YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis (2021)
Figure 2

Figure 2. YAP/TAZ transcriptionally upregulate SLC7A11 expression.

(a) Combinatorial analysis of the genes up-regulated in Sorafenib-resistant cells and the genes down-regulated upon YAP/TAZ depletion uncovered 56 common genes, among which was SLC7A11.

(b) Quantitative RT-PCR analysis confirmed the dependency of SLC7A11 gene expression on YAP/TAZ. HLE cells were transfected with control siRNA (siCtrl) or siRNA against YAP/TAZ (siY/T) and cultured with DMSO or 6μM Sorafenib for 18 hours. RNA was extracted and analyzed by quantitative RT-PCR. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.

(c) SLC7A11 protein levels were upregulated by the exposure to Sorafenib, yet downregulated by siRNA-mediated depletion of YAP/TAZ. HLE cells were transfected with siCtrl or siY/T and cultured with DMSO or 6μM Sorafenib for 18 hours before harvest, followed by immunoblotting for YAP/TAZ and SLC7A1. GAPDH served as loading control. Results represent three independent experiments.

(d) siRNA-mediated ablation of YAP/TAZ significantly reduced SLC7A11 promoter activity, as determined by SLC7A11-promoter-luciferase reporter assay. HLE cells were transfected with SLC7A11-promoter firefly luciferase-reporter construct and a constitutive-active Renilla luciferase reporter construct (pRL-CMV) and with siCtrl or siY/T. Relative luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega E1980). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.

(e) A potential TEAD binding motif was predicted at - 400bp within the promoter region of SLC7A11. PCR primers SLC7A11-CH1 (P1) and SLC7A11-CH2 (P2) were designed to examine the potential binding of transcription factors to the TEAD binding motif by chromatin immunoprecipitation (ChIP).

(f) Binding of YAP and TAZ to a DNA fragment containing the TEAD binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for TEAD binding motif in the SLC7A11 promoter region shown in (e). The non-coding region NC10 served as negative control, and the bona fide TEAD target gene CYR61 served as positive control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.

(g) Knockdown of YAP/TAZ impairs cystine uptake either with or without Sorafenib treatment, and cystine uptake decreased with the exposure to Sorafenib. HLE-shLuc and HLE-shY/T cells were cultured with DMSO or 6μM Sorafenib for 18 hours, cystine-FITC was added to cells and after incubation at 37°C for 30 minutes intracellular Cystine-FITC levels were measured by flow cytometry using a 488 nm laser. Results represent three independent experiments.

(h) Colony formation assay showing that stable overexpression of SLC7A11 reversed Sorafenib-induced cell death in YAP/TAZ-deficient HCC cells. HLE-shLuc and HLE-shY/T were or were not transfected to overexpress SLC7A11 (SLC7A11 OE) and treated with DMSO or 6μM Sorafenib for 2 weeks as indicated. Results represent three independent experiments.

(i) Representative images of immuno-histochemical staining of SLC7A11 and YAP proteins in HCC samples from patients. Scale bars, 50μm.

(j) Quantification of the immunohistochemical stainings shown in (i) revealed a positive correlation of YAP and SLC7A11 expression (N=10). Statistical significance was calculated using Pearson correlation analysis.