TFAP2C drives cisplatin resistance in bladder cancer by upregulating YAP and activating β-catenin signaling

Abstract

Cisplatin-based chemotherapy is a conventional therapy for muscle-invasive bladder cancer (BC); however, its efficacy is often limited by the emergence of resistance to cisplatin. Yes-associated protein (YAP) and β-catenin are involved in this resistance, yet their upstream regulators are not well defined. This study investigates the role of TFAP2C in regulating YAP expression and its impact on cisplatin resistance in BC. The Cancer Genome Atlas (TCGA) gene expression data and GSE231835 dataset were analyzed to identify potential transcription factors regulating YAP. Assessed TFAP2C and YAP expression in clinical samples and cell lines. Functional assays were performed following TFAP2C knockdown. Dual-luciferase reporter assays and Chromatin immunoprecipitation (ChIP) confirmed TFAP2C binding to the YAP promoter. A mouse model evaluated the effects of TFAP2C silencing on tumor growth and cisplatin resistance. The results showed that TFAP2C was identified as an upstream activator of YAP, with elevated expression in cisplatin-resistant BC cell lines and positive correlation with YAP expression. Silencing TFAP2C reduced malignant behaviors, decreased YAP, phosphorylated YAP (p-YAP) and β-catenin levels, and increased apoptosis in both cisplatin-sensitive and cisplatin-resistant BC cells. Besides, TFAP2C directly binds to the YAP promoter, enhancing its transcription. In the xenograft model, TFAP2C silencing significantly inhibited tumor growth and reduced cisplatin resistance. TFAP2C promotes cisplatin resistance and malignant behavior in BC by upregulating YAP and activating the β-catenin signaling pathway. Targeting TFAP2C offers a novel therapeutic strategy to overcome cisplatin resistance in BC, representing a new discovery in combating chemoresistance.

Keywords: YAP; bladder cancer; cisplatin resistance; transcription regulation; β-catenin.

Figure 1

Expression and survival analysis of YAP in BC. A, expression of YAP in TCGA-BLCA in normal and BC tissues. B, expression of YAP in TCGA-BLCA in low and high grade BC tissues. OS (C) and DFS (D) of YAP in TCGA-BLCA, using median as cutoff. OS (E) and DFS (F) of YAP in TCGA-BLCA, using quartiles as cutoffs.

Figure 2

Differential gene analysis of the BC resistance dataset GSE231835. Volcano plot (A) and MA plot (B) showing differentially expressed genes in the GSE231835 dataset. C, Expression of YAP in the GSE231835 dataset. Differential genes were screened using an absolute value of p < 0.05 and | log2FC | >1 as the threshold.

Figure 3

Identification of YAP-related upstream TFs.A, upset plot showing identification of potential TFs for YAP in the integrated KnockTF database and GSE231835 dataset. B, FOXA1 expression in the GSE231835 dataset. C, TFAP2C expression in the GSE231835 dataset. D, Pearson correlation analysis of FOXA1 with YAP in the TCGA-BLCA dataset. E, Pearson correlation analysis of TFAP2C with YAP in the TCGA-BLCA dataset.

Figure 4

Expression and prognostic analysis of FOXA1 and TFAP2C in TCGA-BLCA.A, FOXA1 expression in TCGA-BLCA samples (Red color represented the tumor samples, Black color represented the normal samples). OS (B) and DFS (C) of FOXA1 in TCGA-BLCA. D, TFAP2C expression in TCGA-BLCA samples (Red color represented the Tumor samples, Black color represented the Normal samples). OS (E) and DFS (F) of TFAP2C in TCGA-BLCA.

Figure 5

TFAP2C Promotes Malignant Behavior and Stemness in BC.A, mRNA levels of TFAP2C and YAP in 32 BC patients' cancer tissue samples and their adjacent normal tissue samples.(n = 32). B, Protein levels of YAP and p-YAP expression in 32 BC patients' cancer tissue samples and their adjacent normal tissue samples. C, mRNA and (D) protein expression of TFAP2C in immortalized normal human bladder epithelial cell line SV-HUC-1 and eight bladder cancer cell lines (T24, 5637, J82, RT4, UM-UC-3, SW780, HT-1376, TCCSUP). E, Confirmation of TFAP2C knockdown in UM-UC-3 and T24 cells by qRT-PCR. F, sphere formation assay (scale bar = 100 μm) and (G) apoptosis assay in UM-UC-3 and T24 cells after TFAP2C knockdown. H, quantitative analysis of apoptosis rates. I, CCK-8 assay and (J) transwell invasion assay in TFAP2C-silenced UM-UC-3 and T24 cells. (scale bar = 100 μm), (K) Protein levels of YAP, p-YAP, and β-catenin in UM-UC-3 and T24 cells after TFAP2C knockdown. N = 3 biologically independentsamples. “ns” indicates non-significant, ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 6

TFAP2C Promotes Stemness in BC Cells by Regulating YAP and β-Catenin Pathways.A, relative mRNA levels of TFAP2C and YAP in UM-UC-3-R and T24-R cells. B, Protein levels of YAP and p-YAP in UM-UC-3-R and T24-R cells. C, efficiency of TFAP2C knockdown in UM-UC-3-R and T24-R cells confirmed by qRT-PCR and Western blots. D, sphere formation assay (scale bar = 100 μm), (E) apoptosis assay, (F) CCK-8 assay in TFAP2C-silenced UM-UC-3-R and T24-R cells. G, IC₅₀ of cisplatin in TFAP2C-silenced UM-UC-3-R and T24-R cells compared to the parental cells. H, protein levels of YAP, p-YAP, and β-catenin in TFAP2C-silenced UM-UC-3-R and T24-R cells. I, ChIP-PCR analysis of TFAP2C binding to the YAP promoter region and a distal region (∼1–2 kb upstream of the transcription start site) in UM-UC-3-R and T24-R cells. Input and IgG were used as controls. J, Luciferase activity of the YAP promoter in UM-UC-3-R and T24-R cells co-transfected with YAP promoter-luciferase reporter and either empty vector (blue) or TFAP2C expression vector (red). K, schematic of YAP promoter deletion constructs (P-Δ1–P-Δ5) based on the −2000 to 0 bp region upstream of the YAP coding sequence. L, Luciferase activity of each construct co-transfected with TFAP2C in 293T cells. n = 3 biologically independentsamples. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 7

TFAP2C drives cisplatin resistance and cancer stemness by binds to the YAP promoter.A and B, efficiency of TFAP2C overexpression in UM-UC-3 and T24 cells confirmed by qRT-PCR and Western blots. C, sphere formation assay (scale bar = 100 μm), (D) apoptosis assay, (E and F) CCK-8 assay in TFAP2C-overexpressed UM-UC-3 and T24 cells. G and H, transwell invasion assay in TFAP2C-overexpressed UM-UC-3 and T24 cells. (scale bar = 100 μm). I and J, IC50 of cisplatin in TFAP2C-overexpressed UM-UC-3-R and T24-R cells. ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 8

Effect of TFAP2C on tumor growth and cisplatin resistance in BC xenograft model. A, representative images of tumors excised from each group of mice. B, tumor growth curves showing mean tumor volume (mm³) over time for each group (n = 10 per group). C, final tumor weights measured at the endpoint. D, protein expression levels of YAP, p-YAP, and β-catenin in tumor tissues from each group. E, mRNA expression levels of YAP and TFAP2C in tumor tissues from each group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.