Background
Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults [
1]. Despite considerable advances in the multimodal treatment of tumors, involving surgery followed by radio- and chemotherapy, only a minimal improvement in prognosis has been noted, with a median survival of less than 1 year [
2]. The factors responsible for the limited efficacy of the current treatments include the highly invasive nature of GBMs, rendering them intractable to complete surgical resection, and resistance to conventional radiotherapy and chemotherapies [
3,
4]. The development of novel drugs or overcoming the chemoresistance may therefore comprise a new line of research into the treatment of GBM.
Bruton’s tyrosine kinase (BTK) is a member of 11 tyrosine kinases, including the TEC family kinases, epidermal growth factor receptor (EGFR, ErbB1), ErbB2, ErbB4, Janus kinase 3 (Jak3), and BLK, that carry a conserved cysteine residue adjacent to an ATP-binding site; this residue is critical for covalent inhibition of these enzymes by tyrosine kinase inhibitors [
5,
6]. Ibrutinib, formerly known as PCI-32765, selectively and irreversibly inhibits BTK, and is administered once-daily to prevent B-cell differentiation, proliferation, and survival [
7]. Ibrutinib exerts a potent anti-cancer effect by inhibiting BCR signaling and down-regulating NF-кB signaling, rapidly reducing tumor growth by inhibiting tumor proliferation and increasing apoptosis [
8‐
10]. Recently, ibrutinib has also been used as a novel anticancer drug for several other types of cancers, such as human ovarian, breast, and lung cancer, and also gastric carcinoma, and glioma [
11‐
14]. Ibrutinib may also function as a novel small molecule inhibitor in GBM patients.
Autophagy (macroautophagy) is a “self-eating” process that enables the cell to engulf parts of its cytoplasm, organelles, and/or membrane through the formation of double-membrane vesicles (autophagosomes), and eventually targeting them to the lysosomes; this process is important for cell homeostasis, development, and/or nutrient recycling [
15]. Under cellular stress conditions, such as nutrient deprivation, hypoxia, oxidative stress, DNA damage, etc., autophagy provides energy for the maintenance of essential cellular metabolism and enables cellular survival [
16]. By promoting the survival of tumor cells under unfavorable conditions, autophagy may be involved in an alternative mechanism of drug resistance during cancer therapy. Recent extensive evidence indicates that autophagy is enhanced as a cytoprotective mechanism when cancer cells are subjected to unfavorable conditions, such as nutrient deficiency or treatment with chemotherapeutic drugs, aiding cancer cell survival. Temozolomide (TMZ) is widely used for treating primary and recurrent high-grade gliomas. Recent studies have shown that TMZ treatment can induce autophagy, which contributes to therapy resistance in glioma, and this has received considerable attention [
17]; autophagy may also contribute to GBM resistance to anticancer therapies.
Autophagy is regulated by the main autophagy repressor, mammalian target of rapamycin (mTOR) complex 1 (mTORC1) [
18]. It is inhibited by the intracellular energy sensor AMP-activated protein kinase (AMPK) [
19]. Signaling pathways downstream of BTK, such as the PI3K/Akt pathway, are involved in the regulation of autophagy, indicating a potential link between ibrutinib and autophagy. The question of whether autophagy plays a role in cell death or constitutes a survival mechanism in GBM has not been investigated in detail.
In the current study, we investigated whether the modulation of autophagy may be used as an adjuvant modality to improve the effects of chemotherapy during GBM treatment. We performed a detailed analysis of the effect of ibrutinib on GBM cells. We demonstrated that ibrutinib exerts an antitumor effect and induces autophagy by targeting the Akt/mTOR signaling pathways in GBM. In addition, inhibiting macroautophagosome formation enhanced the GBM antitumor activity of ibrutinib. These findings provide important insights that may aid in the development of novel strategies to enhance the response of cancer cells to ibrutinib by exploiting the role of autophagy in GBM therapy.
Methods
Cell culture and chemicals
Human glioblastoma cell lines LN229, U87, T98, and U251 were purchased from the American Type Culture Collection (ATCC, Shanghai, China). All cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS, 10%; Gibco BRL, Grand Island, NY), nonessential amino acids (100 μM), sodium pyruvate (1 mM), streptomycin (100 μg/mL), and penicillin (100 U/mL, Gibco BRL) at 37 °C, in an atmosphere of 5% CO2. Ibrutinib and LY294002 were obtained from Selleck Chemicals (Houston, TX) and were dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, USA) at a concentration of 10 mM. The final concentration of DMSO in treatment did not exceed 0.1% (v/v). 3-Methyladenine (3MA) was purchased from Sigma-Aldrich (St. Louis, USA) and dissolved in phosphate-buffered saline (PBS, Gibco BRL) at a concentration of 100 mM. Before use, stock solutions were diluted to the required concentrations in culture medium.
Cell viability assay
Cell viability was analyzed using a WST-8 Cell Counting Kit-8 (CCK-8, Beyotime, Jiangsu, China). U87, U251, and LN229 cells were suspended in DMEM medium containing 10% of FBS (3 × 103 cells/100 μL) and were seeded in 96-well plates and treated with different concentrations of chemicals, as specified. At the indicated time points, the cells were stained with CCK-8 (10 μL/well) and the cultures were incubated at 37 °C for 90 min. The absorbance at 450 nm was measured using an immunoreader (Infinite M200; Tecan, Männedorf, Switzerland).
Cells (200 cells per well) were counted and 1.0 × 104 cells were seeded in 6 × 6 cm plates in DMEM supplemented with 10% of FBS. The cells were treated with the indicated agents for 10 day. Colonies were stained with 0.2% crystal violet solution (Beyotime) and counted after 10 day of incubation at 37 °C and 5% CO2. Clusters of cells containing over 50 cells were counted as a colony. For each clone, three independent plates were examined.
5-ethynyl-2′-deoxyuridine (EdU) proliferation assay
GBM cell proliferation was determined in vitro using the Cell-Light™ EdU DNA cell proliferation kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions.
Small interfering RNA (siRNA) and plasmid construction
Cells were seeded (2 × 105 cells/well) in 6-well plates. After a 24-h incubation, the cells were transfected with siRNA targeting Atg7 (GenePharma, Shanghai, China), using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The sequences of interference were as follows: si-Atg7, 5′-CAGCCUGGCAUUUGAUAAATT-3′ (sense) and 5′-UUUAUCAAAUGCCAGGCUGTT-3′ (antisense); si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′. Constitutively-active Akt (CA-Akt) and dominant-negative Akt (DN-Akt) plasmids were constructed by Sunbio (Shanghai, China).
Cell migration assay
Cell migration was assessed in wound healing assays and trans-well migration assays. For the wound healing assays, 5 × 105 cells/well were plated in 6-well dishes, and incubated with various concentrations of ibrutinib at 37 °C overnight. A cell-free gap was generated by scratching dishes with a 10-μL pipette tip. For trans-well migration assay, the cells were re-suspended in a serum-free DMEM medium (3× 105 cells/200 μL) with ibrutinib and then seeded into the upper chamber, over 8-μm pore polycarbonate filters (Millipore, Massachusetts, USA). A serum-containing DMEM medium (600 μL) was placed in the lower chamber. After 24 h of incubation, the cells that migrated to the bottom of the membrane were attached and fixed, and stained with 0.2% crystal violet solution.
Western blot analysis
Drug- or vehicle-treated cells, or mouse tissue samples were lysed in a lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% Na3VO4, 0.5 μg/mL of leupeptin, and 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime). Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The samples were then scraped and transferred into microfuge tubes, centrifuged at 12,000 rpm for 15 min, and heated in an SDS-PAGE protein loading buffer (Beyotime) at 95 °C for 10 min. Equal amounts of protein were separated on 10 or 15% SDS-PAGE gels (Beyotime). After the electrophoresis, the separated proteins were transferred to a PVDF membrane (Beyotime); the membranes were then blocked in 5% of nonfat milk for 60 min. Next, the membranes were incubated overnight at 4 °C with the following primary antibodies raised against: phospho-GSK3β (Ser9) (#5558), phospho-BTK (#5082P), BTK(#8547), phospho-Akt(#9271), Akt (#9272), LC-3A/B (#12741), Atg (#8558), cyclin D1 (#2922), p-Rb (#3590), p-mTOR(#5536), mTOR(#2972), p-ULK1(#12753), ULK1(#8054), p-p70S6K (#9208), p70S6K(#14130), cleaved caspase 3 (#9661), cleaved caspase 9 (#9502), and Bcl-xL (#2764), from Cell Signaling technology (Danfoss, USA); GAPDH (AG019), from Beyotime; or E2F1 (ab179445), from Abcam (Cambridge, UK). Following a 1-h incubation with horseradish peroxidase (HRP)-labeled secondary antibodies, the blots were developed using a western blot chemiluminescence reagent system (Perkin-Elmer, NEL103001EA, Waltham, USA). Three replicates were performed for each experiment.
Transmission electron microscopy (TEM)
To assess cell morphology by electron microscope, the treated cells were fixed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide solution, dehydrated with acetone, and embedded in Epon resin (Agar Scientific, Stansted, UK). Ultrathin sections were prepared with an Ultracut microtome (Leica, Oskar-Barnack, Germany) and then stained with 4% uranyl acetate and lead citrate. The sections were examined using a JEM-100cxII electron microscope (JEM-1010, JEOL, Tokyo akishima, Japan).
Immunocytochemistry
GBM cells were fixed and permeabilized in 0.2% Triton X-100 (). After washing with xx, the cells were blocked with 5% BSA, incubated with specific antibodies against LC-3A/B (1:50, Neomarkers, Fremont, CA), overnight at room temperature, followed by an incubation with Cy3-labeled goat anti-rabbit antibodies (1:200, Beyotime). Finally, the coverslips were removed and mounted onto glass slides in Vectashield mounting medium containing DAPI (Vecta Laboratories, Burlingame, CA). Images were acquired with a laser scanning microscope (Infinite M200 Pro, Tecan); LSM510 software was used to capture the images (Zeiss, Aobokeheng, Germany).
Flow cytometry analysis
GBM cells treated with drugs or DMSO were trypsinized, suspended in ice-cold PBS, and fixed in 70% ethanol at −20 °C. Cell cycle progression was evaluated using BD Cycletest Plus kit and BD FACS Calibur flow cytometer (BD, Franklin Lakes, NJ). After fixing, the cells were washed twice with PBS, stained in 250 μL of trypsin buffer for 15 min, and eventually added to 200 μL of trypsin inhibitor with RNase buffer. The samples were finally stained with 200 μL of PI solution and analyzed.
Cell apoptosis was analyzed using BD annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit. Harvested cells were washed with cold PBS, resuspended in 50 μL of annexin binding buffer, stained with 5 μL of annexin V-FITC and 5 μL of PI solution for 15 min at room temperature in the dark, and then diluted in 400 μL of 1× binding buffer.
Tumorigenicity in nude mice
BALB/C nude mice (4–5 week-old) were provided by the animal center at the Cancer Institute at the Model Animal Research Center of Nanjing University (Nanjing, China) and randomly divided into four groups (control group, Ib group, 3MA group, and Ib + 3MA group). U87 cells (2 × 106) in 100 μL of serum-free DMEM were injected into the right flank of mice. Tumor volume was assessed every 3 days. Mice were injected intraperitoneally (i.p.) every other day, starting on day 3, with PBS alone (control), ibrutinib (6 mg/kg/d), 3MA (30 mg/kg/d), or ibrutinib (6 mg/kg/d) and 3MA (30 mg/kg/d). The tumor and body weights were determined. Tumor volume was calculated by the following formula: (short diameter)2 × (long diameter)/2. Mice were humanely sacrificed on day 22. For immunohistochemical analysis, samples from each group of mice were stained with H&E and a primary antibody (rabbit anti-LC3A/B or rabbit anti-Ki67, both at 1:200 dilution).
Statistical analysis
Data are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Student’s t test was performed to assess statistical significance using GraphPad Prism (GraphPad, San Diego, CA). A value of p < 0.05 was regarded as statistically significant.
Discussion
The poor prognosis of GBM under current therapeutic regiments has necessitated the development of novel therapeutic agents. In the current study, we explored the potential anticancer effect of ibrutinib in GBM. Our results indicate that ibrutinib indeed has a pronounced anticancer potential in GBM. Furthermore, we also confirmed that ibrutinib induces autophagy by targeting Akt/mTOR pathway. Finally, we provided evidence that the blockage of autophagy can potentiate the effect of ibrutinib on GBM in vitro and in vivo.
Autophagy has generally been considered to facilitate cancer survival during growth factor withdrawal or under metabolic stress, e.g., gamma-radiation, exposure to toxic stimuli, and chemotherapy [
26,
27]. This process plays an important role not only during different stages of tumorigenesis but also during disease, creating a tumorigenesis-promoting microenvironment [
28]. In certain cellular settings, however, it was reported that autophagy might suppress tumorigenesis by inducing autophagic cell death [
29,
30]. Thus, the current view of autophagy in tumorigenesis is as of a double-edged sword that can either act as a tumor suppressor or promoter; this issue is receiving increasing scientific attention. Recently, a number of cancer therapeutics in cancer indicated that autophagy can be activated and protect tumor cells when they are exposed to targeted therapies, e.g., Philadelphia chromosome-positive cells and imatinib mesylate [
31], breast cancer and trastuzumab [
32], prostate cancer and Src family kinase inhibitors [
33], and prostate cancer and proteasome inhibitors [
34]. Autophagy-related genes, ATGs, function at several discrete but continuous steps of autophagy. Upon the induction of autophagy, some LC3(
Atg8)-I (LC3-I) synthesized in the cytoplasm is evenly converted to LC3-II, which is tightly bound to the autophagosomal membranes, forming ring-shaped structures in the cytosol. LC3 expression is positively correlated with GBM patient survival and performance status, whereas in patients with normal performance scores, low LC3 expression correlates with better survival [
35]. The combination of TMZ, the most effective drug for GBM treatment, and autophagy inhibitors [e.g., chloroquine (CQ) and its analogs] has attracted attention in a rational development of therapeutic approaches, and is under clinical trials as GBM treatment [
17]. These suggested that autophagy maybe be activated as a cellular response to GBM therapy. In the current study, we confirmed that autophagy is induced by ibrutinib, as determined by TEM and immunocytochemistry. Western blot analysis showed that ibrutinib increases LC3-II protein levels in a concentration- and time-dependent manner, in two independent GBM cancer cell lines. Hence, autophagy may indeed be activated as a cellular response to GBM therapy.
Autophagy is a complex process, fine-tuned by several environmental signals involved in nutrient signaling, growth factor status, energy sensing, hypoxia, oxidative and ER stress, and infection [
36]. AMPK and mTOR signaling pathways have been revealed as the central checkpoints in the regulation of autophagy [
37]. It has been reported that ibrutinib suppresses GBM tumorigenesis by inhibiting BTK and its downstream Akt/mTOR signaling [
14]. Our results revealed that ibrutinib treatment inhibits BTK activation and phosphorylation of its downstream targets, including Akt, mTOR, and p70S6K. Accumulating evidence has highlighted the notion that the inhibition of Akt and its downstream targets mTOR and p70S6K contributes to the initiation of autophagy [
38]. In the current study, overexpression of constitutively-active Akt markedly decreased ibrutinib-induced autophagy. In contrast, PI3K/Akt/mTOR signaling pathway inhibitor LY294002 enhanced ibrutinib-induced autophagy. The Akt/mTOR signaling pathway is therefore a critical mediator regulating ibrutinib-induced autophagy. Aberrant EGFR signaling, expression of EGFR vIII mutant interact with the PI3K/Akt/mTOR pathway were frequently observed in GBM patients, promoting survival and chemo-resistance [
39]. Gao et al. [
40] recently reported that ibrutinib selectively inhibits growth of mutated NSCLC cells, including T790M mutant and erlotinib-resistant H1975 cells, by inhibiting EGFR phosphorylation [
40]. Hence, ibrutinib may also induce autophagy along the RTK-PI3K-Akt-mTOR axis. Autophagy induction by targeting the components of the PI3K-Akt-mTOR axis has been typically suggested to play a cytoprotective role in GBM. Combination of bafilomycin A1 or monensin, which inhibits lysosomal protease activity, with PI-103 or Ku-0063794, mTOR kinase inhibitors, promoted GBM cell death by inducing apoptosis [
41]. A combination of PI3K/mTOR/Akt inhibitors PI-103 and Akt-1/2 with the lysosomotrophic agent CQ enhanced cell death in GBM [
42]. Additionally, a combination of a dual PI3K and mTOR inhibitor, NVPBEZ235, with CQ induced apoptosis of glioma cells [
41]. Similarly, suppression of autophagy has been reported to synergize with Tyrosine Kinase Inhibitor (TKI), such as erlotinib [
43] or imatinib [
44], to increase the cytotoxic effect on GBM cells. In the current study, knocking-down
Atg7 significantly enhanced the ibrutinib-induced apoptosis of glioma cells in vitro. Moreover, we also confirmed that the blockage of autophagy by 3MA increased the anti-cancer effect of ibrutinib on GBM in vivo.
Acknowledgements
We thank Cheng Xu and Changsheng Yan for technical assistance.