Expression levels of p53 and p73 isoforms in stage I and stage III ovarian cancer

Expression levels of p53 and p73 isoforms in stage I and stage III ovarian cancer
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  Expression levels of p53 and p73 isoforms in stage I andstage III ovarian cancer Mirko Marabese a, * ,e , Sergio Marchini a,e , Eleonora Marrazzo a , Pietro Mariani a ,Dario Cattaneo a , Roldano Fossati b , Anna Compagnoni b , Mauro Signorelli c ,Ute M. Moll d , A. Maria Codegoni a , Massimo Broggini a a Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan, Italy b Laboratory of Translational Research and Clinical Outcome, Department of Oncology, Istituto di Ricerche Farmacologiche ‘Mario Negri’,Milan, Italy c Ospedale San Gerardo, Universita` di Milano Monza, Italy d Department of Pathology, State University of New York at Stony Brook, Stony Brook, New York, United States A R T I C L E I N F O Article history: Received 21 June 2007Received in revised form 20September 2007Accepted 9 October 2007Available online 26 November 2007 Keywords: p53p73Ovarian cancerSplice variantsA B S T R A C TThe p53 gene has been investigated for its role in epithelial ovarian cancer but data col-lected until now are contradictory. The evidence that p53 belongs with p63 and p73 to afamily of transcription factors re-opened interest in this gene family.Here, we used quantitative real time RT-PCR to determine expression levels of TAp53,TAp73 and their N-terminal splice variants in a cohort of 169 ovarian cancer patients withstage I and stage III disease. The TAp73 levels in stage III biopsies differed by 100-folddepending on the p53 status and overall survival appears to be significantly related to D Np73 expression. Kaplan–Meyer analyses did not suggest a correlation between overallsurvival and levels of TAp73,  D Np73 or the  D Np73/TAp73 ratio. In conclusion, these datasuggest that at least in our patient cohort p53 and p73 expression levels are not correlatedto malignant progression of ovarian cancer. They might, however, play a role in tumourinitiation.   2007 Elsevier Ltd. All rights reserved. 1. Introduction p53 has long been considered a unique tumour suppressor,whose prominent position in cell cycle regulation, apoptosisand DNA repair has spurred extensive research into both itsbasic and clinical aspects. 1,2 However, since this protein isat the crossroad of an extensive and complex network of stress response pathways, the role of wild-type p53 in onco-genesis extends far beyond our current knowledge. This isparticularly evident in epithelial ovarian cancer (EOC) whichis at present the most deadly cancer in women of westerncountries. 3 Although platinum-based therapy improved EOCoverall survival, more than 75% of patients are diagnosed atlate stages of the disease and relapse after an initial response.This high rate of mortality is generally ascribed to a lackof molecular biomarkers for monitoring early diseaseonset and response to therapy. 3 The role of p53 in EOC malig-nant transformation, clinical course and responsiveness totherapy is not completely clear and data are to some extentcontradictory. 4–6 p53 is the founding member of a gene family that includesp63 and p73, which share with p53 a high degree of structural 0959-8049/$ - see front matter    2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.ejca.2007.10.011*  Corresponding author:  Fax: +39 02 3546277.E-mail address: marabese@marionegri.it (M. Marabese). e Both authors contributed equally to this work. E U R O P E A N J O U R N A L O F C A N C E R  44 (2008) 131  –  141 available at www.sciencedirect.comjournal homepage: www.ejconline.com  similarities. This finding re-opened the debate on the role of these transcription factors in driving malignant transforma-tion, in particular in EOC. 7–9 However, structural similaritiesdo not necessarily translate into functional similarity. p63and p73 share a consensus DNA binding domain with p53and in ectopic overexpression can act as a transcription factoron p53 target genes. However, they also have unique tissuedistribution and functions during development and malig-nant transformation. 10–12 The scenario is further complicatedby the presence of alternative promoters and/or alternativesplicing at the N-termini or C-termini, resulting in a plethoraof different isoforms. Moreover, these isoforms can interactwith each other in a dominant negative fashion. In addition,they might be endowed with unique transcriptional activ-ity. 13–17 Thus, analysis of a single isoform is not sufficientfor drawing any conclusion on their role in human tumours.Instead, it is mandatory to study the relationship among thedifferent isoforms, in particular between the full length iso-forms (called TA, transactivation active), which are character-ised by a p53 like anti-oncogenic activity, and their dominantnegative ones (collectively called  D TA), which are character-ised by pro-oncogenic potential. 16 The aim of this study was to assess a possible correlationbetween the expression levels of p53, p73 and their dominantnegative isoforms and disease progression in a cohort of 169EOC patients at an early and advanced stage of disease. 2. Materials and methods 2.1. Sample collection Patient biopsies were collected during surgery at the depart-ment of Oncology and Gynecology, San Gerardo Hospital(Monza, Italy) from September 1992 to March 2004: 83 werediagnosed as stage I ovarian cancer and 86 as stage III. Theirhistopathologic features are summarised in Table 1. Stage Table 1 – Clinical parameters and p53 status according to stage, histotype and grading Clinical parameters p53 statusHistopatological parameters No. of cases Wild-type Mutated ND Stage I 83a 26 26 0 0b 5 5 0 0c 52 52 0 0 Histotype Serous 33 33 0 0Mucinous 16 16 0 0Endometroid 18 18 0 0Undifferentiated 1 1 0 0Clear cell 14 14 0 0Not available 1 1 0 0 Grade 1 17 17 0 02 18 18 0 03 33 33 0 0Bl 15 15 0 0Stage III 86a 3 2 1 0b 5 2 2 1c 77 36 36 5Not available 1 1 0 0 Histotype Serous 66 31 30 5Mucinous 3 1 1 1Endometroid 10 4 6 0Undifferentiated 6 4 2 0Clear cell 1 1 0 0 Grade 1 5 4 0 12 18 8 8 23 58 25 31 2Bl 5 4 0 1Not available 0 0 0 0Total sample size 169ND, not detected. 132  E U R O P E A N J O U R N A L O F C A N C E R  44 (2008) 131  –  141  and histological grade of the primary tumours were definedaccording to International Federation of Gynecology andObstetrics (FIGO) staging. Fresh tumour tissues were obtainedat the first laparotomy prior to any treatment. The tissueswere freed from necrotic, haemorrhagic and connective tis-sues, minced and stored frozen at  ) 80   C in cryotubes untilprocessed. Histopathological analysis revealed that tumourcells content accounted for more than 70% of tumour sample.The collection and use of tumour samples was approved bythe local Scientific Ethical Committee, and patients gave theirwritten consent. 2.2. Clinical data Tumour histological classification was carried out according to the WHO system. A pathologist confirmed the diagnosisof ovarian cancer, the histological subtype and pathologicalstage (FIGO staging). Conventional clinical features including age and menopausal status were evaluated. 2.3. RNA isolation and cDNA preparation From each frozen sample, a tumour fragment of approxi-mately 30 mg was taken with a sterile and RNase-free scalpel.The division procedure lasted between 0.5 and 2 min, and in-cluded weighing of the aliquots to determine the amount of lysis solution needed for RNA isolation. Tissues were homog-enised by ultraturrax at 4   C and total RNA purified using theSV-Total RNA isolation system (Promega, Milan, Italy). TotalRNA was measured by spectrophotometer. Aliquots werestored at  ) 80   C until use. Two hundred nanogram of totalRNAwas reverse transcribed in 20  l L of reaction mix with Ar-chive Kit (Applied Biosystems, Foster City, USA) and randomprimers, and then stored at  ) 80   C until use. 2.4. Primer design Primer pairs for TAp73, Ex2p73, Ex2/3p73  D Np73,  D N 0 p73, 28 Sand cyclophillin A (Cyclo A) were as previously described. 18 For the other p53 family isoforms and housekeeping genes(glyceraldehyde-3-phosphate dehydrogenase (GAPDH) andactin), optimal primer pairs were chosen spanning splice junctions, using PRIMER-3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). All primers were queriedagainst the non-redundant Human Genome Database (Na-tional Center for Biotechnology Information). Specificity of primers was verified by detecting single bands of ampliconsand sequencing the PCR products. Primers sequences arelisted in Supplemental Table 1. 2.5. Real time RT-PCR Absolute copy numbers for all isoforms were determined byreal time RT-PCR (ABI-7900, Applied Biosystems, Monza, Italy)using the SYBR Green protocol (Applied Biosystems). Briefly,PCR reaction was performed in a 10  l L reaction mix with2  l L of previously prepared cDNA (from 200 ng total RNA)and primer pairs at a final concentration of 200 nM. 384-wellplates were prepared by automatic liquid handling (epMotion5075, Eppendorf, Milan Italy). Annealing temperatures are re-ported in Supplemental Table 1. Melting curves for eachamplicon were automatically generated to evaluate the spec-ificity of the amplified products. The absolute copy number of target and housekeeping genes were calculated by interpola-tion using a standard curve generated from a serial dilution,ranging from 2 to 20 million copies, of a cDNA prepared froma plasmid expressing the specific transcript. Standard con-centration was measured with Nanodrop (Celbio, Milan Italy)and gene copy numbers were determined according to thefollowing formula:  X (molecules/ l l) = 6.023  ·  10 23  /molecularweight  ·  concentration of the plasmid( l g/ l l)  ·  10 ) 6 . 2.6. Western blot Total protein extracts were prepared by lysing tumour tissuesin 50 mM Tris–HCl, pH 7.4, 250 mM NaCl, 0.1% Nonidet NP-40,5 mM EDTA, 50 mM NaF with aprotinin, leupeptine and phe-nyl-methyl-sulfonyl-fluoride (PMSF) as protease inhibitors,for 30 min on ice after sample homogenisation with an ultra-turrax. Insoluble material was pelleted at 13,000 g  for 10 minat 4   C and the protein concentration was determined byusing Biorad kit (BioRad, Milan Italy). Total cellular proteins(100  l g) was separated on SDS–PAGE and transferred to aPVDF membrane. Immunoblotting was carried out with p73monoclonal antibodies (GC-15 mAb, Oncogene Research, CA,USA). RAN, a highly conserved and ubiquitously expressedGTPase was used as housekeeping gene (clone 20, BD trans-duction laboratories, USA). Antibody binding was revealedby peroxidase labelled secondary antibodies and visualisedusing enhanced chemiluminescence (Amersham, Italy). 2.7. Mutational status of p53 The p53 mutational status in exons 4–9 was determined intumour specimens. cDNA, obtained as previously described,was further PCR-amplified with the following primers:p53Fw 5 0 -GGGACAGCCAAGTCTGTGACT and p53Rw: 5 0 -CCTGGGCATCCTTGAGTT. Amplification was performed in a ther-mocycler (PTC-200 MJ, Biorad) with 33 cycles at 95   C for 1 0 ;60   C for 30 00 ; 72   C for 1 0 and TAQ DNA polymerasewith proof-reading activity (LA-Taq, Takara) and, Cambrex Milan, Italy).Sequencing was performed by ABI 3730 DNA sequencer. 2.8. Data and statistical analysis Using the R 2.1.0 software (R Development Core Team 2005;R: A language and environment for statistical computing.R Foundation for Statistical Computing Vienna, Austria. URLhttp://www.R-project.org ), we wrote a dedicated script basedon ‘car’ and ‘stats’ R libraries to normalise the data and to plotthe graphics. Four genes (actin, cyclophillin A, GAPDH and28S) were chosen for data normalisation. A normalisation in-dex was generated foreach patient by computing the geomet-ric mean of the four housekeeping genes. The reliabilityof thechoice of those genes has been evaluated by determining the correlations among the genes and between each geneand the normalisation index, verifying that an acceptable cor-relation level exists. The calculated index for each patientwas then used to normalise data of the target genes. Sincethe values of gene expression for both ovarian cancer E U R O P E A N J O U R N A L O F C A N C E R  44 (2008) 131  –  141  133  stage I and ovarian cancer stage III displayed a non-normaldistribution, all statistical analyses were performed using non-parametric tests.We evaluated the correlation between each patient subsetand also between full length and their relative splice variantsusing the Spearman correlation test.  D TA and  D N  were ana-lysed separately and also as a ratio. In the first case, we splitthe  D TA and  D N  distribution in three groups defined by ter-tiles: patients who had expression range lower than the firsttertile patients whose expression range was between firstand second tertile and patients with expression range higherthan the second tertile. Tertiles were calculated on patientswho did not experience death at the end of the study.The ratio between  D N  and TA isoforms of each target genewas analysed both as a continuous and as a dichotomous var-iable. The cut-off used to split the ratio into two categorieswas set to 1. Gaussian distribution of data was tested withthe Shapiro–Wilk test for normality; as this test gave a non-significant result, a non-parametric analysis of variance (AN-OVA) was used to evaluate the associations between  D N  /TAratio and categorical variables such as stage, grade, residualtumour and histology.Overall survival was classified as outcome measure andwas defined as the length of time from the first surgery tothe last follow-up date or death irrespective of the cause.Overall survival curves were plotted with the Kaplan–Meiermethod. The log-rank test and Cox proportional hazard mod-els were used to compare time-to-event distributions be-tween pre-defined groups. The estimates from the Coxregression model are presented as hazard ratios (HRs) and95% confidence intervals (CIs).Three different Cox multivariate models were built tostudy the prognostic effect of the  D N  /TA ratio, consideredboth as dichotomous and continuous variable: the first modelanalysed the whole sample of patients and included stage ascovariate; the second model analysed stage I patients and in-cluded tumour grade, the third model analysed stage III pa-tients and included the residual tumour as covariate.Statistical significance was set at 0.05 and all tests weretwo-tailed. Analysis was performed using SAS software v9.0(SAS Institute Inc, Cary, USA). 3. Results A cohort of 169 patients with ovarian cancer biopsies pro-cured at the time of diagnosis and a median follow-up of 6.8years was selected for this study. Their histopathologic andclinical parameters are summarised in Table 1: 50% of pa-tients had FIGO stage I disease and 50% stage III disease;53% had a grade 3 tumour. The median age at the time of diagnosis was 53.6 years for the whole sample (median ageof 49.4 years for stage I and 54.9 for stage III) and the medianfollow-up for the whole sample was 6.84 years (median age of 6.75 for stage I and 6.88 for stage III). The p53 mutational sta-tus was determined by sequencing the DNA binding regionbetween exons 4 and 9. All stage I ovarian tumours harbouredwild-type p53, while 45% of stage III (39 out of 86) harbouredmutations (Table 1). Mutations were found more frequentlyin exon 5 (30%), followed by exons 7 and 8 (25%). For 7% of them (6 out 86), we were unable to obtain sequence. Kap-lan–Meyer analysis showed that patients harbouring mutp53 showed a reduced overall survival compared to those har-bouring wild-type (wt) p53 (  p  = 0.052, Supplemental Fig. 1).mRNA levels for TA, the  D TA and the  D Np73 isoforms weremeasured by real time RT-PCR in all 169 biopsies. Throughoutthis text,  D TAp73 refers collectively to the three NH2-termi-nally truncated variants  D NEx2p73,  D NEx2/3p73 and  D N 0 p73. D Np73 refers exclusively to the specific transactivation-defi-cient isoform that derives from the second alternative pro-moter. Absolute copy numbers per 200 ng total RNA for eachisoform were normalised against 4 internal control house-keeping genes: cyclo A, actin B, GAPDH and 28S. Data valida-tion for each specific real time RT-PCR was performed aspreviously described. We used this comprehensive data setto detect possible systematic expression changes in the TA/ D N  ratio of p53 and p73 family variants.The box plots in Fig. 1 indicate in log scale the absolutemRNA copy number for full length TAp53, p53 D 40 andp53 D 133 (panel A) and for TAp73,  D TAp73 and  D Np73 (panelB), separated by FIGO stage. In stage III, p53 and its two mainisoforms p53 D 40 and p53 D 133 had comparable levels of expression (median value of 9.4  ·  10 ) 3 , 2.2  ·  10 ) 3 and1.06  ·  10 ) 3 for full length p53,  D N40 and  D N133, respectively,see Table 2). No differences were evident between tumourswith or without p53 mutations (Table 2).Stage I biopsies express 100-fold higher levels of TAp53compared to stage III, while expression levels of p53 D N40and p53 D 133 were comparable between stage I and stage III(median value of 1.15  ·  10 ) 1 , 7.2  ·  10 ) 4 and 8.2  ·  10 ) 3 for fulllength p53, p53 D N40 and p53 D 133 respectively, Table 2). As al-ready reported, 18 our EOC cohort also showed that Ex2/3p73and Ex2p73 remain largely unchanged. Concerning theexpression levels of TAp73,  D TAp73 and  D Np73 (Fig 1B, rightand left panels), specific differences were apparent. Withinthe  D TAp73 isoforms, the median values are comparable be-tween the two stages and even if there are substantial stagedifferences (Table 2), the box plots in Fig. 1 suggest that there is substantial overlap between stage I and stage III. When wesubdivided stage III patients on the basis of their p53 status,we observed that samples lacking functional p53 had reducedlevels of TAp73, whosevalues dropped 100-fold to a medianof 2.93  ·  10 ) 2 in the p53-mutated subgroup. A different profilewas observed for  D Np73 expression, whose median valuedropped a 1000-fold from 1.44  ·  10 ) 2 in stage I to 1.4  ·  10 ) 5 in stage III (Table 2). No differences were apparent betweenp53 wild-type or mutated subgroups.Western blot analysis on a subset of patients showed thatamong the different C-terminal splice variants,  D Np73 b  iso-form represents the main form. Of note, its expression levelis comparable between the two stages and consistent withthe mRNA measurements in the same tumours. (Fig. 2). The a  isoform was undetectable (Fig. 2). When we correlatedexpression levels of TAp73 with  D TAp73 and  D Np73 for eachpatient (Table 3), we found a significant correlation betweenthe TA and the  D Np73 forms in stage I ( r  = 0.97,  p  < 2.2e ) 16 ).Moreover, in stage III we found a good level of correlationfor the isoformsencodedby the P1 promoter, but notfor thoseencoded by the P2 promoter. Table 3 shows a level of correla-tion of 0.76 (  p  < 2.2e ) 16 ), 0.930 (  p  < 2.2e ) 16 ), 0.96 (  p  < 2.2e ) 16 ) forEx2/3, Ex2 and  D N 0 , respectively. 134   E U R O P E A N J O U R N A L O F C A N C E R  44 (2008) 131  –  141  We next attempted to correlate the expression levels of TAp73 with overall survival (OS). Tumour expression levelswere subdivided into tertiles: the first tertile is defined asthe lowest one-third; the second tertile as the middle one-third and the third tertile as the top one-third of theexpression range. However, Kaplan–Meier curves (Fig. 3a–c)failed to show differences between patients with highexpression of TAp73 compared to those with low expres-sion. This was evident in the full cohort of 169 biopsies(  p  = 0.15, Fig. 3a) as well as in the two subsets of stage Iand stage III patients (panels b and c,  p -value 0.80 and0.66, respectively).When expression of  D Np73 was analysed, we had an inter-esting trend (Fig. 4). In the whole cohort, a lower expressionlevel of   D Np73 strongly associates with poor survival(Fig. 4a,  p  = 0.00001). In fact, after three years follow-up, theOS for patients above the 2nd tertile was 92%, compared tothose between the 1st and the 2nd tertile with 84% and forthose within the 1st tertile with 64%. This difference becamemore consistent after 5 and 7 years of follow-up, when the OSwas 89% above the 2nd tertile, 77% and 65% in the 1st and the2nd tertile and fell to 44% or 36%, respectively, for patientswithin the 1st tertile. When we analysed stage I and stageIII separately, we observed the same trend albeit only in stageIII, although the difference due to the reduced number of samples was no longer significant (Fig. 4c). We calculated thatat least 230 patients would be needed to confirm at a statisti-cal significant level the trend seen in Fig. 4c.Given the fact that the  D Np73 protein acts in a dominantnegative fashion on the TAp73 network, we tested the Fig. 1 – (a) Box plot diagrams showing the expression levels of p53,  D N40 and  D 133 isoforms in 84 stage I (left side) and 86stage III (right side) ovarian cancer biopsies. (b) Box plot diagrams showing the expression levels of TAp73,  D TAp73 and D Np73 in 84 stage I (left) and 86 stage III (right) ovarian cancer biopsies. After normalisation of each sample to its own set of housekeeping genes, data are expressed as absolute copy numbers on a logarithmic scale. The line within the boxesindicates the median. The top edge of the boxes represents the 75th percentile, the bottom edge the 25th percentile. Therange is shown as a vertical line. Outliers (circles) are defined as 1.5-fold above or below the 75th and 25th percentile values. E U R O P E A N J O U R N A L O F C A N C E R  44 (2008) 131  –  141  135
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