Hippo pathway effectors are associated with glioma patient survival, control cell proliferation and sterol metabolism through TEAD3

Abstract

Glioblastomas represent the most common and lethal primary brain tumors in the world. Despite therapeutic advances during the last two decades, patient prognosis remains very poor. The Hippo signaling pathway effectors YAP/TAZ-TEADs play a crucial role in tumor progression and represent promising therapeutic targets in gliomas. In this study, we identified and investigated the clinical and biological significance of TEAD transcription factors. Through comprehensive analyses of TCGA glioma data and patient samples, we identified TEAD3-4 transcription factors as robust prognostic markers of patient outcome. Using up to five different patient-derived glioblastoma stem cell cultures, we confirmed the preferential expression and activation of TEAD3-4 along with their transcriptional coactivators YAP/TAZ. Pharmacological inhibition of YAP/TAZ-TEAD interaction by Verteporfin significantly decreased tumor cell growth, whereas specific inhibition of TEAD3 did not impact cell proliferation but affected sterol/cholesterol biosynthetic and metabolic processes. This study contributes to a better understanding of the role of Hippo effectors in glioblastoma pathophysiology. These transcription factors, particularly TEAD3, could potentially serve as therapeutic targets, especially considering recent data on cholesterol homeostasis in glioblastomas.

Keywords: TEAD3; glioblastomas; gliomas; hippo pathway; sterol metabolism.

FIGURE 1

The Hippo pathway transcription factor TEADs are associated with glioma aggressiveness in TCGA cohort. (A) Heatmap depicting transcription factors and associated co‐factors upregulated and downregulated between glioma molecular subtypes. (B) Dot plots of enriched gene ontology terms (biological processes) (B) and (C) KEGG (KEGG: Kyoto Encyclopedia of Genes and Genomes) pathways of differentially expressed genes. (D) Chord plot of the top enriched KEGG pathways illustrating the relationship between the differentially expressed genes and their pathway terms. (E) Violin plots of TEAD expression based on TCGA RNASeq data (RSEM log2) between glioma molecular subtypes (Kruskal‐Wallis, *p < 0.05, **p < 0.01, ***p < 0.001). (F) Kaplan–Meier survival curves showing overall survival (OS) or progression‐free survival (PFS) according to TEAD low/high expression groups in the TCGA glioma cohort (Mantel–Cox log‐rank test; p‐values are indicated on the graphs). (G) Forest plot of univariate Cox regression analysis for TEAD expression in TCGA glioma cohort. Hazard ratio (HR) with 95% confidence interval (CI) and p‐values are indicated on the graph.

FIGURE 2

TEAD transcription factor expression and subcellular location are associated with patient prognosis. (A) Representative images of TEAD1‐4 expression according to glioma molecular subtypes (scale bar for tissue patch = 100 μm; for high magnification = 10 μm). (B) Histograms representing TEAD expression (mean ± SD) or (C) their subcellular locations (frequency of cytoplasmic/nuclear TEADs) in molecular subtypes (Kruskal–Wallis test and Chi‐squared test; *p < 0.05, **p < 0.01, **p < 0.001). (D) Kaplan–Meier survival curves plotting overall survival or progression‐free survival according to TEAD expression and localization groups (Mantel–Cox log‐rank test; p‐values are indicated on the graphs).

FIGURE 3

YAP/TAZ‐TEAD complex activation regulates proliferation in glioblastoma patient‐derived cell cultures. (A) Western Blot analysis of Hippo pathway core proteins in five different GSCs. (B) Subcellular location analysis of the Hippo pathway effectors by Western Blot after cell fractionation and (C) immunofluorescence in GSCs. (D) Dose–response assessment of verteporfin after 7 days of treatment in three GSC cultures (N = 3). (E) Western blot analysis of YAP target gene inhibition (AXL, CTGF, Survivin) after 24 h with 1 μM of verteporfin. (F) GSC‐viability after 7 days of 1 μM verteporfin treatment using MTS assay (N = 4; Mann–Whitney test; *p < 0.05).

FIGURE 4

TEAD3 inhibition affects sterol metabolism without impacting cell proliferation. (A) Immunofluorescence analysis of TEAD3 transcription factor (green color) and YAP transcriptional coactivator (red color) subcellular location. Nuclei are stained with DAPI (blue color). (B) Western blot analysis of YAP and TEAD3 after immunoprecipitation of flagged YAP with or without TEAD3 overexpression. The shift of TEAD3 molecular weight was due to the overexpression of GAL4‐TEAD3 fusion protein. (C) qRT‐PCR analysis of TEAD1‐4 expression 24 h after TEAD3 silencing by two different siRNA (siRNA1, 2) compared to non‐silencing siRNA (NS). (D) Growth curves of GSCs after TEAD3 siRNA transfection compared to NS control. (E) Volcano plot of differentially expressed genes after TEAD3 inhibition in GSC‐2, GSC‐6 and GSC‐11. The downregulated and upregulated genes are shown in blue and red respectively. (F) Dot plots of enriched KEGG pathways and (G) gene ontology terms (biological processes) of differentially expressed genes. (H) CNET plot of upregulated and downregulated genes among biological processes affected by TEAD3 inhibition.