Identification of PBK as a hub gene and potential therapeutic target for medulloblastoma

Abstract

Medulloblastoma (MB) is the most frequent malignant brain tumor in pediatrics. Since the current standard of care for MB consisting of surgery, cranio‑spinal irradiation and chemotherapy often leads to a high morbidity rate, a number of patients suffer from long‑term sequelae following treatment. Targeted therapies hold the promise of being more effective and less toxic. Therefore, the present study aimed to identify hub genes with an upregulated expression in MB and to search for potential therapeutic targets from these genes. For this purpose, gene expression profile datasets were obtained from the Gene Expression Omnibus database and processed using R 3.6.0 software to screen differentially expressed genes (DEGs) between MB samples and normal brain tissues. A total of 282 upregulated and 436 downregulated DEGs were identified. Functional enrichment analysis revealed that the upregulated DEGs were predominantly enriched in the cell cycle, DNA replication and cell division. The top 10 hub genes were identified from the protein‑protein interaction network of upregulated genes, and one identified hub gene [PDZ binding kinase (PBK)] was selected for further investigation due to its possible role in the pathogenesis of MB. The aberrant expression of PBK in MB was verified in additional independent gene expression datasets. Survival analysis demonstrated that a higher expression level of PBK was significantly associated with poorer clinical outcomes in non‑Wingless MBs. Furthermore, targeting PBK with its inhibitor, HI‑TOPK‑032, impaired the proliferation and induced the apoptosis of two MB cell lines, with the diminished phosphorylation of downstream effectors of PBK, including ERK1/2 and Akt, and the activation of caspase‑3. Hence, these results suggest that PBK may be a potential prognostic biomarker and a novel candidate of targeted therapy for MB.

Keywords: PDZ binding kinase; apoptosis; medulloblastoma; proliferation; therapeutic target.

Figure 1.

Workflow for identifying the hub genes with an upregulated expression in MB and examining the potential of PBK as a therapeutic target. MB, medulloblastoma; PBK, PDZ binding kinase; GEO, Gene Expression Omnibus; NC, normal control; DEGs, differentially upregulated genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 2.

Identification of upregulated hub genes in MB following enrichment analysis and protein-protein interaction network construction. (A) Volcano plots illustrating upregulated DEGs (red dots) and downregulated DEGs (blue dots) identified in the three datasets with the criteria of adjusted P<0.05 and |log2FC| ≥1 (–13). (B) Venn diagram of overlapping upregulated or downregulated DEGs among three datasets. (C-E) GO analysis of upregulated DEGs presents the top 10 significant terms of GO analysis in (C) biological process, (D) molecular function, and (E) celluar component. (F) Significant KEGG pathway enrichment terms. (G) The PPI network was constructed with upregulated genes. Red circular nodes represent the top 10 hub genes. MB, medulloblastoma; DEGs, differentially upregulated genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction; PBK, PDZ binding kinase; CDC20, cell division cycle 20; KIF2C, kinesin family member 2C; NUSAP1, nucleolar and spindle associated protein 1; TTK, TTK protein kinase; KIF20A, kinesin family member 20A; TOP2A, DNA topoisomerase II alpha; CDK1, cyclin dedendent kinase 1; ASPM, assembly factor for spindle microtubules; AURKA, Aurora kinase A.

Figure 3.

Validation of the aberrant expression of the hub gene, PBK, in MB. (A-D) Significant upregulation of PBK in MB relative to normal brain control examined in expression datasets GSE74195 (27 MB vs. 5 NC) (20), GSE50161 (22 MB vs. 13 NC) (21), GSE42656 (16 MB vs. 9 NC) (22) and GSE19360 (3 MB vs. 3 NC) (23). (E-G) PBK was highly expressed in all MB subgroups compared with normal brain tissue. Sample size: GSE109401 (4 NC, 5 WNT, 5 SHH, 5 G3, 4 G4) (24), GSE86574 (10 NC, 5 SHH, 5 G3, 5 G4) (25) and GSE62600 (1 NC, 2 WNT, 7 SHH, 6 G3, 4 G4) (26). (H-J) PBK expression varied among MB subgroups, with group 4 MBs appearing to have the lowest expression level. Sample size: GSE85217 (70 WNT, 223 SHH, 144 G3, 326 G4) (27), GSE37418 (8 WNT, 10 SHH, 16 G3, 39 G4) (28) and GSE21140 (8 WNT, 33 SHH, 27 G3, 35 G4) (29). Quantitative results are presented as the mean ± SD. Statistical significance was determined using (A-D) an unpaired Student's t test or (E-J) one-way ANOVA with Tukey's HSD post hoc test. *P<0.05. MB, medulloblastoma; PBK, PDZ binding kinase; NC, normal control; WNT, Wingless; SHH, Sonic hedgehog; G3, group 3; G4, group 4.

Figure 4.

Prognostic significance of PBK in MB. Survival analysis revealed poorer clinical outcomes in (B) SHH, (C) group 3, and (D) group 4MBs with high expression of PBK. The third quartile was used for defining high or low expression. The log-rank (Mantel-Cox) test was utilized to determine statistically significant differences. MB, medulloblastoma; PBK, PDZ binding kinase; WNT, Wingless; SHH, Sonic hedgehog.

Figure 5.

Targeting PBK inhibits the proliferation of MB cells and reduces the phosphorylation levels of downstream signaling molecules. (A and B) Reverse transcription-quantitative PCR and western blot analysis illustrating the robust expression of PBK in Daoy and D341 cells. (C and D) The viability of Daoy and D341 cells was inhibited by the PBK inhibitor, HI-TOPK-032, with IC50 values of 1.241 and 1.335 µM, respectively. (E and F) Cell proliferation was suppressed by HI-TOPK-032 in a concentration-dependent manner. (G) MB cell lines exhibited a lower proliferation rate in the EdU assay following treatement with HI-TOPK-032. Scale bar, 200 µm. (H) A marked decrease in the levels of p-ERK1/2 and p-Akt was observed in the HI-TOPK-032-treated MB cells. Quantitative results are presented as the mean ± SD. Statistical significance was tested using (E and F) two-way ANOVA or (G) one-way ANOVA with Tukey's HSD post hoc test. *P<0.05. MB, medulloblastoma; PBK, PDZ binding kinase.

Figure 6.

Pharmacological inhibition of PBK promotes the apoptosis of Daoy cells with the activation of caspase-3. (A and B) The percentage of apoptotic cells was significantly higher in the HI-TOPK-032-treated group compared with the control group. Scale bar, 150 µm. Quantitative results are presented as the mean ± SD. Statistical significance according to an unpaired Student's t-test is indicated: *P<0.05. (C) Western blot analysis revealed that the level of cleaved caspase-3 in Daoy cells was substantially increased following treatment with HI-TOPK-032. PBK, PDZ binding kinase.

Figure 7.

Targeting PBK with HI-TOPK-032 induces the apoptosis of D341 cells in vitro. (A and B) The percentage of apoptotic cells was significantly increased in the HI-TOPK-032-treated D341 cells. Scale bar, 100 µm. Quantitative results are presented as the mean ± SD. Statistical significance according to an unpaired Student's t-test is indicated: *P<0.05. (C) Western blot analysis revealed that the level of cleaved caspase-3 in D341 cells was substantially increased following treatment with HI-TOPK-032.