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p73 is over-expressed in vulval cancer principally as the Δ2 isoform

p73 is over-expressed in vulval cancer principally as the Δ2 isoform British Journal of Cancer(2001) 85(10), 1551–1556 © 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.2138, available online at http://www.idealibrary.com on http://www.bjcancer.com p73 is over-expressed in vulval cancer principally as the ∆2 isoform 1 1 1 2 2 3 1 4 5 1 J O’Nions, LA Brooks, A Sullivan, A Bell, B Dunne, M Rozycka, A Reddy, JA Tidy, D Evans, PJ Farrell, 6 7 2 1 A Evans, M Gasco, B Gustersonand T Crook 1 2 Ludwig Institute for Cancer Research, St Mary’s Hospital Medical School, Norfolk Place, London W2 1PG; University Department of Pathology, Glasgow University, Western Infirmary, Glasgow; Department of Molecular Medicine, King’s College School of Medicine and Dentistry, Coldharbour Lane, London SE5; 4 5 Department of Gynaecological Oncology, University of Sheffield, Northern General Hospital, Sheffield, S5 7AU; Department of Histopathology, St Mary’s 6 7 Hospital Medical School, Norfolk Place, London W2; Department of Surgery, Poole Hospitals NHS Trust, Poole, Dorset; UO Oncologia Medica, Azienda Ospedaliera S. Croce e Carle, Via Coppino 26, 12100, Cuneo, Italy Summary p73 was studied in squamous cancers and precursor lesions of the vulva. Over-expression of p73 occurred commonly in both human papillomavirus (HPV)-positive and -negative squamous cell cancers (SCC) and high-grade premalignant lesions. Whereas expr ession in normal vulval epithelium was detected only in the basal and supra-basal layers, expression in neoplastic epithelium increased with grade of neoplasia, being maximal at both protein and RNA levels in SCC. p73 ∆2 was the principal over-expressed isoform in the majority of cases of vulval SCC and often the sole form expressed in SCC. Over-expression of p73 was associated with expression of HPV-encoded E7 or with INK4a ARF hypermethylation or mutation of p16 in HPV-negative cases. There was a close correlation between expression of p73 and p14 in cancers with loss of p53 function. The frequent over-expression of p73 ∆2 in neoplastic but not normal vulval epithelium, and its co-ordinate deregulation with other E2F-1 responsive genes suggests a role in the oncogenic process. © 2001 Cancer Research Campaign http://www.bjcancer.com Keywords: vulval cancer; HPV; p73 Although vulval SCC is less common than cervical cancer, it is of a number of isoforms which arise by alternative splicing of exons major mechanistic interest since cancers are either HPV positive encoding the –COOH terminus of the protein and which exhibit (principally HPV 16) or lack detectable HPV DNA sequences. differences in trans-activating and growth suppressor functions HPV-positive cancers have an association with vulval intraepithe- (De Laurenzi et al, 1998; Ueda et al, 1999). The expression of a lial neoplasia (VIN) (reviewed by Crum, 1992). Clear pathobio- further spliced variant of p73 which lacks exon 2 (p73 ∆2) has logical differences between HPV-positive and HPV-negative been described in ovarian cancer (Ng et al, 2000) and in breast cancers have been described (Crum, 1992). Allelotype analysis cancer cell lines (Fillippovich et al, 2001). Recently, p73 has been has revealed that no significant differences in sites of loss of shown to be directly induced by E2F-1 and thereby to contribute to heterozygosity (LOH) exist between the 2 forms of the disease E2F-1-mediated apoptosis (Irwin et al, 2000; Lissy et al, 2000). (Pinto et al, 1999). However, a number of studies have revealed Furthermore, both myc and adenovirus E1A can activate expres- that mutation in p53 is more common in HPV-negative cancers sion of p73 (Zaika et al, 2000). (Lee et al, 1994; Marin et al, 2000; Brooks et al, 2001). Mechan- Although p73 is subject to methylation-dependent transcriptional istically, it is hypothesised that mutation in p53 functionally silencing in some B-cell malignancies, consistent with a role as a compensates for the absence of HPV 16E6, since this protein putative tumour-suppressor protein (Corn et al, 1999), mutational mediates inactivation of p53 via promotion of ubiquitin-dependent analyses have suggested that neither p63 nor p73 is frequently proteolysis. Despite the more common mutation of p53 in HPV- mutated in human cancers (Yoshikawa et al, 1999). Furthermore, negative cases, a substantial number of vulval SCC occur which p73 is over-expressed in some cancers, although the mechanism of lack both mutation and HPV. The mechanism, if any, by which p53 this is unknown (Chi et al, 1999; Zaika et al, 1999). Assessment of function may be compromised in such cases is not known. the biological significance of the ability of p73 ∆2 to transdomi- p73 has structural and functional homologies to p53, including nantly inhibit both p53 and full-length p73 clearly requires analysis sequence-specific DNA binding and transactivation functions of the expression of this variant in a range of both normal and malig- (Kaghad et al, 1997). Over-expression of p73 is able to induce nant tissues (Fillippovich et al, 2001). In the current study we have apoptosis in some human cancer lines (Jost et al, 1997) and investigated the structure and expression of p73 in vulval neoplasia. expression of p73 has been shown to have a role in the differentia- tion of keratinocytes (De Laurenzi et al, 2000). p73 is expressed as MATERIALS AND METHODS Received 17 May 2001 Tissues Revised 23 July 2001 Accepted 7 August 2001 SCC of the vulva and VIN III tissue samples were collected at Correspondence to:T Crook operation. In each case the diagnosis was confirmed by routine 1551 1552 J O’Nions et al histopathological analysis of resected tissue. Tissues were collected Mutu was used as a positive control. Analysis of p73 coding immediately into liquid nitrogen and stored until isolation of nucleic sequences in the regions corresponding to the mutational hot spots acids. Genomic DNA was isolated by proteinase K digestion and of p53 was done by RT-PCR SSCP using conditions described by total RNA by RNAzol B. Cancers and VIN III were HPV-typed Kawano et al (1999). using standard PCR methodology. Paraffin sections of vulval neoplasia were retrieved from the Department of Histopathology at RESULTS St Mary’s Hospital, Paddington, London. Wild-type p73 is frequently over-expressed in vulval Analysis of gene expression neoplasia cDNA was synthesised with the ProStar system (Stratagene) from The expression of p73 was investigated in a series of vulval SCC and 3 µg of total RNA. Analysis of expression was performed by RT- in VIN, previously analysed for the presence of HPV DNA ARF PCR as described previously for p14 (Gazzeri et al, 1998), and sequences. In initial experiments, expression was analysed using RT- p73 (De Laurenzi et al, 1998). For semi-quantitative analysis of PCR methodology (De Laurenzi et al, 1998). This assay allows gene expression, PCR was performed using the primers and discrimination between alternatively spliced forms encoding thermal cycling conditions described and was for 22 cycles for different –COOH variants of p73 protein. Expression of p73 mRNA ARF p14 , and 28 cycles for p73. In some experiments, amplification was detectable by RT-PCR in each of the 36 normal vulval epithelial was extended to 40 cycles to analyse expression in tissues samples available for analysis using 40 cycles of amplification. expressing a lower level of p73. Following PCR, reactions were Expression was predominantly of α and γ variants in each of the resolved on agarose gels, transferred to Hybond-N nylon and normal vulval samples analysed. In cancers, expression was also hybridised with P γ-ATP-labelled oligonucleotide probes specific restricted to the α and γ forms, but using limiting PCR conditions, for the amplified fragments. Analysis of N-terminal splice variants was markedly increased relative to matched normal vulval epithe- of p73 was performed using the primers described by Ng et al lium in 29/36 cases examined (Figure 1). Interestingly, over- (2000). Identity of these was confirmed by cloning and sequencing expression of p73 mRNA was detected in cancers both positive and and by hybridisation analysis of amplified products with oligo- negative for HPV DNA (Table 1A). To confirm that the elevated p73 nucleotide probes specific for exon 2 and exon 3 of p73. The pres- mRNA levels were reflected in protein expression and to investigate ence of equivalent amounts of cDNA in each PCR was verified by possible associations between p73 expression and grade of neoplasia, amplification of β-actin under similar limiting conditions. RT-PCR we performed immunocytochemical analysis of tissue sections of INK4a analysis of p16 was performed as described previously VIN and SCC cut from paraffin blocks. These studies revealed that (Gonzalez-Zulueta et al, 1995). expression of p73 was detectable in each case of normal epithelium, consistent with RT-PCR analysis performed under extended cycling conditions (Figure 2). Expression was restricted to the basal and Immunocytochemistry supra-basal layers in normal tissue, but this restriction was lost with 5 µm sections were cut from formalin-fixed, paraffin-embedded, increasing grade (Figure 2). To further demonstrate the increase in tissue sections. The diagnosis in each case was confirmed by expression with grade of neoplasia, expression was scored using a examination of haematoxylin and eosin-stained sections. For semi-quantitative immunocytochemical technique in VIN. A mean immunocytochemistry, sections were pressure-cooked in citrate expression index was calculated for each of VIN I, VIN II and VIN ARF buffer, then stained with antibodies: p14 goat polyclonal ((C20) III. This clearly demonstrated the increase in expression from VIN I Santa Cruz, SC-8613) was used at 1/100 dilution; p73 mouse to VIN III (Table 1B). The presence of mutations in the over- monoclonal antibody Neomarkers, MS-764-P0, affinity-purified expressed p73 mRNA was sought using RT-SSCP (Kawano et al, and diluted 1/150. Sections were scored independently by at least 1999). No mobility shifts suggestive of mutation were detected in 24 2 pathologists. To demonstrate increase in p73 expression with vulval SCC analysed. increasing grade of neoplasia, sections were scored according to the following scheme. 1: basal staining only; 2: suprabasal staining p73 ∆2 is the predominant over-expressed form of p73 where less than 50% of nuclei were positive in the strongest staining area in a high power field; 3: suprabasal staining where The high frequency of over-expression of p73α and γ in vulval greater than 50% of nuclei were positive in the strongest stained SCC, in the absence of mutations, was unexpected in view of the area in a high-power field. pro-apoptotic and negative growth-regulatory functions of these proteins. We therefore performed additional RT-PCR analysis to examine the N-termini of the mRNA species. There was over- Analysis of gene structure expression of p73 ∆2 in the majority of vulval SCC shown to Mutations in exon 1α and 2 of the INK4 locus were sought using over-express p73α (Figure 1 and Table 1A). Full-length p73 was single-strand conformation polymorphism analysis (SSCP) with only rarely simultaneously over-expressed, p73 ∆2 being the primers and PCR conditions described by Zhang et al (1994). PCR only form expressed in the majority of cases (Figure 1 and reactions were resolved on 6% native acrylamide gels with and Table 1A). without 5% glycerol. Methylation of CpG sequences in the INK4a p16 promoter was performed using methylation-specific PCR INK4a Inactivation of p16 correlates inversely with the (MSP) as described by Herman et al (1996). To examine exon 1β presence of HPV for mutations, SSCP was performed using cDNA as the substrate and resolution on 6% gels as described above (Gazzeri et al, The ability of HPV E7 to deregulate p73 expression via E2F-1 1998). cDNA prepared from the Burkitt’s lymphoma cell line and pRb provides a mechanistic explanation for over-expression British Journal of Cancer (2001) 85(10), 1551–1556 © 2001 Cancer Research Campaign p73 is deregulated in vulval cancer1553 A B N1 T1 N2 T2 N3 T3 N4 T4 N1 T1 N2 T2 N3 T3 N4 T4 N5 T5 N6 T6 N7 T7 N8 T8 .. p73 .. p73 ARF INK4a p14 p16 actin actin N1 T1 N2 T2 N3 V3 N4 V4 N5 T5 N6 T6 full length p73 p73 ∆ 2 actin Figure 1 (A) p73 expression in vulval neoplasia. RT-PCR analysis of p73 expression in paired normal (N) and tumour (T). PCR was performed for 28 cycles. ARF p73 is predominantly expressed as the α and γ isoforms (upper and lower arrows respectively). The middle panel shows that expression of p14 is ARF deregulated with p73 in a subset of vulval SCC. Cancers T3, T4, T5 and T6 which do not have deregulated p14 lack HPV DNA and mutant p53. The lower INK4a panel is actin. ( B) Expression of p73 in vulval neoplasia can be deregulated in the presence of either HPV DNA or transcriptional silencing of p1 6 . T1 and T2 INK4a (each with matched normal N1 and N2) are HPV positive vulval SCC and over-express p16 mRNA. T3 and T4 (also with matched normal) are vulval SCC INK4a with hypermethylated p16 sequences and concomitant absence of p16 transcript. p73 RNA is over-expressed in both groups of cancer. PCR was performed INK4a for 28 cycles for both p73 and p16. (C) Vulval cancers over-express p73 ∆2. T1 is a vulval SCC which over-expresses both full-length and ∆2 forms, whereas T5 and T6 express only the ∆2 form. V3 and V4 are VIN I and do not express detectable p73 RNA under these limiting PCR conditions (28 cycles of PCR). Note also the absence of detectable p73mRNA in each matched normal (N1 –N6) under these conditions A B Figure 2 Immunocytochemical analysis of expression of p73 in normal and neoplastic vulval epithelium. Sections were prepared from formalin-fixed, parrafin- embedded tissue samples as described in Materials and Methods.( A) Expression of p73 in normal vulval epithelium is in the basal and immediately supra-basal cells. RT-PCR showed that this was predominantly p73 α and p73 γ. (B) Expression of p73 in VIN II of p73 in HPV-positive cancers. We were interested to identify address this issue, we analysed the structure and expression of INK4a alternative mechanisms by which pRb function might be inhibited, p16 to determine whether mutation and/or epigenetic to determine how p73 was deregulated in cases lacking HPV. To silencing of the gene was related to deregulation of p73 in © 2001 Cancer Research Campaign British Journal of Cancer (2001) 85(10), 1551–1556 1554 J O’Nions et al Table 1A Expression of p14, p16 and p73 in vulval cancers INK4a INK4a ARF Cancer p53 status HPV p16RNA p16 meth. FL p73 p73 ∆2 p14 1 Mt – – Yes – + + 2 Mt – – Yes – + + 3 Mt – – Yes + + + 4 Mt – – – + + + 5 Mt – – – – + + 6 Mt – – Yes – + – 7 Mt – – Yes – + – 8 Mt – + – – – – 9 Mt – – Yes – + + 10 Mt – – Yes – – + 11 Mt – – – – – – 12 Mt 16 + – + + + 13 Mt – – Yes – – + 14 Mt 16 + – + + + 15 Mt – – – – – + 16 Mt – – – – – + 17 Mt – Mt – – + + 18 Mt – – Yes – + – 19 Mt – + – – + + 20 Mt 16 + – – + + 21 Mt 16 + – – + + 22 Wt 16 + – – + + 23 Wt – – – + + + 24 Wt 16 + – – + + 25 Wt 16 + – – + + 26 Wt – + – – – – 27 Wt 16 + – + + + 28 Wt 16 + – – + + 29 Wt 16 + – – + + 30 Wt – – Yes – + – 31 Wt – – Yes – + – 32 Wt 16 + – – + + 33 Wt – Mt – – + – 34 Wt 16 + Yes – + – 35 Wt – Mt – – + – 36 Wt 16 – Yes – + + Expression was determined by RT-PCR as described in Methods and Materials. Mt = mutant, Wt = wild-type. FL p73 = full-length p73. ARF ∆2 p73 = deleted exon 2 p73. + denotes expression of p73 and p14 increased relative to matched normal tissue. + denotes detectable INK4a INK4a a b c expression of p16 . The mutations in p16 are: codon 80 Cga >TgA = Arg >Ter; codon 11 CCT > CTT = Pro > Leu; codon 48 CCg >TCg = Pro > Ser. The polymorphism at codon 148 Ala > Thr was detected in 4/36 individuals analysed. Table 1B Effect of grade of neoplasia on expression of p73 VIN I VIN II VIN III SCC Mean score 1.2 +/– 0.46 1.42+/– 0.37 2.25 +/–0.68 2.59+/–0.44 Data shown are staining indices, determined as described in Methods, +/ – standard deviation. HPV negative cases. Using MSP, the presence of hypermethyla- 1M 1U 2M 2U 3M 3U 4M 4U 5M 5U 6M 6U 7M 7U INK4a tion in the p16 gene promoter was detected in 13/36 cases INK4a of vulval SCC (Figure 3, Table 1A). Mutations in p16 were detected in 3/36 vulval SCC (Figure 4), all 3 cases INK4a being HPV-negative. No mutations were detected in p16 in 21 HPV16-positive vulval SCC or in 78 HPV16-positive cervical SCC. Taken together, of the 16 cases of vulval SCC with inactiva- INK4a tion of p16 , only one was HPV16-positive. Over-expression of INK4a Figure 3 MSP analysis of CpG methylation in the p16gene promoter in p73 occurred in 15/16 cases of HPV-negative vulval SCC with vulval neoplasia. Each paired lane represents either methylated (M) or INK4a p16 inactivation (Table 1A, Figure 1). These data imply that unmethylated (U) DNA for each vulval SCC. Note the presence of INK4a methylated DNA in cancers 3 and 5. The presence of unmethylated DNA in inactivation of p16 and expression of HPV16 E7 may be func- both cases is attributable either to the presence of normal tissue within the tionally interchangeable events in vulval neoplasia and that either tumour biopsy or hemimethylation of the CpG sequences. M = DNA can result in p73 over-expression. molecular weight markers British Journal of Cancer (2001) 85(10), 1551–1556 © 2001 Cancer Research Campaign p73 is deregulated in vulval cancer1555 where p73-positive cancers are associated with poor prognosis A B (Tannapfel et al, 1999). 123456 78 910 1234567 8 9 10 11 In view of the hypothesised role of p73 as a tumour-suppressor protein, it was perhaps surprising to observe over-expression in such a high proportion of cases. Sequence analysis did not detect mutations in p73 in vulval cancers, findings consistent with other authors’ studies of both solid and haematological malignancies (Yoshikawa et al, 1999). A variant of p73 which excludes exon 2 (p73 ∆2) was recently described in ovarian carcinomas (Ng et al, 2000) and in some breast cancer cell lines (Filippovich et al, 2001). The authors of the latter study stressed the importance of analysing expression of the ∆2 form in a range of normal and INK4a Figure 4 Mutations occur in exon 2 of p16 in vulval but not cervical malignant tissues. In the present study, we make the interesting SCC. The autoradiographs show SSCP analysis of exon 2 of INK4 in observation that, in vulval cancer, p73 over-expression is indeed anogenital neoplasia. (A) 5 ′ portion of exon 2. Lanes 1 –8 are cervical SCC. predominantly of the ∆2 form, and in some cases in our series this Lane 9 is vulval cancer with mutation. ( B) 3 ′ portion of exon 2. Lanes 1 –9 are cervical SCC. Lanes 10 and 11 are vulval SCC, each with polymorphism at was the only form of p73 detectable. p73 ∆2 has been shown to INK4a codon 148 of p16 . The abnormal mobility bands are indicated by arrows inhibit the transactivating function of both p53 and full-length p73 (Filippovich et al, 2001). As such, the over-expression of p73 ∆2 forms suggests a contribution to tumourigenesis in vulval SCC ARF p73 expression is deregulated with p14in cancers by transdominant inhibition of wild-type p53 and full-length p73. with loss of p53 function It is also an interesting possibility that over-expression of p73∆2 may be related to the differentiation status of squamous cancers. Because p73 is deregulated by E2F-1 expression (Irwin et al, 2000; The importance of full-length p73 expression in mediating Lissy et al, 2000), we determined the expression levels of another keratinocyte differentiation has been clearly demonstrated ARF recognised E2F-1 regulated gene, p14 (Bates et al, 1998), in (De Laurenzi et al, 2000). It is, therefore, an attractive hypothesis cancers with p73 over-expression. Using RT-PCR, we determined that impaired differentiation of some squamous cancers may, at ARF whether p73 and p14 were over-expressed together in vulval least in part, result from transdominant inhibition of full-length cancers either positive for HPV, mutant for p53 or having neither p73-dependent differentiation by the ∆2 variant. Additional (Figure 1). 29/36 vulval SCC analysed by RT-PCR over-expressed studies to address this hypothesis would clearly be of interest. ARF p73 and there was concomittant over-expression of p14 in Expression of p73 is driven by E2F-1 (Irwin et al, 2000; Lissy 21/29. Of these, 20 cases were either mutant for p53 or positive for et al, 2000). It was therefore of interest to investigate potential HPV 16. In total, 6 vulval SCC were both negative for HPV and mechanisms by which E2F-1 is itself deregulated in vulval cancer. contained wild-type p53 sequence. Of these 6 cases, p73 was over- A subset of the SCC contained and expressed HPV 16 DNA ARF expressed in 5, but p14 in only a single case. These results sequences. HPV16 E7 associates with pRb and thereby deregu- ARF suggest that deregulation of p14 , but not p73, requires loss of lates expression of E2F-1 responsive genes such as B-myb (Lam p53 function. et al, 1994), providing a mechanistic explanation for p73 deregu- lation in HPV-positive cases. Analysis of HPV-negative cases INK4a revealed frequent abnormalities in p16 , either in the form DISCUSSION of point mutations or methylation-dependent transcriptional INK4a In this work we show that expression of p73 is deregulated at both silencing. Inactivation of p16 was inversely correlated with the protein and mRNA levels in a high proportion of vulval carci- presence of HPV DNA in the majority of cases, implying that loss INK4a nomas and pre-malignant lesions. Our results are consistent with of p16 function can, at least partially, compensate for the studies which have reported over-expression of p73 in other absence of HPV E7 expression. Support for this hypothesis is INK4a common cancers, including breast (Zaika et al, 1998), bladder (Chi afforded by the consistent absence of p16 mutations in a large et al, 1999) and hepatocellular carcinoma (Tannapfel et al, 1999). series of HPV-positive cervical SCC, whereas mutations in INK4a We also make the important observation that deregulation is p16 were detected in 3/16 HPV-negative vulval SCC. frequently of the ∆2 transdominant form of p73. Support for the hypothesis that over-expression of p73 results p73 exists as a number of isoforms, generated by alternative from E2F-1 deregulation was provided by the observation of co- ARF splicing of exons encoding the -COOH terminus of the protein ordinate over-expression with p14 in a large proportion of (De Laurenzi et al, 1998; Ueda et al, 1999). Multiple isoforms vulval cancers with simultaneous loss of p53 function (via muta- are expressed in keratinocytes (De Laurenzi et al, 1998). In tion or HPV 16 positivity). Interestingly, vulval cancers lacking a ARF cervical epithelium the α form is the overwhelmingly predomi- p53 mutation and HPV 16 did not, in general, over-express p14 nant variant expressed (data not shown). It is of interest, there- despite frequent, abundant over-expression of p73. Previous ARF fore, that p73 expression is predominantly of the α and γ forms in studies have revealed that cells over-expressing p14 are almost vulval epithelium. It is also noteworthy that whereas expression always null for p53 (Stott et al, 1998). Our data are consistent with of p73 is restricted to the basal and supra-basal layers in normal this hypothesis and also suggest that deregulation of p73 is not epithelium, this restriction is lost in VIN and in SCC, expression dependent on the p53 status of the cell. frequently encompassing the entire epithelium. A further observa- In conclusion, our results add to the growing evidence that p73 tion of interest was the increase in expression with increasing over-expression is a common event in human neoplasia and grade of neoplasia. Over-expression of p73 protein has been corre- provide direct support from clinical biopsy material for E2F lated with progression of other cancers, for example in the bladder, 1-driven p73 deregulation in vivo. © 2001 Cancer Research Campaign British Journal of Cancer (2001) 85(10), 1551–1556 1556 J O’Nions et al Jost CA, Marin MC and Kaelin WG Jr (1997) p73 is a simian p53-related protein ACKNOWLEDGEMENTS that can induce apoptosis. 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p73 is over-expressed in vulval cancer principally as the Δ2 isoform

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Springer Journals
Copyright
Copyright © 2001 by The Author(s)
Subject
Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
ISSN
0007-0920
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1532-1827
DOI
10.1054/bjoc.2001.2138
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British Journal of Cancer(2001) 85(10), 1551–1556 © 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.2138, available online at http://www.idealibrary.com on http://www.bjcancer.com p73 is over-expressed in vulval cancer principally as the ∆2 isoform 1 1 1 2 2 3 1 4 5 1 J O’Nions, LA Brooks, A Sullivan, A Bell, B Dunne, M Rozycka, A Reddy, JA Tidy, D Evans, PJ Farrell, 6 7 2 1 A Evans, M Gasco, B Gustersonand T Crook 1 2 Ludwig Institute for Cancer Research, St Mary’s Hospital Medical School, Norfolk Place, London W2 1PG; University Department of Pathology, Glasgow University, Western Infirmary, Glasgow; Department of Molecular Medicine, King’s College School of Medicine and Dentistry, Coldharbour Lane, London SE5; 4 5 Department of Gynaecological Oncology, University of Sheffield, Northern General Hospital, Sheffield, S5 7AU; Department of Histopathology, St Mary’s 6 7 Hospital Medical School, Norfolk Place, London W2; Department of Surgery, Poole Hospitals NHS Trust, Poole, Dorset; UO Oncologia Medica, Azienda Ospedaliera S. Croce e Carle, Via Coppino 26, 12100, Cuneo, Italy Summary p73 was studied in squamous cancers and precursor lesions of the vulva. Over-expression of p73 occurred commonly in both human papillomavirus (HPV)-positive and -negative squamous cell cancers (SCC) and high-grade premalignant lesions. Whereas expr ession in normal vulval epithelium was detected only in the basal and supra-basal layers, expression in neoplastic epithelium increased with grade of neoplasia, being maximal at both protein and RNA levels in SCC. p73 ∆2 was the principal over-expressed isoform in the majority of cases of vulval SCC and often the sole form expressed in SCC. Over-expression of p73 was associated with expression of HPV-encoded E7 or with INK4a ARF hypermethylation or mutation of p16 in HPV-negative cases. There was a close correlation between expression of p73 and p14 in cancers with loss of p53 function. The frequent over-expression of p73 ∆2 in neoplastic but not normal vulval epithelium, and its co-ordinate deregulation with other E2F-1 responsive genes suggests a role in the oncogenic process. © 2001 Cancer Research Campaign http://www.bjcancer.com Keywords: vulval cancer; HPV; p73 Although vulval SCC is less common than cervical cancer, it is of a number of isoforms which arise by alternative splicing of exons major mechanistic interest since cancers are either HPV positive encoding the –COOH terminus of the protein and which exhibit (principally HPV 16) or lack detectable HPV DNA sequences. differences in trans-activating and growth suppressor functions HPV-positive cancers have an association with vulval intraepithe- (De Laurenzi et al, 1998; Ueda et al, 1999). The expression of a lial neoplasia (VIN) (reviewed by Crum, 1992). Clear pathobio- further spliced variant of p73 which lacks exon 2 (p73 ∆2) has logical differences between HPV-positive and HPV-negative been described in ovarian cancer (Ng et al, 2000) and in breast cancers have been described (Crum, 1992). Allelotype analysis cancer cell lines (Fillippovich et al, 2001). Recently, p73 has been has revealed that no significant differences in sites of loss of shown to be directly induced by E2F-1 and thereby to contribute to heterozygosity (LOH) exist between the 2 forms of the disease E2F-1-mediated apoptosis (Irwin et al, 2000; Lissy et al, 2000). (Pinto et al, 1999). However, a number of studies have revealed Furthermore, both myc and adenovirus E1A can activate expres- that mutation in p53 is more common in HPV-negative cancers sion of p73 (Zaika et al, 2000). (Lee et al, 1994; Marin et al, 2000; Brooks et al, 2001). Mechan- Although p73 is subject to methylation-dependent transcriptional istically, it is hypothesised that mutation in p53 functionally silencing in some B-cell malignancies, consistent with a role as a compensates for the absence of HPV 16E6, since this protein putative tumour-suppressor protein (Corn et al, 1999), mutational mediates inactivation of p53 via promotion of ubiquitin-dependent analyses have suggested that neither p63 nor p73 is frequently proteolysis. Despite the more common mutation of p53 in HPV- mutated in human cancers (Yoshikawa et al, 1999). Furthermore, negative cases, a substantial number of vulval SCC occur which p73 is over-expressed in some cancers, although the mechanism of lack both mutation and HPV. The mechanism, if any, by which p53 this is unknown (Chi et al, 1999; Zaika et al, 1999). Assessment of function may be compromised in such cases is not known. the biological significance of the ability of p73 ∆2 to transdomi- p73 has structural and functional homologies to p53, including nantly inhibit both p53 and full-length p73 clearly requires analysis sequence-specific DNA binding and transactivation functions of the expression of this variant in a range of both normal and malig- (Kaghad et al, 1997). Over-expression of p73 is able to induce nant tissues (Fillippovich et al, 2001). In the current study we have apoptosis in some human cancer lines (Jost et al, 1997) and investigated the structure and expression of p73 in vulval neoplasia. expression of p73 has been shown to have a role in the differentia- tion of keratinocytes (De Laurenzi et al, 2000). p73 is expressed as MATERIALS AND METHODS Received 17 May 2001 Tissues Revised 23 July 2001 Accepted 7 August 2001 SCC of the vulva and VIN III tissue samples were collected at Correspondence to:T Crook operation. In each case the diagnosis was confirmed by routine 1551 1552 J O’Nions et al histopathological analysis of resected tissue. Tissues were collected Mutu was used as a positive control. Analysis of p73 coding immediately into liquid nitrogen and stored until isolation of nucleic sequences in the regions corresponding to the mutational hot spots acids. Genomic DNA was isolated by proteinase K digestion and of p53 was done by RT-PCR SSCP using conditions described by total RNA by RNAzol B. Cancers and VIN III were HPV-typed Kawano et al (1999). using standard PCR methodology. Paraffin sections of vulval neoplasia were retrieved from the Department of Histopathology at RESULTS St Mary’s Hospital, Paddington, London. Wild-type p73 is frequently over-expressed in vulval Analysis of gene expression neoplasia cDNA was synthesised with the ProStar system (Stratagene) from The expression of p73 was investigated in a series of vulval SCC and 3 µg of total RNA. Analysis of expression was performed by RT- in VIN, previously analysed for the presence of HPV DNA ARF PCR as described previously for p14 (Gazzeri et al, 1998), and sequences. In initial experiments, expression was analysed using RT- p73 (De Laurenzi et al, 1998). For semi-quantitative analysis of PCR methodology (De Laurenzi et al, 1998). This assay allows gene expression, PCR was performed using the primers and discrimination between alternatively spliced forms encoding thermal cycling conditions described and was for 22 cycles for different –COOH variants of p73 protein. Expression of p73 mRNA ARF p14 , and 28 cycles for p73. In some experiments, amplification was detectable by RT-PCR in each of the 36 normal vulval epithelial was extended to 40 cycles to analyse expression in tissues samples available for analysis using 40 cycles of amplification. expressing a lower level of p73. Following PCR, reactions were Expression was predominantly of α and γ variants in each of the resolved on agarose gels, transferred to Hybond-N nylon and normal vulval samples analysed. In cancers, expression was also hybridised with P γ-ATP-labelled oligonucleotide probes specific restricted to the α and γ forms, but using limiting PCR conditions, for the amplified fragments. Analysis of N-terminal splice variants was markedly increased relative to matched normal vulval epithe- of p73 was performed using the primers described by Ng et al lium in 29/36 cases examined (Figure 1). Interestingly, over- (2000). Identity of these was confirmed by cloning and sequencing expression of p73 mRNA was detected in cancers both positive and and by hybridisation analysis of amplified products with oligo- negative for HPV DNA (Table 1A). To confirm that the elevated p73 nucleotide probes specific for exon 2 and exon 3 of p73. The pres- mRNA levels were reflected in protein expression and to investigate ence of equivalent amounts of cDNA in each PCR was verified by possible associations between p73 expression and grade of neoplasia, amplification of β-actin under similar limiting conditions. RT-PCR we performed immunocytochemical analysis of tissue sections of INK4a analysis of p16 was performed as described previously VIN and SCC cut from paraffin blocks. These studies revealed that (Gonzalez-Zulueta et al, 1995). expression of p73 was detectable in each case of normal epithelium, consistent with RT-PCR analysis performed under extended cycling conditions (Figure 2). Expression was restricted to the basal and Immunocytochemistry supra-basal layers in normal tissue, but this restriction was lost with 5 µm sections were cut from formalin-fixed, paraffin-embedded, increasing grade (Figure 2). To further demonstrate the increase in tissue sections. The diagnosis in each case was confirmed by expression with grade of neoplasia, expression was scored using a examination of haematoxylin and eosin-stained sections. For semi-quantitative immunocytochemical technique in VIN. A mean immunocytochemistry, sections were pressure-cooked in citrate expression index was calculated for each of VIN I, VIN II and VIN ARF buffer, then stained with antibodies: p14 goat polyclonal ((C20) III. This clearly demonstrated the increase in expression from VIN I Santa Cruz, SC-8613) was used at 1/100 dilution; p73 mouse to VIN III (Table 1B). The presence of mutations in the over- monoclonal antibody Neomarkers, MS-764-P0, affinity-purified expressed p73 mRNA was sought using RT-SSCP (Kawano et al, and diluted 1/150. Sections were scored independently by at least 1999). No mobility shifts suggestive of mutation were detected in 24 2 pathologists. To demonstrate increase in p73 expression with vulval SCC analysed. increasing grade of neoplasia, sections were scored according to the following scheme. 1: basal staining only; 2: suprabasal staining p73 ∆2 is the predominant over-expressed form of p73 where less than 50% of nuclei were positive in the strongest staining area in a high power field; 3: suprabasal staining where The high frequency of over-expression of p73α and γ in vulval greater than 50% of nuclei were positive in the strongest stained SCC, in the absence of mutations, was unexpected in view of the area in a high-power field. pro-apoptotic and negative growth-regulatory functions of these proteins. We therefore performed additional RT-PCR analysis to examine the N-termini of the mRNA species. There was over- Analysis of gene structure expression of p73 ∆2 in the majority of vulval SCC shown to Mutations in exon 1α and 2 of the INK4 locus were sought using over-express p73α (Figure 1 and Table 1A). Full-length p73 was single-strand conformation polymorphism analysis (SSCP) with only rarely simultaneously over-expressed, p73 ∆2 being the primers and PCR conditions described by Zhang et al (1994). PCR only form expressed in the majority of cases (Figure 1 and reactions were resolved on 6% native acrylamide gels with and Table 1A). without 5% glycerol. Methylation of CpG sequences in the INK4a p16 promoter was performed using methylation-specific PCR INK4a Inactivation of p16 correlates inversely with the (MSP) as described by Herman et al (1996). To examine exon 1β presence of HPV for mutations, SSCP was performed using cDNA as the substrate and resolution on 6% gels as described above (Gazzeri et al, The ability of HPV E7 to deregulate p73 expression via E2F-1 1998). cDNA prepared from the Burkitt’s lymphoma cell line and pRb provides a mechanistic explanation for over-expression British Journal of Cancer (2001) 85(10), 1551–1556 © 2001 Cancer Research Campaign p73 is deregulated in vulval cancer1553 A B N1 T1 N2 T2 N3 T3 N4 T4 N1 T1 N2 T2 N3 T3 N4 T4 N5 T5 N6 T6 N7 T7 N8 T8 .. p73 .. p73 ARF INK4a p14 p16 actin actin N1 T1 N2 T2 N3 V3 N4 V4 N5 T5 N6 T6 full length p73 p73 ∆ 2 actin Figure 1 (A) p73 expression in vulval neoplasia. RT-PCR analysis of p73 expression in paired normal (N) and tumour (T). PCR was performed for 28 cycles. ARF p73 is predominantly expressed as the α and γ isoforms (upper and lower arrows respectively). The middle panel shows that expression of p14 is ARF deregulated with p73 in a subset of vulval SCC. Cancers T3, T4, T5 and T6 which do not have deregulated p14 lack HPV DNA and mutant p53. The lower INK4a panel is actin. ( B) Expression of p73 in vulval neoplasia can be deregulated in the presence of either HPV DNA or transcriptional silencing of p1 6 . T1 and T2 INK4a (each with matched normal N1 and N2) are HPV positive vulval SCC and over-express p16 mRNA. T3 and T4 (also with matched normal) are vulval SCC INK4a with hypermethylated p16 sequences and concomitant absence of p16 transcript. p73 RNA is over-expressed in both groups of cancer. PCR was performed INK4a for 28 cycles for both p73 and p16. (C) Vulval cancers over-express p73 ∆2. T1 is a vulval SCC which over-expresses both full-length and ∆2 forms, whereas T5 and T6 express only the ∆2 form. V3 and V4 are VIN I and do not express detectable p73 RNA under these limiting PCR conditions (28 cycles of PCR). Note also the absence of detectable p73mRNA in each matched normal (N1 –N6) under these conditions A B Figure 2 Immunocytochemical analysis of expression of p73 in normal and neoplastic vulval epithelium. Sections were prepared from formalin-fixed, parrafin- embedded tissue samples as described in Materials and Methods.( A) Expression of p73 in normal vulval epithelium is in the basal and immediately supra-basal cells. RT-PCR showed that this was predominantly p73 α and p73 γ. (B) Expression of p73 in VIN II of p73 in HPV-positive cancers. We were interested to identify address this issue, we analysed the structure and expression of INK4a alternative mechanisms by which pRb function might be inhibited, p16 to determine whether mutation and/or epigenetic to determine how p73 was deregulated in cases lacking HPV. To silencing of the gene was related to deregulation of p73 in © 2001 Cancer Research Campaign British Journal of Cancer (2001) 85(10), 1551–1556 1554 J O’Nions et al Table 1A Expression of p14, p16 and p73 in vulval cancers INK4a INK4a ARF Cancer p53 status HPV p16RNA p16 meth. FL p73 p73 ∆2 p14 1 Mt – – Yes – + + 2 Mt – – Yes – + + 3 Mt – – Yes + + + 4 Mt – – – + + + 5 Mt – – – – + + 6 Mt – – Yes – + – 7 Mt – – Yes – + – 8 Mt – + – – – – 9 Mt – – Yes – + + 10 Mt – – Yes – – + 11 Mt – – – – – – 12 Mt 16 + – + + + 13 Mt – – Yes – – + 14 Mt 16 + – + + + 15 Mt – – – – – + 16 Mt – – – – – + 17 Mt – Mt – – + + 18 Mt – – Yes – + – 19 Mt – + – – + + 20 Mt 16 + – – + + 21 Mt 16 + – – + + 22 Wt 16 + – – + + 23 Wt – – – + + + 24 Wt 16 + – – + + 25 Wt 16 + – – + + 26 Wt – + – – – – 27 Wt 16 + – + + + 28 Wt 16 + – – + + 29 Wt 16 + – – + + 30 Wt – – Yes – + – 31 Wt – – Yes – + – 32 Wt 16 + – – + + 33 Wt – Mt – – + – 34 Wt 16 + Yes – + – 35 Wt – Mt – – + – 36 Wt 16 – Yes – + + Expression was determined by RT-PCR as described in Methods and Materials. Mt = mutant, Wt = wild-type. FL p73 = full-length p73. ARF ∆2 p73 = deleted exon 2 p73. + denotes expression of p73 and p14 increased relative to matched normal tissue. + denotes detectable INK4a INK4a a b c expression of p16 . The mutations in p16 are: codon 80 Cga >TgA = Arg >Ter; codon 11 CCT > CTT = Pro > Leu; codon 48 CCg >TCg = Pro > Ser. The polymorphism at codon 148 Ala > Thr was detected in 4/36 individuals analysed. Table 1B Effect of grade of neoplasia on expression of p73 VIN I VIN II VIN III SCC Mean score 1.2 +/– 0.46 1.42+/– 0.37 2.25 +/–0.68 2.59+/–0.44 Data shown are staining indices, determined as described in Methods, +/ – standard deviation. HPV negative cases. Using MSP, the presence of hypermethyla- 1M 1U 2M 2U 3M 3U 4M 4U 5M 5U 6M 6U 7M 7U INK4a tion in the p16 gene promoter was detected in 13/36 cases INK4a of vulval SCC (Figure 3, Table 1A). Mutations in p16 were detected in 3/36 vulval SCC (Figure 4), all 3 cases INK4a being HPV-negative. No mutations were detected in p16 in 21 HPV16-positive vulval SCC or in 78 HPV16-positive cervical SCC. Taken together, of the 16 cases of vulval SCC with inactiva- INK4a tion of p16 , only one was HPV16-positive. Over-expression of INK4a Figure 3 MSP analysis of CpG methylation in the p16gene promoter in p73 occurred in 15/16 cases of HPV-negative vulval SCC with vulval neoplasia. Each paired lane represents either methylated (M) or INK4a p16 inactivation (Table 1A, Figure 1). These data imply that unmethylated (U) DNA for each vulval SCC. Note the presence of INK4a methylated DNA in cancers 3 and 5. The presence of unmethylated DNA in inactivation of p16 and expression of HPV16 E7 may be func- both cases is attributable either to the presence of normal tissue within the tionally interchangeable events in vulval neoplasia and that either tumour biopsy or hemimethylation of the CpG sequences. M = DNA can result in p73 over-expression. molecular weight markers British Journal of Cancer (2001) 85(10), 1551–1556 © 2001 Cancer Research Campaign p73 is deregulated in vulval cancer1555 where p73-positive cancers are associated with poor prognosis A B (Tannapfel et al, 1999). 123456 78 910 1234567 8 9 10 11 In view of the hypothesised role of p73 as a tumour-suppressor protein, it was perhaps surprising to observe over-expression in such a high proportion of cases. Sequence analysis did not detect mutations in p73 in vulval cancers, findings consistent with other authors’ studies of both solid and haematological malignancies (Yoshikawa et al, 1999). A variant of p73 which excludes exon 2 (p73 ∆2) was recently described in ovarian carcinomas (Ng et al, 2000) and in some breast cancer cell lines (Filippovich et al, 2001). The authors of the latter study stressed the importance of analysing expression of the ∆2 form in a range of normal and INK4a Figure 4 Mutations occur in exon 2 of p16 in vulval but not cervical malignant tissues. In the present study, we make the interesting SCC. The autoradiographs show SSCP analysis of exon 2 of INK4 in observation that, in vulval cancer, p73 over-expression is indeed anogenital neoplasia. (A) 5 ′ portion of exon 2. Lanes 1 –8 are cervical SCC. predominantly of the ∆2 form, and in some cases in our series this Lane 9 is vulval cancer with mutation. ( B) 3 ′ portion of exon 2. Lanes 1 –9 are cervical SCC. Lanes 10 and 11 are vulval SCC, each with polymorphism at was the only form of p73 detectable. p73 ∆2 has been shown to INK4a codon 148 of p16 . The abnormal mobility bands are indicated by arrows inhibit the transactivating function of both p53 and full-length p73 (Filippovich et al, 2001). As such, the over-expression of p73 ∆2 forms suggests a contribution to tumourigenesis in vulval SCC ARF p73 expression is deregulated with p14in cancers by transdominant inhibition of wild-type p53 and full-length p73. with loss of p53 function It is also an interesting possibility that over-expression of p73∆2 may be related to the differentiation status of squamous cancers. Because p73 is deregulated by E2F-1 expression (Irwin et al, 2000; The importance of full-length p73 expression in mediating Lissy et al, 2000), we determined the expression levels of another keratinocyte differentiation has been clearly demonstrated ARF recognised E2F-1 regulated gene, p14 (Bates et al, 1998), in (De Laurenzi et al, 2000). It is, therefore, an attractive hypothesis cancers with p73 over-expression. Using RT-PCR, we determined that impaired differentiation of some squamous cancers may, at ARF whether p73 and p14 were over-expressed together in vulval least in part, result from transdominant inhibition of full-length cancers either positive for HPV, mutant for p53 or having neither p73-dependent differentiation by the ∆2 variant. Additional (Figure 1). 29/36 vulval SCC analysed by RT-PCR over-expressed studies to address this hypothesis would clearly be of interest. ARF p73 and there was concomittant over-expression of p14 in Expression of p73 is driven by E2F-1 (Irwin et al, 2000; Lissy 21/29. Of these, 20 cases were either mutant for p53 or positive for et al, 2000). It was therefore of interest to investigate potential HPV 16. In total, 6 vulval SCC were both negative for HPV and mechanisms by which E2F-1 is itself deregulated in vulval cancer. contained wild-type p53 sequence. Of these 6 cases, p73 was over- A subset of the SCC contained and expressed HPV 16 DNA ARF expressed in 5, but p14 in only a single case. These results sequences. HPV16 E7 associates with pRb and thereby deregu- ARF suggest that deregulation of p14 , but not p73, requires loss of lates expression of E2F-1 responsive genes such as B-myb (Lam p53 function. et al, 1994), providing a mechanistic explanation for p73 deregu- lation in HPV-positive cases. Analysis of HPV-negative cases INK4a revealed frequent abnormalities in p16 , either in the form DISCUSSION of point mutations or methylation-dependent transcriptional INK4a In this work we show that expression of p73 is deregulated at both silencing. Inactivation of p16 was inversely correlated with the protein and mRNA levels in a high proportion of vulval carci- presence of HPV DNA in the majority of cases, implying that loss INK4a nomas and pre-malignant lesions. Our results are consistent with of p16 function can, at least partially, compensate for the studies which have reported over-expression of p73 in other absence of HPV E7 expression. Support for this hypothesis is INK4a common cancers, including breast (Zaika et al, 1998), bladder (Chi afforded by the consistent absence of p16 mutations in a large et al, 1999) and hepatocellular carcinoma (Tannapfel et al, 1999). series of HPV-positive cervical SCC, whereas mutations in INK4a We also make the important observation that deregulation is p16 were detected in 3/16 HPV-negative vulval SCC. frequently of the ∆2 transdominant form of p73. Support for the hypothesis that over-expression of p73 results p73 exists as a number of isoforms, generated by alternative from E2F-1 deregulation was provided by the observation of co- ARF splicing of exons encoding the -COOH terminus of the protein ordinate over-expression with p14 in a large proportion of (De Laurenzi et al, 1998; Ueda et al, 1999). Multiple isoforms vulval cancers with simultaneous loss of p53 function (via muta- are expressed in keratinocytes (De Laurenzi et al, 1998). In tion or HPV 16 positivity). Interestingly, vulval cancers lacking a ARF cervical epithelium the α form is the overwhelmingly predomi- p53 mutation and HPV 16 did not, in general, over-express p14 nant variant expressed (data not shown). It is of interest, there- despite frequent, abundant over-expression of p73. Previous ARF fore, that p73 expression is predominantly of the α and γ forms in studies have revealed that cells over-expressing p14 are almost vulval epithelium. It is also noteworthy that whereas expression always null for p53 (Stott et al, 1998). Our data are consistent with of p73 is restricted to the basal and supra-basal layers in normal this hypothesis and also suggest that deregulation of p73 is not epithelium, this restriction is lost in VIN and in SCC, expression dependent on the p53 status of the cell. frequently encompassing the entire epithelium. A further observa- In conclusion, our results add to the growing evidence that p73 tion of interest was the increase in expression with increasing over-expression is a common event in human neoplasia and grade of neoplasia. Over-expression of p73 protein has been corre- provide direct support from clinical biopsy material for E2F lated with progression of other cancers, for example in the bladder, 1-driven p73 deregulation in vivo. © 2001 Cancer Research Campaign British Journal of Cancer (2001) 85(10), 1551–1556 1556 J O’Nions et al Jost CA, Marin MC and Kaelin WG Jr (1997) p73 is a simian p53-related protein ACKNOWLEDGEMENTS that can induce apoptosis. Nature 389: 191–194 Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon Research in the laboratory of B Gusterson is supported by P, Lelias JM, DuMont X, Ferrara P, McKeon F and Caput D (1997) Breakthrough Breast Cancer. We are most grateful to Dr W Kaelin Monoallelically expressed gene related to p53 at 1p36, a region for providing the monoclonal antibody to p73. frequently deleted in neuroblastoma and other human cancers. Cell 90: 809–819 Kawano S, Miller CW, Gombart AF, Bartram CR, Matsuo Y, Hiroya A, Akiko S, REFERENCES Said J, Tatsumi E and Koeffler HP (1999) Loss of p73 gene expression in leukemias/lymphomas due to hypermethylation. Blood 94: 1113–1120 Bates S, Phillips AC, Clarke PA, Stott F, Peters G, Ludwig RL and Vousden KH Lam EW, Morris JD, Davies R, Crook T, Watson RJ and Vousden KH (1994) (1998) p14ARF links the tumour suppressors RB and p53. 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British Journal of Cancer – Springer Journals

Published: Nov 13, 2001

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