Endothelial cells-derived SEMA3G suppresses glioblastoma stem cells by inducing c-Myc degradation

Abstract

The poor prognosis of glioblastoma (GBM) patients is attributed mainly to abundant neovascularization and presence of glioblastoma stem cells (GSCs). GSCs are preferentially localized to the perivascular niche to maintain stemness. However, the effect of abnormal communication between endothelial cells (ECs) and GSCs on GBM progression remains unknown. Here, we reveal that ECs-derived SEMA3G, which is aberrantly expressed in GBM patients, impairs GSCs by inducing c-Myc degradation. SEMA3G activates NRP2/PLXNA1 in a paracrine manner, subsequently inducing the inactivation of Cdc42 and dissociation of Cdc42 and WWP2 in GSCs. Once released, WWP2 interacts with c-Myc and mediates c-Myc degradation via ubiquitination. Genetic deletion of Sema3G in ECs accelerates GBM growth, whereas SEMA3G overexpression or recombinant SEMA3G protein prolongs the survival of GBM bearing mice. These findings illustrate that ECs play an intrinsic inhibitory role in GSCs stemness via the SMEA3G-c-Myc distal regulation paradigm. Targeting SEMA3G signaling may have promising therapeutic benefits for GBM patients.

Fig. 1. Expression profiles of SEMA3G in GBM patients.

A The differentially expressed genes (DEGs) in various cell types (GSE162631). B Volcano plot of DEGs in endothelial cells (GSE162631). C Heatmap of SEMA3G expression in non-tumor and GBM tissue of different GEO data sets. D Bivariate correlation analysis of SEMA3G and markers for various types of cells (GSE162631). EC: endothelial cell; AC: astrocyte; MG: microglia; OL: oligodendrocyte; PC: pericyte; SMC: smooth muscle cell; FB: fibroblast. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, the P values were calculated by the Pearson correlation test. E SEMA3G expression profile in the various type of cells (GSE162631). F The expression level of SEMA3G in non-tumor brain (n = 10) and GBM tissues (n = 521) from the TCGA GBM dataset. ^***P < 0.001, as determined by the Mann-Whitney U test. G–I Expression levels of SEMA3G in non-tumor (from traumatic brain injury patients) and GBM tissues from patients. The relative mRNA level of SEMA3G were determined by qPCR (G). The representative bands (H) and quantification (I) of protein level of SEMA3G as determined by western blot analysis. Data are shown as mean ± s.e.m. n = 5, ^**P < 0.01, ^***P < 0.001, as determined by two-tailed unpaired t test. J The protein level of SEMA3G in the cerebrospinal fluid (CSF) of non-tumor (from traumatic brain injury patients) and GBM tissues assessed by ELISA. Data are shown as mean ± s.e.m. n = 5, ^**P < 0.01, as determined by two-tailed unpaired t-test.

Fig. 2. SEMA3G suppresses intracranial GBM growth.

A Intercellular communication networks of SEMA3s signaling pathways analyzed by CellChat (GSE162631). B Immunofluorescent staining of NRP2 in brain sections of tumor-bearing mice. Scale bar, 10 μm. C BCIP/NBT color development reveals AP-Ctrl or AP-SEMA3G binding in brain sections of tumor-bearing mice. Scale bar, 5 μm. D Schematic diagram of GBM model establishment and monitor using Sema3G^f/f and Cdh5-cre; Sema3G^f/f mice. In vivo bioluminescent image (E) and quantification (F) of tumor growth on days 7 and 21 post cell implant. Data are shown as mean ± s.e.m. n = 6, ^***P < 0.001. Data were analyzed by two-way ANOVA. Representative images (G) and the tumor-to-brain area ratio (H) based on the H&E staining mouse brains collected 21 days after transplantation of GL261 cells. Scale bar, 1 mm. Data are shown as mean ± s.e.m. n = 6, ^***P < 0.001, as determined by two-tailed unpaired t test. I Kaplan-Meier survival curves of the tumor bearing Sema3G^f/f mice and Cdh5-cre; Sema3G^f/f mice. The P values were calculated by the log-rank test. n = 6. J Schematic diagram of overexpressing SEMA3G in nude mice and the construction of a GBM model using GSC07-Luc cells. In vivo bioluminescent image (K) and quantification (L) of tumor growth in GBM bearing mice on days 7 and 21 post cell implant. Data are shown as mean ± s.e.m. n = 10, ^*P < 0.05, ^**P < 0.01. Data were analyzed by two-tailed unpaired t test. M H&E staining of mouse brains collected 21 days post GSC07 cell transplantation. Scale bar, 1 mm. n = 10. N The tumor-to-brain area ratio based on the H&E staining (M) was calculated using ImageJ software. Data are shown as mean ± s.e.m. n = 10, ^***P < 0.001, as determined by two-tailed paired t test. O Kaplan-Meier survival curves of the tumor bearing nude mice. n = 10. The P values were calculated by the log-rank test. n = 10.

Fig. 3. SEMA3G inhibits GSCs growth and self-renewal.

GSC07 (A) or GSC27 (B) cells were incubated with recombinant hSEMA3G protein (200 ng/ml) for the indicated days. Cell viability was measured by CCK8. Data are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by two-way ANOVA. GSC07 (C) or GSC27 (D) cells were incubated with recombinant hSEMA3G protein (200 ng/ml) for the indicated days. The sphere formation was determined by cell counting. Data are shown as mean ± s.e.m. ^**P < 0.01, ^***P < 0.001. Data were analyzed by two-way ANOVA. Representative images (E) and quantification (F) of neurospheres formed by GSC07 and GSC27 cells treated with 200 ng/ml hSEMA3G for 7 days. Scale bar, 100 μm (E). The number of neurospheres was calculated. Data are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed two-tailed unpaired t test. Sphere formation in GSC07 (G) and GSC27 (H) cells incubated with recombinant hSEMA3G protein (200 ng/ml) for 7 days by extreme limiting dilution assays. Representative images (I) and quantification (J) of neurospheres formed by GSC07 and GSC27 cells treated with CM or CM combination with NRP2 neutralizing antibodies for 7 days. Scale bar, 100 μm (I). The number of neurospheres was calculated. Data are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by one-way ANOVA. Sphere formation in GSC07 (K) and GSC27 (L) cells incubated with CM or combination with NRP2 neutralizing antibodies for the 7 days by extreme limiting dilution assays. Scale bar, 100 μm. Data represent the mean ± s.e.m. All data were collected from three independent experiments.

Fig. 4. SEMA3G reduces c-Myc protein stability in GSCs.

A Gene signatures enrichment of c-Myc targets in SEMA3G expressed GBM specimens. mRNA levels of c-Myc target genes in GSC07 (B) and GSC27 (C) cells treated with or without recombinant hSEMA3G (200 ng/ml) for 72 h. Data are shown as mean ± s.e.m. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Data were analyzed by two-way ANOVA. The representative bands (D) and quantification (E) of c-Myc in GSC07 and GSC27 cells treated with or without recombinant hSEMA3G (200 ng/ml) for 72 h, as determined by western blot analysis. Data are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by two-tailed unpaired t test. F mRNA levels of c-Myc in GSC07 and GSC27 cells treated with or without recombinant hSEMA3G (200 ng/ml) for 72 h. Data are shown as mean ± s.e.m. Data were analyzed by two-tailed unpaired t test. G Correlation of SEMA3G and c-Myc at transcriptional level in GBM specimens from TCGA_GBMLGG dataset. The P values were calculated by the Pearson correlation test. H The representative bands of c-Myc in GSC07 and GSC27 cells treated with or without recombinant hSEMA3G (200 ng/ml) for 72 h, followed by CHX (10 µg/ml) for various time point. I, J Data summary of (H) are shown as mean ± s.e.m. ^**P < 0.01. Data were analyzed by two-way ANOVA. K GO terms analysis of the DEGs between GBM datasets (GSE162631) with different SEAM3G expression levels. L The representative bands of ubiquitination in GSC07 cells treated with SEMA3G (200 ng/ml) for 66 h, followed by MG132 (10 μmol/l) or the same volume DMSO for an additional 6 h. M The representative bands of the ubiquitination level of c-Myc in GSC07 cells treated with SEMA3G (200 ng/ml) for 72 h. The representative bands (N) and quantification (O, P) of c-Myc in GSC07 and GSC27 incubated with SEMA3G (200 ng/ml) for 66 h, followed by MG132 (10 μmol/l) for an additional 6 h. Data are shown as mean ± s.e.m. ^**P < 0.01. Data were analyzed by one-way ANOVA. All the western blot bands represent one of the three independent experiments.

Fig. 5. SEMA3G promotes c-Myc degradation in a WWP2-dependent manner.

A HEK-293 cells were transfected with Flag-tagged c-Myc and/or MYC-tagged WWP2 for 48 h. The interaction of c-Myc and WWP2 were detected using Co-immunoprecipitation. B The binding of endogenous WWP2 to c-Myc in GSC07 cells by co-immunoprecipitation analysis. C HEK-293 cells were transfected with Flag-tagged c-Myc domains and MYC-tagged WWP2 for 48 h. The interaction of c-Myc and WWP2 were detected using co-immunoprecipitation. D The overexpressed GSC07 cells (+) were transfected with WWP2-overexpresson lentivirus again (++) to induced the higher expression of WWP2. The protein level of c-Myc in the GSC07 cells with various WWP2 overexpression was determined by western blot. E Data summary of (D) are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by one-way ANOVA. F GSC07 cells were transfected with various WWP2 silence lentivirus for 72 h and screened using puromycin (1 μg/ml) to induced the stable knockdown cell. The protein level of c-Myc was determined by western blot. G Data summary of (F) are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by one-way ANOVA. H The WWP2 overexpressed GSC07 cells were treated with CHX (10 µg/ml) for the indicated time points. The protein level of c-Myc was determined using western blot. I Data summary of (H) are shown as mean ± s.e.m. ^**P < 0.01. Data were analyzed by two-way ANOVA. J The WWP2 overexpressed GSC07 cells were treated with MG132 (μmol/l) for 6 h. The protein level of c-Myc was determined using western blot. K Data summary of (J) are shown as mean ± s.e.m. ^**P < 0.01. Data were analyzed by one-way ANOVA. L The ubiquitination level of c-Myc in WWP2 overexpressed GSC07 was determined by co-immunoprecipitation analysis. M The WWP2 knockdown GSC07 and GSC27 cells were treated with or without SEMA3G for 72 h. The protein level of c-Myc was determined using western blot. N, O Data summary of (M) are shown as mean ± s.e.m. ^**P < 0.01, ^***P < 0.001. Data were analyzed by one-way ANOVA. Representative images (P) and the number (Q) of neurospheres of WWP2 knockdown GSC07 that were treated with or without SEMA3G (200 ng/ml) for 7 days. Images of neurospheres were captured using microscope. Scale bar, 100 μm. Data are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by one-way ANOVA. All the western blot bands represent one of the three independent experiments.

Fig. 6. SEMA3G inactivates Cdc42 through NRP2/PLXNA1 in GSCs.

A Schematic illustration of the TurboID-tagged plasmid constructure and proximity labeling experiment. B PLXNA1, PLXNA2, PLXNA3, PLXNA4 and PLXND1 protein levels in whole cell lysates (input) or streptavidin bead–enriched protein samples from T98G cells transfected with NRP2-TurboID construct and treated with or without biotin. C HEK-293 cells were transfected with HA-tagged NRP2 and/or MYC-tagged PLXNA1. The interaction was determined using co-immunoprecipitation. D The PLXNA1 knockdown and control GSC07 and GSC27 cells treated with or without SEMA3G (200 ng/ml) for 72 h. The protein level of c-Myc was determined using western blot. E, F Data summary of (D) are shown as mean ± s.e.m. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Data were analyzed by one-way ANOVA. G PLXNA1 knockdown GSC07 cells were treated with or without SEMA3G (200 ng/ml) for 7 days. Images of neurospheres were captured using microscope. Scale bar, 100 μm. H Data summary of (G) are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by one-way ANOVA. I The gene signatures of negative regulation GTPase enrichment analysis. J–O GSC07 and GSC27 cells were treated with or without SEMA3G (200 ng/ml) for 72 h. the activation of Cdc42 (J), RhoA (L), Rac1 (N) were determined using GST pulldown assay. K, M, O Data summary of (J, L, N) are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by two-tailed paired t test. All the western blot bands represent one of the three independent experiments.

Fig. 7. Cdc42 acts as a molecular switch regulating c-Myc degradation by WWP2.

A The ubiquitination level of c-Myc in GSC07 cells treated with or without ML141 (10 μmol/l) for 24 h. B, C c-Myc level in T98G cells transfected with GFP-Tagged Cdc42^WT, Cdc42^T17N or Cdc42^Q61L. D Schematic diagram depicting the plasmid construction for bimolecular fluorescence complementation assays. E Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between Cdc42 and WWP2. F HEK-293 cells were transfected with CrN173-tagged Cdc42^T17N or Cdc42^Q61L, and VC155-tagged WWP2 for 48 h. CFP signal that represents Cdc42 binding to WWP2 were determined using confocal. Scale bar, 5 μm. G HEK-293 cells were transfected with Flag-tagged c-Myc, HA-tagged ubiquitin, MYC-tagged WWP2 and GFP-tagged Cdc42^T17N or Cdc42^Q61L for 48 h. The ubiquitin level of c-Myc and the binding of Cdc42 to WWP2 were determined by co-immunoprecipitation. H Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between WWP2 and Cdc42 or c-Myc. I HEK-293 cells were incubated with or without SEMA3G (200 ng/ml) for 48 h after co-transfected with CrN173-tagged Cdc42^WT or VN173-tagged c-Myc and VC155-tagged WWP2 for 12 h. CFP signal that represents Cdc42 binding to WWP2 and Venus signal that represents WWP2 binding to c-Myc were captured using confocal. Scale bar, 5 μm. J HEK-293 cells that were co-transfected with GFP-tagged Cdc42^WT, MYC-tagged WWP2, Flag-tagged c-Myc, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The binding of Cdc42^WT to WWP2 and the ubiquitination level of c-Myc were determined by co-immunoprecipitation. K HEK-293 cells were co-transfected with GFP-tagged Cdc42^Q61L and MYC-tagged WWP2, Flag-tagged c-MYC, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The protein level of c-Myc and ubiquitination level of c-Myc were determined by co-immunoprecipitation. L Schematic diagram illustrating the potential mechanism by which Cdc42 regulates WWP2 binding to c-Myc and promotes c-Myc degradation. All the western blot bands represent one of the three independent experiments.