FBXL18 is required for ovarian cancer cell proliferation and migration through activating AKT signaling

Abstract

Background: F-box and leucine-rich repeat protein 18 (FBXL18) is an F-box protein that functions as an E3-ubiquitin ligase, and it plays pivotal roles in multiple disease processes. However, its role and underlying mechanism in ovarian cancer (OC) are still unknown. We investigated the impact and mechanism of FBXL18 in OC cell growth and tumorigenesis.

Methods: Silent interfering RNAs and overexpression plasmids were employed to knock down and overexpress FBXL18 in OC cells (A2780 and OVCAR3). CCK-8, colony formation, cell migration, and nude mouse xenograft assays were used to assess the effect of FBXL18 on OC cell proliferation and migration. Western blotting and co-immunoprecipitation followed by ubiquitination assays were performed to detect the mechanism of the FBXL18/AKT axis in OC.

Results: FBXL18 knockdown inhibited OC cell proliferation and migration, whereas FBXL18 overexpression showed the opposite results. Phosphorylated-AKT (S473) protein expression was increased by FBXL18 overexpression and markedly decreased after phosphorylated-AKT inhibitor (MK-2206) treatment. Co-immunoprecipitation assays demonstrated that FBXL18 strongly interacted with AKT in OC cells. Ubiquitination assays revealed that FBXL18 promoted K63-linked AKT ubiquitination to activate AKT. MK-2206 treatment reversed the increase in proliferation and migration of OC cells induced by FBXL18 overexpression.

Conclusions: FBXL18 promoted OC cell proliferation and migration and facilitated OC tumorigenesis. Mechanically, FBXL18 interacted with AKT and promoted K63-linked ubiquitination of AKT to activate AKT in OC cells. Our study revealed that the FBXL18/AKT axis plays a crucial role in the OC process, indicating that FBXL18 may be a valuable target for OC diagnosis and treatment.

Keywords: AKT; FBXL18; cell proliferation; migration; ovarian cancer.

Figure 1

F-box and leucine-rich repeat protein 18 (FBXL18) is a risk factor for and is negatively associated with the prognosis of ovarian cancer (OC). A. E3 ubiquitin protein ligase genes associated with overall survival (OS) of OC in The Cancer Genome Atlas database (TCGA). B. E3 ubiquitin protein ligase genes associated with OS of OC in the GSE63885 database. C-F. The Kaplan-Meier curves of four meaningful genes from the intersecting portion of the two gene data sets. G, H. The expression levels of Lysine-specific demethylase 2A (KDM2A) and FBXL18 in OC tissue (n = 426) and normal ovarian tissue (n = 88) from the Gene Expression Profiling Interactive Analysis database (t-test). *P < 0.05.

Figure 2

Knockdown of FBXL18 suppresses OC cell (A2780 and OVCAR3) proliferation and migration in vitro. (A, B) Real-time quantitative PCR was used to detect the relative expression of FBXL18 mRNA in A2780 and OVCAR3 cells after FBXL18 knockdown (analysis of variance, ANOVA). (C) Western blotting was employed to verify the efficiency of FBXL18 knockdown. (D, E) A CCK-8 assay (n = 4 for each group) was conducted to determine the viability of A2780 and OVCAR3 cells (ANOVA). (F) A colony formation assay (n = 3) was performed to examine the proliferation of A2780 and OVCAR3 cells. Scale bar: 5 mm. (G) Quantification of the results from (F) (ANOVA). (H) A Transwell assay (n = 3) was conducted to assess the migratory abilities of A2780 and OVCAR3 cells. Scale bar: 200 μm. (I) Quantification of (H) (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, compared with the si-NC condition.

Figure 3

Decreasing FBXL18 attenuates OC cell proliferation in vivo. (A) OVCAR3 cells were treated with sh-FBXL18 and sh-NC and then subcutaneously injected into the armpits of nude mice (n = 6). The macroscopic appearance of tumors is shown. (B) Tumor sizes were calculated every 3 days and are represented as the mean tumor size ± standard deviation (n = 6) (t-test). (C) The weights of subcutaneous tumors were measured (n = 6) (t-test). (D) Ki67 expression in tumors was examined using immunofluorescence. n = 3. Scale bar: 10 μm. (E) Quantification of Ki67-positive cells in (D) (t-test). *P < 0.05, **P < 0.01, ***P < 0.001, compared with the sh-NC condition.

Figure 4

Overexpression of FBXL18 promotes OC cell proliferation and migration in vitro. (A) Western blotting of FBXL18 protein expression in A2780 and OVCAR3 cells transfected with pcDNA3.1-Flag-FBXL18 and pcDNA3.1-NC (EV). (B, C) The proliferative viability of OC cells was detected using a CCK-8 assay after OC cells were treated with the FBXL18 and EV overexpression plasmids (n = 4) (t-test). A colony formation assay (D, E) (n = 3) (t-test, scale bar: 5 mm) was conducted, and the migratory ability was assessed using a cell migration assay (F, G) (n = 3) (t-test). Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the EV condition.

Figure 5

AKT activation is implicated in OC cell proliferation and migration induced by FBXL18 overexpression. (A-D) A2780 and OVCAR3 cells were treated with si-FBXL18s and si-NC for 48 h, followed by western blotting analysis of phosphorylated-AKT (p-AKT) (Ser473) protein expression (n = 3). (B) and (D) show the quantification of (A) and (C) (ANOVA). (E-H) OC cells were transfected with FBXL18 overexpression plasmids with or without MK-2206 treatment (p-AKT inhibitor, 10 μM), and the levels of AKT phosphorylation at Ser473 were determined by western blot analysis in A2780 (E, F) (t-test) and OVCAR3 (G, H) cells (t-test, n = 3). CCK-8 (I, J) (n = 4) (t-test) and colony formation (K, L) (n = 3) (t-test, scale bar: 5 mm) assays were used to assess the effect of p-AKT inhibition on the proliferative viability of OC cells induced by FBXL18. (M, N) A cell migration assay (n = 3) (t-test, scale bar: 100 μm) was performed to reveal the relationship between AKT activation and the migratory ability of OC cells induced by FBXL18. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the si-NC or EV condition. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the Flag-FBXL18 condition.

Figure 6

FBXL18 interacts with AKT and promotes AKT K63-linked ubiquitination. A, B. GFP-labeled AKT was co-transfected with Flag-labeled FBXL18 in OVCAR3 cells followed by a co-immunoprecipitation assay. C. The OVCAR3 cells were transiently transfected with Flag-labeled FBXL18, GFP-labeled AKT, and HA-labeled-Ub-WT/K48/K63/K0 plasmids, followed by a ubiquitin assay.