Mechanism of RIP2 enhancing stemness of glioma cells induces temozolomide resistance

Abstract Aims We aimed to investigate the role of receptor‐interacting protein 2 (RIP2) in regulation of stemness of glioma cells and chemotherapy resistance. Methods Plasmid transfection was used to overexpress RIP2. Chemical inhibitors were used to inhibit RIP2 or NF‐κB activity. Cancer stemness of glioma cells was investigated by sphere formation assays, clone formation assays, and xenograft tumor formation assays. The expression of RIP2, p‐NF‐κB, IκBα, CD133, or SOX‐2 was detected by Western blotting and immunofluorescence. Apoptosis was detected by flow cytometry. Immunohistochemical staining was used to detect the expression of RIP2, CD133, and SOX‐2 in xenograft tumor tissue. The effect of the RIP2/NF‐κB pathway on temozolomide (TMZ) resistance was evaluated by xenograft tumor assay. Results Transfection with RIP2 plasmid enhanced the sphere formation capability of U251 cells, clone formation capability, and xenograft tumor formation capability. RIP2 could mediate TMZ resistance by upregulating the expression of CD133 and SOX‐2 by activating the NF‐κB pathway. Both RIP2 inhibitor GSK583 and the NF‐κB inhibitor SC75741 could reverse the resistance of U251 cells to TMZ. Conclusion RIP2 mediates TMZ resistance by regulating the maintenance of stemness in glioma cells through NF‐κB. Interventions targeting the RIP2/NF‐κB pathway may be a new strategy for TMZ‐resistant gliomas.

plays an important role in the treatment of gliomas. 6 Inevitably, after a period of TMZ treatment, the treatment often fails due to development of acquired drug resistance, which is one of the major reasons for tumor progression. 7 Therefore, the prevention of TMZ resistance is of significance for treating glioma. The causes of acquired drug resistance in gliomas are complex. Current studies have found that it is mainly related to the increased expression of ATP-binding cassette transporters, 8 enhanced stem cell characteristics, 9 abnormal DNA damage repair systems, 10 and antiapoptosis, up-regulation of protein expression, 11 and epithelial-mesenchymal transition. 12 There is increasing evidence that enhanced glioma stem cell properties are associated with TMZ resistance, but the signaling mechanisms are not fully clarified.
RIP2 is a kinase that is mainly involved in mediating immunity, inflammation, and apoptosis. 13 In recent years, studies have found that RIP2 is expressed in various malignant tumor cells such as liver cancer, 14 breast cancer, 15 and bladder cancer 16 and can mediate malignant biological behaviors such as tumorigenesis, cell migration and invasion, and metastasis. Increasing evidence shows that RIP2 plays an important role in mediating resistance of malignant tumor cells to drug therapy, such as resisting paclitaxel and ceramide-induced apoptosis of breast cancer cells, 17 reversing B-lymphoproliferative disorders, and bortezomib resistance. 18 The previous work of this study showed that RIP2 was involved in the resistance of gliomas to TMZ by inducing the expression of DNA repair enzyme MGMT. 19 However, the relationship between RIP2-mediated TMZ resistance and the stemness of glioma cells was still unclear, which was the focus of this study. were cultured in DMEM high glucose medium (Invitrogen) containing 10% FBS and 1% penicillin-streptomycin. All cells were cultured in a 5% CO 2 , 37°C cell incubator.
After TMZ treatment, 10 μl of CCK-8 (Beyotime, Shanghai, China) solution was added to each well and incubated in an incubator for 3 h. The absorbance at 450 nm was read using an ELx800 microplate reader (BioTek, Winooski, VT, USA).

| Plate cloning experiments
The cells were seeded in 6-well plates at 500 cells per well and cultured for 12-14 days. Cells were fixed with 4% paraformaldehyde (Beyotime), stained with 1% crystal violet staining solution (Beyotime), washed with water to remove the staining solution, and photographed with a camera.

| Sphere formation assays
A serum-free suspension medium was prepared using DMEM/F12 medium +20 ng/ml EGF + 20 ng/ml bFGF +2% B27. Cells were collected and seeded at 3000 cells/well in a low-adsorption 12-well plate for 10 consecutive days. Cell pellets were collected, trypsinized to prepare a single-cell suspension, centrifuged to remove trypsin, and the suspension was prepared with serum-free suspension medium. Per well 3000 cells were inoculated into a low-adsorption 12well plate and cultured for 10 days to observe the cell spheroidization.

| Western blotting
Cells were collected in centrifuge tubes and lysed with RIPA lysis buffer (Beyotime) to obtain total protein. The total protein concentration was measured in a BCA protein quantification kit (Beyotime), and a sample containing 3 μg/μl total protein was prepared. Proteins

| Cell apoptosis analysis by flow cytometry
The cells to be tested were resuspended in binding buffer at 5 ×

| Immunofluorescence analysis
Cells were fixed with pre-cooled methanol at 0-5°C for 20 min. After washing off the fixative, the cells were blocked with 3% BSA for 30 min

| Animal experiments
Male nude mice (BALB/c-nu) were purchased from GemPharmatech

| Statistical analysis
Statistical analysis was performed using SPSS 19.0 and GraphPad Prism 8.0 for Windows. All data conform to the normal distribution by Shapiro-Wilk test. Statistics are presented as mean ± standard deviation (SD). Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Fisher's multiple comparison test. P < 0.05 indicated statistical significance.

| RIP2 induces TMZ resistance in glioma cells
To study the role of RIP2 in TMZ drug resistance of gliomas, we first used Raji cells with high RIP2 expression as a positive control to observe the endogenous expression of RIP2 in four gliomas (SW1783, T98G, U87, and U251). The results showed that all four gliomas could express RIP2, but the expression level was significantly weaker than that of Raji cells ( Figure 1A). We further pretreated four types of cells with rRIP2 and observed the inhibitory effect of TMZ on cell proliferation. With increasing doses, rRIP2 significantly resisted the TMZ-induced decrease in cell proliferation ( Figure 1B-E). Among them, U251 cells were the most sensitive to TMZ. We then observed the sensitivity of cells to TMZ by exogenously upregulating RIP2 protein expression in four cell types. After RIP2 overexpression, all four cells were significantly less sensitive to TMZ ( Figure 1F-J). Similarly, U251 cells remained the most sensitive to TMZ.

| RIP2 enhances stemness of glioma cells
Considering that the effect of RIP2-induced U251 cells on TMZ sensitization was the most obvious among the four types of cells, we took U251 cells as the research object for further study. Currently, it is unclear whether RIP2 is involved in the maintenance of stemness of F I G U R E 1 RIP2 induces glioma cells to resist TMZ. (A) Western blot assay to detect the expression of RIP2 in Raji, SW1783, T98G, U87, U251 cells. **P < 0.01. (B-E) Different glioma cells were treated with gradient concentrations of rRIP2 for 24 h and then TMZ was added (75, 150, 300, 600, 1200, 2400, and 4800 μM) for 72 h, and cell viability was detected by CCK-8 assay. (F) T98G, SW1783, U87, and U251 cells were transfected with RIP2 plasmid and vector, respectively, and untransfected cells were used as negative controls. The expression of RIP2 was detected by Western blotting. **P < 0.01. (G-J) Different glioma cells transfected with RIP2 plasmid and vector were treated with TMZ (75, 150, 300, 600, 1200, 2400, and 4800 μM) for 72 h, and cell viability was measured by CCK-8 assay. Normalization was performed with GAPDH as an internal reference. glioma cells. We performed a sphere formation assay and found that overexpression of RIP2 enhanced sphere formation capability of U251 cell lines (Figure 2A). Then, plate cloning experiments found that overexpression of RIP2 significantly enhanced the colony forming ability of U251 cells. In vivo experiments showed that U251 xenografts overexpressing RIP2 had stronger colony forming ability ( Figure 2F). These suggested that RIP2 might enhance the stemness of U251 cells. For further verification, we observed the expression changes in stem cell marker proteins (CD133 and SOX-2) by Western blotting experiments.
The results showed that both rRIP2 treatment and exogenous overexpression of RIP2 up-regulated CD133 and SOX-2 protein expression ( Figure 2C and D). The immunofluorescence experiments also found that the expression of CD133 and SOX-2 in U251 cells overexpressing RIP2 was significantly up-regulated ( Figure 2E).

| RIP2 enhances stemness of glioma cells dependent on the NFκ B pathway
To observe whether NF-κB pathway is involved in RIP2-mediated maintenance of stemness in glioma cells, we first detected the expression of NF-κB pathway-related proteins. Western blot results showed that rRIP2 up-regulated p-NF-κB p65 expression and down-regulated IκBα expression in U251 cells ( Figure 3A). At the same time, similar changes in expression also occurred in p-NF-κB p65 and IκBα in U251 cells transfected with RIP2 plasmid ( Figure 3B). Subsequently, we pre-

| Inhibition of RIP2/NFκ B signaling pathway inhibits RIP2-induced TMZ resistance in glioma cells
Our study revealed that RIP2 enhanced glioma cell stemness through the NF-κB pathway and induced cellular TMZ resistance. To confirm that RIP2 induces TMZ resistance in glioma cells dependent on NF-κB, we used flow cytometry to observe the effect of TMZ on apoptosis of RIP2-overexpressing U251 cells pretreated with GSK583 or SC75741. Transfection of the RIP2 plasmid inhibited TMZ-induced apoptosis in U251 cells, while either GSK583 or SC75741 reversed the effect of RIP2 ( Figure 4A). Detecting the effect of TMZ on the proliferation of cells in each group found that GSK583 or SC75741 could enhance the cytotoxic effect of TMZ on RIP2-overexpressing U251 cells ( Figure 4B). Our further validation with rRIP2 also found similar results to the above ( Figure 4C,D).

| RIP2 enhances the stemness of glioma xenografts through the NFκ B pathway
We had found from in vitro studies that RIP2 enhances the expression of glioma stem cell marker proteins CD133 and SOX-2 through the NF-κB pathway. To further confirm the biological role of RIP2 in vivo, we used a xenograft tumor model. First, we observed the expression of CD133 and SOX-2 in U251 cell xenografts transfected with RIP2 plasmid. Immunohistochemical staining showed that the expression of CD133 and SOX-2 in U251 tumor tissue transfected with RIP2 plasmid was significantly up-regulated ( Figure 5A).
Subsequently, we treated RIP2-overexpressing U251 cells with SC75741 and GSK583 and found that both could down-regulate CD133 and SOX-2 protein expression ( Figure 5B).

| Inhibition of RIP2/NFκ B can inhibit RIP2overexpressing glioma xenografts resistant to TMZ
We finally observed RIP2/NF-κB-induced TMZ resistance in a xenograft tumor model. First, we treated normal U251 cells and empty vector-transfected U251 cell tumor-bearing mice with TMZ. The results showed that the sensitivity of the two transplanted tumors to TMZ was strong and that there was no significant difference, with T/C ratios of 37.5% and 39.7%, respectively (Figure 6A-C,G).

| DISCUSS ION
Multidisciplinary comprehensive treatment has gradually emerged with the development of medical and health services. The treatment of brain gliomas (especially glioblastomas), however, is still not optimal. 20,21 The clinical application of the alkylating agent TMZ shows promise for the treatment of malignant gliomas; however, acquired drug resistance eventually leads to treatment failure. 7 RIP2 kinase has been shown to play a role in acquired drug resistance. [17][18][19] This study found that four glioma cell lines.
(SW1783, T98G, U87, and U251) stimulated by rRIP2 demonstrated enhanced resistance to TMZ. We found similar results after the four cell lines. Transfected with RIP2 plasmids exogenously overexpressed RIP2. This suggests that RIP2 plays a role in promoting glioma resistance to TMZ. We selected U251 cells for further study because RIP2 induced the strongest effect against TMZ in U251 cells among the four cell types we used.
Cancer stem cells are a significant cause of cancer occurrence, development, drug resistance, and recurrence due to their capacity for self-renewal, limitless proliferation, and treatment resistance. [22][23][24] In recent years, research has focused on regulating stem cell characteristics and the mechanism of drug resistance in tumor cells. Glioma stem cells are also characterized by self-renewal Furthermore, we found that CD133 and SOX-2 were up-regulated in U251 cells treated with rRIP2 or transfected with RIP2 plasmids.
We found that CD133 and SOX-2 were up-regulated in U251 cells and xenograft tumor tissue treated with rRIP2 or transfected with F I G U R E 3 RIP2 enhances the stemness of glioma cells dependent on the NF-κB pathway. (A) U251 cells were treated with 100, 200, and 400 ng/mL rRIP2 for 24 h, and the expressions of p-NF-κB and IκBα were detected by Western blotting. Compared with control, **P<0.01. (B) Using untransfected cells as negative control, U251 cells were transfected with RIP2 plasmid and vector, respectively, and the expression of p-NF-κB and IκBα was detected by Western blotting. Compared with Vector, **P < 0.01. (C) U251 cells were pretreated with a chemical inhibitor of RIP2 (GSK583, 1 μM) or a chemical inhibitor of NF-κB (SC75741, 1 μM) and then stimulated with rRIP2. Western blotting was used to detect p-NF-κB, CD133 and SOX-2 expression. **P < 0.01 compared with Control. ## P < 0.01 compared with rRIP2 group. (D) U251 cells overexpressing RIP2 were pretreated with GSK583 or SC75741, and the expressions of p-NF-κB, CD133, and SOX-2 were detected by western blotting. **P < 0.01 compared with the Vector group. ## P < 0.01 compared to the RIP2 plasmid group. (E) U251 cells overexpressing RIP2 were pretreated with GSK583 or SC75741, and the expressions of CD133 and SOX-2 were detected by immunofluorescence assay. **P < 0.01 compared with the RIP2 plasmid group. (F) Sphere formation assay for detection of RIP2-overexpressing U251 cells pretreated with GSK583 or SC75741. **P < 0.01, # P < 0.05, ## P < 0.01, && P < 0.01, compared with the solvent control group. (G) Clone formation assays to detect RIP2-overexpressing U251 cells pretreated with GSK583 or SC75741. **P < 0.01 compared with the solvent control.
RIP2 plasmids. However, the RIP2 inhibitor GSK583 inhibited the up-regulation effects. We also found that GSK583 could reverse TMZ resistance induced by RIP2 overexpression. CD133, a 5-transmembrane glycoprotein, is an important marker of various cancer stem (including glioma) cells, 26,27 and is associated with chemoresistance of glioma cells. Studies have shown that the resistance of hypoxia-induced glioblastoma to cisplatin is related to CD133, and silencing CD133 expression can reverse the induction effect of hypoxia. 28 SOX-2 plays a vital role in the early development of mammalian organs and is one of the necessary transcription factors for regulating mammalian embryonic development. SOX-2 is also a common marker in cancer stem cells, which may be involved in F I G U R E 4 Inhibition of RIP2/NF-κB signaling pathway inhibits RIP2-induced TMZ resistance in glioma cells. (A) Vector, RIP2 plasmid, RIP2 plasmid+SC75741, and RIP2 plasmid+GSK583 cells were treated with TMZ for 72 h, and cell apoptosis was detected by flow cytometry. **P < 0.01 compared with vector+TMZ group. ## P < 0.01 compared with the RIP2 plasmid+TMZ group. (B) Vector, RIP2 plasmid, RIP2 plasmid+SC75741, and RIP2 plasmid+GSK583 groups were treated with gradient concentrations of TMZ for 72 h, and cell viability was detected by CCK-8 experiment. (C) Cells in control, rRIP2, rRIP2 + SC75741, and rRIP2 + GSK583 groups were treated with TMZ for 72 h, and cell apoptosis in each group was detected by flow cytometry. **P < 0.01 compared with the control+TMZ group. ## P < 0.01 compared with the rRIP2 + TMZ group. (D) Cells in control, rRIP2, rRIP2 + SC75741, and rRIP2 + GSK583 groups were treated with gradient concentrations of TMZ for 72 h, and cell viability was detected by CCK-8 assay.
the chemoresistance of glioma cells. Studies have shown that SOX2 plays a crucial role in regulating the chemoresistance of CD133(+) glioblastoma. 29 It has also been reported that long non-coding RNA PVT1 induces up-regulation of SOX2 expression through the miR-365/ELF4 axis, thereby promoting the maintenance of stemness and TMZ resistance in glioma cells. 30 Knockdown of PVT1 and overexpression of miR-365 has been found to inhibit stemness and TMZ resistance of glioma cells. 30 This suggests that RIP2 may maintain the stemness of U251 cells by up-regulating CD133 and SOX-2, thereby resisting TMZ.
The NF-κB signaling pathway is the cascade that initiates nuclear transcription, which plays a key role in the cellular inflammatory, immune, and emergency responses. 31,32 Studies have shown that RIP2 promotes triple-negative breast cancer cell migration and invasion by activating NF-κB and c-Jun N-terminal kinase pathways. 15 RIP2 can activate the NF-κB signaling pathway to resist paclitaxel-and ceramide-induced apoptosis in breast cancer cells. 17 RIP2 also interacts with PAX5 to promote resistance of B-lymphoproliferative disorders to bortezomib by activating NF-κB. 18 In glioma cells, RIP2 promotes cell growth by regulating TRAF3 and activating NF-κB and p38 signaling pathways. 33 Previously, we found that RIP2 regulates MGMT expression through the NF-κB signaling pathway, thereby mediating the resistance of glioma cells to TMZ. 19 This suggests that NF-κB is a downstream signaling target of RIP2, and activation of NF-κB may be involved in RIP2-induced enhancement of stemness of glioma cells. Therefore, we observed the activation state of the NF-κB signaling pathway and found that rRIP2 or transfection of the RIP2 plasmids activated the NF-κB signaling pathway. This is consistent with the published literature 15,[17][18][19]33 Studies have shown that activating stem cell developmental pathways such as Wnt, 34 Notch, 35  In cell and xenograft studies, we found that SC75741 treatment inhibited TMZ resistance induced by RIP2 overexpression. These findings suggest that RIP2 can induce U251 cells to resist TMZ, and this effect is related to the activation of the NF-κB signaling pathway and maintenance of stemness of U251 cells (Figure 7).
A retrospective analysis of many studies found that stem cell therapy may present an effective treatment for metastatic brain cancer and glioblastoma. 37 Therefore, eliminating glioma cell stemness is one of the critical strategies for clinical treatment.
Some signaling molecules for the maintenance of stemness, such as Hedgehog, CD133, and NF-κB, have been confirmed as potential therapeutic targets. 38 Some natural products and their chemical derivatives have been shown to play a role in regulating glioma cell stemness. 39 Our study suggests that RIP2/NF-κB is a potential target for treating glioma stemness. This may provide theoretical support for clinical treatment. However, the regulatory mechanism of RIP2/NF-κB is not fully understood and needs further study.

| CON CLUS ION
In conclusion, our study shows that RIP2 is involved in the induction of TMZ resistance in gliomas. Furthermore, the RIP2/NF-κB signaling pathway induces enhanced stemness and plays an important role in TMZ resistance. Therefore, the combined application of RIP2 or NF-κB inhibitors and TMZ may be a new strategy for treating RIP2related drug-resistant gliomas.

AUTH O R CO NTR I B UTI O N S
All listed authors designed the study, performed the experiments and the statistical analysis, and wrote the manuscript. All authors have read the manuscript and approved the final version.

FU N D I N G I N FO R M ATI O N
This work was supported by the National Natural Science Foundation of China (No. 81241076).

CO N FLI C T O F I NTE R E S T
The authors do not have any possible conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the