The p53 family member p63 exists as two major protein variants (TAp63 and ΔNp63) with distinct expression patterns and functional properties. Whilst downstream target genes of p63 have been studied intensively, how p63 variants are themselves controlled has been relatively neglected. Here, we review advances in understanding ΔNp63 and TAp63 regulation, highlighting their distinct pathways. TAp63 has roles in senescence and metabolism, and in germ cell genome maintenance, where it is activated post‐transcriptionally by phosphorylation cascades after DNA damage. The function and regulation of TAp63 in mesenchymal and haematopoietic cells is less clear but may involve epigenetic control through DNA methylation. ΔNp63 functions to maintain stem/progenitor cells in various epithelia and is overexpressed in squamous and certain other cancers. ΔNp63 is transcriptionally regulated through multiple enhancers in concert with chromatin modifying proteins. Many signalling pathways including growth factors, morphogens, inflammation, and the extracellular matrix influence ΔNp63 levels, with inconsistent results reported. There is also evidence for reciprocal regulation, including ΔNp63 activating its own transcription. ΔNp63 is downregulated during cell differentiation through transcriptional regulation, while post‐transcriptional events cause proteasomal degradation. Throughout the review, we identify knowledge gaps and highlight discordances, providing potential explanations including cell‐context and cell–matrix interactions. Identifying individual p63 variants has roles in differential diagnosis and prognosis, and understanding their regulation suggests clinically approved agents for targeting p63 that may be useful combination therapies for selected cancer patients. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background ΔNp63 overexpression is a common event in squamous cell carcinoma (SCC) that contributes to tumorigenesis, making ΔNp63 a potential target for therapy. Methods We created inducible TP63-shRNA cells to study the effects of p63-depletion in SCC cell lines and non-malignant HaCaT keratinocytes. DNA damaging agents, growth factors, signaling pathway inhibitors, histone deacetylase inhibitors, and metabolism-modifying drugs were also investigated for their ability to influence ΔNp63 protein and mRNA levels. Results HaCaT keratinocytes, FaDu and SCC-25 cells express high levels of ΔNp63. HaCaT and FaDu inducible TP63-shRNA cells showed reduced proliferation after p63 depletion, with greater effects on FaDu than HaCaT cells, compatible with oncogene addiction in SCC. Genotoxic insults and histone deacetylase inhibitors variably reduced ΔNp63 levels in keratinocytes and SCC cells. Growth factors that regulate proliferation/survival of squamous cells (IGF-1, EGF, amphiregulin, KGF, and HGF) and PI3K, mTOR, MAPK/ERK or EGFR inhibitors showed lesser and inconsistent effects, with dual inhibition of PI3K and mTOR or EGFR inhibition selectively reducing ΔNp63 levels in HaCaT cells. In contrast, the antihyperlipidemic drug lovastatin selectively increased ΔNp63 in HaCaT cells. Conclusions These data confirm that ΔNp63-positive SCC cells require p63 for continued growth and provide proof of concept that p63 reduction is a therapeutic option for these tumors. Investigations of ΔNp63 regulation identified agent-specific and cell-specific pathways. In particular, dual inhibition of the PI3K and mTOR pathways reduced ΔNp63 more effectively than single pathway inhibition, and broad-spectrum histone deacetylase inhibitors showed a time-dependent biphasic response, with high level downregulation at the transcriptional level within 24 h. In addition to furthering our understanding of ΔNp63 regulation in squamous cells, these data identify novel drug combinations that may be useful for p63-based therapy of SCC.