TOP2A modulates signaling via the AKT/mTOR pathway to promote ovarian cancer cell proliferation

Abstract

Ovarian cancer (OC) is a form of gynecological malignancy that is associated with worse patient outcomes than any other cancer of the female reproductive tract. Topoisomerase II α (TOP2A) is commonly regarded as an oncogene that is associated with malignant disease progression in a variety of cancers, its mechanistic functions in OC have yet to be firmly established. We explored the role of TOP2A in OC through online databases, clinical samples, in vitro and in vivo experiments. And initial analyses of public databases revealed high OC-related TOP2A expression in patient samples that was related to poorer prognosis. This was confirmed by clinical samples in which TOP2A expression was elevated in OC relative to healthy tissue. Kaplan-Meier analyses further suggested that higher TOP2A expression levels were correlated with worse prognosis in OC patients. In vitro, TOP2A knockdown resulted in the inhibition of OC cell proliferation, with cells entering G1 phase arrest and undergoing consequent apoptotic death. In rescue assays, TOP2A was confirmed to regulate cell proliferation and cell cycle through AKT/mTOR pathway activity. Mouse model experiments further affirmed the key role that TOP2A plays as a driver of OC cell proliferation. These data provide strong evidence supporting TOP2A as an oncogenic mediator and prognostic biomarker related to OC progression and poor outcomes. At the mechanistic level, TOP2A can control tumor cell growth via AKT/mTOR pathway modulation. These preliminary results provide a foundation for future research seeking to explore the utility of TOP2A inhibitor-based combination treatment regimens in platinum-resistant recurrent OC patients.

Keywords: AKT/mTOR signaling pathway; Ovarian cancer; TOP2A; prognosis; proliferation; rescue experiments.

Figure 1.

Screening and analysis of DGEs in OC. (a) 14 DEGs were up-regulated and 22 DEGs were down-regulated in four datasets (log2-fold change > 2, p < .05). (b) The 14 up-regulated genes and 22 down-regulated genes. (c-f) KEGG, GO-BP, GO-CC and GO-MF analysis of up-regulated genes. (g) High TOP2A expression in dataset 201,291-s-at and 201,292-at was associated with poor overall survival (OS) of OC patients. (h) High TOP2A expression in dataset 201,291-s-at was associated with poor progression-free survival (PFS) of OC patients.

Figure 2.

TOP2A expression levels in OC. (a) TOP2A mRNA expression levels were significantly increased in OC patients in the GEPIA database. (b-c) TOP2A mRNA expression levels in OC patients were significantly increased in GTEx combined with TCGA database and in GTEx combined with ICGC database. (d) Expression of TOP2A mRNA in different OC cell lines in CCLE database. (e) The localization of TOP2A protein in cells was obtained through the subcell section of the human protein atlas. (f) Total TOP2A protein expression was increased in OC tissues compared with normal tissues. (g) Compared with normal tissues, the expression of p-TOP2A protein was enhanced in OC tissues. *p < .05, ****p < .0001.

Figure 3.

To investigate the expression and prognostic significance of TOP2A in OC by clinical samples. (a-b) TOP2A protein expression level was increased in 5 OC tissues compared with 5 normal tissues by western blotting. (c) The immunohistochemistry atlas of different FIGO stages of 94 ovarian cancer tissues. 50× and 200× (partial enlargement) microscopic views were shown. (d) TOP2A protein expression was enhanced with the increase of FIGO stage and histological grade. (e) TOP2A high expression is associated with poor PFS in OC patients(p = .034). (f) TOP2A high expression is associated with poor OS in OC patients(p = .024). *p < .05, ****p < .0001.

Figure 4.

The effect of TOP2A knockdown on OC cell proliferation. (a) The transfection efficiency of SH-TOP2A in the two cell lines was observed by fluorescence microscope. (b-d) TOP2A mRNA and protein levels were significantly decreased after transfection in SKOV3 cells. (e-g) TOP2A mRNA and protein levels were significantly decreased after transfection in HEYA8 cells. (h-j) CCK-8 and colony formation assays suggested that the proliferation ability of SKOV3 cells was decreased after TOP2A knockdown. (k-m) CCK-8 and colony formation assays suggested that the proliferation ability of HEYA8 cells was decreased after TOP2A knockdown. Number of experiments (N) = 3, *p < .05, **p < .01, ***p < .001, ****p < .0001.

Figure 5.

The effect of TOP2A knockdown on OC cell cycle and apoptosis. (a) TOP2A knockdown increased the proportion of cells in G1 phase in both OC cell lines. (b) TOP2A knockdown promoted apoptosis in both OC cell lines. N = 3, *p < .05, **p < .01, ****p < .0001.

Figure 6.

The effect of TOP2A knockdown on related proteins. (a-c) after TOP2A knockdown, the expression levels of C-myc, cyclin D1, CDK4 and Bcl2 were decreased, while the expression levels of P21 and Bax were increased. (d-f) after TOP2A knockdown, the expression levels of p-AKT/AKT and p-mTOR/mTOR were decreased. N = 3, *p < .05, **p < .01, ***p < .001, ****p < .0001.

Figure 7.

Rescue experiments were conducted to verify the mechanism of TOP2A regulating OC cell behavior. (a-c) after SC79 treatment of cells in the SH-TOP2A group, WB results suggested that the levels of p-AKT/AKT and p-mTOR/mTOR were increased. (d-e) CCK-8 assays showed that the cell proliferation ability of the two SH-TOP2A groups was enhanced after SC79 treatment. (f-h) flow cytometry confirmed that SC79 induced the G1-S phase progression of the cell cycle in both SH-TOP2A groups. N = 3, *p < .05, **p < .01, ***p < .001, ****p < .0001.

Figure 8.

Expression of related proteins after activation of AKT/mTOR pathway. (a-c) the protein levels of C-myc, CyclinD1 and CDK4 in the SH-TOP2A#1+SC79 and SH-TOP2A#1+SC79 groups were higher than those before treatment of SC79. N = 3, *p < .05, **p < .01, ***p < .001, ****p < .0001.

Figure 9.

The effect of TOP2A knockdown on the growth of OC cells in vivo. (a-d) TOP2A knockdown significantly decreased the HEYA8 cell-derived tumor volume and weight. (e) IHC staining revealed reductions in TOP2A and ki-67 levels in both SH-TOP2A groups relative to tumors from NC group. ****p < .0001.