THE SPLICING FACTOR PTBP1 REPRESSES TP63 γ ISOFORM PRODUCTION IN SQUAMOUS CELL CARCINOMA

Abstract

ABSTRACTThe TP63 gene encodes the transcription factor p63. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, β, γ and δ. The regulatory functions of p63 may be isoform-specific. The α isoform inhibits the epithelial to mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Here, we observed in TCGA data that a high ratio of the TP63γ isoform to the other isoforms is a pejorative factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC). We therefore addressed the regulation of the γ isoform. In several tissues (GTEX data), the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines leads to an increase in abundance of the γ isoform. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. The region around the TP63γ-specific exon was sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Finally, we demonstrated that the regulation of TP63γ production by PTBP1 is conserved in amphibians, revealing that it encounters a strong evolutionary pressure. Together, these results identify TP63γ as a prognostic marker in HNSCC, and identify PTBP1 as a direct negative regulator of its production.