Background
Gliomas account for approximately 70 % of all primary malignant brain tumors [
1]. On the basis of histological features, gliomas are classified into four distinct subtypes by the World Health Organization [
2]: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma). Glioblastomas account for approximately 60 to 70 % of malignant gliomas. Despite advances in the understanding of glioma biology and the development of new treatment options, the median survival for patients with glioblastomas is merely 12 to 15 months [
3]. Thus, there is an urgent need for the development of novel targeted therapeutics.
Two decades of molecular studies together with recent large-scale cancer gene sequencing efforts have identified three critical signaling pathways that are misregulated in human glioblastomas: the RTK/PI3K/AKT/Foxos signaling pathway and the p53 and Rb1 tumor suppressor-pathways [
4‐
6]. Consistent with these findings, the simultaneous deletion of
Pten-,
p53-, and
Nf1-mediated CRISPR/Cas9 can promote the development of highly aggressive tumors resembling human glioblastomas in the mouse brain [
7]. Furthermore, nearly all subtypes of gliomas are associated with the amplification and overexpression of the epidermal growth factor receptor (EGFR) gene [
8]. However, inhibitors of receptor tyrosine kinases (RTK) [
9‐
11], PI3K [
12], and mTOR [
13,
14] have exhibited only modest activity in glioma, with response rates of 0 to 15 % and no prolongation of 6-month progression-free survival [
15,
16]. Given the complexity and redundancy of the signaling networks associated with glioma, the simultaneous targeting of critical oncogenic pathways might constitute a promising treatment approach.
Chromosomal region maintenance 1 (CRM1), also referred to as exportin 1 (XPO1) [
17], is a promising therapeutic target for gliomas. Increased CRM1 expression has been observed in gliomas and is correlated with a poor prognosis and higher grade of malignancy [
18]. CRM1 is a key member of the karyopherin β superfamily of nuclear transport receptors, which mediate the transport of specific proteins from the nucleus to the cytoplasm in eukaryotic cells [
19]. CRM1 is the key exporter of multiple tumor-suppressor proteins [
20], including Foxos, Rb1, p53, p21, p27, and survivin. Accumulating lines of evidence suggest that the misregulation of nuclear protein export dynamics is involved in cancer cell survival, tumor progression, and drug resistance [
21,
22]. These observations have stimulated considerable interest in drugs targeting the nuclear export of proteins.
Leptomycin B (LMB), the first natural inhibitor of CRM1 to be identified, can covalently bind the Cys528 residue in the cargo-binding region of CRM1 [
23]. However, the phase I clinical trial of LMB was terminated due to its toxic effects and lack of efficacy at tolerable doses [
24]. Recently, a novel class of selective inhibitors of nuclear export (SINE) has been developed [
25‐
27]. One member of this class, selinexor (KPT-330), is currently undergoing phase I/II clinical trials to evaluate its effect in several solid and hematologic malignancies [
28]. KPT-330 is a CRM1 inhibitor that forms a slowly reversible covalent bond with CRM1, and preliminary evidence indicates that it exhibits a relatively favorable drug tolerability profile. These findings suggest that a selective and reversible inhibitor might offer an improved tolerability profile. However, most of the currently available CRM1 inhibitors function by irreversibly binding to Cys528. Recently, we developed the novel reversible CRM1 inhibitor S109, which can induce CRM1 protein degradation [
29].
In the present study, we evaluated the mechanism and therapeutic potential of a reversible CRM1 inhibitor, S109, in the treatment of human glioma. Specifically, we investigated the therapeutic efficacy of S109 in vitro and in intracranial mouse models of malignant glioma and elucidated the mechanism underlying S109-mediated anti-glioma activity. This study provides a basis for further clinical investigations of S109.
Methods
Glioma and non-tumor human brain tissues
Human glioma specimens (obtained through surgical resection) and non-tumorous brain tissues (obtained from patients with internal decompression in cerebral trauma) were obtained from the Affiliated Hospital of Xuzhou Medical College (Xuzhou, China). Written informed consent was obtained from all of the participants, and this study was approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University.
Cell lines and culture conditions
The human glioma cell lines U251 and SHG-44 and glioblastoma cell lines U118 and U87 were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. All of the cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco) in a humidified incubator with 5 % CO2 at 37 °C.
Antibodies and reagents
Primary antibodies against CRM1, GAPDH, actin, RanBP1, and p53 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against cyclin D1, Cdc25B, p27, p21, Foxo1, p-Foxo1, Akt, p-Akt, p-Rb1, and histone H3 were purchased from Cell Signaling Technology (CST, Beverly, MA, USA).
Cytotoxicity assay
The cell counting kit-8 (CCK-8) assay was used to assess cell viability following S109 treatment. Briefly, glioma cells were plated in triplicate in 96-well plates (2 × 103 cells per well). After overnight incubation, the cells were treated with various concentrations of S109 for 72 h. The cells were subsequently washed three times with phosphate-buffered saline (PBS), and 10 μL of CCK8 was added to each well. Following a 3-h incubation, the absorbance was measured using a microplate reader at a wavelength of 450 nm.
EdU incorporation assays
Cell proliferation was assessed using the Cell-Light™ EdU Cell Proliferation Detection Kit (RiboBio, China) according to the manufacturer’s instructions. The cells were treated with various concentrations of S109 for 12 h. Subsequently, the cells were incubated with 50 μM EdU for 2 h and then fixed in 4 % paraformaldehyde for 30 min. After permeabilization with 0.5 % Triton X-100, the cells were incubated with Apollo® reaction cocktail for 30 min in the dark. The cellular DNA was stained with DAPI for 15 min. Following three washes with PBS, the cells were examined and imaged using an inverted microscope (Olympus, Japan).
The cells were seeded at a density of 500 cells/well in 6-well culture plates. The plated cells were treated with different concentrations of S109 for 12 h, and fresh medium was subsequently added. After a 14-day incubation, the cells were fixed in methanol for 15 min and stained with 0.1 % crystal violet solution. Positive colony formation, defined as colonies with more than 50 cells, was confirmed by manual counting.
Co-immunoprecipitation
Immunoprecipitation assays were performed as previously described [
30]. Briefly, cells treated with 0.1 % DMSO or S109 were lysed in cold lysis buffer. The supernatant was incubated with anti-CRM1 antibody for 8 h and then with protein G-Sepharose 4B (Roche, Basel, Switzerland) overnight at 4 °C while rocking. The immunoprecipitated complexes were washed three times with lysis buffer and analyzed by western blot.
Immunofluorescence microscopy
The cells were then treated with S109 and subsequently fixed for 20 min with 4 % paraformaldehyde in PBS. The cell membranes were subsequently permeabilized in 0.1 % Triton X-100 and blocked with 1 % bovine serum albumin (BSA) in PBS. The cells were then incubated with the indicated antibody. DAPI was used for nuclei labeling (blue), and the stained cells were visualized and imaged through fluorescence microscopy (Olympus, Japan).
Establishment of CRM1-WT and CRM1-C528S stable cell lines
The cDNA encoding human CRM1-WT or CRM1 with the C528S mutation was inserted into the pWPXLd-puro lentiviral vector containing a sequence encoding a flag tag. The viruses were produced in 293FT cells by co-transfecting the recombinant plasmids with the helper plasmids pSPXA2 and pMD2.G. The U87 cells were then transfected with the CRM1-WT or CRM1-C528S lentivirus for 48 h and then continuously cultured in medium containing 2.5 μg/mL puromycin. The surviving cells were cultured and used to generate cell lines that stably expressed CRM1-WT or CRM1-C528S.
CRM1 expression and survival analysis in patients with glioma
CRM1 gene expression datasets were obtained from R2: microarray analysis and visualization platform (
http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). The prognosis analysis was conducted online, and cutoff values for separating high and low expression groups were determine by auto scan.
In vivo studies
All animal experimental protocols were approved by the Ethics Committee of the Xuzhou Medical University. Male athymic BALB/c nude mice aged 5 to 6 weeks were obtained from the Experimental Animal Center of Xuzhou Medical College. Firefly luciferase-labeled U87 cells (5 × 10
5 cells per mouse) were intracranially injected into the right striatum of nude mice using a small animal stereotactic apparatus as described in our previous report [
31,
32]. Once the presence of a tumor was confirmed by imaging system, the tumor-bearing mice were randomly divided into one of the following three treatment groups (
n = 8 per group): S109 at 20 mg/kg, S109 at 50 mg/kg, and vehicle. The drugs and vehicle were delivered daily via intraperitoneal injections. Tumor growth was monitored at regular intervals by injecting D-luciferin 10 min prior to imaging using a NightOWL LB 983 small-animal in vivo imaging system (Berthold Technologies, Germany).
Histopathology and immunofluorescence staining
The whole brain and vital organs (lung, liver, testis, kidney, and heart) of the control and treated mice were harvested on day 21, fixed in 4 % paraformaldehyde and dehydrated sequentially in 20 and 30 % sucrose at 4 °C until they sank. The frozen glioma tissues were serially sectioned at a thickness of 12 μm, and the slide with the largest tumor area was stained with hematoxylin and eosin (H&E).
Statistical analysis
The statistical analyses were performed using the GraphPad Prism 5 software package. All of the data are presented as the means ± SEM of three independent experiments. Comparisons of the mean values between the control and treated groups were performed using Student’s t test. A Kaplan-Meier survival curve and the log-rank test were used for the in vivo survival analysis. P values <0.05 were considered statistically significant.
Discussion
Effective chemotherapies for gliomas are limited, and improving long-term survival in glioma patients is imperative [
36]. Therefore, new modalities that can improve or replace the current treatments for gliomas are highly desirable. This study demonstrates that S109 exerts anti-tumor effects in glioma models in vitro and in vivo. Furthermore, we discovered that S109 exerts its anti-tumor effects by perturbing the three core pathways implicated in glioma: the RTK/AKT/Foxos signaling pathway and the p53 and Rb1 tumor-suppressor pathways.
CRM1 overexpression in human gliomas is associated with a poor prognosis and higher grade of malignancy [
18]. Therefore, targeting CRM1 is a promising therapeutic strategy for gliomas. Our data support this hypothesis because we found that the CRM1 inhibitor S109 significantly suppresses the proliferation of glioma cells both in vitro and in vivo. The irreversible inhibitor of CRM1, KPT-330, also exerts anti-tumor activity in preclinical models of glioblastoma [
37], but the molecular mechanism and cellular signaling pathways mediating the anti-glioma activity of KPT-330 remain unknown. The anti-glioblastoma effects of KPT-330 treatment result from the induction of apoptosis but are not associated with cell-cycle arrest [
37]. In contrast, the anti-proliferative activity of S109 results from the induction of cell-cycle arrest in the G1 phase but does not appear to be associated with apoptosis. This discrepancy between the mechanism of KPT-330 and that of S109 might be related to the reversible binding of S109 to CRM1 or to a greater selectivity for CRM1. CRM1 can recognize and transport the leucine-rich nuclear export signal (NES) of cargo proteins, including transcription factors and tumor suppressor proteins such as p53, p21, p27, and Foxos. The inhibition of CRM1 function could disrupt and retain tumor suppressor proteins in the nucleus to induce cancer cell cycle arrest or apoptosis.
A number of genetic alterations have been implicated in processes that promote glioma progression, such as increased proliferation, resistance to apoptosis, and robust invasive capability [
38]. The EGFR pathway has generated particular interest as a drug target. However, EGFR kinase inhibitors have demonstrated disappointing results in patients with gliomas [
39]. The resistance of gliomas to this treatment approach might be due to the role of oncogenic mutations in genes that function up- and mid-stream of the EGFR/Akt pathway. This hypothesis raises the possibility that molecules that function upstream of the EGFR/Akt pathway might not be optimal therapy targets. Foxo transcription factors, the critical downstream transcription factors of the EGFR/Akt pathway, are inactivated by cytoplasmic mislocalization in glioma cells [
40,
41]. High cytoplasmic Foxo1 expression in human gliomas is associated with a higher grade of malignancy [
42]. However, it is difficult to develop direct inhibitors of transcription factors. In this manuscript, we report that the inhibition of CRM1 by S109 might significantly promote the nuclear retention of Foxo1, and our results indicate that redirecting Foxo1 to the nucleus by inhibiting CRM1 might be an effective approach for treating gliomas.
Inactivation of the p53 and Rb1 tumor-suppressor pathways is frequently observed in glioma cells. The proteins p14/ARF and p16/INK4A are the key regulators of the p53 and Rb1 signaling pathways, respectively [
43]. In human gliomas, one of the most common genetic aberrations is the homozygous deletion of
CDKN2A. Because
CDKN2A encodes both p14/ARF and p16/INK4A, the deletion of
CDKN2A results in the disruption of both the p53 and Rb1 pathways [
4]. We found that S109 treatment induces the nuclear accumulation of p53 and decreases the level of Rb1 phosphorylation. Rb1 is a well-characterized protein that functions as a key negative regulatory protein of cell-cycle progression from the G1 phase. Thus, one of the mechanisms through which S109 induces G1 cell-cycle arrest is by promoting the nuclear retention of Rb1. Interestingly, we observed that high-grade glioma cells are more sensitive to S109 treatment than low-grade cells. This difference might be related to the observation that the three core oncogenic pathways are more frequently disrupted in high-grade glioma cells [
4]. Taken together, our results indicate that S109 treatment can simultaneously target the three core pathways implicated in glioma. Therefore, the targeted inhibition of CRM1 is an attractive strategy for the treatment of gliomas.
Acknowledgements
We thank Qiong Shi and Zengtian Sun for the technical assistance.