Introduction
Glioblastoma multiforme (GBM) is the most common malignant and aggressive tumor of the central nervous system (CNS) [
1,
2]. Almost all GBM patients experience relapse despite the usual combination of surgery, chemotherapy and radiation therapy, and the median survival time has been approximately 12 to 15 months for decades [
3,
4]. Obstacles to glioma treatment are due not only to the limited extent of the tumor that can be safely removed but also to resistance to adjuvant therapy after surgical resection [
5]. Temozolomide (TMZ), a second-generation oral alkylating agent, is the first-line chemotherapeutic agent for patients with GBM [
6,
7]. However, nearly all patients develop resistance to TMZ and relapse after a progression-free survival period of 7 to 10 months [
8]. Therefore, it is urgent to elucidate the underlying mechanisms of TMZ resistance to treat and prevent GBM recurrence.
Long non-coding RNAs (LncRNAs) is a class of heterogeneous RNA that are more than 200 nucleotides in length and limit protein coding potential [
9]. LncRNAs have been proven to perform diverse cellular functions, including transcriptional regulation in cis or trans, organization of nuclear domains, and posttranscriptional regulation by interacting with miRNAs, mRNAs, or proteins [
10‐
12]. Emerging evidence has shown that lncRNAs are associated with multiple features of cancer, such as proliferation, apoptosis, metastasis, metabolism, and therapy resistance [
13,
14]. Recent studies have demonstrated that lncRNAs regulate numerous signaling pathways through interactions with proteins [
15‐
17]. However, the regulation of posttranslational modifications by lncRNAs and the subsequent impact on TMZ treatment resistance in GBM remain largely uncharacterized.
Various mechanisms contribute to TMZ resistance in GBM, of which GBM cell heterogeneity and plasticity are thought to be key factors driving treatment resistance and tumor recurrence [
18]. Based on bulk RNA sequencing findings, intertumor heterogeneity is manifested by at least three GBM subtypes, including proneural (PN), classical (CL) and mesenchymal (MES) [
19]. Heterogeneity is also manifested by differences in the developmental status of GBM cells in tumors. Glioma stem cells (GSCs) are a group of cells with the capacity for self-renewal and asymmetric differentiation [
20]. The presence of GSCs is thought to be a driving force in tumorigenesis, tumor propagation and preferential resistance to radiotherapy and chemotherapy; thus, GSCs are considered a valuable model for studying GBM [
21]. GBMs of PN and MES subtypes correspond to PN and MES GSCs, respectively, but no GSCs corresponding to the CL subtype of GBM have been identified [
22]. Recent studies have shown that GBM undergoes proneural-to-mesenchymal transition (PMT) as the disease progresses and the tumor recurs [
23‐
25]. PMT is therefore considered a marker of tumor tolerance in response to multiple treatments and tumor recurrence. A variety of possible mechanisms drive the occurrence of PMT, including intracellular signaling pathways and the extracellular tumor microenvironment (TME). For instance, Carro et al. identified STAT3 and C/EBPβ as two master regulators (MRs) of PMT [
26]. In addition, the impact of treatment and the subsequent selective pressure within the tumor may contribute PMT [
27]. However, the mechanisms of treatment-induced PMT and modulation of MRs by lncRNAs remain unclear.
In this study, we identified a key lncRNA, PDIA3P1, which is closely associated with GBM TMZ therapy resistance and recurrence. In vitro and in vivo assays revealed that knockdown of PDIA3P1 resulted in decreased resistance to TMZ in glioma cells; in contrast, overexpression of PDIA3P1 resulted in increased resistance of glioma cells to TMZ. Mechanistically, PDIA3P1 promoted PMT by stabilizing CEBPβ, enabling GSCs to acquire preferential resistance to TMZ treatment. Even more valuable, we identified a drug called neflamapimod (NEF) that specifically targets p38α and has the ability to easily cross the blood–brain barrier (BBB). We demonstrated that NEF inhibits TMZ-induced upregulation of PDIA3P1 and enhances the sensitivity of glioma cells to TMZ treatment.
Materials and methods
Differential expression analysis
The limma R package was leveraged to identify differentially expressed genes (DEGs) between TMZ resistance and sensitive cell lines. The top 30 upregulated genes sorted according to p-value in TMZ resistant group were visualized using the pheatmap R package.
Single sample gene set enrichment analysis (ssGSEA)
To determine the abundance of GBM immune infiltration levels, immune gene signatures were obtained from data of Bindea et al. [
30] to perform ssGSEA. The immune cell infiltration levels were estimated using “GSVA” R package based on deconvolution algorithm.
Gene set enrichment analysis (GSEA)
The gene sets of “c2.cp.kegg.v7.4”, “c5.go.bp.v7.4”, “verhaak glioblastoma mesenchymal” and “verhaak glioblastoma proneural” were obtained from The Molecular Signatures Database (MSigDB;
http://www.gsea-msigdb.org/gsea/login.jsp) database for running GSVA.
P-value < 0.05 indicates statistical significance.
Cell lines and cell culture
Human glioma cell lines U118MG, U87MG, LN229 and U251MG were purchased from the Chinese Academy of Sciences Cell Bank and cultured in DMEM medium (Gibco, USA) with 10% fetal bovine serum (FBS). The neural progenitor cell (NPC) and GBM patient-derived GSC cell lines and were kindly donated by Dr. Krishna P.L. Bhat (The University of Texas, M.D. Anderson Cancer Center, Houston, TX). GSC lines (GSC20, GSC267, GSC8–11, GSC11) have been used extensively in previous studies and the subtypes of GSCs have been clarified according to the Verhaak or Philips gene signatures, respectively. GSCs and NPC were cultured in DMEM/F12 (Gibco, USA) with 2% B-27 no serum supplement (Gibco, USA), 20 ng/mL human recombinant bFGF (R&D Systems) as well as 20 ng/mL human recombinant EGF (R&D Systems, USA). The GSC or NPC spheres were digested using accutase solution (Sigma-Aldrich, USA). All cell lines were cultured in a humid chamber at 37 °C and containing 5% carbon dioxide and 5% oxygen.
RNA extraction and quantitative real-time PCR (RT-qPCR)
TRIzol (Invitrogen, USA) was used to extract total RNA according to manufacturer’s instruction. The high capacity cDNA Reverse Transcription Kit (Toyobo, China) was leveraged for reverse transcription in accordance with the manufacturer’s protocol. An Mx-3000P Quantitative PCR System (Applied Biosystems, USA) was used for qRT-PCR. The primers used for RT-qPCR were: 5′-GGAAAACCACTGGGGAGGAC-3′ (forward) and 5′-CAGTGCAGCTAAGAAATGGCT-3′ (reverse) for PDIA3P1; 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse) for GAPDH; 5′-TTTGTCCAAACCAACCGCAC-3′ (forward) and 5′-GCATCAACTTCGAAACCGGC-3′ (reverse) for CEBPB.
Plasmids, viral transfections and cloning
Human full-length PDIA3P1 as well as sh-PDIA3P1 plasmids were used in the current study for stable overexpression and knockdown, respectively, whereas empty plasmid was used as a control. Lentiviral particles were constructed by transfecting 293 T cells with the packaging vectors psPAX2 and pMD2G. Lentiviral particles were collected 24 and 48 hours after transfection of 293 T cells, filtered through a 0.45 μm filter (Corning), and then used to treat cells in culture. After 48 hours, cells were selected by Puromycin (2 μg/mL). All small interfering RNAs (siRNA) and overexpression plasmids were purchased from Genepharma (shanghai, China). For short-term knockdown and overexpression of GBM cells, cells were transfected of siRNAs and plasmids using the Lipofectamine 3000 kit (Invitrogen, USA) according to the manufacturer’s instruction.
Reagents and antibodies
TMZ and NEF (Synonyms: VX-745) were purchased from MedChemExpress (MCE,
https://www.medchemexpress.cn/). TMZ and NEF were dissolved in dimethyl sulfoxide (DMSO) at concentrations of 100 mM and 10 mM, respectively. TMZ and NEF in solvent are stored at − 20 °C and used up within 1 month. The primary antibodies used in this study are listed as follows: β-actin (Cell Signaling Technology, 8480), CD44 (Cell Signaling Technology, 3570), C/EBPβ (Abcam, ab32358), YKL-40 (Cell Signaling Technology, 47,066), SOX2 (Cell Signaling Technology, 3579), γ-H2AX (Cell Signaling Technology, 7631), MDM2 (Abcam, ab259265), JUN (Cell Signaling Technology, 9165), p-JUN (Cell Signaling Technology, 3270), ubiquitin (Cell Signaling Technology, 3933), P38 (Cell Signaling Technology, 8690), p-P38 (Cell Signaling Technology, 4511).
CCK-8 assay and drug treatment
CCK-8 reagent (RiboBio, China) was used to assess GBM cells viability. We seeded GBM cells in 96-well plates at a density of 2 × 103 cells per well in 100 μl of Gibco DMEM containing 10% FBS. The cells were incubated at 37 °C 12 h for cells adhesion and then treated with different concentrations of TMZ or NEF. After incubation for 48 h, 10 μl of CCK-8 solution was added to each well for 1 h before measurement. Absorbance (OD value) at 450 nm was measured using a microplate.
Alkaline comet assay
The alkaline comet assay was used to detect the damaged DNA with high sensitivity [
31]. GBM cells in different groups were harvested in PBS at a 1–3*10
5 cells/ml density. Cells were mixed with molten LM agarose at a ratio of 1:10 (V/V) and 50 μl of the mixture was immediately pipetted onto a CometSlide. Then cells were lysed in alkaline lysis solution at 4 °C for 12 h for lysis. After that, the slides were soaked with alkaline electrophoresis buffer for 20 minutes away from light and electrophoresis for 30 min at 25 V. After precipitation and washing, the slides were stained with Green-DNA Dye and images were captured by fluorescence microscopy.
Immunofluorescence (IF) assay
GBM cells were fixed in 4% paraformaldehyde for 15 min and washed three times in PBS. Then cells were permeabilized in 0.3% Triton X-100 for 10 min and blocked with 5% Goat serum for 1 h. Then the cells were incubated with indicated primary antibodies overnight at 4 °C. Cells were then incubated with fuorescent second antibody at room temperature for 1 h. DAPI was used to counterstain nuclei for 15 min. Images were captured using a LeicaSP8 confocal microscope.
EdU assay
EdU cell proliferation assay kit (RiboBio, China) was used to determine cell proliferation. Cells were incubated with 200 μl of 5-ethynyl-20-deoxyuridine at 37 °C for 2 h. After fixed and permeabilized, the cells were incubated with Apollo reagent for 30 min and the Hoechst were used to stain nuclei. The images were viewed and obtained using fluorescence microscope.
Flow cytometry
Both suspended and adherent GBM cells were obtained for apoptosis analysis after treating with TMZ or DMSO (solvent control of TMZ) for 48 h. Annexin VFITC and PI staining (BD Biosciences, USA) was leveraged for apoptosis analysis according to the instruction. The number of cells were counted by BD Accuri C6 flow cytometer.
We seeded about 2000 GBM cells in 6-well plates per well in 1.5 ml of Gibco DMEM containing 10% FBS. The cells were incubated in a humidified chamber containing 5% CO2 and 5% O2 at 37 °C for 2 weeks. After that, colonies were fixed and stained with crystal violet (Solarbio, China) for 20 min. The colonies were washed with PBS for at least three times and the number of colonies were counted.
We seeded about 1000 GSCs per well in 6-well plates with 1.5 ml DMEM/F12 containing 2% B-27. After 7 days incubation at 37 °C, the images were acquired and the relative diameters of neurospheres were calculated.
Extreme limiting dilution assay (ELDA)
We implanted GSCs into ultralow-attachment 96-well plates at densities of 0, 2, 4, 8, 16, 32, 64 and 128 cells per well in 10 replicates. The number of wells with neurospheres formation was counted after 7 days incubation. Collected data was analyzed using (
http://bioinf.wehi.edu.au/software/elda/).
Protein half-life assay
CHX was used to inhibit new proteins synthesis. Cells were treated with 100 μg/ml CHX for 0 h, 2 h, 4 h, 6 h and 8 h prior to protein collection. The proteins levels were detected by western blot assay.
RNA pull-down assay and RNA immunoprecipitation (RIP) assay
Biotinylated PDIA3P1 and its anti-sense sequence were synthesized by RiboBio (GenePharma, China). Pierce™ Magnetic RNA-protein pull-down kit (Thermo Fisher Scientific, SA) was used for RNA pull-down assay. Cell lysates of GSC267 were firstly incubated with a biotin-labelled PDIA3P1 probe. Then the conjugated magnetic beads were added to cell lysates and the interacting proteins were separated by western blot and then the silver staining was used for visualization.
Magna RIP kit (Millipore, USA) was leveraged for RIP assay according to manufacturer’s instruction. RT-qPCR was used for detecting the relative expression of immunoprecipitated RNA. The IgG antibody (from Magna RIP kit) was used for negative control.
Immunoprecipitation (IP) assay
The IP assay was performed using Pierce Classic Magnetic immunoprecipitation (IP)/Co-IP Kit (Thermo Fisher, USA) according to the manufacturer’s instruction. Firstly, the different antibodies were incubated with protein A/G magnetic beads. Then the cell lysates from GSCs were collected and incubated with antibody coupled beads. The beads interacting proteins were washed and denatured and the proteins were examined using western blotting.
Drug combination analysis
To assess the combination of effect of TMZ and NEF, GBM cells were treated with different concentrations of TMZ and NEF for 48 h in 3 replicates. CompuSyn software (Biosoft, Ferguson, MO, USA) was leveraged to evaluate drug synergism. The combination index (CI) values were calculated using non-constant ratios drug combination analysis according to instruction of the software. CI < 0.75, CI = 0.75–1.25, and CI > 1.25 were defined as synergistic, additive, and antagonistic effects, respectively.
Animal studies
Luciferase labeled and stably transfected sh-PDIA3P1-U118MG cells or sh-Control-U118MG, or ov-PDIA3P1-U251 or ov-vector-U251 were injected into the brains of randomly grouped 4-week male BALB/c nude mice (5 × 105 cells/mouse). On the fifth postoperative day, the mice were randomly divided into TMZ treatment or control groups. Mice were treated with or without TMZ by oral gavage per week (5 mg/kg, p.o., 5 times per week). For evaluating the anti-tumor effect of TMZ in combination with NEF in vivo, luciferase-labeled GSC267 cells (1 × 106 cells/mouse) were implanted into the brains of 4-week male BALB/c nude mice. After 7 days post-operative, the mice were randomly divided into four groups, control, TMZ only (5 mg/kg, p.o.,5 times per week), NEF only (5 mg/kg, p.o.,5 times per week) and combination group. To evaluate the intracranial tumor, bioluminescence imaging was used to quantify tumor burden using an IVIS Lumina Series III (PerkinElmer). All procedures used for animal treatments and experiments were approved by and under the requirements of the Animal Care and Use Committee of the Qilu Hospital of Shandong University.
Statistical analysis
All statistical analysis was conducted by R 4.1.1 and GraphPad Prism 8.0 software. Acquired data were certified as normal distribution through Shapiro-Wilk Normality test and homogeneity of variances through Bartlett test. Then t-tests and one-way ANOVA were used for comparisons between two independent samples and comparisons among multiple samples, respectively. The Wilcoxon test was used for non-parametric data. P-value < 0.05 was considered statistically significant (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001). The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of PDIA3P1, and the area under the curve (AUC) was quantified using the pROC R package. Pearson correlation was used to calculate the correlation between two or more groups. Kaplan-Meier curve and log-rank test were used to evaluate survival between different groups.
Discussion
In this study, we screened the lncRNA PDIA3P1, which is closely related to TMZ resistance in GBM, based on a comprehensive analysis of the CCLE and GDSC databases. Bioinformatics analyses of public databases combining qRT–PCR results indicated that expression of PDIA3P1 was upregulated in TMZ-resistant cell lines and predicted a higher risk of tumor recurrence. Combining in vitro and in vivo assays, we further confirmed that PDIA3P1 reduces the TMZ sensitivity of glioma cell lines. Mechanistically, PDIA3P1 promoted PMT by disrupting the C/EBPβ/MDM2 complex to inhibit the ubiquitination of C/EBPβ, enabling glioma cells to obtain stronger TMZ therapy resistance. To our knowledge, this is the first report showing the function and mechanism of PDIA3P1 in promoting TMZ resistance in GBM.
The primary obstacle to GBM therapy is the development of TMZ resistance. Increasing evidence suggests that excessive activation of O6-methylguanine-DNA methyltransferase (MGMT), which removes TMZ-induced alkylation from different nucleotides, is the most important cause of TMZ resistance in GBM [
43‐
45]. However, studies have recently shown that MGMT overexpression is not the only determinant contributing to GBM resistance to TMZ [
5]. Other factors, such as the advent of GSCs, overactivation of DNA repair pathways, favorable autophagy, decreased drug influx and increased drug efflux, facilitate drug resistance in TMZ in addition to MGMT overexpression [
46‐
51]. GSCs exhibit the capacity for self-renewal, immortal propagation and multilineage differentiation [
52]. GSCs can be divided into PN and MES subtypes according to their transcriptional program, genotype and epigenetic status [
19,
53,
54]. PN GSC is characterized by relatively faster proliferation and sensitivity to adverse stimulation, whereas MES GSC is characterized by the secretion of various factors and the ability to maintain relative stability under adverse conditions [
33]. The PN subtype transition to the MES subtype is considered a crucial process for tumor recurrence and treatment tolerance in GBM [
55]. It was reported that immune infiltration in the TME is associated with PMT. However, our analysis indicated that expression of PDIA3P1 was not associated with tumor immunity (Fig. S
3B and Table. S
3), suggesting the impact of PDIA3P1 on PMT based on an endogenous pathway.
C/EBPβ is highly expressed and activated in MES subtype GSCs and is the MR in the process of PMT. Given its role in the PMT, C/EBPβ has great potential as a therapeutic target for GBM [
26]. However, the mechanisms for C/EBPβ regulation in GBM have not been completely clarified. Based on RNA pulldown and mass spectrometry analysis, we concluded that PDIA3P1 promotes PMT by targeting C/EBPβ. We found that PDIA3P1 had no effect on mRNA expression but did increase C/EBPβ protein expression in GSCs by increasing C/EBPβ protein stability and decreasing C/EBPβ ubiquitination. Therefore, our results suggest that PDIA3P1 functions as a regulator of PMT by restricting C/EBPβ degradation. PDIA3P1 has been postulated to primarily function as a competitive endogenous RNA (ceRNA) that competes for microRNA (miRNA) binding, playing an important role in gene regulation [
56‐
58]. In this study, PDIA3P1 did not function as a ceRNA but was able to physically bind to C/EBPβ protein, reducing its ubiquitination and subsequent degradation. Ubiquitin-dependent protein degradation plays a critical role in the posttranscriptional regulation of most proteins [
59]. It has been reported that C/EBPβ can be degraded by the E3 ubiquitin ligase MDM2 to promote myogenesis [
60]. Therefore, we hypothesized and verified that PDIA3P1 affects the ubiquitination and degradation of C/EBPβ through MDM2. Our data suggest that PDIA3P1 binds proteins that function to disrupt the C/EBPβ/MDM2 complex rather than binding to miRNAs.
The function of the p38α-MAPK pathway is to relay, amplify and integrate a variety of extracellular stresses, such as radiotherapy, chemotherapy, hypoxia and hunger, thereby regulating the genomic and physiological response of cells to their environment [
61]. It has been reported that acute treatment with TMZ induces DNA damage and transitory activation of MAPK14/p38α [
62,
63]. In addition, activation of the MAPK pathway has been associated with poor survival in GBM patients during the TMZ era [
64]. The p38α-MAPK pathway is markedly activated during TMZ treatment and resists the killing effect of TMZ. We found that expression of PDIA3P1 increased after treatment of cells with TMZ in a concentration- and time-dependent manner. Further analyses indicated that the p38α-MAPK signaling pathway mediated TMZ-induced upregulation of PDIA3P1. There is a loop in which TMZ treatment activates the p38α-MAPK signaling pathway, which then promotes the expression of PDIA3P1, finally resulting in PDIA3P1 promoting PMT to attenuate the adverse effects of TMZ treatment. We next aim to test interventions to break this loop and provide potential therapeutic strategies for overcoming TMZ resistance.
Currently, TMZ combined with other antitumor agents has become the primary strategy for treating refractory glioma [
65]. The basic principle of combination treatment is to leverage different agents that target key pathways by different mechanisms to reduce drug-resistant cancer cells. There is evidence that combination therapy with TMZ prolongs the overall survival of GBM patients [
66]. Based on the strategy of combination therapeutics, we selected a specific p38α inhibitor, NEF, which exhibits BBB permeability. We revealed that NEF in combination with TMZ exhibits synergistic effects at the indicated concentrations. Moreover, we confirmed the efficacy of the combined treatment strategy using both in vitro and in vivo experiments. In summary, we determined the mechanism by which PDIA3P1 mediates TMZ resistance. More importantly, we demonstrated a new treatment strategy in which the combined use of TMZ and NEF has the potential to overcome TMZ resistance.
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