FBXO22 promotes osteosarcoma progression via regulation of FOXO1 for ubiquitination and degradation

Abstract

Accumulating evidence has demonstrated that F-box protein 22 (FBXO22) participates in tumour development and progression in various types of human malignancies. However, the functions and detailed molecular mechanisms of FBXO22 in osteosarcoma tumorigenesis and progression remain elusive. In this study, we aimed to determine the effects of FBXO22 on the cell proliferation, migration and invasion of osteosarcoma cells using cell counting kit-8 and Matrigel Transwell approaches. Moreover, we explored the molecular mechanisms by which FBXO22 mediated oncogenesis and progression in osteosarcoma via Western blotting, immunoprecipitation and ubiquitination. We found that FBXO22 depletion inhibited the proliferation, migration and invasion of osteosarcoma cells, whereas FBXO22 overexpression increased the proliferation and motility of osteosarcoma cells. Mechanistically, FBXO22 promoted the ubiquitination and degradation of FoxO1 in osteosarcoma cells. FBXO22 depletion reduced cell proliferation and motility via regulation of FoxO1. Taken together, our findings provide new insight into FBXO22-induced osteosarcoma tumorigenesis. The inhibition of FBXO22 could be a promising strategy for the treatment of osteosarcoma.

Keywords: FBXO22; FoxO1; degradation; osteosarcoma; ubiquitination.

FIGURE 1

Silencing of F‐box protein 22 (FBXO22) inhibits the proliferation, migration and invasion of osteosarcoma cells. (A) RT‐PCR was performed to measure the FBXO22 mRNA levels in SaOS‐2 and U2OS cells after shFbxo22 transfection for 72 h. (B) Western blotting analysis was performed to measure the protein levels of FBXO22 in SaOS‐2 and U2OS cells after shFbxo22 transfection for 72 h. (C) A CCK‐8 assay was conducted to measure the viability of SaOS‐2 and U2OS cells after shFbxo22 transfection for different durations. (D) An EdU assay was performed to measure the proliferation of SaOS‐2 and U2OS cells after shFbxo22 transfection for 72 h. **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control.

FIGURE 2

Silencing of F‐box protein 22 (FBXO22) inhibits the migration and invasion of osteosarcoma cells. (A) A wound healing assay was utilized to measure the rate of wound closure in SaOS‐2 and U2OS cells after shFbxo22 transfection for 20 h. (B) Transwell migration and invasion assays were used to detect the migratory and invasive ability of SaOS‐2 and U2OS cells after shFbxo22 transfection for 20 h. (C) Quantitative data are shown in Panel B. **p < 0.01, ***p < 0.001 versus control.

FIGURE 3

F‐box protein 22 (FBXO22) overexpression promotes the proliferation of osteosarcoma cells. (A) Western blotting analysis was performed to measure the protein levels of FBXO22 in osteosarcoma cells after Fbxo22 cDNA transfection for 72 h. (B) A CCK‐8 assay was conducted to measure the viability of SaOS‐2 and U2OS cells after FBXO22 cDNA transfection for different durations. ***p < 0.001 versus control. (C) An EdU assay was performed to measure the proliferation of osteosarcoma cells after FBXO22 cDNA transfection for 72 h. (D) Right panel: Quantitative data are shown for the EdU assay. ***p < 0.001 versus control.

FIGURE 4

F‐box protein 22 (FBXO22) overexpression promotes the migration and invasion of osteosarcoma cells. (A) Transwell migration and invasion assays were used to detect the migratory and invasive ability of osteosarcoma cells after FBXO22 cDNA transfection for 20 h. (B) Quantitative data are shown for Panel A. **p < 0.01, ***p < 0.001 versus control. (C) A wound healing assay was used to measure the rate of wound closure in osteosarcoma cells after FBXO22 cDNA transfection for 10 h.

FIGURE 5

F‐box protein 22 (FBXO22) targets FoxO1 for ubiquitination and degradation. (A) Western blotting analysis was performed to measure the protein levels of FBXO22 and FoxO1 in SaOS‐2 and U2OS cells after shFbxo22 transfection for 72 h. (B) Western blotting analysis was performed to measure the protein levels of FBXO22 and FoxO1 in osteosarcoma cells after Fbxo22 cDNA transfection for 72 h. (C) RT‐PCR was performed to measure the FoxO1 mRNA levels in SaOS‐2 and U2OS cells after shFbxo22 transfection for 72 h. (D) Immunoblotting (IB) of immunoprecipitates and whole‐cell lysates (WCLs) was conducted to detect the interaction between FBXO22 and FoxO1. (E) IB analysis of WCLs and productions of ubiquitination derived from U2OS cells transfected with different constructs. (F) IB analysis of WCLs derived from U2OS and SaOS‐2 cells transfected with different constructs. MG132 (10 μM) was used to treat the cells before harvesting.

FIGURE 6

Silencing of F‐box protein 22 (FBXO22) inhibits proliferation by regulating FoxO1. (A) Western blotting analysis was performed to measure the half‐life of FoxO1 protein in SaOS‐2 cells after shFbxo22 transfection for different durations. (B) Quantitative data are shown for the half‐life of FoxO1. (C) Western blotting analysis was performed to measure the protein levels of FoxO1 in SaOS‐2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection. (D) A CCK‐8 assay was conducted to measure the viability of SaOS‐2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection for different durations. (E) Left panel: An EdU assay was performed to measure the proliferation of SaOS‐2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection for 72 h. Right panel: Quantitative data are shown for the EdU assay. **p < 0.01, ***p < 0.001 versus control, # p < 0.05 versus shFBXO22 or shFoxO1 alone.

FIGURE 7

Silencing of F‐box protein 22 (FBXO22) inhibits cell migration, invasion and tumour growth. (A) Left panel: Transwell migration assays were utilized to detect the migratory ability of SaOS‐2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection for 20 h. Right panel: Quantitative data are shown for left panel. (B) Quantitative data are shown for the migration ability of osteosarcoma cells. (C) Left panel: Transwell invasion assays were utilized to detect the invasive ability of SaOS‐2 and U2OS cells after shFbxo22 transfection plus shFoxO1 transfection for 20 h. Right panel: Quantitative data are shown for left panel. *p < 0.05, **p < 0.01, ***p < 0.001 versus control, # p < 0.05 versus shFBXO22 or shFoxO1. (D) ShFBXO22‐transfected SaOS‐2 cells were injected subcutaneously into BALB/c‐nu/nu mice. After 32 days, the tumours were dissected, and images were obtained. (E) The weights of the dissected tumours are shown. (F) The tumour volumes were measured over the different time periods. (G) Western blotting analysis was performed to measure the expression of FBXO22 and FoxO1 in the dissected tumours. ***p < 0.001 versus control.