Homozygous deletions scanning in tumor cell lines detects previously unsuspected loci

Authors

  • Pascal Pineau,

    Corresponding author
    1. Unité de Recombinaison et Expression Génétique, INSERM, Département de Médecine Moléculaire, Institut Pasteur, Paris, France
    • Unité de Recombinaison et Expression Génétique, INSERM U163, Département de Médecine Moléculaire, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15, France
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    • Fax: +33-1-45-68-89-43

  • Agnès Marchio,

    1. Unité de Recombinaison et Expression Génétique, INSERM, Département de Médecine Moléculaire, Institut Pasteur, Paris, France
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  • Emilie Cordina,

    1. Unité de Recombinaison et Expression Génétique, INSERM, Département de Médecine Moléculaire, Institut Pasteur, Paris, France
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  • Pierre Tiollais,

    1. Unité de Recombinaison et Expression Génétique, INSERM, Département de Médecine Moléculaire, Institut Pasteur, Paris, France
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  • Anne Dejean

    1. Unité de Recombinaison et Expression Génétique, INSERM, Département de Médecine Moléculaire, Institut Pasteur, Paris, France
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Abstract

High rates of loss of heterozygosity commonly affect multiple chromosomes in individual tumor types, yet the number of known tumor suppressor genes (TSGs) systematically mutated in the corresponding tumors is usually low. The search for homozygously deleted genome segments in tumor samples or cell lines has become a method of choice to identify major TSGs or to reveal their influence on the development of a given tumor type. Here, we report a detailed homozygous deletion (HD) profiling for 246 critical loci on a panel of 89 tumor cell lines containing significant subsets of lung, ovarian and head and neck squamous cell carcinomas. We found a total of 53 HDs affecting 17 loci. The major target for HDs was p16-INK4A/p14-ARF (23/89, 26% of cases). Among the remaining alterations, HDs affecting TP73 or telomeric markers have never been previously described, whereas other HDs represent the first examples associating lesions of certain TSGs with a given tumor type (NF2 in lung and ovarian cells, STK11 in HELA cells). Overall, tumor cell lines established from ovarian or lung carcinomas displayed a surprising diversity of loci targeted by HDs with 7 and 6 loci involved, respectively. Our data suggest that, beside allelotyping or transcriptome/proteome studies, extensive HD profiling represents a promising approach for the detection of hitherto not implicated signalling pathways of tumorigenesis. © 2003 Wiley-Liss, Inc.

Tumorigenesis implies alterations of diverse genetic factors that impact numerous biochemical pathways. Although cancer cell genomes display a high degree of loss of heterozygosity (LOH), only a limited number of known tumor suppressor genes (TSGs) have been found systematically mutated in most sporadic cancer types. In lung cancer, high rates of mutations (>30%) affect only TP53 and RB, whereas for ovarian cancer, TP53 and KRAS are the only genes recurrently mutated.1, 2 Recent findings on epigenetic inactivation through promoter hypermethylation may explain the relatively low rates of mutations in various TSGs.3 However, this phenomenon does not apply to all known TSGs and is often not exclusive of other genetic hits at a given gene locus.4 Thus, the search for genetic alterations associated with the carcinogenic processes in sporadic tumors is required and may provide additional therapeutic targets. Indeed, several recent TSG identifications commenced, at least partially, from homozygous deletion (HD) detection in primary tumors and/or cell lines. Such was the case for WT1, INK4A, BRCA2, PTEN, SMAD4 and SNF5.5, 6, 7, 8

In an attempt to identify tumorigenic pathways, we examined 89 tumor cell lines for HDs throughout the human genome. Although cell lines differ from both normal and cancerous tissues, a recent study has shown that they represent a faithful image of the organs from which they were derived.9 Assuming that the same should be true for tumorigenic processes, we investigated 230 loci either at well-established or putative TSGs. Furthermore, to assess genomic instability, the survey included several fragile sites, telomeric single-tagged sequence (STS) and housekeeping genes. Seventeen different loci were affected by at least one HD. Interestingly, our results demonstrate the occurence of HDs at novel loci, and we provide evidence for previously unknown associations of certain TSGs with particular types of cancers. Finally, we noticed that some subsets of cell lines display HDs at multiple sites, suggesting that, in the corresponding tumors, the carcinogenic process implies several alternative and/or parallel pathways.

MATERIAL AND METHODS

Tumor cell lines and DNA extraction

In total, 89 tumor cell lines were used for HD analysis (Table I). The panel includes 69 cells derived from epithelial tumors and 20 from sarcomas, hematopoietic malignancies, brain tumors and neuroblastomas. The precise source of each of these cell lines is available upon request. After growth in appropriate culture media, cells were harvested and nucleic acids extracted. Genomic DNA was extracted from cells after a treatment with lysis buffer (2% SDS, 10 mM Tris, HCl pH 8,10 mM NaCl, 10 mM EDTA, 10 mg/ml proteinase K) and a single phenol-chloroform. DNA was ethanol precipitated and resuspended in TE. RNA was extracted using the Tri-Reagent® solution (Euromedex, Souffelweyersheim, France).

Table I. Names and Tissular Origins of the 89 Cell Lines Profiled
Tissue of originCell lines
  1. The 37 cell lines of the first panel analyzed for the 246 loci are marked by an asterisk (*).

Lung cancer (LC), n = 22A549*, CALU1*, CALU6*, GILI5C, NCIH69, NCIH178, NCIH209, NCIH378, NCIH441*, NCIH446, NCIH526, NCIH711, NCIH748, NCIH865, NCIH1299*, SC6*, SC10*, SC74*, SC91, SC101, SC108, SKMES1*
Ovarian carcinoma (OC), n = 1959M*, A2780*, BG1*, CAVEOC2*, ES2*, HEY*, IGROV1*, OAW28*, OAW42*, OC2008*, OC2000C13, OV1063*, OVCAR3*, PA1*, PEO4*, PEO14*, PEO16*, SKOV3*, SW626
Head and neck squamous cell carcinoma (SCC), n = 13BB49, CAL60, CAL165, CAL166, FADU*, ISUL, PCI52, RPMI2650*, SCC4*, SCC9*, SCC15, SCC25*, SW579
Breast carcinoma (BC), n = 4MCF7*, MDAMB231*, T47D*, ZR75.1*
Brain tumors (BT)/neuroblastomas (NB), n = 4IMR32, SKBN2, SKNSH, U373
Leukemias-lymphomas (LL), n = 12BJAB, BL41, CEM, DAUDI, HL60, JIJOYE, JURKAT, K562, MT4, NB4, RAJI, U937
Prostate carcinoma (PRC), n = 3DU145*, LNCAP, PC3*
Cervix carcinoma (CC), n = 3HELA, C33A, CASKI
Other tumors (OT), n = 9A431, CHQ5B, HACAT, HEK293, HT29, LOVO, SAOS2, SKVTU, U2OS

Genomic PCR

The analysis of tumor cell lines' genomic DNAs for HD at candidate loci was performed by PCR using the STS listed in Table II. Most oligonucleotide sequences were determined from the published human genome project working draft on the GoldenPath server (http://genome.ucsc.edu/goldenPath/hgTracks.html). For markers aiming at specific genes, when no HD was previously published, coding exons were preferentially chosen. When HDs in a given region were already reported, the marker (exonic sequence or anonymous STS) was chosen in the consensus region of HD overlap. For HOXA5 (7p12), primer sequences detecting indicated STS were found in UniGene (http://www.ncbi.nlm.nih.gov/UniGene). Primers for β-globin (11p15) and β-actin (7p22), commonly used to test genomic DNA quality, have been published.10, 11 When exon size did not exceed 600 bp, both primers were chosen in intronic sequences, whereas for larger exons (>600 bp), one primer was chosen in exonic sequences and the other in a flanking intron. Telomeric markers have been described by Rosenberg et al.12 and Knight et al.13 All primer sequences are available upon request. Oligonucleotides were obtained from Eurogentec (Seraing, Belgium), Genset (Paris, France) and Life Technologies (Cergy-Pontoise, France). Reactions were performed as previously described.14 A step-down amplification was performed in most cases with a minimum annealing temperature ranging from 48–58°C.15 All primer sets mapping at the INK4A/ARF locus were used following the initially reported method of Kamb et al.6 Amplification products were separated on a 2% agarose gel and visualized on a UV transilluminator.

Table II. Candidate Genes and Loci Scanned for Homozygous Deletions
  1. For each gene, the number of the exon analyzed is indicated. The asterisk (*) indicates that homozygous deletions have already been described for this locus in the literature.

Chr. 1 (n = 16) 
 1PTEL061ptel
 ISG15ex21p36
 p73ex11p36
 CTNNBIP1ex41p36
 RIZex51p36
 CASP9ex21p36
 MIZ1ex91p36
 14-3-3cex11p35
 HDAC1ex31p35
 p18ex11p32
 GADD45ex31p31
 BCL10ex31p22
 IL6Rex21q21
 D1S2768*1q23
 SIPex21q25
 PARPex51q42
Chr. 2 (n = 10) 
 MSH2ex3*2p16
 MADex12p13
 BUB1ex42q14
 LRP-DITex22q21
 LRP-DITex4*2q21
 LRP-DITex422q21
 LRPDITex852q21
 PLC5ex2*2q33
 CFLARex42q33
 KUBOex62q35
Chr. 3 (n = 10) 
 D3S1297*3p26
 VHLex13p26
 PPARGex23p25
 RARBex23p24
 RASSF1ex4*3p21
 FHITex93p14
 D3S1300-FHIT3p14
 D3S3*3p12
 GSK3Bex23q13
 RUVBL1ex33q21
Chr. 4 (n = 36) 
 CTBP1ex34p16
 LAGYex24q12
 PDGFRAex124q12
 ALBex14q13
 PRKG2ex14q21
 FAP1ex64q21
 PKD2ex14q21
 SMARCADex94q21
 LIMex34q22
 ALK6ex84q22
 RAP1ex64q22
 DAPP1ex24q22
 MP1ex24q22
 PPP3CAex34q24
 NFKB1ex114q24
 CENPEex114q24
 IDAXex14q24
 DKK2ex24q24
 LEF1ex44q25
 CASP6ex24q25
 PITX2ex14q25
 MAD2ex34q27
 SPRY1ex24q27
 CCNA2ex44q27
 SMAD1ex24q31
 CDC4ex44q31
 TLR2ex24q32
 SAP30ex14q33
 FBX8ex24q34
 IRF2ex44q35
 CASP3ex34q35
 ALPex24q35
 ARGBP2ex34q35
 TLR3ex24q35
 FATHex44q35
 4QTEL114qtel
Chr. 5 (n = 7) 
 NKD2ex55p15
 CDH6ex3*5p14
 MEKK1ex35q11
 BACTIL5q11
 APCex155q21
 IRF1ex4*5q23
 CSKN1Aex35q33
Chr. 6 (n = 8) 
 DAXXex26p21
 BAK1ex36p21
 p21ex26p21
 D6S3146q24
 LOT1ex36q24
 ESR-CA6q25
 IGF2R-M6Pex286q25
 6QTEL546qtel
Chr. 7 (n =7) 
 HOXA5-W178547p15
 ACHEex2*7q22
 ST7ex77q31
 HBP1aex27q31
 D7S522/FRA7G7q31
 D7S6857q31
 FASTKex27q36
Chr. 8 (n = 16) 
 8PTEL918ptel
 PG1ex28p23
 DLC1ex3*8p22
 DLC1ex148p22
 N33ex2*8p22
 DHHC2ex38p22
 PRLTSex28p22
 ATIP1ex28p22
 FEZex18p21
 DR5ex28p21
 NKX3ex28p21
 CLUex28p21
 WRNex25*8p12
 VDAC3ex38p12
 SFRP1ex28p11
 NBSex48q21
Chr. 9 (n = 13) 
 SMARCA2ex29p24
 p16ex1*9p21
 p14ex2*9p21
 p15ex2*9p21
 1B9G21T7*9p21
 D9S171*9p21
 TLE1ex79q21
 DAPK1ex49q22
 SYKex39q22
 PTC1ex4–59q22
 ALK5ex39q22
 DBCCRex29q33
 TSC1ex39q34
Chr. 10 (n = 7) 
 KLF6ex2s10p15
 ANX7ex310q21
PTENex2*10q22
 PTENex4*10q22
 PTENex9*10q22
 FASex210q24
 BTRCPex410q24
Chr. 11 (n = 10) 
 KIP2ex211p15
 BGLOBex111p15
 WEEex3–411p15
 TSG101ex411p15
 WT1ex10*11p13
 MEN1ex3*11q13
 FADDex211q13
 D11S2179-ATM11q23
 PPP2R1Bex411q23
 TSLC1ex511q23–24
Chr. 12 (n = 6) 
 GAPDHex812p13
 p27ex112p13
 ACVR1Bex212q13
 CK18ex2–312q13
 APAFex2–312q23
 HNF1αex212q24
Chr. 13 (n = 13) 
 TUBA2ex213q12
 CX26ex113q12
 RNF6ex513q12
 CDX2ex413q12
 BRCA2ex3*13q12
 RB1ex213q14
 RB1ex1713q14
 RB1ex20*13q14
 D13S319*13q14
 D13S272*13q14
 SPRY2ex213q34
 ING1ex113q14
 13QTEL5613qtel
Chr. 14 (n = 3) 
 DUPLex414q11
 CDKN3ex2s14q22
 PSENex2–314q24
Chr. 15 (n = 5) 
 HNF6ex115q21
 MAP2K5ex115q22
 TLE3ex315q22
 PMLex815q24
 BLMex315q26
Chr. 16 (n = 20) 
 AXIN1ex2*16p13
 AXIN1ex4*16p13
 AXIN1ex616p13
 TSC2ex416p13
 PKD1ex416p13
 MYT1ex216p13
 NK4ex316p13
 SOCS116p13
 SIAHex216q12
 NKD1ex316q12
 CYLDex416q12
 RBL2ex916q12
 TRADDex216q22
 CADHEex1016q22
 TATex316q22
 WWOXex4*16q23
 WI2755/FRA16D*16q23
 WWOXex7*16q23
 WWOXex9*16q23
 16QTEL01316qtel
Chr. 17 (n = 17) 
 17PTEL8017ptel
 HCCS1ex517p13
 14-3-3ϵex2*17p13
 P53ex2*17p13
 P53ex7*17p13
 P53ex11*17p13
 MAP2K4ex4*17p13
 MAP2K4ex717p12
 MAP2K4ex1117p12
 MAP2K3ex2–3*17p12
 MAP2K3ex6–717p11
 MAP2K3ex1217p11
 NLKex217q11
 NF1ex28–29*17q11
 NM23ex117q21
 BRCA1ex617q21
 AXIN2ex117q25
Chr. 18 (n = 5) 
 SMAD4ex2*18q21
 SMAD4ex10*18q21
 SMAD4ex13*18q21
 DCCex418q21
 MBD2ex218q21
Chr. 19 (n = 11) 
 STK11ex2*19p13
 STK11ex719p13
 STK11ex919p13
 APC2ex2–319p13
 INK4Dex119p13
 BRG1ex2*19p13
 CEBPA19q13
 E1Bex9–1019q13
 BAXex419q13
 BAXex719q13
 PUMAex219q13
Chr. 20 (n = 5) 
 RBL1ex820q11
 PTPRTex1220q12–13
 HNF4αex320q13
 CEEPB20q13
 TNFRSF6Bex120q13
Chr. 21 (n = 4) 
 D21S1433-ANA*21q11
 TMPRSS2ex3*21q22
 TMPRSS2ex721q22
 TMPRSS2ex1321q22
Chr. 22 (n = 14) 
 BIDex322q11
 SNFSex7*22q12
 D22S429*22q12
 SEZ6Lex222q12
 CHK2ex122q12
 KREMex222q12
 BAM22ex422q12
 LARGEex3*22q12
 p300ex222q12
 NF2ex1*22q13
 D22S939–NF2*22q13
 NF2ex822q12
 NF2ex1622q13
 BIKex222q13
Chr. X (n = 3) 
 DYSTex4Xp21
 CX32ex2Xq13
 HPRTex3Xq26

A first panel of 37 cell lines was tested for all 246 loci (Table II). Each negative PCR was performed twice. Bona fide homozygous deletions were confirmed using external, nonoverlapping primer pairs. HD confirmation by duplex PCR was not used as it does not rule out negative PCR attributable to mismatches on primer annealing sequences. The extent of the different HDs were then established step-by-step by amplification of flanking exons or flanking STS.

Reverse transcriptase and PCR

Samples with HD were studied at the RNA level. Five micrograms of total RNA were reverse transcribed at 37°C using 200 units of Superscript reverse transcriptase (Life Technologies) and 300 ng of pd(N)6 random hexameres (Pharmacia, Orsay, France) in 12 μl final. One microliter of the produced cDNA was amplified according to a nested PCR procedure. Amplified fragments were purified from agarose gels (Qiaex II kit, Qiagen, Courtaboeuf, France) and ligated in the TOPO-TA cloning vector (InVitrogen, Cergy-Pontoise, France). Sequencing was performed using Thermosequenase (USB, Amersham, Orsay, France) on an ABI automated fluorescent sequencer (Applied Biosystems, Courtaboeuf, France) at the ESGS sequencing facilities (Evry, France).

RESULTS

Homozygous deletion screen

In a first step, a panel of 37 tumor cell lines was tested for HDs at 246 distinct loci (Tables I and II). Seventeen of these loci showed HDs and were further examined in a second panel of 52 cell lines. We decided to perform HD profiling in various tumor types for two reasons: (i) many TSGs are ubiquitously expressed and active in most human tissues and (ii) some TSGs frequently altered in certain tumors were identified through abnormalities detected in cancer types marginally involved (e.g., BRCA2 was localized in part by an HD in a pancreatic cancer).8 A large proportion of the cell lines studied were established from tumors of epithelial origin (n = 69/89, 77%) with, among them, important subsets of lung (n = 22), ovarian (n = 19) and head and neck squamous cell carcinoma (HNC; n = 13). Other carcinomas included tumors of the breast, prostate, colon and cervix. The remaining cell lines were mostly derived from mesenchymal tumors (blood, muscle, kidney, bone) (Table I). All chromosomes were investigated at 3 to 36 loci. The vast majority (230/246, 92%) of the studied loci were either previously shown to be homozygously deleted or considered as harboring candidate TSGs. The remaining 16 loci can be considered as markers of genomic stability (7 subtelomeric STS, 6 housekeeping gene exons and 3 STS at fragile sites) (Table II). When no amplification product was detected, we performed a confirmatory PCR using an external primer pair to rule out the possibility that the lack of a detectable PCR product was the result of a sequence polymorphism at one of the primer's sequence. In total, 53 HDs (53/9,986, 5.3% PCR) were found at 17 independent loci in 40 cell lines of 89 (44%) (Table III).

Table III. Homozygous Deletions (HDs) in Tumor Cell Lines1
 P73MSH2LRPDITFHIT4QTEL11INK4A/ARFPTENPBWWOX17PTEL80MAP2K4P53MAP2K3SMAD4STK11TMPR5S2NF2
 1p362p162q213p144qtel9p2110q2313q1416q2417ptel17p1317p1317p1218q2119p1321q2222q11
  • LC, lung carcinoma; OC, ovarian carcinoma; SCC, head and neck squamous cell carcinoma; BC, breast carcinoma; BT/NB, brain tumor/neuroblastoma; LL, leukemia-lymphoma; PC, prostate carcinoma; CC, cervix carcinoma; Misc, miscellanous tumors.

  • 1

    The ratio of HDs for each cell type is indicated in brackets. Homozygously deleted exons are indicated.

  • 2Exons 1 of INK4A and ARF are indicated as exon 1a (alpha) and exon 1 b (beta), respectively.–−/−, HDs at STS on 4q and 17p.

LC (8/22)     ex1b–ex3           
 A549     ex1b–ex3     ex2–11    ex2–3
 CALU1     ex1b–ex3           
 GILI5C     ex1b–ex3           
 NCIH69        ex6–R        
 NCIH378                 
 NCIH1299           ex2–9     
 SC10ex1–6                
 SKMES1  ex4–5  ex1b–ex3           
OC (7/19)                 
 59M     ex1a–ex2           
 ES2         −/−       
 OVCAR3  ex4–6              
 OAW28          ex1–11 ex1–12    
 PEO4     ex1b–ex3  ex4–8       ex1
 PEO16         −/−       
 SKOV3     ex1b–ex2           
SCC (9/13)                 
 BB49     ex1a–ex3           
 CAL165     ex1a–ex3           
 CAL166     ex1a–ex3           
 FADU             ex1–11   
 PCI52     ex1b–ex3           
 SCC9   ex5 ex1b–ex1a           
 SCC15   ex5             
 SCC25    −/−ex1a–ex3         ex1–13 
 SW579     ex1b–ex3           
BC (2/4)                 
 MDAMB231     ex1b–ex3           
 MCF7     ex1a–ex3           
BT/NB (2/4)                 
 SKNSH     ex1b–ex3           
 U373     ex1b           
LL (5/12)                 
 CEM     ex1a–ex3ex2–5          
 HL60     ex2     ex2–11     
 JURKAT     ex1b–ex3           
 K562     ex1b–ex3   −/−       
 NB4     ex1b–ex3           
PC (1/3)                 
 PC3      ex3–9          
CC (2/3)                 
 C33A   ex5             
 HELA              ex1–4  
Misc. (4/9)                 
 LOVO ex3–8 ex5             
 HT29   ex5             
 SAOS2       ex23–25   ex2–11     
 U2O5  ex4–7              

General instability in tumor cell lines

Six housekeeping genes (albumin on 4q, β-actin on 7p, β-globin on 11p, α-tubulin on 2q, GAPDH on 12p, HPRT on Xq) were tested to assess the importance of HDs as potentially nonspecific events in tumor cell lines. No HD was detected at these loci. Unbalanced translocations resulting from terminal deletions and subsequent chromosome fusions represent recurrent alterations in epithelial tumors.16 Therefore, we suspected that in some cases both telomeres may be lost at major targets of LOH. We tested subtelomeric markers (<300 kb from telomeres) at 7 chromosomes (1p, 4q, 6q, 8p, 13q, 16q, 17p) that were previously found deleted in numerous carcinomas.17, 18 Two of these markers, 4QTEL11 and 17PTEL80, were found to be homozygously deleted in 1 (SCC25) and 3 (PEO16, ES2, K562) cell lines, respectively, indicating that homozygous loss of chromosome ends can occur occasionally, albeit at a low frequency (4 HDs of 363 telomeres tested; Table III). Abnormalities at 17PTEL80 were not associated with homozygous deletions of p53. To our knowledge, this is the first time that such alterations are detected by a molecular method in human cells. HD in 4qtel was then studied in detail since 4q35 is a prominent target for loss of heterozygosity in liver cancer, one of our main focuses of interest. According to the last versions of human genome sequence, in SCC25, a 4.7 Mb segment was biallelically removed from the 4q35 region. This relatively gene-poor region contains only a few candidate TSGs, among them the gene encoding the melatonin receptor 1A (MNTR1A) known to exert antiproliferative effects on various tumor cells.19 The 89 fragile sites (FS) mapped on human chromosomes represent additional candidate regions for HD search.20 Three of them were tested in our survey. D7S522 mapping at FRA7G (7q31) did not exhibit any instability in cell lines. In contrast, FRA3B (D3S1300 on 3p14) was homozygously deleted along with exon 5 of the FHIT gene in 5 cases (6%) (Table III). The WWOX gene, a putative tumor suppressor mapping at close range of another common fragile site, FRA16D (16q23), was deleted in 2 samples (2.5%) (Table III).

Variations of HD incidence

A high proportion (9/13, 69%) of HNC cell lines showed at least 1 HD (Table III), whereas the proportion was lower (approx. 40%) for lung or ovarian carcinoma cell lines as well as for malignant blood cells. In ovarian and lung cell lines, however, a multiplicity of targets were involved (7 and 6, respectively), indicating an apparent complexity of carcinogenesis in these tumor types (Table III). Remarkably, 11 cell lines (11/89, 12%) displayed more than 1 HD (as many as 3 in SCC25 and PEO4), whereas the other samples appear more stable. This suggests the existence of variable sensibilities to limited biallelic loss among the different tumors.

p16-INK4A/p14-ARF/p15-INK4B locus

Four STS (p14 exon 1β, p16 exonα and p14-p16 exons 2 and 3) mapping to the p16-INK4A/p14-ARF locus were systematically studied in the 89 samples to cover the region in detail. In 23/89 cell lines (approx. 26%), an HD was detected (Table III and Fig. 1).6 A significantly higher prevalence of HDs was detected in leukemia-lymphoma and in HNC cells (41–61%) when compared to ovarian and lung cancer cell lines (18–21%, χ,2p < 0,05). P16-INK4A and p14-ARF genes were co-inactivated in most cases (22/23, 95%) (Fig. 1). However, in the astrocytoma cell line, U373, a lesion was found only in p14-ARF.

Figure 1.

Homozygous deletions at the p16-INK4A/p14-ARF/p15-INK4B locus. Fine deletion map of the locus. Filled boxes represent an HD and empty boxes symbolize a retained locus. Tissue origins of the different cell lines are indicated on the left. The abbreviations are explained in Table I.

Other targets

The second most frequently deleted target was FHIT in 3p14 (5/89 cases, 5.5% of the cell lines). The double inactivation of this gene, which encodes a diadenosine triphosphate hydrolase involved in stress response signaling, is common in carcinomas.21 However, we found it to be restricted to only 3 specific cell types (HCN, colorectal and cervix cells) (Table III). All HDs were limited to a single exon (exon 5) of FHIT mapping at a close range from FRA3B. Such an alteration results in the removal of the initiation codon and the first 32 amino acids (Table III). Multiple HDs were also observed at the TP53, LRPDIT, PTEN, WWOX and NF2 genes (4, 3 and 2 for the 3 latter, respectively) in cell lines established from several tumor types (Table III). Mutations in the WWOX gene have been reported previously.22 In the case of the TP53 tumor suppressor, initiation codon (exon 2) and most or all of the coding region were homozygously deleted in 4 cell lines of lung, bone and blood origin. The recently identified low-density lipoprotein-related protein 1B (LRPDIT) gene was homozygously deleted in 3 cell lines (Table III).23 The 3 samples (SKMES1, OVCAR3 and U2OS) established from different tissue types were altered through various internal deletions involving exons 4–7 that did not affect the reading frame (Fig. 2). Our findings represent the first reports of HD affecting LRPDIT in ovarian cancer as well as osteosarcoma. The PTEN gene is a potent growth suppressor negatively regulating cell-matrix interactions as well as phosphoinositide-3-phosphate kinase signalling pathways. Initially found mutated in glioblastomas as well as prostate and breast cancers, PTEN mutations were detected later in leukemias and endometrial cancers.24 HDs were detected in the PC3 prostate cancer cells and in the CEM lymphoblastic leukemia. For the already known double deletion of PC3, no PTEN transcript was detectable due to a deletion of all or most of the coding region, whereas in CEM, removal of exons 2–5 resulted in abnormal transcripts with introduction of a premature stop codon (Fig. 2).25 The NF2 gene, which encodes the cytoskeleton-associated merlin protein,26 was homozygously lost in a lung (CALU1) and an ovarian (PEO4) cell line. Exon 1, containing the initiation codon, is removed in PEO4, whereas in CALU1 cells internal deletion of exons 2–3 preserves the frame of the transcript (Fig. 2). Interestingly, a homologous murine mutant form lacking exons 2–3 was shown to be transforming rather than tumor suppressive in transgenic mice.27 HDs reported in the current survey represent the first cases of mutation affecting NF2 in lung or ovarian cancers.28, 29

Figure 2.

Representative examples of RT-PCR analysis on samples harboring HD in selected genes. (a) Lane MWM contains a 100 bp ladder from Bohringer. Genes and cell lines studied are indicated. PTEN, nested amplification of PTEN with primers mapping in exons 1 and 7. No transcript is detected in PC3. CEM cells display a 443 bp amplimere corresponding to homozygous deletions of exons 2–5. The additional 588 bp band in CEM corresponds to an aberrant splicing product including the initial 145 bp from intron 1. The 857 bp amplification product in HELA cells represents the wild-type transcript. NF2, nested amplification of NF2 with primers mapping in exons 1 and 5. A 188 bp amplification product corresponding to homozygous loss of exons 2–3 is detected in CALU1. No transcript is detected in PEO4 cells. HELA provides the 423 bp fragment obtained from the normal transcript. LRPDIT, nested amplification of LRPDIT with primers mapping in exons 2 and 8. The 846 bp product detected in OVCAR3 corresponds to the homozygous deletions of exons 4–6. No transcript is detectable in SKMES1, whereas in U2OS, 2 products are amplified: The 942 bp band corresponds to the biallelic loss of exons 4–5 and 7, while the 545 bp RT-PCR product is an additional isoform resulting from splicing between exons 3 and 7. All abnormal amplimeres were sequenced. STK11, nested amplification of STK11 with primers mapping in exons 1 and 4. No transcript is detectable in HELA cells. PEO4 provides the 489 bp fragment obtained from the wild-type transcript. (b) Vinculin (VCL) amplification (480 bp) was performed as a control of cDNA integrity.

Single HDs were found at 8 additional loci, RB, SMAD4, MSH2, MAP2K3, MAP2K4, TMPRSS2, STK11 and TP73, genes in ovarian, colorectal, lung, cervix or bone tumor cells (Table III). HDs at the 3 former loci confirmed previous work.30, 31, 32 A double deletion of STK11 was found in HELA cells (Table III). The STK11 gene encodes a serine-threonine kinase interacting with p53 and involved in mediating the p53-dependent apoptotic program.33 It is found mutated in patients suffering from Peutz-Jeghers syndrome as well as in selected sporadic tumors.34 In HELA cells, HD removes the initiation codon together with the 155 N-terminal amino acids, thus likely precluding the production of the wild-type protein. It is well known that cross-contamination of cell lines by HELA is a widespread phenomenon.35, 36 Consequently, we tested 2 HELA cross-contaminants, CHANG-CCL13 and HEP2 for HD at STK11. Both cells lines were homozygously deleted for exons 1–4, indicating that this alteration is not restricted to bona fide HELA cells but rather represents an ancient event preceding cross-contaminations (data not shown). No STK11 transcript was detected in HELA by RT-PCR (Fig. 2). This is the first example of a mutation affecting STK11 in a cervical carcinoma. TMPRSS2, encoding a plasma membrane serine protease, was formerly found deleted in a pancreatic cell line, whereas we detected an HD in the SCC25 HNC cell line in which all exons are removed (Table III).37 The exact functions of TMPRSS2 as well as the other members of the hepsin family of serine proteases remains unknown in the neoplastic process.38, 39 Two HDs affecting the signal transduction machinery were found in OAW28 cells established from an ovarian carcinoma. Both for MAP2K3 and for MAP2K4 all exons were deleted. MAP2K4 is suspected to be a tumor suppressor for pancreatic and breast cancer, where it is found mutated in a small proportion of cases. The role of MAP2K3 in cancer is still largely unknown.40 Our current study is the first to report HD affecting MAPKs in ovarian carcinoma. Finally, an HD affecting TP73 in a small cell lung carcinoma cell line (SC10) was detected. A single mutation among 114 cases of lung cancer screened has been reported previously.41 Significantly, HD affected the 5′ region of the gene encoding the transactivation domain responsible for cell cycle arrest and for the apoptotic properties of p73.42

We found no abnormalities in 40 of 57 loci that had tested positive previously in at least 1 tumor or cell line specimen. The most significant of these negative loci are BRCA2, CASP8, CDH6, MEN1, NF1, BRG1, SNF5, TGFBR1 and WT1. No recurring associations of HDs were noticed among the 11 cell lines displaying at least 2 HDs (Table III).

DISCUSSION

The search for homozygous deletions in tumors and cell lines is considered a powerful tool to identify TSGs. This prompted us to perform a comprehensive scan for HD in a large panel of cell lines with the aim of revealing genes targeted for mutation in the course of carcinogenesis. We detected a significant number of HDs (n = 53) on 17 different loci of the 246 tested. Some of the data presented here confirm previously published cases of HDs (SMAD4 in FADU, MSH2 in LOVO, RB in SAOS2, both WWOX deletions and some of the p53, FHIT and p16-INK4A cases).22, 32, 43 However, we provide evidence for novel examples of HDs at some of these loci and, more significantly, we report deletions at new loci.

In most cases, TSG loss of function results from a combination of genetic abnormalities including LOH, point mutations or promoter methylation, whereas HD generally represents an infrequent mechanism of TSG inactivation. The only notable exceptions are the p16-INK4A/p14-ARF/p15-INK4B and SMAD4 loci where HDs are frequent. Remarkably, for a given gene, the rate of HDs is not necessarily representative of the global incidence of biallelic inactivation, and certain TSGs appear to be almost never homozygously deleted. For example, SOCS1 and IGF2/M6PR, previously shown to be inactivated by methylation and LOH/point mutation in more than half of the liver cancers studied,44, 45 have as yet not been shown to be affected by HDs. Furthermore, significant variations in HD prevalence among tumor types exist for a given TSG. For example, mutation rates of SMAD4 are similar in pancreatic and colorectal cancers (40–50%). Yet, in pancreatic xenografts, HDs of SMAD4 represent 65% of all mutations, whereas in colorectal cell lines, HDs are found in only 30% of mutated samples.5, 46 Thus, these data suggest that defining a cutoff of significance for HDs at a given TSG may be misleading. Therefore, global biallelic inactivation rate, taking into account LOH, point mutations, methylation and HD, may be the only value providing accurate information about the importance of a TSG in a given tumor type.

Regarding bona fide and candidate TSGs associated with particular neoplasms in our present work, further studies are required to assess the actual percentages of mutations in the corresponding cancers. We already know that there is only a low prevalence of abnormalities at TP73 and MAP2K4 in most tumors studied so far,40, 41 and it is thus possible that this situation also applies to lung, ovarian or head and neck carcinomas for other candidates identified in our present study (NF2, LRPDIT, STK11, MAP2K3). However, in tumorigenic pathways, all intervening partners are not evenly mutated. In lung cancer, for example, RB, p16-INK4A and cyclin D1, which interact at the same step of cell cycle, are altered at different rates.47 Therefore, we can hypothesize that HDs detected in our present survey may represent only fractions of all abnormalities affecting their respective growth control pathways. There is, in addition, a possibility for HDs found in cell lines to be in vitro-selected events. However, most of the works leading to TSG identification associate fresh tumors and established cell lines. In these studies, differences between both kinds of samples rely on the magnitude of mutation rates detected rather than on discrepancies such as absence/presence of mutations.5, 7, 9, 48

Our study revealed the presence of deletions in well-established TSGs not previously associated with certain types of cancers. Here, we describe for the first time HDs at NF2 in lung and ovarian carcinomas as well as at STK11 in a cervical carcinoma. In addition, our present survey disclosed potential roles for LRPDIT, MAP2K3, MAP2K4 and TMPRSS2 in ovarian, bone and head and neck tumors (Table III). NF2 is known to be mutated in familial or sporadic neoplasms of the nervous system.49NF2 has, however, been associated with mutations in seemingly unrelated tumors such as breast and colon carcinomas, melanomas and mesotheliomas.50 According to our data, it is possible that NF2 might also be implicated in lung and ovarian tumors. Therefore, it is reasonable to assume that the spectrum of NF2 mutations is larger than previously believed. STK11 (also termed LKB1) was recently shown to have a key role in p53-induced apotosis or in BRG1-induced growth arrest.33, 51 STK11 is mutated in a restricted set of sporadic tumors (testicular tumors, melanoma, biliary and pancreatic cancer).52, 53, 54 Our data suggest that STK11 may also be involved in cervical carcinoma since HELA cells display a double deletion affecting exons 1–4. Surprisingly, the HD at STK11 locus was hitherto unknown despite the conspicuous absences of STK11 mRNA and protein in previous expression and methylation studies.55, 56LRPDIT encodes for a large plasma membrane receptor protein of 4,599 amino acids. HDs in LRPDIT were found originally in lung, kidney and bladder cancer cell lines and were shown more recently in high-grade bladder cancers.23, 57 Here we provide evidence suggesting that LRPDIT is also an HD target in ovarian carcinoma as well as in osteosarcoma. The impact of LRPDIT deletion on tumorigenesis is unknown, but its preferential alterations in advanced tumors suggest that it is not a rate-limiting event but rather involved in tumor progression. Finally, we describe the first homozygous deletion in TP73. p73 has significant sequence and functional similarities with p53. However, together with the third member of the family, p63, p73 is involved in development rather than acting as a tumor suppressor. The TP73 gene encodes different protein variants, 2 major forms of which differ by the presence or the absence of the first 3 exons and thus display highly divergent activities.41 Significantly, in the SC10 cell line, the 5′ region of p73 has been homozygously lost, thus inactivating the impact of p73 on apoptosis and cell cycle. Our data suggest, therefore, that in selected cases, lung cancer may be partially controlled by p73 activity. Another finding lies in the homozygous losses of telomeres 17p and 4q. It has been recently hypothesized that, preceding telomerase activation in tumors, chromosome attrition may lead, in some circumstances, to chromosome breaks subsequently stabilized through unbalanced translocations.16 According to the present screen, such attrition-driven homozygous loss occurs for more than 1% of telomeres tested. In the panel of cell lines used, chromosome 17p is a prominent target for such phenomenon, being involved in 3 HDs (Table III). In addition, chromosome 17p as a whole appears to undergo a surprisingly wide spectrum of alterations with 3 additional loci affected by HD (TP53, MAP2K3 and MAP2K4).

The main purpose of our present survey was to identify previously unknown associations between genetic pathways and certain types of carcinomas as well as to find novel TSG. Among the 197 different genes tested for HD, only 17 (8.6%) were positive, with all but TP73 previously reported to undergo double deletion. Although we cannot rule out the possibility that HDs may occur at random during tumorigenesis, our data suggest that such alterations preferentially involve unstable regions of the genome or, alternatively, are selected when they affect targets that control cellular growth. The findings of HDs at bona fide TSGs not typically associated with certain tumors suggest that some pathways, thus restricted to specific neoplasms, may have a more general involvement in tumorigenesis (e.g., STK11 in cervical carcinoma). Another salient feature of our current study is the diversity of HD targets in lung, ovarian or even head and neck carcinomas, thus emphasizing the multiplicity of pathways implicated in sporadic cancers. Finally, our data provide novel information for tumor cell lines widely used as in vitro models in cancer research (e.g., CALU1 or HELA). More generally, the present HD scanning of a tumor cell line panel represents a pilot study toward a detailed genetic annotation of common cellular models. These results should thus complement existing and future data on the transcriptome, on the proteome, the allelic status at critical loci as well as on the mutation and methylation status of recurrently altered genes.

Acknowledgements

We thank Dr. P. Coulie (Université Catholique de Louvain, Faculté de Médecine, Unité de Génétique Cellulaire, Bruxelles, Belgium), Dr. O. Delattre (Laboratoire de Pathologie Moleculaire des Cancers, INSERM U509, Institut Curie, Paris, France), Dr. J.-L. Fischel (Unité d'Oncopharmacologie, Centre Antoine Lacassagne, Nice, France), Dr. H. Gabra (Medical Oncology Unit, Western General Hospital, Edinburgh, United Kingdom), Dr. P. Gauduchon (Laboratoire de Cancérologie Expérimentale, Centre François Baclesse, Caen, France), Dr. S. Jozan (Laboratoire d'Histologie-Embryologie, Faculte de Medecine Toulouse-Rangueil, Toulouse, France), Dr. B.M. Kacinski (Department of Therapeutic Radiology and Dermatology, Yale University School of Medicine, New Haven, CT), Dr. J.-L. Merlin (Laboratoire de Recherche en Oncologie, Centre Alexis Vautrin, Vandoeuvre-les-Nancy, France) and Dr. H. Rochefort (Laboratoire de Biologie Cellulaire, INSERM U148, Faculté de Médecine-CHU, Montpellier, France) for providing us with cell line samples. We are grateful to Dr. J.S. Seeler for careful reading and helpful discussion of the manuscript.

Ancillary