Clinical significance and potential regulatory mechanism of overexpression of pituitary tumor-transforming gene transcription factor in bladder cancer

Abstract

Background: Pituitary tumor transforming gene-1 (PTTG1) transcription factor is identified as carcinogenic and associated with tumor invasiveness, but its role in bladder cancer (BLCA) remains obscure. This research is intended to analyze the aberrant expression and clinical significance of PTTG1 in BLCA, explore the relationship between PTTG1 and tumor microenvironment characteristics and predict its potential transcriptional activity in BLCA tissue.

Methods: We compared the expression discrepancy of PTTG1 mRNA in BLCA and normal bladder tissue, using the BLCA transcriptomic datasets from GEO, ArrayExpress, TCGA, and GTEx. In-house immunohistochemical staining was implemented to determine the PTTG1 protein intensity. The prognostic value of PTTG1 was evaluated using the Kaplan-Meier Plotter. CRISPR screen data was utilized to estimate the effect PTTG1 interference has on BLCA cell lines. We predicted the abundance of the immune cells in the BLCA tumor microenvironment using the microenvironment cell populations-counter and ESTIMATE algorithms. Single-cell RNA sequencing data was applied to identify the major cell types in BLCA, and the dynamics of BLCA progression were revealed using pseudotime analysis. PTTG1 target genes were predicted by CistromeDB.

Results: The elevated expression level of PTTG1 was confirmed in 1037 BLCA samples compared with 127 non-BLCA samples, with a standardized mean difference value of 1.04. Higher PTTG1 expression status exhibited a poorer BLCA prognosis. Moreover, the PTTG1 Chronos genetic effect scores were negative, indicating that PTTG1 silence may inhibit the proliferation and survival of BLCA cells. With PTTG1 mRNA expression level increasing, higher natural killer, cytotoxic lymphocyte, and monocyte lineage cell infiltration levels were observed. A total of four candidate targets containing CHEK2, OCIAD2, UBE2L3, and ZNF367 were determined ultimately.

Conclusions: PTTG1 mRNA over-expression may become a potential biomarker for BLCA prognosis. Additionally, PTTG1 may correlate with the BLCA tumor microenvironment and exert transcriptional activity by targeting CHEK2, OCIAD2, UBE2L3, and ZNF367 in BLCA tissue.

Keywords: BLCA; PTTG1; Transcriptional regulation; Tumor microenvironment.

Fig. 1

Over-expression of PTTG1 protein in bladder carcinoma tissue. In-house immunohistochemistry was conducted to explore the protein expression level of PTTG1, and PTTG1 protein was overexpressed in bladder carcinoma (BLCA) tissue samples when compared with non-BLCA tissue samples

Fig. 2

Over-expression of PTTG1 protein in the urothelial carcinoma of the bladder. The protein expression level of PTTG1 was confirmed by the immunohistochemistry result from the human protein atlas. PTTG1 protein was moderately stained in the urothelial carcinoma of the bladder and was lowly stained or not detected in normal or inflammatory bladder tissue (Antibody: HPA045034 and CAB008373)

Fig. 3

Significant over-expression of PTTG1 mRNA in bladder carcinoma tissue samples. A The transcriptome data from TCGA, GEO, and ArrayExpress implied an increased expression trend of PTTG1 mRNA in bladder carcinoma tissue samples. B PTTG1 was significantly up-regulated in global bladder carcinoma tissue specimens. C Begg’s funnel plot. Insignificant bias was detected (P > 0.05). D Forest plot of sensitive analysis. The standardized mean difference result may be unstable

Fig. 4

Over-expression verification of PTTG1 in bladder carcinoma cell lines. A The relative expression levels of PTTG1 mRNA were compared in pan-cancer cell lines. B PTTG1 mRNA was broadly expressed in a series of bladder carcinoma cell lines

Fig. 5

A strong discriminatory ability of PTTG1 in bladder carcinoma tissue samples. PTTG1 showed a strong discriminatory ability between bladder carcinoma tissue samples and normal bladder tissue samples, with a high sensitivity (A) and a high specificity (B). The accuracy of PTTG1 in differentiating bladder carcinoma from normal tissue samples was confirmed by a high positive likelihood ratio (C) and a low negative likelihood ratio (D)

Fig. 6

PTTG1 as an unfavorable prognostic biomarker in bladder carcinoma. A summary receiver operating characteristic curve was used to evaluate the comprehensive discriminatory ability of PTTG1 in bladder carcinoma. Intriguingly, PTTG1 displayed a strong ability in differentiating bladder carcinoma tissue samples from normal bladder tissue samples (AUC > 0.9) (A). Kaplan-Meier survival analysis was performed to determine the prognostic value of PTTG1 in bladder carcinoma. Higher PTTG1 mRNA levels presaged worse overall survival probability (HR > 1, P < 0.05) (B) and poorer disease-free survival outcomes (HR > 1, P > 0.05) (C). A time-dependent receiver operating characteristic curve was utilized to evaluate the 1-year, 2-year, and 3-year survival rates of bladder carcinoma patients (D)

Fig. 7

Potential association between PTTG1 and tumor microenvironment in bladder cancer tissue samples. The immune infiltration levels of immune and stromal cells of TCGA-BLCA patients were quantified by using the Microenvironment cell populations-counter algorithm. A The immune microenvironment and clinical phenotypes were compared in normal bladder tissues, PTTG1 low expression bladder cancer tissue samples, and PTTG1 high expression bladder cancer tissue samples. B Higher PTTG1 mRNA expression levels indicated higher natural killer, cytotoxic lymphocyte, and monocyte lineage cell infiltration levels, and indicated lower neutrophils and endothelial cell infiltration levels. C The immune score was significantly lower in low PTTG1 mRNA expression bladder cancer tissue samples than that in high PTTG1 mRNA expression bladder cancer tissue samples or normal bladder tissue samples. However, no difference was detected in the stromal score of low PTTG1 mRNA expression bladder cancer tissue samples than that in high PTTG1 mRNA expression bladder cancer tissue samples (P < 0.05). D A high stromal score predicted poor prognosis in bladder carcinoma patients. E The prognostic value of the immune score in bladder carcinoma was insignificant (P < 0.05). **, P < 0.01; ****, P < 0.0001

Fig. 8

Potential transcriptional regulatory mechanisms of PTTG1 in bladder cancer at single-cell resolution. GSE135337 dataset was utilized for analyzing the transcriptional regulatory mechanisms of PTTG1 in seven bladder cancer patients. Single cells from bladder cancer patients were clustered and annotated. Epithelial cells were the predominant cell type (A). The expression distributions of PTTG1 were shown in single cells. PTTG1 was predominantly expressed in cancerous epithelial cells (B). C Epithelial cells from bladder cancer tissues were filtered for pseudotime analysis. D Gene expression heatmap along the pseudotime direction. The gene expression alterations at the first branch were analyzed and the differentially expressed genes were functionally annotated. Cell fate 1 and cell fate 2 in panel D refer to state 4 and state 5 in panel C, respectively

Fig. 9

Transcriptional targets of PTTG1 were identified by using chromatin immunoprecipitation followed by sequencing data from CistromeDB. A The interplay between the PTTG1 transcriptional factor and four transcriptional targets was analyzed in a protein-protein interaction network. B Chromatin immunoprecipitation followed by sequencing data was used to explore the binding peak of PTTG1 in the promoter regions of transcriptional targets. CHEK2, OCIAD2, UBE2L3, and ZNF367 were predicted as transcriptional targets of PTTG1 in bladder carcinoma. The data were downloaded from CistromeDB (ID: 63264 and 63265)

Fig. 10

Global expression trends of PTTG1 transcriptional targets in BLCA tissue. CHEK2, OCIAD2, UBE2L3, and ZNF367 were significantly up-regulated in BLCA tissue samples. BLCA, bladder carcinoma

Fig. 11

Functional enrichment of PTTG1 transcriptional targets in BLCA tissue. Differentially expressed genes (DEGs) associated with CHEK2, OCIAD2, UBE2L3, and ZNF367 were identified from the TCGA-BLCA cohort study. The prospective biological functions of these PTTG1 targets were annotated. A. CHEK2-related DEGs were significantly enriched in the antibacterial humoral response. B. OCIAD2-related DEGs were significantly enriched in the complement activation (classical pathway). C. UBE2L3-related DEGs were significantly enriched in the phagocytosis and recognition. D. ZNF367-related DEGs were significantly enriched in the cell cycle pathway. BLCA, bladder carcinoma