-Pereira M, Jones TA, Williams RD, Grigoriadis A, Vassal G, et al: Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development. PLoS One. 4:e52092009. View Article : Google Scholar : PubMed/NCBI | |
|
Chou TC: Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 15:440–446. 2011. | |
|
Adjei AA, Cohen RB, Franklin WB, Morris C, Wilson D, Molina JR, Hanson LJ, Gore L, Chow L, Leong S, et al: Phase I pharmacokinetic and pharmacodynamic study of the oral, small-molecule mitogen-activated protein kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers. J Clin Oncol. 26:2139–2146. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Chen YH and Gutmann DH: The molecular and cell biology of pediatric low-grade gliomas. Oncogene. 33:2019–2026. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Khatua S, Gutmann DH and Packer RJ: Neurofibromatosis type 1 and optic pathway glioma: Molecular interplay and therapeutic insights. Pediatr Blood Cancer. 65:2018. View Article : Google Scholar : PubMed/NCBI | |
|
Yang L, Jackson E, Woerner BM, Perry A, Piwnica-Worms D and Rubin JB: Blocking CXCR4-mediated cyclic AMP suppression inhibits brain tumor growth in vivo. Cancer Res. 67:651–658. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Warriton NM, Gianino SM, Jackson E, Goldhoff P, Garbow JR, Piwnica-Worms D, Gutmann DH and Rubin JB: Cyclic AMP suppression is sufficient to induce gliomagenesis in a mouse model of neurofibromatosis-1. Cancer Res. 70:5717–5727. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Xing F, Luan Y, Cai J, Wu S, Mai J, Gu J, Zhang H, Li K, Lin Y, Xiao X, et al: The anti-Warburg effect elicited by the cAMP-PGC1α pathway drives differentiation of glioblastoma cells into astrocytes. Cell Rep. 23:2832–2833. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Goldhoff P, Warrington NM, Limbrick DD Jr, Hope A, Woerner BM, Jackson E, Perry A, Piwnica-Worms D and Rubin JR: Targeted inhibition of cyclic AMP phosphodiesterase-4 promotes brain tumor regression. Clin Cancer Res. 14:7717–7725. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Lucchi S, Calebiro D, de Filippis T, Grassi ES, Borghi MO and Persani L: 8-Chloro-cyclic AMP and protein kinase A I-selective cyclic AMP analogs inhibit cancer cell growth through different mechanisms. PLoS One. 6:e207852011. View Article : Google Scholar : PubMed/NCBI | |
|
Cheng YM, Zhu Q, Yao YY, Tang Y, Wang MM and Zou LF: 8-Chloroadenosine 3′,5′-monophosphate induces cell cycle arrest and apoptosis in multiple myeloma cells through multiple mechanisms. Oncol Lett. 4:1384–1388. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Grassi ES, Dicitore A, Negri I, Borghi MO, Vitale G and Persani L: 8-Cl-cAMP and PKA I-selective cAMP analogs effectively inhibit undifferentiated thyroid cancer cell growth. Endocrine. 56:388–398. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Garcia MA, Solomon DA and Haas-Kogan DA: Exploiting molecular biology for diagnosis and targeted management of pediatric low-grade gliomas. Future Oncol. 12:1493–506. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Banerjee RI, Jakacki A, Onar-Thomas S, Wu T, Nicolaides T, Young Poussaint J, Fangusaro J, Phillips A, Perry A, Turner D, et al: A phase I trial of the MEK inhibitor selumetinib (AZD6244) in pediatric patients with recurrent or refractory low-grade glioma: A pediatric brain tumor consortium (PBTC) study. Neuro Oncol. 19:1135–1144. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Smith AM, Zhang CRZ, Cristino AS, Grady JP, Fink JL and Moore AS: PTEN deletion drives acute myeloid leukemia resistance to MEK inhibitors. Oncotarget. 10:5755–5767. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Hur EH, Goo BK, Moon J, Choi Y, Hwang JJ, Kim CS, Bae KS, Choi J, Cho SY, Yang SH, et al: Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells. Oncotarget. 20:41387–41400. 2017. View Article : Google Scholar | |
|
Lim SY, Menzies AM and Rizos H: Mechanisms and strategies to overcome resistance to molecularly targeted therapy for melanoma. Cancer. 123:21–29. 2017. View Article : Google Scholar | |
|
Nörz D, Grottke A, Bach J, Herzberger C, Hofmann BT, Nashan B, Jücker M and Ewald F: Discontinuing MEK inhibitors in tumor cells with an acquired resistance increases migration and invasion. Cell Signal. 27:2191–2200. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Simeone E, Grimaldi AM, Festino L, Vanella V, Palla M and Ascierto PA: Combination treatment of patients with BRAF-mutant melanoma: A new standard of care. BioDrugs. 31:51–61. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Welsh SJ, Rizos H, Scolyer RA and Long GV: Resistance to combination BRAF and MEK inhibition in metastatic melanoma: Where to next? Eur J Cancer. 62:76–85. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A, Ye Q, Lobo JM, She Y, Osman I, et al: BRAF mutation predicts sensitivity to MEK inhibition. Nature. 439:358–362. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Dizdar L, Werner TA, Drusenheimer JC, Möhlendick B, Raba K, Boeck I, Anlauf M, Schott M, Göring W, Esposito I, et al: BRAFV600E mutation: A promising target in colorectal neuroendocrine carcinoma. Int J Cancer. 144:1379–1390. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Gilmartin AG, Bleam MR, Groy A, Moss KG, Minthorn EA, Kulkarni SG, Rominger CM, Erskine S, Fisher KE, Yang J, et al: GSK1120212 (JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokinetic properties for sustained in vivo pathway inhibition. Clin Cancer Res. 17:989–1000. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Catalanotti F, Solit DB, Pulitzer MP, Berger MF, Scott SN, Iyriboz T, Lacouture ME, Panageas KS, Wolchok JD, Carvajal RD, et al: Phase II trial of MEK inhibitor selumetinib (AZD6244, ARRY-142886) in patients with BRAFV600E/K-mutated melanoma. Clin Cancer Res. 19:2257–2264. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Burger MC, Ronellenfitsch W, Lorenz NI, Wagner M, Voss M, Capper D, Tzaridis T, Herrlinger U, Steinbach JP, Stoffels G, et al: Dabrafenib in patients with recurrent, BRAF V600E mutated malignant glioma and leptomeningeal disease. Oncol Rep. 38:3291–3296. 2017.PubMed/NCBI | |
|
Ranzani M, Alifrangis C, Perna D, Dutton-Regester K, Pritchard A, Wong K, Rashid M, Robles-Espinoza CD, Hayward NK, McDemott U, et al: BRAF/NRAS wild-type melanoma, NF1 status and sensitivity to trametinib. Pigment Cell Melanoma Res. 28:117–119. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Ming Z, Lim SY, Kefford RF and Rizos H: Mitogen-activated protein kinase dependency in BRAF/RAS wild-type melanoma: A rationale for combination inhibitors. Pigment Cell Melanoma Res. 33:345–357. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Fangusaro J, Onar-Thomas A, Pussaint TY, Wu S, Ligon AH, Lindman N, Banerjee A, Pacher RJ, Kilburn LB, Goldman S, Polack IF, et al: Selumetinb in paedriatic patients with BRAF-aberrant or neurofibromatosis type 1-associated recurrant, refractory, or progressive low-grade glioma: A multicenter, phases 2 trial. Lancet Oncol. 20:1011–1022. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Grasso S, Tristante E, Saceda M, Carbonell P, Mayor-López L, Carballo-Santana M, Carrasco-García E, Rocamora-Reverte L, García-Morales P, Carballo F, et al: Resistance to Selumetinib (AZD6244) in colorectal cancer cell lines is mediated by p70S6K and RPS6 activation. Neoplasia. 16:845–860. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Tentler JJ, Nallapareddy S, Tan AC, Spreafico A, Pitts TM, Morelli MP, Selby HM, Kachaeva MI, Flanigan SA, Kulikowski GN, et al: Identification of predictive markers of response to the MEK1/2 inhibitor selumetinib (AZD6244) in K-ras-mutated colorectal cancer. Mol Cancer Ther. 9:3351–3362. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Kerstjens M, Driessen EM, Willekes M, Pinhanços SS, Schneider P, Pieters R and Stam RW: MEK inhibition is a promising therapeutic strategy for MLL-rearranged infant acute lymphoblastic leukemia patients carrying RAS mutations. Oncotarget. 8:14835–14846. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Meng J, Peng H, Dai B, Guo W, Wang L, Ji L, Minna JD, Chresta CM, Smith PD, Fang B and Roth JA: High level of AKT activity is associated with resistance to MEK inhibitor AZD6244 (ARRY-142886). Cancer Biol Ther. 8:2073–2080. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Sweetlove M, Wrightson E, Kolekar S, Rewcastle GW, Baguley BC, Shepherd PR and Jamieson SM: Inhibitors of pan-PI3K signaling synergize with BRAF or MEK inhibitors to prevent BRAF-mutant melanoma cell growth. Front Oncol. 5:1352015. View Article : Google Scholar : PubMed/NCBI | |
|
Tsubaki M, Takeda T, Noguchi M, Jinushi M, Seki S, Morii Y, Shimomura K, Imano M, Satou T and Nishida S: Overactivation of Akt contributes to MEK inhibitor primary and acquired resistance in colorectal cancer cells. Cancers (Basel). 25:11–12. 2019. | |
|
Balmanno K, Chell SD, Gillings AS, Hayat S and Cook SJ: Intrinsic resistance to the MEK1/2 inhibitor AZD6244 (ARRY-142886) is associated with weak ERK1/2 signalling and /or strong PI3K signalilling in colorectal cancer cell lines. Int J Cancer. 125:2332–2341. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Chen Z, Cheng K, Walton Z, Wang Y, Ebi H, Shimamura T, Liu Y, Tupper T, Ouyang J, Li J, et al: A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response. Nature. 483:613–617. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Chang T, Krisman K, Theobald EH, Xu J, Akutagawa J, Lauchle JO, Kogan S, Braun BS and Shannon K: Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice. J Clin Invest. 123:335–339. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
See WL, Tan IL, Mukherjee J, Nicolaides T and Pieper RO: Sensitivity of glioblastomas to clinically available MEK inhibitors is defined by neurofibromin 1 deficiency. Cancer Res. 72:3350–3359. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Nissan MH, Pratilas CA, Jones AM, Ramirez R, Won H, Liu C, Tiwari S, Kong L, Hanrahan AJ, Yao Z, et al: Loss of NF1 in cutaneous melanoma is associated with RAS activation and MEK dependence. Cancer Res. 74:2340–2350. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Whittaker SR, Theurillat JP, Van Allen E, Wagle N, Hsiao J, Cowley GS, Schadendorf D, Root DE and Garraway LA: A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition. Cancer Discov. 3:350–362. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Li Y, Dong Q and Cui Q: Synergistic inhibition of MEK and reciprocal feedback networks for targeted intervention in malignancy. Cancer Biol Med. 16:415–434. 2019.PubMed/NCBI | |
|
Chen CH, Hsia TC, Yeh MH, Chen TW, Chen YJ, Wei YL, Tu CY and Huang WC: MEK inhibitors induce Akt activation and drug resistance by suppressing negative feedback ERK-mediated HER2 phosphorylation at Thr701. Mol Oncol. 11:1273–1287. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Iizuka-Ohashi M, Watanabe M, Sukeno M, Morita M, Hoang NTH, Kuchimaru T, Kizaka-Kondoh S, Sowa Y, Sakaguchi K, Taguchi T, et al: Blockage of the mevalonate pathway overcomes the apoptotic resistance to MEK inhibitors with suppressing the activation of Akt in cancer cells. Oncotarget. 9:19597–19612. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Coussy F, El Botty R, Lavigne M, Gu C, Fuhrmann L, Briaux A, de Koning L, Dahmani A, Montaudon E, Morisset L, et al: Combination of PI3K and MEK inhibitors yields durable remission in PDX models of PIK3CA-mutated metaplastic breast cancers. J Hematol Oncol. 13:132020. View Article : Google Scholar : PubMed/NCBI | |
|
Liu X, Hu J, Song X, Utpatel K, Zhang Y, Wang P, Lu X, Zhang J, Xu M, Su T, et al: Combined treatment with MEK and mTOR inhibitors is effective in in vitro and in vivo models of hepatocellular carcinoma. Cancers (Basel). 3:9302019. View Article : Google Scholar | |
|
Ewald F, Nörz D, Grottke A, Bach J, Herzberger C, Hofmann BT, Nashan B and Jücker M: Vertical targeting of AKT and mTOR as well as dual targeting of AKT and MEK signaling is synergistic in hepatocellular carcinoma. J Cancer. 16:1195–1205. 2015. View Article : Google Scholar | |
|
Meng J, Dai B, Fang B, Bekele BN, Bornmann WG, Sun D, Peng Z, Herbst RS, Papadimitrakopoulou V, Minna JD, et al: Combination treatment with MEK and AKT inhibitors is more effective than each drug alone in human non-small cell lung cancer in vitro and in vivo. PLoS One. 29:e141242010. View Article : Google Scholar | |
|
Arnold A, Yuan M, Price A, Harris L, Eberhart CG and Raabe EH: Synergistic activity of mTORC1/2 kinase and MEK inhibitors suppresses pediatric low-grade glioma tumorigenicity and vascularity. Neuro Oncol. 22:563–574. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Choi KY, Cho YJ, Kim JS, Ahn YH and Hong SH: SHC1 sensitizes cancer cells to the 8-Cl-cAMP treatment. Biochem Biophys Res Commun. 463:673–678. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Grbovic O, Jovic V, Ruzdijic S, Pejanovic V, Rakic L and Kanazir S: 8-Cl-cAMP affects glioma cell-cycle kinetics and selectively induces apoptosis. Cancer Invest. 20:972–982. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Langeveld CH, Jongenelen CA, Heimans JJ and Stoof JC: Growth inhibition of human glioma cells induced by 8-chloroadenosine, an active metabolite of 8-chloro cyclic adenosine 3′:5′-monophosphate. Cancer Res. 52:3994–3999. 1992.PubMed/NCBI | |
|
Xing F, Luan Y, Cai J, Wu S, Mai J, Gu J, Zhang H, Li K, Lin Y, Xiao X, et al: The anti-Warburg effect elicited by the cAMP-PGC1α pathway drives differentiation of glioblastoma cells into astrocytes. Cell Rep. 18:468–481. 2017. View Article : Google Scholar : PubMed/NCBI |
