Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma
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   Journal of Pathology  J Pathol   2010;  220 : 97–107Published online  4 August 2009  in Wiley InterScience(  DOI:  10.1002/path.2609 Original Paper  Identification of a novel 12p13.3 ampliconin nasopharyngeal carcinoma Yvonne Y-Y Or, 1# Grace T-Y Chung, 1 , 2# Ka-Fai To, 1 , 2 Chit Chow, 1 Kwong-Wai Choy, 3 Carol Y-K Tong, 1 Alice W-C Leung, 1 Angela B-Y Hui, 4 Sai-Wah Tsao, 5 Ho-Keung Ng, 1 , 2 Timothy T-C Yip, 6 Pierre Busson 7 and Kwok-Wai Lo 1 , 2 * 1 Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The ChineseUniversity of Hong Kong, Hong Kong SAR 2 Li Ka Shing Institute of Health Science, The Chinese University of Hong Kong, Hong Kong SAR 3 Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong SAR 4 Division of Applied Molecular Oncology, Ontario Cancer Institute, University of Toronto, Canada 5 Department of Anatomy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong SAR 6 Department of Clinical Oncology, Queen Elizabeth Hospital, Hong Kong SAR 7 Laboratoire de Biologie des Tumeurs Humaines, Institut Gustave Roussy, Villejuif, France*Correspondence to:Kwok-Wai Lo, Department of  Anatomical and Cellular Pathology, Prince of WalesHospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR.E-mail:  # These authors contributed equally to this study.No conflicts of interest weredeclared. Received: 21 April 2009Revised: 24 July 2009Accepted: 26 July 2009 Abstract Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonlyoccurring in southern China. To decipher the molecular basis of this cancer, we performedhigh-resolution array CGH analysis on eight tumour lines and 10 primary tumours toidentify the genes involved in NPC tumsrcenesis. In this study, multiple regions of gainwere consistently found at 1q21-q24, 7q11-12, 7q21-22., 11q13, 12p13, 12q13, 19p13 and19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft,xeno-2117. By FISH mapping, we have further delineated the amplicon to a 1.24 regionflanked by RP11-319E16 and RP11-433J6. Copy number gains of this amplicon wereconfirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copynumber amplification. Among the 13 genes within this amplicon, three candidate genes,lymphotoxin beta receptor (LT β R), tumour necrosis factor receptor superfamily memeber1A ( TNFRSF1R ) and  FLJ10665 , were specifically over-expressed in the NPC xenograftwith 12p13.3 amplification. However, only LT β R was frequently over-expressed in primarytumours. LT β R is a member of the TNF family of receptors, which can modulate NF- κ Bsignalling pathways. Over-expression of LT β R in nasopharyngeal epithelial cells resulted inan increase of NF- κ B activity and cell proliferation.  In vivo  study showed that suppressionof   LT  β  R  by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification.These findings implied that  LT  β  R  is a potential NPC-associated oncogene within the 12p13.3amplicon and that its alteration is important in NPC tumsrcenesis.Copyright  ©  2009 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.Keywords: nasopharyngeal carcinoma; 12p13.3 amplicon; array CGH; oncogene; LT β R;Epstein–Barr virus Introduction Nasopharyngeal carcinoma (NPC) is a highly preva-lent malignant disease in Southern China and South-East Asia. Its incidence in southern Chinese is upto  ∼ 25–30/100000 persons/year, which is 100-foldhigher than that in Caucasians from Western coun-tries. As a distinct type of head and neck cancer,NPC is closely associated with EBV infection andcommonly appears as poorly differentiated or undif-ferentiated carcinomas [1]. The clonal EBV genomeand latent viral gene products are found in almost allprimary tumours [1,2]. Our previous studies showedthat NPC tumorigenesis is a multi-step process thatinvolved multiple genetic and epigenetic abnormal-ities, resulting in the disruption of various cellularmechanisms [1,3]. Radiation therapy is the mainstayof NPC treatment and the survival rate for patientswith early disease is  >  70%. However, most patientsare diagnosed in the later stages and have a lowersurvival rate. Thus, identifying novel biomarkers andtherapeutic targets is important in the control of thisdeadly disease.Our early molecular genetic studies demonstratedthe consistent genetic losses on chromosomes 3p,9, 11q, 13q, 14q and 16q and recurrent gains on Copyright 󰂩 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons,   98 Y Y-Y Or   et al chromosome 12 in NPC [1,4,5]. In particular, theinactivation of tumour suppressors on 3p21.3 and 9p21was demonstrated to be an early event during thedevelopment of NPC [6,7]. Although abnormalitiesof multiple critical tumour suppressor genes (eg  p16  ,  RASSF1A, TSLC1 ) have been identified, informationabout oncogenes involved in transforming nasopharyn-geal epithelial cells is limited. Recently, using a CGHarray containing 1800 BAC clones, which span thehuman genome at about 1–3 Mb resolution, we haveinvestigated the genomic abnormalities in NPC andrevealed two novel amplicons at 11q13.1–13.3 and12p12.1–13.3. The study further showed that  CCND1 ( cyclin D1 ) is the target oncogene in the 11q13 ampli-con and that its activation plays a role in NPC tumori-genesis [8]. Knockdown of   CCND1  by siRNA in NPCcells led to significant inhibition of cell proliferation.However, this BAC array study was not able to fur-ther define the smaller amplicons consistently foundin NPC, due to its low resolution.In the current study, we aimed to comprehensivelycharacterize the novel amplicon at 12p12.1–13.3 andto identify the candidate oncogenes in a panel of EBV-positive NPC tumours by high-resolution arrayCGH analysis. A minimal region of gain mapped to12p13.31 was further delineated by FISH analysis. Weidentified the candidate oncogenes in this ampliconby comparing the copy number abnormality (CNA)and its expression in the tumour lines and primarytumours. The role of candidate oncogene in regulat-ing signalling pathways, cell proliferation and tumori-genicity was further demonstrated in nasopharyngealepithelial cells. Materials and methods Cell lines, xenografts and tumour specimens Seven EBV-positive NPC xenografts (xeno-2117,xeno-1915, xeno-8, C15, C17, C18 and C19), a cellline (C666-1) and 10 microdissected primary tumoursfrom our previous BAC array CGH study were sub- jected to the current high-resolulation array CGHanalysis [8–12]. Two immortalized normal nasopha-ryngeal epithelial cell lines, NP69 and NP460, wereused as controls [13,14]. The cell lines were main-tained as described in our previous reports. Forimmunohistochemistry and FISH analysis, a total of 41archival formalin-fixed paraffin-embedded NPCs wererecruited from the Department of Anatomical and Cel-lular Pathology at the Prince of Wales Hospital, TheChinese University of Hong Kong. The study protocolwas approved by the Clinical Research Ethics Com-mittee of the Chinese University of Hong Kong. Allthe tumours were reviewed by two independent pathol-ogists as poorly differentiated or undifferentiated NPC,according to the WHO classification. The tumour sam-ples were also confirmed to be EBV-positive by EBER Figure 1.  Identification of 12p13.3 amplicon in NPC byhigh-resolution array CGH analysis. Copy number changes of chromosome 12 were examined in 18 NPC tumours. Gainsare shown in red and losses in green. Red arrow indicates the12p13.3 amplicon in situ  hybridization. The clinical parameters are sum-marized in Table S1 (see Supporting information). Array comparative genomic hybridization (CGH)analysis The Agilent Human Genome CGH Microarray 44B(Agilent Technologies Inc., Palo Alto, CA, USA),which contained about 44000 oligonucleotide probes,was used in this study. Array CGH analysis wasperformed according to the manufacturer’s protocol.In each array, 1  µ g genomic male DNA was used  J Pathol   2010;  220 : 97–107 DOI: 10.1002/pathCopyright 󰂩 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.  12p13.3 amplicon in NPC 99 Figure 2.  Characterization of 12p13.3 amplicon in NPC. (a) The 12p13.3 locus amplified in NPCs. The regions of gains wereshown as grey bars. Black bars represent regions with high-level amplification. A 2.05 Mb minimal common region of gains wasdelineated. (b) Illustration of 12p13.3 amplicon in xeno-2117. The genomic location is shown on the  x   axis and the copy numberchange is shown on the  y   axis. The position and the copy number of the probes contain on the microarray are indicated byred (gains) or green (losses) dots. Two distinct regions of gains (regions 1 and 2) were found. The 2.1 Mb amplicon core withthreshold  >  7.5 at region 1 was defined as reference and labelled with Cy3–dUTP (Perkin-Elmer), while the target genomic DNA was labelledwith Cy5–dUTP. After scanning, data extraction andnormalization, the results were analysed using CGHAnalytics 3.3 software (Human Genome Build 17,Agilent Technologies). The log 2  ratio measured for allprobes of the chromosome was smoothed by using2 Mb moving average. The Z-score algorithm wasapplied for calculating the chromosomal aberration,with a threshold set at 2 for defining regions withsignificant copy number gains and losses, while athreshold of 7.5 was used for amplification. cDNA expression array The Whole Human Genome Oligo Microarray, withover 41 000 probes (Agilent Technologies), was usedfor expression analysis on NPC tumour lines (xeno-2117, C666-1 and C15), with NP69 as the reference.The expression analysis was carried out accordingto the manufacturer’s protocol. NPC sample cRNAwas labelled with 5-CTP (Cy5), while NP69 refer-ence cRNA was labelled with cyanine 3-CTP (Cy-3). Microarrays were scanned on Agilent’s dual-lasermicroarray scanner. The data were normalized andanalysed using GeneSpring GX version 9.0.5 software(Agilent Technologies). Fluorescence  in situ  hybridization (FISH) FISH analysis was performed to confirm the amplifica-tion of 12p13.31 in NPC tumour lines and 41 paraffin-embedded primary tumours as described [8]. FiveBAC clones located at 12p13.32–p13.31 were labelled  J Pathol   2010;  220 : 97–107 DOI: 10.1002/pathCopyright 󰂩 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.  100 Y Y-Y Or   et al Figure 3.  FISH analysis of 12p13.3 amplicon in NPC tumour lines. (a) Representative images from the FISH validation of the12p13.3 amplicon in xeno-2117. Clusters of RP11–451H11 signals (red) and two reference signals (green, chromosome 18centromeric probe) are shown in the xeno-2117 tumour cells. Only 24% of the tumour cells showed the cluster signals of RP11–277E18 in region 2. (b) Illustration of FISH analysis of NPC tumour lines (xeno-2117, xeno-1915 and C666-1) with six BACclones on chromosome 12p13.3. The maxima located at RP11–451H11 are shown in xeno-2117 with Spectrum Orange–dUTP (Vysis, Downers Grove,IL, USA) by nick translation as the test probes.Centromere 18 labelled with Spectrum Green–dUTP(Vysis) was used as the reference probe. Chromosomallocations of these clones were validated in metaphasesof a normal individual. Amplifications and chromo-somal gains were scored when the ratios of test toreference probes were  >  2 and 1.2, respectively. Quantitative reverse transcription polymerasechain reaction (qRT–PCR) Transcriptional expression of the candidate oncogenesat 12p13.3 in NPC samples and normal nasopharyn-geal epithelial cells were evaluated by quantitativeRT–PCR as described [8,12]. The expression levelsof   β -actin  of each sample were determined for nor-malization. The primers used are shown in Table S2(see Supporting information). Western blotting and immunohistochemistryanalysis The expression of TNFRSF1A and LT β R in NPCsamples was investigated by western blotting andimmunohistochemistry analysis. Total cell protein of the xenografts and cell lines was extracted and sub- jected to western blotting analysis as described [8].Primary antibodies used in this study were anti-LT β Rgoat polyclonal antibody (N-15), anti-TNFRSF1Agoat polyclonal antibody (G-20) and anti-actin goatpolyclonal antibody (I-19; Santa Cruz). By immuno-histochemistry analysis, expression of LT β R andTNFRSF1A in the panel of 41 paraffin-embedded pri-mary tumours was also examined. Expression scoreswere defined with positive staining according to thepercentage of cells ( < 20%, score 1; 20–79%, score 2;80–100%, score 3). Scores 2 and 3 were assigned asover-expression. NF- κ B activity The NF- κ B activity of   LT  β  R -transfected NP69 cellsand controls was determined by electrophoretic moli-bity shift assay (EMSA) and p50 ELISA assay. NP69cells were transfected with vector pReceiver–M03 orpReceiver–-LT β R (GeneCopoeia), using Fugene HD(Roche Diagnostics). After 24 h, the culture mediumwas aspirated and replaced with medium supplementedwith lymphotoxin  α 1 β 2 at concentrations of 0, 10, 50and 100 ng/ml, respectively. After 20 h, the cells wereharvested and nuclear protein was extracted. EMSAassay was performed as described previously [19]. p50binding activity was measured using a NF- κ B p50ELISA Kit (Panomics). Cell proliferation analysis The cell proliferation of LT β R-transfected NP69 cellswas determined by WST-1 assay. In brief, NP69 cellswere seeded at 4  ×  10 3 cells/well in a 96-well plateand transfected with pReceiver–M03 (vector) or pRe-ceiver–LT β R (GeneCopoeia). After 24 h, the culturemedium was replaced with medium supplemented withlymphotoxin  α 1 β 2 at concentrations of 0, 10, 50 and100 ng/ml, respectively. Six culture wells were setup for each treatment. WST-1 assay was performedaccording to the manufacturer’s protocol after 44 h.WST-1 (10  µ l; Roche Diagnostics) and 100  µ l mediumwas added to replace the old medium, incubated at37 ◦ C for 30 min and UV absorbance was measuredat 450 and 690 nm.  J Pathol   2010;  220 : 97–107 DOI: 10.1002/pathCopyright 󰂩 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.  12p13.3 amplicon in NPC 101 In vivo  siRNA treatment For  in vivo  siRNA treatment, xeno-2117 was inoc-ulated into athymic nude mice and allowed togrow to about 150 mm 3 . The treatment was doneusing Invivofectamine, according to the manufac-turer’s protocol (Invitrogen, Carlsbad, CA, USA).siRNA (10 mg) against  LT  β  R  (5 ′ -GCACCUAUGUCUCAGCUAA) or control siRNA (Silencer NegativeControl No. 2) were mixed with 1 ml Invivofectamineand diluted in 15 ml 5% glucose solution. After con-centration using Amicon Ultra-15 Centrifugal Devicewith Ultracel-50 membrane, the concentrated complexwas collected and brought up to 1 ml with 5% glucosesolution. Each mouse was injected with 75  µ l Invivo-fectamine complex intratumorally. The injection wasrepeated at day 4 after the first injection. The size of the tumour was measured every 2 days until day 8.The mice were sacrificed at day 8 and the xenograftswere collected for histological examination. Results Identification of 12p13.3 amplicon in NPC by arrayCGH Using array CGH analysis, a high-resolution genomicprofile of each NPC sample was generated. Con-sistent with our previous studies, frequent chromo-some gains at 1q21-q24, 7q11-12, 7q21-22., 11q13,12p13, 12q13, 19p13 and 19q13, and chromosomelosses at 11q13, 11q23, 13q12-13, 14q21, 20p11 and22q13, were detected [4,8]. All these copy numberchanges were found in  >  40% NPC samples (seeSupporting information, Figure S1). Interestingly, anovel region of gain or amplification on chromosome12p13 was identified in 50% NPC samples, includingfive xenografts and four primary tumours (Figure 1).A minimal region of gain mapped to 2.05 Mb at12p13.31 delineated by these tumours is shown inFigure 2a. Importantly, high copy number amplifica-tion of 12p13.31 was found in xeno-2117 and primarytumour T1 (Figure 2a). Figure 2b shows the xeno-2117 array CGH profile at 12p13.31. Two distinctamplification regions, regions 1 and 2, were noted.Region 1 spans a 2.6 Mb region flanked by the probesat the location of 4515530–7135185. Furthermore,an amplicon core was found within region 1 whenthe threshold was increased to 7.5. This maximumis about 2.1 Mb in size, located between 5025464and 7135185. Region 2 is about 0.4 Mb in size andis located at the centrometric part of 12p13.31. Theregion is flanked by the probes at 7708418–8129280. Characterization of 12p13.3 amplicon by FISHanalysis To verify the array CGH findings, we have furtherexamined the amplification of 12p13.31 in NPC sam-ples by FISH analysis, using multiple BAC clones Figure 4.  FISH analysis of 12p13.3 amplicon in primary NPCs.(a) Copy number aberrations of the five BAC clones are plottedfor 41 primary tumours. Red spots represented the cases withcopy number gain or amplification. RP11–451H11 shows thehighest frequency and level of gain and amplification in primarytumours. (b) Examples of 12p13.3 amplification in primarytumours.MultiplecopiesofRP11–451H11weredetectedinthecases A10, A12 and A25. In case A10, no gains of RP11–319E6and RP11–433J6 were detected (Figure 3a). Copy number gains and amplificationof the BAC clones within the two distinct ampli-cons were detected in xeno-1915 and xeno-2117(Figure 3a, b). In xeno-2117, high-level amplificationwas detected in RP11–451H11, which locates withinthe core region but not in the two flanking BACclones, RP11–319E16 and RP11–433J6. Figure 3ashows examples of FISH analysis on xeno-2117,showing clusters of RP11–451H11 signals in thetumour cells. This RP11–451H11 amplifcation wasdetected in almost all tumour cells of xeno-2117. Theresult further refined the amplicon core to a 1.24 Mbregion flanked by RP11–319E16 and RP11–433J6.However, only 24% xeno-2117 tumour cells showedcluster signals of RP11–277E18 in region 2. This find-ing suggested that the amplification of region 2 onlyoccurred in a subset of the tumour cells during cancerdevelopment.Copy number gains of the 12p13.3 ampliconwere further examined in 41 undifferentiated primary  J Pathol   2010;  220 : 97–107 DOI: 10.1002/pathCopyright 󰂩 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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