ORIGINAL ARTICLE PIR2/Rnf144B regulates epithelial homeostasis by mediating degradation of p21 WAF1 and p63 F Conforti 1 , Ai Li Yang 2 , M Cristina Piro 3 , M Mellone 1 , A Terrinoni 3 , E Candi 3 , P Tucci 2,3 , GJ Thomas 1 , RA Knight 2 , G Melino 2,3 and BS Sayan 1 DNp63 is a transcription factor that is critical for the development of stratified epithelia and is overexpressed or amplified in 480% of squamous cell carcinomas (SCCs). We identified the RING finger E3 ubiquitin ligase PIR2/Rnf144b as a direct transcriptional target of DNp63a and showed that its expression parallels that of DNp63a in keratinocytes, SCC cell lines and SCCs. We used primary keratinocytes as a model system to investigate the function of PIR2/Rnf144b in stratified epithelia. Depletion of PIR2/Rnf144b severely impaired keratinocyte proliferation and differentiation, associated with accumulation of p21 WAF1/CIP1 ; a known target of PIR2/Rnf144b. More importantly, we found that PIR2/Rnf144b binds and mediates proteasomal degradation of DNp63a, generating a hitherto unknown auto-regulatory feedback loop. These findings substantiate PIR2/Rnf144b as a potentially critical component of epithelial homeostasis, acting downstream of DNp63a to regulate cellular levels of p21 WAF1/CIP1 and DNp63a. Oncogene (2013) 32, 4758–4765; doi:10.1038/onc.2012.497; published online 5 November 2012 Keywords: PIR2; Rnf144B; p63; p21 WAF1 ; squamous cell carcinoma (SCC); skin INTRODUCTION Squamous cell carcinomas of the head and neck (HNSCCs) are among the most-frequent cancers worldwide, counting over 650 000 new cases each year. 1 A common molecular abnor- mality detected in these cancers is the genomic amplification and/or overexpression of the transcription factor DNp63a. p63 is a member of the p53 family of tumour suppressor genes 2,3 and similar to other family members is expressed as different isoforms. Transcription from two different promoters generates proteins with or without the N-terminal transactivation domain (TA and DN isoforms, respectively) and alternative splicing at the 3 0 terminus produces the a, b, g and d variants. 4 DNp63a is the predominant p63 isoform expressed in the basal layer of stratified epithelia, where mitotically active cells reside, while TAp63a is expressed mainly in oocytes. 5–7 The p63 À / À mouse models indicate an essential role for p63 in epithelial development with mice born lacking epidermis, displaying severe alterations in epithelial appendages and with profound defects in other stratified epithelia. 5,7 This phenotype is largely rescued following complementation of the mice with DNp63a but not TAp63a, further substantiating the critical role of DNp63a in the biology of stratified epithelia. 8 Unlike p53, p63 is rarely mutated in tumours; however, its deregulated expression is commonly observed in a variety of malignancies. While overexpression of TAp63 has been described, so far, in follicular lymphomas (54–99%) and diffuse large B-cell lymphomas (15–34%), 9 DNp63 overexpression is observed in a large range of epithelial malignancies, in particular the squamous cell carcinomas (SCCs) of the oesophagus, lung and head-and- neck. Among these, may be the most striking is the amplification and/or overexpression of the DNp63a isoform in 480% of HNSCCs. A number of studies have highlighted the oncogenic potential of the DNp63a isoform and its expression is associated with more aggressive, less differentiated tumours that are resistant to therapy. In spite of the established role of p63 to sustain proliferation and survival in HNSCC cells, 9–11 its biochemical regulation and functional contribution to HNSCC remains to be fully clarified. One of the main molecular pathways regulated by DNp63a to sustain proliferation involves inhibition of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 (p21). In normal stratified epithelia, p21 has an essential role in inducing cell-cycle withdrawal allowing for differentiation to proceed. When basal layer keratinocytes commit to differentiate, DNp63a is rapidly down- regulated allowing p21 to accumulate. This results in exit from the cell cycle and enables initiation of the differentiation programme. However, as differentiation proceeds, p21 levels are also down- regulated in a proteasome-dependent manner, as sustained p21 expression acts as an inhibitor of differentiation. 12 In tumours, loss of cell-cycle control via reduction of p21 expression is regarded as one of main drivers of cellular transformation. Many human cancers, including HNSCC, are associated with reduced p21 expression, which is not attributed to inactivating mutations, but rather to a complex molecular network that regulates its transcription, stability and cellular localization. 13 The epidermis has been a valuable tool to dissect the p63-regulated pathways in stratified epithelia. It is a multi-layered epithelium that protects the underlying tissues against physical, chemical and biological insults and acts as a waterproof barrier to prevent dehydration. It is a three-dimensional matrix of tightly adhering cells consisting of four distinct cell layers: basal layer, spinous layer, granular layer and stratum corneum. 14 Most cells at the basal layer are transit amplifying cells that are generated by asymmetric divisions of the keratinocyte stem cells. After limited 1 Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK; 2 Medical Research Council, Toxicology Unit Leicester University, Leicester, UK and 3 Biochemistry Laboratory, IDI-IRCCS, c/o Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy. Correspondence: Dr BS Sayan, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Somers Cancer Research Building, Tremona road, Southampton, MP824, SO16 6YD, UK. E-mail: b.s.sayan@soton.ac.uk Received 20 June 2012; revised 6 September 2012; accepted 13 September 2012; published online 5 November 2012 Oncogene (2013) 32, 4758–4765 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc