RND1 inhibits epithelial-mesenchymal transition and temozolomide resistance of glioblastoma via AKT/GSK3-β pathway

Human GBM cell lines U87 and U251 were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Authentication of GBM cells was conducted by short tandem repeat (STR) in Procell Life Science & Technology (Wuhan, China). GBM cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (10099141C, Gibco, USA) at 37°C with 5% CO₂. The TMZ-resistant U87 cell line (U87-TR) was established by exposing normal U87 cell lines to TMZ at gradient ascending doses (final dose at 400 μM) for 6 months. The establishment of TMZ-resistant U87 cell line was confirmed via cell viability assay (Supplemental Figure 1).

DMSO was purchased from Servicebio (GC203005↗). The AKT inhibitor SC66 was obtained from MedChemExpress (HY-19832). TMZ was purchased from Selleck (S1237).

The following antibodies were used: anti-RND1 (GTX83709, GeneTex), anti-GAPDH (#5174, Cell Signalling Technology, CST), anti-Flag (M185, Medical Biological Laboratories), anti-E-cadherin (20874–1-AP, Proteintech), anti-N-cadherin (22018–1-AP, Proteintech), anti-Vimentin (10366–1-AP, Proteintech), anti-Snail1 (sc -28,199, Santa Cruz), anti-mmp2 (10373–2-AP, Proteintech), anti-AKT (GTX121937, GeneTex), anti-Phospho-AKT (#4060, CST), anti-GSK-3β (#12456, CST), anti-Phospho-GSK-3β (#9323, CST), anti-β-catenin (51067–2-AP, Proteintech), anti-MAPK (#4511, CST), anti-Phospho-MAPK (#9212, CST) and anti-Smad3 (66516–1-Ig, Proteintech).

The association between RND1 and a series of EMT-related genes (MMP2, MMP3, MMP9, SNAI1, SNAI2, TJP1, CDH1, CDH2, and VIM) was analyzed based on RNAseq data of 703 glioma tissues from TCGA database. P-values were obtained from Pearson correlation.

Cell samples were lysed in Radio Immunoprecipitation Assay (RIPA) buffer (P0013B, Beyotime) containing the protease inhibitor Cocktail (4693116001, Roche). Protein concentration was measured by the BCA Kit (P0012S, Beyotime) and 5×loading buffer (P0015, Beyotime) was added. Protein samples (20 μg/lane) were electrophoretic in 10–12% SDS-PAGE gels and transferred to poly vinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies and HRP-conjugated secondary antibodies. Finally, the membranes were prepared with ECL Western blotting substrate Kit (BL520A, Biosharp) and captured using a ChemiDoc Imaging System (Bio-Rad, USA). The analysis was conducted using Image J.

The construction of the overexpression plasmid pCDNA3.1-Flag-RND1 and specific shRNA targeting of human RND1 (shRND1) was helped with Miaoling Biology (Wuhan, China) and described previously.²⁰ The sequences of pCDNA3.1-Flag-RND1 and shRND1 were provided in Supplemental Table S1.

Cell viability of U87 and U251 was measured by a Cell Counting Kit-8 (CCK-8) Kit (CK04, Dojindo Molecular Technologies). Cells were cultured in 96-well plates with plasmid transfection or drug action and detected at specified time points (0 h, 24 h, 48 h and 72 h) with the help of CCK8 Kits. After CCK8 reagent was added, cells were placed in an incubator at 37°C for 2 h. The OD value was measured at 450 nm using a Multimode Plate Reader (PerkinElmer, Germany).

U87 and U251 cells were seeded into six-well plates (500 cells per well) and incubated at 37°C with 5% CO₂ for 2 weeks. The colonies were fixed with 4% formaldehyde for and stained with .2% crystal violet at room temperature. The representative colonies were captured and quantified.

U87 and U251 cells in 6-well plates were scraped with a 200 μL pipette tip to create the wounds. To exclude the effect of cell proliferation, only 1% FBS was added in DMEM. Cells that migrated to the wound area were photographed at 0 h and 48 h. Representive boundaries of the wound were drawn with straight lines. Wound healing ratio was calculated and analyzed.

U87 and U251 cells were seeded into the upper chamber of polycarbonate transwell filters in serum-free DMEM. DMEM containing 10% FBS was added in lower chambers as chemoattractant. Cells were incubated for 24 h and fixed with 4% paraformaldehyde. The cells on the lower chamber were dyed with crystal violet (Beyotime). Images were captured by an inverted microscope (Olympus BX51, Japan) and the number of cells was counted.

To establish the stable U87 cell line with RND1 overexpression and carrying luciferase, pLVX-CMV-Luciferase-RND1-puro was transfected with pMD2.G and psPAX2 into 293T cells to produce lentiviruse. Then, U87 cell line was cultured in lentiviruse for 48 h and selected by puromycin.

Four-week-old male Balb/c nude mice were purchased from Shulaibao (Wuhan, China) Biotechnology Co., Ltd. In the subcutaneous xenograft model, 1 × 10⁶ stable cells (pLVX-luciferase and pLVX-RND1-luciferase) were subcutaneously injected into armpits of nude mice. TMZ treatment started 7 days after injection, the mice were injected intraperitoneally with two cycles of TMZ (50 mg/kg) for 5 days. All nude mice were captured by IVIS (In Vivo Imaging System) at the 21st day after tumor injection and sacrificed at were sacrificed at 28th day. Animal experiment of this research was approved by the institutional animal care and use committee of Renmin Hospital of Wuhan University.

Tumor-bearing mice were injected with 10 mg D-Luciferin potassium (ST196, Beyotime, China) via caudal vein. Luminescence was captured and calculated by an In Vivo Imaging System (IVIS) (Bruker Xtreme BI, Bruker, USA).

Experiments were repeated three times and data are presented as the mean ± standard deviation. Data were analyzed using SPSS (Version 19.0) and GraphPad Prism (Version 8.0). Student’s t-test was used for data comparisons between two groups. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test. p < .05 was considered as statistical significance.

We first overexpressed RND1 via the transfection of Flag-RND1 plasmid in U87 and U251 cells (Figure 1h). Colony formation assay and cell viability assay were used to assess the growth of U87 and U251 cells when RND1 was overexpressed. Results indicated that the overexpression of RND1 obviously suppressed the growth of U87 and U251 cells (Figure 1a–c). We evaluated the EMT status of U87 and U251 cells based on transwell and wound-healing assays. From the results, we observed that the cell invasion ability of U87 and U251 was significantly inhibited when RND1 was up-regulated (Figure 1d–g). Hence, RND1 suppressed the growth and invasion status of U87 and U251 cells.

We further knockdown RND1 via the transfection of shRND1 plasmid in U87 and U251 cells and the efficiency of knockdown was confirmed by western blot analysis (Figure 2h). Colony formation assay and cell viability assay were used to assess the growth of U87 and U251 cells when RND1 was knocked down. Results indicated that the knockdown of RND1 obviously facilitated the growth of U87 and U251 cells (Figure 2a–c). Meanwhile, we evaluated the EMT status of U87 and U251 cells based on transwell and wound-healing assays. We found that the cell invasion of U87 and U251 was significantly promoted when RND1 was down-regulated (Figure 2d–g). Thus, knockdown of RND1 promoted GBM invasion and progression.

To further confirm the effect of RND1 on EMT state of GBM cells, we first analyzed the association between RND1 and a series of EMT-related genes (MMP2, MMP3, MMP9, SNAI1, SNAI2, TJP1, CDH1, CDH2 and VIM) based on RNAseq data of 703 glioma tissues from TCGA database. The results significantly indicated that RND1 was positively correlated with anti-EMT genes (TJP1 and CDH1) and negatively correlated with pro-EMT genes (MMP2, MMP3, MMP9, SNAI1, SNAI2, CDH2 and VIM) (Figure 3a–i).

In vitro experiments, we analyzed the expression level of EMT-related genes using western blot analysis when RND1 was up-regulated or down-regulated in U87. Results showed that E-cadherin was upregulated when RND1 was overexpressed in U87 (Figure 3j). Meanwhile, N-cadherin, Vimentin, Snail1 and mmp2 was downregulated (Figure 3j). Consistently, when RND1 was knocked down, E-cadherin was downregulated and N-cadherin, Vimentin, Snail1, and mmp2 was upregulated (Figure 3k). Thus, through the detection and analysis of the expression of EMT-related genes, we discovered that RND1 inhibited the EMT status in GBM

To investigate the potential mode of RND1 on GBM invasion, we detected the several signals regulating tumor EMT. Considering TGF-β-Smad, PI3K-AKT, MAPK-p38, and Wnt-β-catenin signals were core pathways regulating tumor EMT,^21,22 we researched the protein levels of these signals via western blot analysis when the expression of RND1 was changed. The results showed that the overexpression of RND1 did not affect the protein level of p-MAPK, Smad3, and β-catenin but inhibit the phosphorylation of AKT (p-AKT) (Figure 4a). Meanwhile, to confirm the effect of RND1 on AKT signal, we also detected the protein level of total and phosphorylated GSK3-β, an important signal downstream of AKT.²³ We discovered a significant decrease of phosphorylated GSK3-β (p-GSK3-β) protein when RND1 was overexpressed in U87 cells (Figure 4a, d), indicating a convincing negative regulation of RND1 on AKT signal.

Moreover, we also detected the AKT signal activity when RND1 was knocked down in U87 cells. Results from western blot analysis indicated that downregulation of RND1 obviously increased the phosphorylation of AKT and GSK3-β and regulated EMT-related genes including E-cadherin, Vimentin, Snail1, and mmp2 (Figure 4b). Meanwhile, we discovered that the use of SC66 (an inhibitor of AKT phosphorylation²⁴) significantly decreased phosphorylated AKT and GSK3-β and reversed the effect of RND1 on EMT-related proteins (Figure 4b). Furthermore, transwell assay was performed in U87 cells with the treatment of SC66. Results showed that knockdown of RND1 facilitated the invasion of U87 cells, which could be reversed by SC66 (Figure 4c, e).

Next, to determine the impact of RND1 on the TMZ sensitivity of GBM cells, we overexpressed or knocked down RND1 in U87 cells and treated U87 cells with 400 μM TMZ (Figure 5a). The results of cell viability assay showed that RND1 enhanced the TMZ sensitivity of normal U87 cells. While in clinical practice, resistance to TMZ in GBM is a common phenomenon. To continue the research on the role of RND1 in GBM resistance, we established the TMZ-resistant U87 cell line (U87-TR) by exposing normal U87 cell lines to TMZ at a high dose of 400 μM for 6 months. The establishment of U87-TR was confirmed via cell viability assay (Supplemental Figure 1). The cell viability assay in U87-TR indicated that RND1 overexpression significantly enhanced the anti-tumor activity of TMZ in drug-resistant U87-TR (Figure 5b). Consistently, in the transwell and wound-healing assays, upregulation of RND1 combined with the use of TMZ showed the strongest inhibition of cell invasion in U87-TR (Figure 5c–f).

As a next step, the specific mechanism of RND1’s regulation on the TMZ sensitivity of GBM cells should be identified. We first detected the activity of AKT signal and EMT status in U87-TR cells. The results of western blot analysis showed that U87-TR cells obviously had a stronger AKT signaling pathway activity and more malignant EMT status compared to U87-WT cells (Figures 6a and 6c). Moreover, in U87-TR cells, RND1 also showed a function of inhibiting the activity of AKT signaling pathway and improving the EMT status (Figures 6b and 6d).

Furthermore, we overexpressed RND1 and treated U87-TR cells with SC79 (an activator of AKT phosphorylation²⁵). The results of cell viability assay showed that upregulation of RND1 suppressed the growth of U87-TR cells, which could be reversed by SC79 (Figure 6e). Meanwhile, from western blot analysis, we discovered that the use of SC79 significantly reversed the effect of RND1 on AKT signaling pathway (Figure 6f).

To better evaluate the potential application of RND1 as a clinical target for GBM, treatment targeted RND1 combined with TMZ therapy was conducted in vivo. Stable U87 cell line (U87-Luc-RND1) overexpressing RND1 was established using lentivirus and injected into armpits for the generation of subcutaneous xenograft model. 7 days after tumor injection, two groups (Luc+TMZ and Luc-RND1+TMZ) of mice received two cycles of intraperitoneal treatment of TMZ (50 mg/kg/day, 5 day/cycle). Subsequently, tumor growth was evaluated by IVIS and the fluorescence showed that progression of subcutaneous tumor was suppressed when RND1 was overexpressed in U87 (Figure 7a, b). Meanwhile, it was observed that RND1 overexpression enhanced the TMZ sensitivity of treatment in U87-tumor-bearing mice (Figure 7a, b). By comparing the relative size and weight of tumors from each group, it was observed that tumor growth was significantly inhibited in Luc-RND1 group and RND1 enhanced the TMZ sensitivity in GBM (Figure 7c, d).

Qian Sun, is studying for an M.D. at Wuhan University, specializing in targeted treatment of glioma.

Junjie Xu, is currently working in Office of Director, Renmin Hospital of Wuhan University.

Fan’en Yuan, is graduated from Wuhan University with a major in neurosurgery, and is currently a postdoctoral fellow in University of Pittsburgh.

Yan Liu, is currently working in Department of Anesthesiology, Renmin Hospital of Wuhan University.

Qianxue Chen, MD, Professor, Supervisor, is currently the Director of Department of Neurosurgery, Renmin Hospital of Wuhan University.

Lirui Guo, is currently the head nurse of Department of Neurosurgery, Renmin Hospital of Wuhan University.

Huimin Dong, MD, is currently the associate senior doctor of Department of Neurology, Renmin Hospital of Wuhan University.

Baohui Liu, MD, Associate Professor, is currently the senior doctor of Department of Neurosurgery, Renmin Hospital of Wuhan University.

The data used to support the findings of this study are available from the corresponding author upon request.