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TP63 P2 Promoter Functional Analysis Identifies Β-Catenin As Oncogene (2011) 30, 4656–4665 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE TP63 P2 promoter functional analysis identifies b-catenin as a key regulator of DNp63 expression C Ruptier1,2,3,10,11, A De Gaspe´ris1,2,3,10,SAnsieau1,2,3,AGranjon1,2,3,12,PTanie` re4,13, I Lafosse1,2,3,11, HShi4,14,APetitjean4,15, E Taranchon-Clermont4,16, V Tribollet1,2,3,17,TVoeltzel1,2,3, J-Y Scoazec1,2,3,5, V Maguer-Satta1,2,3, A Puisieux1,2,3,6,7, P Hainaut4, C Cavard8,9 and C Caron de Fromentel1,2,3 1Universite´ de Lyon, Lyon, France; 2INSERM U1052, Centre de Recherche en Cance´rologie de Lyon, Lyon, France; 3CNRS5286, Centre de Recherche en Cance´rologie de Lyon, Lyon, France; 4International Agency for Research on Cancer, Lyon, France; 5Hospices Civils de Lyon, Hoˆpital E. Herriot, Service central d’Anatomie et de Cytologie Pathologiques, Lyon, France; 6Centre Le´on Be´rard, Lyon, France; 7Institut Universitaire de France, Paris, France; 8Institut Cochin, De´partement Endocrinologie Me´tabolisme et Cancer, Universite´ Paris Descartes, CNRS (UMR 8104), Paris, France and 9INSERM U567, Paris, France The DNp63 protein, a product of the TP63 gene that lacks Introduction the N-terminal domain, has a critical role in the maintenance of self renewal and progenitor capacity in p53 is the founding member of a family of proteins that several types of epithelial tissues. DNp63 is frequently also comprises p63 and p73. All three proteins are overexpressed in squamous cell carcinoma (SCC) and in transcription factors that control partially overlapping some other epithelial tumours. This overexpression may and interconnected genomic programs, such as cell-cycle contribute to tumour progression through dominant- arrest or apoptosis (Moll and Slade, 2004). These negative effects on the transcriptionally active (TA) proteins are expressed as multiple isoforms due to isoforms of the p53 family (TAp63, TAp73 and p53), as alternative splicing and promoter usage (Yang et al., well as through independent mechanisms. However, the 1998; Yang and McKeon, 2000; Marcel and Hainaut, molecular basis of DNp63 overexpression is not fully 2009). Most of the isoforms possess a central DNA- understood. Here, we show that the expression of DNp63 binding domain, but differ in their N- and C-terminal is regulated by the Wnt/b-catenin pathway in human regions. The N-terminal region contains a transactiva- hepatocellular carcinoma (HCC) and SCC cell lines. This tion domain present in the so-called transcriptionally regulation operates in particular through TCF/LEF sites active (TA) isoforms, but absent in the truncated (DN) present in the P2 promoter of TP63. In addition, we show ones. In the case of TP63, TA isoforms are produced that DNp63 and b-catenin are frequently coexpressed and from P1 promoter, whereas P2 promoter, located in accumulated in oesophageal SCC, but not in HCC. These intron 3, generates DN isoforms (Yang et al., 1998) results suggest that activation of the b-catenin pathway (Figure 1). By competing with TA isoforms for the may contribute to overexpression of DNp63 during binding to their responsive elements (REs), DN isoforms tumour progression, in a cell type-specific manner. prevent target-gene activation, thus behaving as trans- Oncogene (2011) 30, 4656–4665; doi:10.1038/onc.2011.171; dominant-negative isoforms (Yang et al., 1998; Petitjean published online 6 June 2011 et al., 2008). Given the similarities in DNA-binding properties between p63, p73 and p53, non-physiological Keywords: b-catenin; p53 family; promoter; transcrip- upregulation of DNp63 or DNp73 expression may tional regulation; tumour progression contribute to the functional inactivation of p53, thereby promoting tumourigenesis in the absence of mutation in p53. DNp63 or DNp73 upregulation can also turn down Correspondence: Dr C Caron de Fromentel, UMR INSERM U1052 TAp63 and TAp73 activity, favouring tumour progres- CNRS5286, 151 cours Albert Thomas, 69008 Lyon, France. sion independently of p53 inactivation. The notion that E-mail: [email protected] the three factors operate through partially independent 10These authors contributed equally to this work. mechanisms is supported by the cooperative effects of 11Current address: Genoway, 181 avenue Jean Jaure` s, 69007 Lyon, France. 12Current address: CNRS UMR5123 Physiologie Inte´grative Cellulaire TP63, TP73 and TP53 gene inactivation on tumour et Mole´culaire, 43 bd du 11 novembre 1918, 69622 Villeurbanne formation in transgenic mice (Flores et al., 2002; Flores Cedex, France. et al., 2005). Thus, the transcriptional activity of each 13Current address: Department of Cellular Pathology, The Medical p53 family member may depend upon a delicate balance School, University of Birmingham, Edgbaston, Birmingham B115 between TA and DN isoforms. 2TT, UK. 14Current address: Knobelstrasse 3, 5611 Anglikon, Switzerland. Despite their structural and functional similari- 15Current address: Re´sidence Roche Arnaud, 27 rue Emile Reynaud, ties, TP53, TP63 and TP73 present different genetic 43000 Le Puy en Velay, France. alterations in human cancer. Although p53 is frequently 16Current address: CEPH, 27 rue Juliette Dodu, 75010 Paris, France. inactivated by mutation and loss of heterozygosity in 17Current address: CNRS UMR5242- IGFL, ENS Lyon, 46 alle´e d’Italie, 69364 Lyon cedex 07, France. many cancer types, mutations in TP73 and TP63 Received 31 July 2010; revised and accepted 8 April 2011; published are rare. In contrast, deregulated expression of TP63, online 6 June 2011 resulting in an increased DNp63/TAp63 ratio, is common b-catenin-DNp63 axis in tumour progression C Ruptier et al 4657 γ P1 P2 Here, we demonstrate that the longest DN p63 TP63 gene α isoform (DNp63a) and b-catenin bind to and modulate β the TP63 P2 promoter activity. Moreover, we show STAT3 TBE 2 SP1 that b-catenin-mediated P2 activation implies multiple (-678/-659) (-347/-340) (-55/-49) +1 non-canonical TBEs. Finally, in line with these results, P2 we describe that DNp63 overexpression correlates with (-2 928) TBE p53RE CAAT boxes TATA (+139) (-2 083/-2 074) (-615/-593) (-164/-159) box b-catenin delocalization in oesophageal SCC. These (-112/-107) (-27/-23) results provide novel insights into the mechanism by (-78/-73) which TP63 expression is altered in tumours. Figure 1 Structure of the P2 promoter. Schematic representation of TP63 gene organization and of the P2 promoter REs reported in the literature or identified by our in silico analysis. TP63 gene, white, UTR; black, exon 30 coding region; dotted light grey, DNA binding domain; Results hatched, oligomerization domain; dotted dark grey, SAM domain; light grey, post SAM; a, b, g, C-terminal alternative splicing products. Structure of P2 promoter The putative transcription factor-binding sites present in a 3000 bp region of P2 promoter were identified by using in epithelial tumours, including all forms of squamous the Matinspector (http://www.genomatix.de) software. cell carcinomas (SCCs) and some forms of adeno- Figure 1 shows the structure of the promoter and the carcinoma (Moll and Slade, 2004). In these tissues, the position of putative RE (detailed in Supplementary expression of TP63 is a hallmark of normal proliferating Figure 1). Several regulatory elements located within the cells of the basal or myoepithelial layers. In stratified TP63 P2 promoter, including a p53RE (Lanza et al., epithelium, p63 is essential for the self renewal of 2006), a TATA box, three CAAT boxes and a SP1/SP3 progenitors, as well as their rate of progression towards et al. differentiation (Yang et al., 1999). Thus, in tumours, the site (Romano , 2006), were confirmed in our in silico analysis. However, we failed to identify the increased DN/TAp63 ratio is likely to provide growth STAT3 RE previously reported (Chu et al.,2008).Onthe and survival advantages by maintaining cells in a proli- ferative status that retains some of the characteristics of other hand, we identified two putative TBEs and four Pitx2-binding sites (PBSs). The presence of p53RE and progenitors (Senoo et al., 2007; Wu et al., 2005; Rocco TBEs/PBSs suggests that expression of DNp63 may be et al., 2006). However, the molecular mechanisms that controlled through two mechanisms of particular rele- lead to DNp63 overexpression are only partially charac- vance in carcinogenesis, one involving auto- and hetero- terized. DNp63 overexpression has been associated with regulatory loops by the p53 family members and the 3q27–29 amplification, a region encompassing the TP63 other involving the Wnt/b-catenin pathway and TCF/ gene, in about 20–25% of SCC, but it is also observed in cancers without such an amplification, suggesting that LEF factors. On the basis of these considerations, we set out to assess the functionality of TBEs and of the p53RE. other mechanisms may operate to upregulate this gene in a pathological context (Taniere et al., 2001). In the present study, we carried out a survey of Regulation of P2 promoter by DNp63a protein itself TP63 P2 promoter in search of candidate factors for We previously reported that DNp63 and p53 expression transcriptional regulation of DNp63. In silico analysis are inversely correlated in hepatocytes and hepatocel- revealed the presence of a potential p53RE, previously lular carcinoma (HCC) cell lines (Petitjean et al., 2005), identified by others (Waltermann et al., 2003; Lanza suggesting that p53 might repress DNp63. In fact, et al., 2006), and two TCF/LEF-binding elements repression of the P2 promoter by p53 has further been (TBEs). TCF/LEF transcription factors are activated confirmed in keratinocytes (Lanza et al., 2006). As through a signalling cascade involving b-catenin, a DNp63 and p53 share similar DNA-binding specificities key protein of the Wnt/b-catenin pathway frequently (Yang et al., 1998), we investigated the possibility that deregulated in many epithelial cancers, including SCC DNp63a regulates its own promoter.
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