Skp2 modulates proliferation, senescence and tumorigenesis of glioma

Abstract

Background: Gliomas represent the largest class of primary central nervous system neoplasms, many subtypes of which exhibit poor prognoses. Surgery followed by radiotherapy and chemotherapy has been used as a standard strategy but yielded unsatisfactory improvements in patient survival outcomes. The S-phase kinase protein 2 (Skp2), a critical component of the E3-ligase SCF complex, has been documented in tumorigenesis in various cancer types but its role in glioma has yet to be fully clarified. In this study, we investigated the function of Skp2 in the proliferation, stem cell maintenance, and drug sensitivity to temozolomide (TMZ) of glioma.

Methods: To investigate the role of Skp2 in the prognosis of patients with glioma, we first analyzed data in databases TCGA and GTEx. To further clarify the effect of Skp2 on glioma cell proliferation, we suppressed its level in glioblastoma (GBM) cell lines through knockdown and small molecule inhibitors (lovastatin and SZL-P1-41). We then detected cell growth, colony formation, sphere formation, drug sensitivity, and in vivo tumor formation in xenograft mice model.

Results: Skp2 mRNA level was higher in both low-grade glioma and GBM than normal brain tissues. The knockdown of Skp2 increased cell sensitivity to TMZ, decreased cell proliferation and tumorigenesis. In addition, Skp2 level was found increased upon stem cells enriching, while the knockdown of Skp2 led to reduced sphere numbers. Downregulation of Skp2 also induced senescence. Repurposing of lovastatin and novel compound SZL-P1-41 suppressed Skp2 effectively, and enhanced glioma cell sensitivity to TMZ in vitro and in vivo.

Conclusion: Our data demonstrated that Skp2 modulated glioma cell proliferation in vitro and in vivo, stem cell maintenance, and cell sensitivity to TMZ, which indicated that Skp2 could be a potential target for long-term treatment.

Keywords: Glioma; Glioma stem-like cells; Lovastatin; S-phase kinase-associated protein 2 (Skp2); SZL-P1-41.

Fig. 1

High expression level of Skp2 predicted poor prognosis in glioma. a Skp2 was overexpressed in various types of tumor tissues compared with paired normal tissues. b Skp2 mRNA levels were high in both LGG and GBM, but only significant in GBM. c In glioma (including both LGG and GBM), the OS was shorter for patients with high-level of Skp2 (n = 330) than those with low-level of Skp2(n = 331) (p < 0.0001). d In LGG, the OS was poorer in patients with high-level of Skp2 (n = 254) than those with a low-level of Skp2 (n = 256) (p < 0.0001). e There was no significant difference on OS between different mRNA levels of Skp2 in GBM. f The OS was analyzed according the IDH mutation status and Skp2 expression level (n = 507). g The OS was analyzed in LGG (n = 94) and GBM (n = 137) patients with IDH^wt and different Skp2 levels. h The OS was analyzed by status of 1p19q co-deletion and Skp2 expression level in IDH^mut LGG patients (n = 413) (BLCA: Bladder Urothelial Carcinoma; CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD: Colon adenocarcinoma; DLBC: Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; GBM: Glioblastoma; ESCA: Esophageal carcinoma; LUSC: Lung squamous cell carcinoma; READ: Rectum adenocarcinoma; STAD: Stomach adenocarcinoma; TGCT: Testicular Germ Cell Tumors; THYM: Thymoma; UCS: Uterine Carcinosarcoma)

Fig. 2

Downregulation of Skp2 attenuated in vitro and in vivo cell proliferation. a On Western blot analyses, the protein level of Skp2 in glioma cell lines (A172, U87, U118, U373, LNZ308, U138 and U343), as compared to normal astrocytes (AST), was enhanced. b Skp2 was successfully knocked-down by two shRNA fragments in U87, U138, and LNZ308 cells compared with the negative control shLuc. The protein levels of the downstream molecules p21^Cip1/Waf1 and p27^Kip1 were increased upon the knockdown of Skp2 in all three cell lines. c Cell proliferation was retarded slightly in U87, U138 and LNZ308 cells after the knockdown of Skp2. d The colony formation ability of the three cell lines was reduced after Skp2 knockdown. e, f The tumor weight and volume were smaller than the control group upon Skp2 knockdown in xenograft mouse models. **p < 0.01, ***p < 0.001

Fig. 3

Downregulation of Skp2 sensitized glioma cells to TMZ. a After Skp2 knockdown, the cell sensitivity to TMZ (inhibition rate) was enhanced in all three cell lines (except sh532 of LNZ308 cells). b The IC50 of TMZ was reduced when Skp2 was knocked down in three cell lines. c The time course of tumor cell sensitivity to TMZ in vivo. d Tumor progression starting from the day TMZ was added. e, f Tumors and their corresponding weights after removal from the mice, sacrificed 24 days after TMZ treatment. ***p < 0.001

Fig. 4

Downregulation of Skp2 reduced sphere formation ability of glioma stem-like cells and induced cellular senescence. a Skp2 protein levels were enhanced together with the glioma stem cell markers Nestin and Sox2 after enrichment with stem cell conditional medium in three glioma cell lines. b The sphere formation ability was reduced in all three cell lines upon Skp2 knockdown. c Cellular senescence was enhanced in the three cell lines upon Skp2 knockdown. **p < 0.01, ***p < 0.001

Fig. 5

Combination therapy of TMZ and lovastatin or SZL-P1-41 was more effective than TMZ alone. a, b The chemical structures of lovastatin and SZL-P1-41. c–e The inhibitory effect was increased by the combined treatment of TMZ with lovastatin or SZL-P1-41, as shown in the left panels; in the right panels, the protein level of Skp2 and downstream targets p21^Cip1/Waf1, p27^Kip1 upon different treatments. f Tumor progression upon TMZ and lovastatin or SZL-P1-41 treatment. g, h Tumors and their corresponding weights from sacrificed mice. LV: lovastatin, SP: SZL-P1-41. *p < 0.05***p < 0.001