Prognostic value of members of NFAT family for pan-cancer and a prediction model based on NFAT2 in bladder cancer

Abstract

Bladder cancer (BLCA) is one of the common malignant tumors of the urinary system. The poor prognosis of BLCA patients is due to the lack of early diagnosis and disease recurrence after treatment. Increasing evidence suggests that gene products of the nuclear factor of activated T-cells (NFAT) family are involved in BLCA progression and subsequent interaction(s) with immune surveillance. In this study, we carried out a pan-cancer analysis of the NFAT family and found that NFAT2 is an independent prognostic factor for BLCA. We then screened for differentially expressed genes (DEGs) and further analyzed such candidate gene loci using gene ontology enrichment to curate the KEGG database. We then used Lasso and multivariate Cox regression to identify 4 gene loci (FER1L4, RNF128, EPHB6, and FN1) which were screened together with NFAT2 to construct a prognostic model based on using Kaplan-Meier analysis to predict the overall survival of BLCA patients. Moreover, the accuracy of our proposed model is supported by deposited datasets in the Gene Expression Omnibus (GEO) database. Finally, a nomogram of this prognosis model for BLCA was established which could help to provide better disease management and treatment.

Keywords: NFAT; bladder cancer; nomogram; overall survival; prognostic risk score.

Figure 1

The expression of NFAT family gene in BLCA. Blue represents the expression of normal tissues in the GTEx database, and red represents the expression of BLCA patients in the TCGA database.

Figure 2

Genetic alterations of NFAT family genes. (A) Oncoprint visual summary of genetic alterations in NFAT family members. (B) Summary of genetic alterations in NFAT family members. (C) Kaplan-Meier survival curves for OS in cancer patients with genetic alterations. (D) Kaplan-Meier survival curves for DFS in cancer patients with genetic alterations.

Figure 3

Prognostic value of NFAT members in BLCA patients. Kaplan-Meier survival curves for OS of BLCA patients with expression of NFAT1, NFAT2, NFAT3, NFAT4 and NFAT5.

Figure 4

Expression of NFAT2 in BLCA patients with different clinical and pathological features. Kaplan-Meier survival curves for OS of BLCA patients and clinical factors. (A) M Stage. (B) Gender. (C) Age. (D) Stage. (E) N Stage. (F) T Stage. (G) Grade Stage.

Figure 5

NFAT2 is an independent prognostic factor of BLCA. Multivariate Cox proportional hazard model of the expression of NFAT2 and clinical factors.

Figure 6

DEGs between high and low expression of NFAT2 groups. (A) Volcanic map for the DEGs identified by R software with limma package. The abscissa represented log2FC, and the ordinate represented the negative logarithm of the P-value. The red, green, and black nodes represented upregulated mRNA, downregulated mRNA, and non-differentially expressed mRNA. (B) Heatmap for the DEGs identified by R software with limma package.

Figure 7

Gene ontology pathway enrichment analysis of DEGs. The rich factor demonstrates the degree of enrichment by GO. The node size represents the number of selected genes, and color represents the P-value of the enrichment analysis. CC, cellular component; MF, molecular function; BP, biological process.

Figure 8

KEGG pathway enrichment analysis of DEGs. (A) The rich factor demonstrates the degree of enrichment by GO. The Node size represents the number of selected genes, and color represents the P-value of the enrichment analysis. (B) Network diagram provides the KEGG pathway interaction in the DEGs.

Figure 9

Network of protein-protein interactions (PPI) analysis. (A) Protein-protein interaction network was constructed for the DEGs using Cytoscape. (B) Subnetwork with the highest score using MCODE tool.

Figure 10

Construction of overall survival risk score model. (A) LASSO coefficient profiles of the genes associated with the DEGs. (B) Partial likelihood deviance was plotted versus log (Lambda). The vertical dotted line indicates the lambda value with the minimum error and the largest lambda value. (C) Risk scores of the patients in the high (red) and low (green) risk groups. (D) Patients of the validation set from TCGA were divided by risk score into high risk and a low risk groups. OS between two risk groups were analyzed and compared by Kaplan-Meier analysis. Red lines represent the high-risk group samples, and blue lines represent the low-risk group samples. (E) ROC curves in the validation set. The abscissa represents sensitivity, and the ordinate represents specificity.

Figure 11

Evaluation of risk score model. (A) The nomogram is applied by adding up the points identified on the points scale for each variable. (B) The calibration curve for predicting 1-3-5 years OS for patients with BLCA. The Y-axis represents actual survival, as measured by K-M analysis, and the X-axis represents the nomogram-predicted survival (P<0.05).

Figure 12

Validation of prognosis risk model. (A) Kaplan-Meier curves for OS time of patients with expression of NFAT2 in clinical study GSE100926. (B) Patients data from GSE100926 were divided by risk score into a high risk and a low risk groups. OS between two risk groups were analyzed and compared by Kaplan-Meier analysis. (C) 1-3-5 years ROC curves in GSE100926. The abscissa represents sensitivity, and the ordinate represents specificity. (D–F) The calibration curve for predicting 1-3-5 years OS for patients with BLCA. The Y-axis represents actual survival, as measured by K-M analysis, and the X-axis represents the nomogram-predicted survival (P<0.05). (G) The expression profiles of the NFAT2 in the normal bladder tissue and bladder specimens. Images were taken from the HPA.

Figure 13

NFAT2 participates in the regulation of BLCA as an oncogene. (A) The expression of NFAT2 was verified by WB. (B) The expression of NFAT2 was verified by RT-PCR. (C) The knockdown efficiency was verified by RT-PCR. (D) The knockdown efficiency was verified by WB. (E) The cell viability was assessed by cell proliferation assay (see Materials and Methods). (F) Cell proliferation detected by colony formation assay. (G) Cell proliferation was measured by EdU incorporation assay. (H) Cell invasion measured by Transwell migration assay. (I) Cell migration evaluated by wounded cell monolayer closure assay. (J) Western Blot analysis of protein expression of NFAT2 and EMT-linked gene products. (K) Quantitative RT-PCR analysis of mRNA for NFAT2 and EMT-related genes. (L) Tumor weight from control and NFAT2 knockdown mouse tumor groups. (M) Tumor volume from control and NFAT2 knockdown mouse tumor groups. (N) Tumor cell proliferation evaluated by Ki-67 immunohistochemical staining. *P<0.05; **P<0.01; ***P<0.001; #P<0.05. All data are representative of three independent experiments.