ITM2A inhibits the progression of bladder cancer by downregulating the phosphorylation of STAT3

Abstract

Bladder cancer stands as one of the prevalent malignancies in urological clinics, highlighting the pressing need to uncover prognostic or therapeutic avenues. ITM2A, a transmembrane protein, has been identified as a suppressor in tumor progression recently. Our study underscored a significant correlation between low ITM2A expression in bladder cancer tissues and high tumor grade, AJCC stage, and poor overall survival time. Additionally, our findings demonstrated that reinstating ITM2A expression impeded cell proliferation, migration, and invasion, while conversely, its suppression enhanced these malignant behaviors. Furthermore, we elucidated that ITM2A could suppress malignant phenotypes of bladder cancer cells via inhibiting activation of the STAT3 induced by IL-6. In conclusion, our research unveiled the mechanistic role of ITM2A in inhibiting tumor progression, shedding light on its potential as a prognostic predictor and therapeutic target in bladder cancer management.

Keywords: ITM2A; bladder cancer; cell progression; phosphorylation of STAT3; tumor suppressor.

Figure 1

ITM2A expression was decreased in BLCA. A-C. The mRNA level of ITM2A in BLCA tissues and normal bladder tissues according to TCGA and GEO. D, E. The relative expression of ITM2A in forty pairs of BLCA tissues and adjacent normal bladder tissues. F. The relative expression of ITM2A in BLCA tissues and normal bladder tissues using IHC score. G. The expression of ITM2A was correlated with OS in IHC. H, I. ITM2A protein expression in different AJCC stages of BLCA tissues and adjacent normal bladder tissues in IHC assays. J, K. The expression of ITM2A in BLCA cell lines and SV-HUC-1 cells at both the transcriptional and translational levels. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Upregulation of ITM2A inhibited the proliferation of BLCA cells. A. The transcriptional and translational levels of ITM2A in T24 and UMUC-3 cells after infection with empty vector or OE-ITM2A lentivirus. C. The transcriptional and translational levels of ITM2A in T24 and SW780 cells after transfection with siRNAs. B, D. CCK-8 assays showed the proliferation capacity of BLCA cells treated with the indicated lentivirus or siRNAs. E, F. Colony formation assays revealed the colony number of BLCA cells treated with the indicated lentivirus or siRNAs. Each experiment was repeated with three independent replicates.

Figure 3

Upregulation of ITM2A inhibited the migration and invasion of BLCA cells. A, B. Wound healing assays showed the migration capacity of BLCA cells treated with the indicated lentivirus or siRNAs. C, D. Transwell assays revealed the invasion capacity of BLCA cells treated with the indicated lentivirus or siRNAs. Each experiment was repeated with three independent replicates.

Figure 4

ITM2A was related to JAK2/STAT3 signaling pathway. A. Volcano plot of the distribution of DEGs by comparing Ctrl and OE-ITM2A cell samples. B. Venn diagram of upregulated genes and downregulated genes. C. Heatmap of RNA-seq expression data for BLCA cells infected with empty vector or OE-ITM2A lentivirus. D-F. The biological functions of DEGs were explored by GO and KEGG enrichment analysis. G. Western blot showed the expression of p-STAT3 (Tyr705), STAT3, c-Myc, FN1, and ITM2A in BLCA cells.

Figure 5

Overexpression of ITM2A abolished the positive action of the STAT3 activator on the growth of BLCA cells. A. Western blot showed the expression of p-STAT3 (Tyr705), STAT3, c-Myc, FN1, and ITM2A in T24 and UMUC-3 cells treated with OE-ITM2A lentivirus and IL-6. B. CCK-8 assays showed the proliferation capacity of BLCA cells treated with OE-ITM2A lentivirus and IL-6. C. Colony formation assays revealed the colony number of BLCA cells treated with OE-ITM2A lentivirus and IL-6. D. Wound healing assays showed the migration capacity of BLCA cells treated with OE-ITM2A lentivirus and IL-6. E. Transwell assays revealed the invasive capacity of BLCA cells treated with OE-ITM2A lentivirus and IL-6. Each experiment was repeated with three independent replicates.

Figure 6

ITM2A inhibited the growth of BLCA cells in vivo. A. To develop a xenograft tumor model, subcutaneous injections of UMUC-3 cells infected with OE-ITM2A were administered to BALB/c mice (n=5 for each group). The tumor weight and volume were measured. B. The levels of c-Myc, FN1, and ITM2A were measured by western blot. C. The levels of ITM2A, Ki67, c-Myc, and FN1 were measured by IHC. D. Schematic diagram used to explain the role of ITM2A in BLCA.