Prognostic value of TOP2A in bladder urothelial carcinoma and potential molecular mechanisms

Abstract

Background: The prognosis of bladder urothelial carcinoma (BLCA) varies greatly among patients, and conventional pathological predictors are generally inadequate and often inaccurate to predict the heterogeneous behavior of BLCA. This study aims to investigate the prognostic value and function of TOP2A in BLCA.

Methods: TOP2A expression level was examined by RNA-sequencing, quantitative real time polymerase chain reaction and immunohistochemistry from 10, 40 and 209 BLCA samples, respectively. Public databases were analyzed for validation. Cell proliferation, migration, invasion assays were performed to explore potential functions of TOP2A in BLCA. Flow cytometry was performed for cell cycle and apoptosis analysis. Univariable and multivariable Cox regression models were performed to identify independent risk factors for the prognosis of BLCA.

Results: We found TOP2A was significantly upregulated in BLCA samples, especially for high-grade and advanced stage tumors, compared with matched normal epithelial tissue. Univariable COX regression analysis revealed high TOP2A expression was significantly associated with poorer cancer-specific, progression-free and recurrence-free survival, but not independently of clinical characteristics in the multivariable models. Knockdown of TOP2A remarkably inhibited the proliferation of BLCA cells and non-cancerous urothelial cells. Furthermore, migration and invasion capacity of BLCA cells were strongly suppressed after TOP2A knockdown. Moreover, flow cytometry suggested TOP2A had anti-apoptotic function, and knockdown of TOP2A could induce resistance to doxorubicin in J82 cells.

Conclusions: In our study, TOP2A was overexpressed in BLCA and could serve as a prognostic biomarker for BLCA. Moreover, TOP2A is functionally important for the proliferation, invasion and survival of BLCA cells.

Keywords: Biomarker; Bladder urothelial carcinoma; Prognosis; TOP2A.

Fig. 1

Increased TOP2A expression in bladder urothelial carcinoma (BLCA) revealed by RNA-sequencing (a) and RT-qPCR (c-e). a Hierarchical clustering heat map of differentially expressed genes between tumor tissue and matched normal epithelial tissue. b Expression levels of TOP2A in each sample for RNA-sequencing. c TOP2A gene expression was significantly up regulated in tumor tissue compared with matched normal epithelial tissue. d TOP2A gene expression in different stages of BLCA. e TOP2A gene expression in different grades of BLCA. f TOP2A gene expression was significantly higher in MIBC compared with NMIBC in NCBI-GEO GSE31684 dataset. MIBC = muscle-invasive bladder cancer; NMIBC = non-muscle invasive bladder cancer; RPKM = reads per gene per kilobase exon per million mapped reads. **, p < 0.01

Fig. 2

The relationship between TOP2A protein expression and prognosis of patients with BLCA. a-c Representative images of TOP2A protein expression in normal and bladder cancer tissue. d-f Higher TOP2A protein expression was associated with poorer cancer-specific, progression-free and recurrence-free survival from our single institution cohort (n = 209)

Fig. 3

The prognostic value of TOP2A mRNA and its relationship with TOP2A protein in BLCA. a-c Kaplan-Meier survival curve comparing recurrence-free survival between patients with TOP2A mRNA high and low expression in TCGA, NCBI-GEO (GSE31684) and MSKCC datasets, respectively. Patients with TOP2A mRNA expression above 75% or below 25% were included for analysis. d RT-qPCR assay of TOP2A mRNA expression level in 14 bladder cancer samples. e TOP2A protein level of the 14 bladder cancer samples were investigated by western blotting assay

Fig. 4

Cell proliferation, migration and invasion capacity was inhibited after TOP2A knockdown in bladder cancer cells. a TOP2A mRNA and protein expression levels in different bladder cancer cell lines. b The growth curves of J82 and 5637 cells between control and TOP2A knockdown groups. c The effect of TOP2A knockdown on tumor growth in nude mouse xenograft models. d Knockdown of TOP2A significantly decreased cell migration capacity in wound-healing assay. e Knockdown of TOP2A significantly reduced cells invading through Matrigel.NC = negative control. *, p < 0.05, **, p < 0.01. At least three independent replicates were performed for each experiment

Fig. 5

Apoptosis analysis after TOP2A knockdown. a Apoptosis were significantly increased after TOP2A knockdown in J82 and cells after induction of apoptosis by gemcitabine treatment for 24 h. b Western blotting assay revealed increasing expression of cleaved caspase 3 after TOP2A knockdown. c-d Dose response curves of bladder cancer cell lines treated with doxorubicin at different concentrations as determined in 72-h cell viability assays. NE = necrosis; NC = negative control; IC₅₀ = half maximal inhibitory concentration *, p < 0.05. At least three independent replicates were performed for each experiment