Distinct Expression Profiles of p63 Variants during Urothelial Development and Bladder Cancer Progression

Abstract

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ⌬Np63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ⌬Np63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ⌬Np63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ⌬Np63 acting as an oncogene in certain invasive bladder tumors.

Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity

“…Network analysis revealed significant TP63 expression that was associated with a high level of expression of the oncogenic Np63 isoform. It is likely that squamous/basal MIBCs expressing this isoform represent the lethal subset of p63-expressing advanced bladder cancers that were reported by others 169 . The relevance of p63 expression in poor prognosis UroB tumours remains unresolved, but the expression of different p63 isoforms in NMIBC and MIBC may account for this 169 .…”

“…It is likely that squamous/basal MIBCs expressing this isoform represent the lethal subset of p63-expressing advanced bladder cancers that were reported by others 169 . The relevance of p63 expression in poor prognosis UroB tumours remains unresolved, but the expression of different p63 isoforms in NMIBC and MIBC may account for this 169 . It is currently unclear whether analyses that have assessed MIBC only have revealed all biologically relevant heterogeneity as some data suggest that transcriptional subtypes are independent of conventional grade and stage groupings 191 .…”

“…TP63 (which encodes p63) is expressed in the basal and intermediate cell layers of the normal urothelium. Studies of TP63 isoforms show that Np63 expression is linked to EMT in tumours 169 and this is associated with a more 'basal' phenotype with adverse prognosis [169][170][171] . Recent expression profiling of large numbers of MIBC defines a claudin-low basal subtype (discussed below) that is characterised by the enrichment of EMT markers and the expression of low levels of cytokeratins 37,172 .…”

p63-expressing cells are the stem cells of developing prostate, bladder, and colorectal epithelia

“…By contrast, transplants of p63 −/− urogenital sinus (UGS) revealed that luminal cells can form and regenerate in the absence of basal cells, hinting that the two cell types might represent independent cell lineages during development (12,16,25). Similarly, p63-deficient mouse urothelium contains umbrella-like cells in the absence of p63-positive basal/intermediate cells, suggesting that the cells are not related hierarchically (13,16,17). Because epithelial cell lineages in the developing bladder and prostate glands need to be further clarified, we generated knock-in mice expressing Cre recombinase (Cre) under control of the endogenous ΔNp63 promoter and performed a rigorous genetic lineage tracing analysis of ΔNp63-expressing cells in the developing caudal endoderm that gives rise to the prostate, bladder, and colorectal epithelia.…”

“…Heterozygous p63 mutations underlie various human syndromes of ectodermal dysplasia, orofacial clefting, and limb malformation (15), and p63 KO mice show defects in limb, craniofacial, and epithelial development. These mice lack all stratified epithelia and their derivatives (i.e., mammary, lachrymal, and salivary glands), die at birth from dehydration, and have markedly abnormal prostate and bladder epithelia (12,13,16,17). Specific KO mice for the TA and the ΔNp63 isoforms reveal that these anomalies result from ΔNp63 absence (18,19).…”