ANGPTL4 Induces TMZ Resistance of Glioblastoma by Promoting Cancer Stemness Enrichment via the EGFR/AKT/4E-BP1 Cascade

Figure 1. Effect of angiopoietin-like 4 (ANGPTL4) on temozolomide (TMZ) sensitivity in glioblastoma (GBM) (A) Cells were transfected with a Flag-ANGPTL4 plasmid for 2 days and then treated with different doses of TMZ for 4 days. Cell viability was measured by an MTT assay. The experiment was performed independently three times, and data were expressed as a relative value ± SEM. (* p < 0.05 and ** p < 0.01). Western blotting was used to confirm ANGPTL4 overexpression in U87MG cells. (B) Cells treated with recombinant ANGPTL4 (rANGPTL4) and TMZ for 5 days were stained with methylene blue. The size of the colony was quantitated using Image J software. The experiment was performed independently three times, and data were expressed as a relative value ± SEM. (* p < 0.05). (C) After treatment with ANGPTL4 small interfering RNA (siRNA) #2 (siANGPTL4-2) or #4 (siANGPTL4-4) for 3 days, cells were treated with different doses of TMZ for 4 days. Cell viability was measured by an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (** p < 0.01 and *** p < 0.001). The experiment was performed independently three times. Western blotting was used to confirm ANGPTL4 knockdown.…

Figure 2. Effect of ANGPTL4 on glioma stem-like cell (GSC) enrichment. (A) After ANGPTL4 overexpression for 2 days, the protein expression of stemness markers were presented by Western blotting, including the product of PROM1 gene (CD133), SRY (sex determining region Y)-box 2 (SOX2), and polycomb complex protein BMI-1. The experiment was performed independently three times, and quantitative results were expressed as the mean ± SEM (* p < 0.05, ** p < 0.01, and *** p < 0.001). (B) A comparison of ANGPTL4 production in attached and spheroid cells (GSC-like cells). The protein expression of ANGPTL4 in cell lysate and cultured medium was validated by Western blotting. The cultured medium of cells was collected and concentrated for Western blotting. (C) U87MG cells were transfected with a Flag-ANGPTL4 plasmid for 2 days and then treated with the ANGPTL4-neutralizing antibody for an additional 1 day. The protein expression of stemness markers was presented by Western blotting. (D) U87MG cells were induced to spheroid formation for 7 days in the absence or presence of rANGPTL4 (5 μg/mL). The scale bar was 0.2 mm. (E) After 7 days, cell lysates of spheroid GSCs were collected for Western blotting.…

Figure 3. ANGPTL4-regulated phosphorylation cascade for GSC enrichment. (A) The putative target receptor of ANGPTL4 was detected by Human receptor tyrosine kinase (RTK) Phosphorylation Antibody Array C1. (B) The putative phosphorylation signaling pathway of ANGPTL4 was detected by Human and Mouse AKT Pathway Phosphorylation Array C1. (C) Epidermal growth factor receptor (EGFR) phosphorylation (Y1068) and Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1; Thr37/46) phosphorylation in A172 and U87MG were validated by Western blotting. (D) After ANGPTL4 overexpression for 24 h, cells were treated with gefitinib for an additional 24 h. The protein expression was validated by Western blotting. The experiment was performed independently 3 times, and quantitative results were expressed as the mean ± SEM (* p < 0.05). (E) After ANGPTL4 overexpression for 24 h, cells were treated with the AKT or PI3K inhibitor for another 24 h. The protein expressions were validated by Western blotting. The experiment was performed independently three times, and quantitative results were expressed as the mean ± SEM (* p < 0.05).

Figure 4. Effect of specificity protein (Sp)4 on ANGPTL4 and GBM. (A) The protein expression of Sp1, 2, 3, and 4 in normal brain tissue and a brain tumor was validated by Western blotting. Normal brain tissue lysate: GTX27918 (Genetex); Brain tumor tissue: SF268 (Human Glioblastoma, Origene). (B) Left panel: heatmap of Sp4-regulated cell movement-related genes. Right panel: The results of microarray analysis after Ingenuity Pathway Analysis (IPA)-mediated function annotation. (C) After Sp4 knockdown for 3 days, the protein expression of ANGPTL4 and Sp4 was validated by Western blotting. (D) Sp4-regulated genes in which Sp4 associates with the promoter region revealed by chromatin immunoprecipitation coupled with sequencing (ChIP-Seq). (E) The defined promoter region of ANGPTL4 and the binding regions of Sp4 determined by ChIP-seq. (F) After transfection with the indicated plasmid and pGL2-ANGPTL4 promoter for 2 days, the promoter activity of ANGPTL4 were measured by a reporter assay. Data were expressed as the relative luciferase activity mean ± SEM. (*** p < 0.001). (G) After Sp4 overexpression for 48 h, the protein expression was validated by Western blotting. The experiments were…

Figure 5. Effect of Sp4 and ANGPTL4 in the clinical relevance of GBM. (A) The protein expression of Sp4 and ANGPTL4 in normal brain tissue and glioma was evaluated by immunohistochemical (IHC) staining. (B) The protein expression of Sp4 in human fetal glial cells (SVG-P12) and GBM (U87MG) was validated by Western blotting (C) The mRNA expression of Sp4 and ANGPTL4 in normal brain tissue and glioma was analyzed using the Gene Expression Omnibus (GEO) dataset (GSE 4290). (D) The mRNA expression of ANGPTL4 in normal brain tissue and GBM was analyzed using the Oncomine database (GSE2223). (E) The association of Sp4 and ANGPTL4 expression with the risk of glioma. (F,G) The correlations of Sp4 and ANGPTL4 with the survival of GBM patients was analyzed using the SurvExpress database (F: brain−Philips Aldape Astrocitome GSE4271 GPL97; G: brain−LGG −TCGA−low grade gliomas).

Figure 6. Sp4-mediated ANGPTL4 production induces GSC enrichment through the EGFR/AKT/4E-BP1 cascade, leading to TMZ resistance in GBM. Sp4 induces the expression of ANGPTL4 through binding to the putative promoter region of ANGPTL4, −979 to −606. The secretion of ANGPTL4 induces stemness development by inducing the phosphorylation of EGFR, AKT, and 4E-BP1, leading to the activation of eukaryotic translation initiation factor 4 (eIF4)-mediated cap-dependent translation. The activation of the EGFR/AKT/4E-BP1 cascade by ANGPTL4 results in the upregulation of stemness markers, such as BMI-1, followed by the acquired TMZ resistance.