PDIA3P1 is markedly upregulated in TMZ-resistant cell lines and promotes TMZ resistance.
To search for potential lncRNAs involved in GBM resistance to TMZ chemotherapy, the information of 10 glioma cell lines paired with specific half-maximal inhibitory concentration (IC50) values of TMZ were obtained from GDSC and the RNA-seq data of corresponding cell lines was downloaded from the CCLE. We divided the glioma cell lines into TMZ resistance group and TMZ sensitive group based on IC50 values. The limma package was utilized to analyze the DEGs between the two groups. The clustered heatmap (Fig. 1A) exhibited the top 30 upregulated genes in TMZ resistant group sorted according to p-value. Among the DEGs, PDIA3P1 is markedly upregulated in TMZ resistance cell lines (log2Fold change = 1.6, P < 0.001) (Fig. S1A and Table. S1). Based bioinformatic analysis, we found that the expression of PDIA3P1 was significantly upregulated in most recurrent gliomas (Fig. 1B). Survival analysis exhibited that higher PDIA3P1 levels were related to poorer progression-free survival (PFS) in GBM patients (Fig. S1B). In addition, the low-PDIA3P1 group showed a significant survival advantage in GBM patients either receiving or not receiving chemotherapy (Fig. 1C). Next, qRT-PCR on two TMZ-sensitive (U251 and LN229) and TMZ-resistant GBM cell lines (U118MG and U87MG) verified that PDIA3P1 was upregulated in TMZ-resistant cell lines (Fig. S1C).
To investigate the functional role of PDIA3P1 in promoting TMZ resistance, PDIA3P1 was knocked down using two independent shRNAs in U118MG and U87MG and overexpressed in U251 and LN229. The expression of PDIA3P1 was detected using qRT-PCR (Fig. S1D). Knockdown of PDIA3P1 in resistant cell lines (U118MG and U87MG) resulted in a notably reduction in IC50 and further inhibition of tumor cell growth rate upon TMZ treatment (Fig. 1D and Fig. S1E). In contrast, overexpression of PDIA3P1 in sensitive cell lines (U251 and LN229) resulted in a significant increase of IC50 values and counteracted inhibitory effect of TMZ on tumor cells growth (Fig. 1E and Fig. S1E).
To evaluate the effect of PDIA3P1 on the TMZ-resistant phenotype in vivo, 5 × 105 luciferase-labeled U118MG-shPDIA3P1 or U118-shNC, and U251-PDIA3P1 or U251-Vector cells were injected into nude mice, respectively. We tracked tumor proliferation through in vivo bioluminescence imaging. Despite the initial tumor size being similar (Fig. S2A and Fig. S2B), xenografts bearing U118MG-shPDIA3P1 cells showed significant tumor growth inhibition, whereas xenografts bearing U251-PDIA3P1 cells showed tumor growth promotion. As expected, TMZ treatment (5 mg/kg, p.o., 5 times per week) reduced tumor burden. Tumor size in U118MG-shPDIA3P1 group was reduced compared to control group (Fig. 1F and Fig. 1G), while tumor size in U251-PDIA3P1 group was relatively increased compared to control group (Fig. 1H and Fig. 1I). Consistently, Kaplan-Meier curves revealed that the overall survival time of mice in PDIA3P1 knockdown group was prolonged with and without TMZ treatment (Fig. S2C). Although TMZ treatment significantly prolonged the survival time of mice in U251-Vector group, PDIA3P1 overexpression shortened the survival time of mice in both the treatment and control groups (Fig. S2D). H&E-stained mice brain sections showed that knockdown of PDIA3P1 greatly reduced tumor invasiveness, with or without TMZ treatment, whereas overexpression of PDIA3P1 promoted tumor invasiveness (Fig. S2E and Fig. S2F). Taken together, these findings indicated that PDIA3P1 promoted glioma cells resistance to TMZ both in vitro and in vivo.
Knockdown of PDIA3P1 exacerbates DNA damage and proliferation inhibition induced by TMZ intervention.
To explore the biological behaviors of PDIA3P1, we performed GSVA enrichment. PDIA3P1 high expression group was significantly enriched in damage repair and stress response pathways such as the regulation of DNA repair and cellular response to chemical stress, suggesting that PDIA3P1 may play a role in damage repair and stress response (Fig. S3A and Table. S2). As TMZ exerts its antitumor effects mainly by damaging DNA and inducing programmed cell death (PCD), we performed COMET and ɣH2AX assays to detect DNA damage. Alkaline COMET assay could be used for sensitive detection of DNA double-strand breaks (DSBs) and single-strand breaks. We found increased DNA damage in shPDIA3P1 cells after TMZ treatment, whereas knockdown of PDIA3P1 had very little effect on DNA damage in the absence of TMZ in U118MG and U87MG cells (Fig. 2A, Fig. S4A and Fig. 2B). Phosphorylated histone H2AX (ɣH2AX) is an indicator of the DNA damage response (DDR). When DNA damage occurs, ɣH2AX can be recruited to the lesions. Using IF staining assay, we confirmed that knockdown of PDIA3P1 increased nuclear ɣH2AX levels after TMZ treatment, while nuclear ɣH2AX levels remained virtually unchanged in the absence of TMZ intervention. (Fig. 2C, Fig. S4B and Fig. 2D). We further performed EdU, colony formation and apoptosis assays to explore the function of PDIA3P1. EdU and colony formation assays showed that knockdown of PDIA3P1 inhibited cell proliferation, whereas in the case of TMZ treatment, knockdown of PDIA3P1 inhibited cell growth even more (Fig. 2E, Fig. S4C and Fig. S4D). Then we recorded apoptosis rate using flow cytometry and the proportion of apoptotic cells significantly increased in PDIA3P1 knockdown group after TMZ treatment (Fig. 2F). Collectively, the above results indicated that knockdown of PDIA3P1 exacerbated TMZ intervention induced DNA damage and growth inhibition, thereby restoring the sensitivity of tumor cells to TMZ.
Overexpression of PDIA3P1 counteracted TMZ treatment-induced DNA damage and growth inhibition.
Since knockdown of PDIA3P1 was able to restore the TMZ sensitivity of GBM cells, we investigated whether overexpression of PDIA3P1 could promote TMZ resistance. We overexpressed PDIA3P1 in U251 and LN229 GBM cell lines which are relatively sensitive to TMZ. Comet assay showed that PDIA3P1 overexpression without TMZ intervention had little effect on the DNA damage, while overexpression of PDIA3P1 could salvage TMZ-induced DNA damage (Fig. 3A and Fig. S4E). Similarly, IF staining assay found that PDIA3P1 overexpression decreased nuclear ɣH2AX levels after TMZ treatment, whereas nuclear ɣH2AX levels remained almost constant and at a relatively low level in the absence of TMZ (Fig. 3B and Fig. S4F). The EdU assay exhibited that overexpression of PDIA3P1 had a positive effect on cell proliferation. Furthermore, PDIA3P1 overexpression partially counteracted TMZ-mediated cell growth inhibition (Fig. 3C). We then evaluated the effect of PDIA3P1 overexpression on the apoptosis rate of GBM cells. As shown in Fig. 3D, a slight decrease in apoptosis rate was observed in these cells overexpressing PDIA3P1 compared to control cells without TMZ treatment, whereas PDIA3P1 overexpression greatly counteracted the apoptosis induced by TMZ treatment (Fig. 3D). Hence, through a series of experiments, we demonstrated that overexpression of PDIA3P1 could reduce DNA damage and proliferation inhibition caused by TMZ intervention, thus conferring TMZ resistance to GBM cells.
Elevated expression of PDIA3P1 is associated with Mesenchymal subtype.
We have used gain-of-function and loss-of-function experiments in vitro and in vivo to prove that PDIA3P1 can promote GBM cells resistance to TMZ by reducing DNA damage. We intend to further investigate the mechanism of PDIA3P1-mediated TMZ resistance. Phenotypic heterogeneity and plasticity in GBM drive therapy resistance and recurrence. Compared to PN subtype, which has a better survival prognosis and is sensitive to TMZ treatment, the MES subtype shows a higher resistance to radiotherapy and chemotherapy and a higher risk of recurrence. [25, 33]. We hypothesized that the function of PDIA3P1 to promote TMZ resistance is mediated by affecting GBM subtypes. We first examined the expression of PDIA3P1 in TCGA and CGGA datasets and found that PDIA3P1 expression was significantly higher in the MES subtype than in the PN subtype (Fig. 4A and Fig. S5A). The existence of GSCs is an important factor contributing to GBM heterogeneity and TMZ resistance, and GSCs are used as a valuable experimental model for GBM analysis. By detecting PDIA3P1 expression by qRT-PCR, we concluded that PDIA3P1 expression was markedly upregulated in MES GSCs (GSC20, GSC267) compared with PN GSCs (GSC8-11, GSC11), and PDIA3P1 was least expressed in neuronal precursor cell lines (NPC) (Fig. 4B). To explore the predictive efficiency of PDIA3P1 for GBM subtype, the area under the receiver operating characteristic (ROC) curve (AUC) was calculated and PDIA3P1 expression was found to be accurate in assessing GBM subtypes (AUC = 0.7687) (Fig. 4C and Fig. S5B). Meanwhile, we performed pearson correlation analysis of gene expression and found a significant positive correlation between PDIA3P1 and MES subtype-related genes (CD44, FN1, CHI3L1, SERPINE1), while PDIA3P1 was negatively correlated with PN subtype-related genes (DLL3, NCAM1, ASCL1, OLIG2) (Fig. 4D). We further performed a GSEA analysis of the relationship between PDIA3P1 and GBM subtypes based on the TCGA dataset. As the results showed, the MES-GBM subtype was enriched at high PDIA3P1 expression group, whereas the PN-GBM subtype presented in the low PDIA3P1 expression group (Fig. 4E). The two independent shRNAs were transfected into GSC20 and GSC267, and PDIA3P1 was overexpressed in GSC8-11 to investigate the causal relationship between PDIA3P1 and GBM subtype (Fig. S5C). Stable knockdown of PDIA3P1 in GSC20 and GSC267 resulted in an obvious inhibition of tumorsphere expansion (Fig. S5D) and reduced sphere formation ability (Fig. 4G). The above results demonstrated that PDIA3P1 was associated the ability of tumorigenesis and stemness of GSCs. CD44 and SOX2 are marker proteins of the MES and PN subtypes, respectively. In GSC20 and GSC267, PDIA3P1 knockdown decreased CD44 expression and increased SOX2 expression, which was verified by IF assays (Fig. 4D and Fig. S5E). Besides, two MES marker proteins CD44 and YKL-40 were downregulated after interfering with PDIA3P1 expression (Fig. 4H).
PDIA3P1 promotes PMT and TMZ resistance by affecting C/EBPβ in GSCs.
Given that LncRNAs can directly bind to proteins to exert regulatory functions, we first performed RNA pull-down experiments in GSC267 to explore the molecular interaction mechanism of PDIA3P1. Then we detected the binding proteins of PDIA3P1 by silver staining and mass spectrometry analysis (Table. S4). We found that C/EBPβ was significantly enriched on PDIA3P1 compared to antisense, and RNA pull down assay was performed again to verify their interaction (Fig. 5A and Fig. 5B). Subsequently, RNA immunoprecipitation (RIP) assay further confirmed that C/EBPβ could specifically combine with PDIA3P1 (Fig. 5C). C/EBPβ is thought to be one of the MRs promoting PMT, and the interaction and effect of PDIA3P1 on C/EBPβ expression further validated our finding that PDIA3P1 can be involved in GBM PMT progression. Knockdown of PDIA3P1 decreased the expression of C/EBPβ, CD44 and YKL-40, whereas transfection of C/EBPβ into GSC267 counteracted the effect of PDIA3P1 knockdown on the expression of CD44 and YKL-40 (Fig. 5D). We further performed neurosphere formation and extreme limiting dilution assay (ELDA) to explore the effect of PDIA3P1- C/EBPβ on tumorigenesis. We observed the expansion of tumor spheres and the ability to form spheres were significantly restored when C/EBPβ was transfected into PDIA3P1-knckdown GSC267 cells (Fig. 5E and Fig. 5F). Knockdown of C/EBPβ in PDIA3P1-expressing GSC8-11 resulted in the suppression of tumorsphere expansion (Fig. 5G) and reduced sphere formation ability (Fig. 5H). To investigate that PDIA3P1 promotes the resistance of GSCs to TMZ by affecting C/EBPβ, the comet assay and γ-H2AX IF assay were performed. The results showed that knockdown of PDIA3P1 increased nuclear γ-H2AX levels in GSC267 after TMZ treatment, whereas transfection of C/EBPβ into shPDIA3P1-GSC267 decreased nuclear γ-H2AX expression, implying that overexpression of C/EBPβ restores TMZ resistance of GSCs (Fig. 5I and Fig. S6A). Similar results were obtained for the comet experiment (Fig. 5I and Fig. S5B), which suggests that overexpressing C/EBPβ could counteract the effect of PDIA3P1 knockdown on TMZ resistance in GSCs. For GSC8-11, comet assay and γ-H2AX IF assay revealed that knockdown of C/EBPβ restored the sensitivity of GSC8-11 expressing PDIA3P1 to TMZ (Fig. 5J, Fig. S6C and Fig. S6D). Collectively, we determined that PDIA3P1 promoted PMT and TMZ resistance by affecting C/EBPβ expression.
PDIA3P1 stabilizes C/EBPβ by preventing MDM2 -mediated ubiquitination.
We further investigate the interaction of PDIA3P1-C/EBPβ. PDIA3P1 knockdown decreased protein expression of C/EBPβ (Fig. 5D and Fig. 5K), but not mRNA levels of C/EBPβ (Fig. S6E), suggesting that PDIA3P1 regulates protein level of C/EBPβ through affecting translational or post-translational modification. The ubiquitin-proteasome system (UPS) is the main pathway of protein degradation, and it participates in the degradation of more than 80% of proteins in cells . To confirm the possibility of that PDIA3P1 regulates C/EBPβ through proteasome, the GSCs were treated with the proteasome inhibitor MG132. Knockdown of PDIA3P1 significantly decreased the expression of C/EBPβ, whereas, MG132 treated GSCs with silenced PDIA3P1 showed minimal change in C/EBPβ levels (Fig. 5K). Then we blocked the protein synthesis using cycloheximide (CHX) and found that PDIA3P1 knockdown resulted in a significantly shorter half-life of C/EBPβ protein in GSC267 (Fig. 5L). Consistently, the half-life of C/EBPβ protein in PDIA3P1 stable overexpression GSC8-11 was significantly longer than that of the corresponding control cells (Fig. S6F). Immunoprecipitation (IP) results showed that knockdown of PDIA3P1 significantly increased the ubiquitylation of C/EBPβ in GSC267, whereas overexpression of PDIA3P1 significantly decreased the ubiquitylation of C/EBPβ in GSC8-11 (Fig. 5M). Above all, our data suggested that PDIA3P1 was involved in the post-translational modification of C/EBPβ.
E3 ubiquitin ligase is a family of more than 700 proteins, binding ubiquitin to the target protein and play a major role in protein degradation. To further investigate the E3 ubiquitin ligases involved in the post-translational modification of C/EBPβ, we reviewed numerous references and found that Mouse double minute 2 homolog (Mdm2) could target C/EBPβ for degradation . MDM2 is an E3 ubiquitin ligase of the RING finger family that is known to be involved in the degradation of p53 . Since we have proved that PDIA3P1 directly binds to C/EBPβ to affect the ubiquitination level of C/EBPβ, we supposed that PDIA3P1 may impact on the C/EBPβ-MDM2 complex formation. To test our hypothesis, the interaction between C/EBPβ and MDM2 was investigated by co-IP assays. The results demonstrated that overexpression of PDIA3P1 hampered the interaction of C/EBPβ and MDM2 in GSC267 (Fig. 5N). In addition, knockdown of PDIA3P1 resulted in more MDM2 binding to C/EBPβ in GSC267 (Fig. 5N). Collectively, our present data suggested that PDIA3P1 could stabilize C/EBPβ by disrupting C/EBPβ-MDM2 complex.
Pdia3p1 Is Upregulated In Response To Tmz-induced Activation Of The P38-mapk Signaling Pathway
TMZ treatment and subsequent detrimental stress within tumor cells can change expression levels of multiple genes. We treated GSC20, GSC267, U118MG and U251 using different concentrations of TMZ for 48 h, as well as cells with 400 µM TMZ for different treatment durations. We observed that PDIA3P1 expression was upregulated in a dose-dependent and time-dependent manner following TMZ intervention (Fig. 6A and Fig. 6B). To explore the mechanism of TMZ-induced PDIA3P1 upregulation, we obtained RNA array data from GSE68029, which identified defense profiles of GSC-500uM TMZ. We performed differential analysis of this data and conducted gene oncology (GO) enrichment analysis on the differential genes. Compared with parental GSCs, TMZ treatment resistant GSCs were significantly enriched in the gene sets associated with p38α MAPK biological pathway, which suggested that the p38α MAPK signaling pathway was possibly involved in TMZ resistance and was activated after TMZ treatment of GSCs (Fig. 6C). The p38α MAPK signaling pathway is mainly responsible for transduction of extracellular signals, which can be activated by various environmental stresses and inflammatory cytokines . Activation of the core molecule p38α indirectly regulates the transcriptional process of various genes by regulating multiple transcription factors, which helps cells to respond adequately to changing environmental conditions .
Targeting p38α may block the stress response of tumor cells, thus preventing TMZ-induced upregulation of PDIA3P1. We reviewed small molecule inhibitors specifically targeting p38 from DRUGBANK and MCE. We screened Neflamapimod (NEF) is a potential drug for the treatment of Alzheimer's disease (AD) and has been preliminarily confirmed to be safe for human use . NEF has excellent blood-brain barrier (BBB) permeability, which suggests its value for central nervous system (CNS) disorders and some studies have exhibited the anti-tumor activity of NEF [40–42]. CCK-8 cell proliferation was performed to determine IC50 of four cell lines for NEF (Fig. 6D). GBM cells treated with NEF could inhibited TMZ-induced upregulation of PDIA3P1 (Fig. 6E). Then, we further explored whether TMZ in combination with NEF could synergistically inhibit GBM cells growth. GBM cells were treated with indicated concentrations of TMZ and NEF, respectively and the cell growth inhibition was determined by CCK-8 assay (Fig. 6F). Based the results of proliferation inhibition, we calculated combination index (CI) score to evaluate the combination effect of TMZ and NEF (Fig. 6G and Fig. S7A). CI > 1.25, CI = 0.75–1.25, and CI < 0.75 were defined as antagonistic, additive and synergistic effects, respectively. For instance, in GSC267 cells, a relative low concentration of TMZ (50 µM) and NEF (20 µM) may exhibit better synergistic effect (CI = 0.44), despite their relatively low growth inhibitory effects about 23%. When GSC20 cells were treated with moderate concentrations of TMZ (800 µM) and NEF (80 µM), they showed an additive effect despite their relatively high growth inhibition of about 81%. Collectively, these data revealed that TMZ in combination with NEF exhibited synergistical effects at the indicated concentrations.
Activation of p38-MAPK signaling pathway could further activate some transcriptional factors like JUN. We observed a significantly positive relationship between the expression of JUN and PDIA3P1 in TCGA and CGGA datasets (Fig. 6H). Knockdown of JUN not only reduced the PDIA3P1 expression, but also counteracted TMZ-induced upregulation of PDIA3P1 (Fig. 6I), preliminarily suggesting that JUN is responsible for PDIA3P1 transcription. We constructed four fragments of different lengths located upstream of the TSS based on the JUN binding motif (Fig. 6J). The four luciferase reporter plasmids were transfected into GSC267 individually to verify the JUN binding sites. The luciferase activity of fragment 4 was statistically unchanged after knockdown of JUN, demonstrating that JUN does not bind to fragment 4 (Fig. 6K). To further determine the binding sites in more detail, we designed three pairs of PCR primers and performed CHIP assay. qRT-PCR assay showed approximate 10-fold enrichment and 5-fold enrichment for site #1 and site #2, respectively, while there was no significant enrichment at site #3 (Fig. 6L and Fig. S7B). In conclusion, our data suggest that JUN is involved in TMZ-induced upregulation of PDIA3P1 and can directly bind and initiate PDIA3P1 transcription.
NEF combined with TMZ confers a better anti-tumor effect both in vitro and in vivo.
To evaluate the antitumor effect of TMZ and NEF combination, we conducted a series of experiments in vitro. In GSC20 and GSC267 cells, the comet assay showed that the level of DNA damage was higher when treated with TMZ or NEF alone than in the control group, while DNA damage was more pronounced in the combination treatment group than in the monotherapy, indicating that the combination of TMZ and NEF exhibited a more powerful anti-tumor effect (Fig. 7A and Fig. 7B). Similar results were obtained in the ɣH2AX IF assay, where significantly higher nuclear ɣH2AX was observed in the combined group of TMZ and NEF, suggesting that the combined treatment resulted in a potentially enhanced effect of DNA damage (Fig. 7C and Fig. 7D). The EdU assay showed that both TMZ or NEF treatment alone could inhibit proliferation of tumor cells, whereas the combination group had a more obvious inhibition of proliferation efficiency (Fig. S8A). Next, we detected apoptosis levels using flow cytometry. U118MG with TMZ or NEF treatment alone exhibited 31.35% and 30.22% apoptosis rates, respectively, whereas the apoptosis rate increased to 51.2%, when TMZ combined with NEF was employed (Fig. S8B). Given that NEF targeted p38α and thereby affected the subsequent transcriptional process of PDIA3P1, which have been shown to promote PMT, we intend to verify whether NEF is also involved in the subtype of GSC. As the results showed, the expression of CD44 was significantly reduced in GSC20 and GSC267 after 48 hours of NEF (50 µM) treatment, while the expression of SOX2 was elevated (Fig. 7E and Fig. 7F). In addition, overexpression of PDIA3P1 counteracted the effect of NEF treatment on CD44 and SOX2 expression, indicating that NEF could affect the subtypes of GSC through PDIA3P1 (Fig. 7E and Fig. 7F). To evaluate the antitumor activities of TMZ and/or NEF in vivo, nude mice carrying GSC267 xenografted tumors were administered TMZ (5mg/kg, p.o., 5 days per week), NEF (5 mg/kg/day, p.o., 5 days per week), or both drugs in combination after building the orthotopic xenograft model. The results showed that both TMZ or NEF treatment alone could inhibit proliferation of tumor cells, whereas the combination treatment produced remarkable tumor regression (Fig. 7G and Fig. S8C). Consistently, survival analysis showed that both TMZ or NEF treatment alone could prolong the survival time of mice, whereas the combination treatment group showed a significantly longer survival time (Fig. S8D). H&E-stained mouse brain sections showed that TMZ in combination with NEF limited the invasion ability of the tumor to the greatest extent (Fig. S8E). Taken together, our results exhibited that TMZ in combination with NEF had an excellent synergistic anti-tumor effect both in vitro and in vivo.