[ RESEARCH 60, 6116–6133, November 1, 2000] The 630-kb Lung Cancer Homozygous Deletion Region on Human 3p21.3: Identification and Evaluation of the Resident Candidate Tumor Suppressor Genes1

Michael I. Lerman2 and John D. Minna2 for The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Consortium3 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593 [J. D. M.], and Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [M. I. L.]

ABSTRACT FUS1, HYAL1, FUS2, and SEMA3B. However, none of the 19 tested for mutation showed a frequent (>10%) mutation rate in lung cancer We used overlapping and nested homozygous deletions, contig building, samples. This led us to exclude several of the genes in the region as genomic sequencing, and physical and transcript mapping to further classical tumor suppressors for sporadic lung cancer. On the other hand, define a ϳ630-kb lung cancer homozygous deletion region harboring one the putative lung cancer TSG in this location may either be inactivated by or more tumor suppressor genes (TSGs) on chromosome 3p21.3. This tumor-acquired promoter hypermethylation or belong to the novel class of (location was identified through somatic genetic mapping in tumors, cancer haploinsufficient genes that predispose to cancer in a hemizygous (؉/؊ cell lines, and premalignant lesions of the lung and breast, including the state but do not show a second mutation in the remaining wild-type allele discovery of several homozygous deletions. The combination of molecular in the tumor. We discuss the data in the context of novel and classic cancer manual methods and computational predictions permitted us to detect, gene models as applied to lung . Further functional testing isolate, characterize, and annotate a set of 25 genes that likely constitute of the critical genes by gene transfer and gene disruption strategies should the complete set of -coding genes residing in this ϳ630-kb se- permit the identification of the putative lung cancer TSG(s), LUCA. quence. A subset of 19 of these genes was found within the deleted overlap Analysis of the ϳ630-kb sequence also provides an opportunity to probe region of ϳ370-kb. This region was further subdivided by a nesting and understand the genomic structure, evolution, and functional organi- 200-kb breast cancer homozygous deletion into two gene sets: 8 genes zation of this relatively gene-rich region. lying in the proximal ϳ120-kb segment and 11 genes lying in the distal ϳ250-kb segment. These 19 genes were analyzed extensively by compu- tational methods and were tested by manual methods for loss of expres- INTRODUCTION sion and mutations in lung to identify candidate TSGs from within Lung cancer kills Ͼ150,000 patients each year in the United States and this group. Four genes showed loss-of-expression or reduced mRNA levels many more around the world. These are more deaths than attributable to in non-small cell lung cancer (CACNA2D2/␣2␦-2, SEMA3B [formerly SEMA(V), BLU, and HYAL1] or small cell lung cancer (SEMA3B, BLU, colon, prostate, and breast cancer combined (1–4). The scale of this and HYAL1) cell lines. We found six of the genes to have two or more epidemic has heightened efforts to understand the molecular pathogenesis amino acid sequence-altering mutations including BLU, NPRL2/Gene21, of lung cancer, including genes frequently undergoing somatic mutation. The isolation of such “lung cancer genes” should guide the development Received 5/4/00; accepted 8/29/00. of new therapeutic interventions and new early detection and prevention The costs of publication of this article were defrayed in part by the payment of page strategies. Although tobacco smoking is a well-established environmental charges. This article must therefore be hereby marked advertisement in accordance with etiology in lung carcinogenesis (5), our understanding of the acquired 18 U.S.C. Section 1734 solely to indicate this fact. 1 The University of Texas Southwestern Medical Center Group was supported by genetic changes leading to lung cancer is still rudimentary and incomplete National Cancer Institute Grants CA71618, SPORE P50 CA70907, the G. Harold and (6). The lack of (and difficulty of performing) genetic linkage studies of Leila Y. Mathers Charitable Foundation, and the Leroy “Skip” Malouf Foundation. The lung cancer in families (4, 7) that have the potential of identifying University of Texas Southwestern group received technical support from Sherrie Cundiff, Yvonne Feller, Gina Mele, and Mina Viswanathan. The National Cancer Institute Group initiating cancer causing genes has directed the search toward allele loss has been funded in whole with Federal funds from the National Cancer Institute, NIH, mapping in tumors, cell lines, and premalignant lesions of the lung and under Contract NO1-CO-56000. The United Kingdom Group was supported by grants from Cancer Research Campaign, The Royal Society, and Fundacao para a Cientia e a breast (8–13). A convergence of evidence from these allele loss mapping Technologia. The Karolinska Institute Group was funded by grants from the Swedish studies, including identification of overlapping homozygous deletions, Cancer Society, Karolinska Institute, and the Royal Swedish Academy of Science (to E. Z. strongly suggests the presence of a TSG4 in the chromosome 3p21.3 band and to G. K.). The National Cancer Institute and University of Texas Southwestern groups contributed equally to this work. (6, 9, 10, 14). Allele loss in the 3p21.3 area is the earliest premalignant 2 To whom requests for reprints should be addressed (M. I. L.) at Laboratory of change thus far detected in lung cancer development (12, 15, 16). Bial- Immunobiology, National Cancer Institute, FCRDC, Frederick, MD 21702. Phone: (301) lelic or monoallelic inactivation of this putative TSG gene(s) likely 846-1288; Fax: (301) 846-6145; E-mail: [email protected] or (J. D. M.) at Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical represents a critical (rate-limiting) step in the development of sporadic Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-8593. Phone: (214) 648-4900; lung cancer. As part of our efforts to identify a lung cancer TSG (which Fax: (214) 648-4940; E-mail: [email protected]. 3 University of Texas Southwestern Medical Center at Dallas, United States of Amer- we will provisionally call LUCA) on 3p21.3, we physically mapped and ica Group (Yoshitaka Sekido, Scott Bader, David Burbee, Kwun Fong, Eva Forgacs, cloned the genomic DNA surrounding this as defined by homozy- Boning Gao, Harold Garner, Adi F. Gazdar, Luc Girard, Craig Kamibayashi, Masashi gous deletions (6, 9, 10, 14, 17–20). Subsequently, the ϳ630-kb clone Kondo, Ashwini Pande, Alex Persemlidis, Vivek Ramalingam, Dwight Randle, Yoshio 5 Tomizawa, Arvind Virmani, Ivan Wistuba, Grace Zeng, and John D. Minna); University contig was sequenced jointly by The Washington University and The of Birmingham, United Kingdom Group (Donia Macartney, Sofia Honorio, Eamonn Sanger6 Sequencing Centers. In more recent work, we Maher, and Farida Latif); The Karolinska Institute, Sweden, Group (Vladimir Kashuba, Alexei Protopopov, Jingfeng Li, George Klein, and Eugene Zabarovsky); and National Cancer Institute, United States of America Group [Intramural Research Support Program, 4 The abbreviations used are: TSG, ; SCLC, small cell lung Science Applications International Corporation-Frederick, and Laboratory of Immunobi- cancer; NSCLC, non-small cell lung cancer; NCI, National Cancer Institute; LOH, loss of ology, National Cancer Institute, Frederick Cancer Research and Development Center, heterozygosity; EST, expressed sequence tag; MTN, multiple tissue Northern blots from Frederick, Maryland 21702] (Fuh-Mei Duh, Ming-Hui Wei, Laura Geil, Igor Kuzmin, and ClonTech; SSCP, single strand conformation polymorphism; ORF, open reading frame; Sergei Ivanov; F-M. D. and M-H. W. contributed equally to this work), [Laboratory of NLS, nuclear localization signal; FISH, fluorescence in situ hybridization; VWA, Von Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Willebrand factor type A; PKC, protein kinase C; RT-PCR, reverse transcription-PCR; Center, Frederick, Maryland 21702] (Debora Angeloni, Alla Ivanova, Bert Zbar, and DAG, diacylglycerol. Michael I. Lerman), and [Medicine Branch at the National Naval Medical Center, 5 Internet address: http://genome.wustl.edu/gsc/human/chrom3.shtml. National Cancer Institute, NIH, Bethesda, Maryland 20892] (Bruce E. Johnson). 6 Internet address: http://www.sanger.ac.uk/cgi-bin/hummap?mapϭ3p21.3. 6116

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. 3p21.3 TUMOR SUPPRESSOR GENE POSITIONAL CLONING placed the putative 3p21.3 TSG(s) in a ϳ120-kb segment that was Informatics Tools defined by a homozygous deletion in a breast cancer specimen that was The software package, GENSCAN (27), was licensed from Christopher Burge nested within the three small cell lung cancer homozygous deletions (10). (MIT, Cambridge, MA) and installed at the NCI Advanced Biomedical Comput- In parallel with these genetic and physical studies, we have been con- ing Center at the Frederick Cancer Research and Development Center. The inte- structing a map of transcript sequences with the aim to identify a com- grated informatics package, PANORAMA, incorporating BLAST, GENSCAN, plete set of all transcripts encoded in the region and to define/annotate the GRAIL, and other gene interpretation features, was developed at University of respective genes. Here we report the catalogue of genes we have discov- Texas Southwestern Medical Center at Dallas (TX) by H. Garner and run on a ered to be residing in the 630-kb sequence and their experimental and Hewlett Packard Exemplar supercomputer. PANORAMA is available for internet informatics characterization. Of these, only two “G protein” genes, i.e., use.8 For this analysis, GenBank was downloaded December 1999. GNAI2 (21) and GNAT1 (22), had been cloned and characterized previ- ously, and from this catalog we positioned these two genes within the Manual Molecular Procedures contig DNA sequence. The set of 19 genes found in the overlapping All molecular manipulations (DNA and RNA isolations, screening genomic homozygous deletions in SCLCs NCI-H740, NCI-H1450, and GLC20 and cDNA libraries, Northern and Southern blot analyses, and PCR) were (18), including eight in the smaller critical 120-kb sequence defined by a performed using standard methods according to Sambrook et al. (28). For breast homozygous deletion (10), were analyzed extensively. We used DNA sequencing, cDNA clones were sequenced on an Applied Biosystems both manual experimental methods to study expression and search for 373 or 377 DNA sequencer (Stretch) using Taq Didroxy Terminator Cycle mutations and web-based computational servers to predict possible pro- Sequence kits (Applied Biosystems, Foster City, CA) with either vector or tein functions. Four of the genes by Northern analysis showed frequent clone-specific walking primers. Cosmid and P1 phage DNAs were sequenced reduced or absent mRNA levels in NSCLC (CACNA2D2, SEMA3B, by the Washington University and Sanger Human Genome Sequencing Cen- BLU, and HYAL1) and SCLC (BLU, HYAL1, and SEMA3B) cell lines. ters using the shotgun procedure as described (21, 22). FISH and two-color We found that six of the genes had mutations, but none of the 19 genes FISH were used to locate and orient the cosmid contig on chromosome 3p. Normal metaphase were hybridized simultaneously with showed a high frequency of mutations (Ͼ10%) in the analyzed lung digoxygenin-labeled NotI linking clone NL1–210, part of cosmid LUCA1, tumor samples. This raises the possibility that the putative TSG, LUCA, (green) and biotin-labeled cosmid LUCA20 (red) (29). 4Ј,6-Diamidino-2- may be one of the genes with frequent loss of expression that occurs phenylindole was used as a counterstain. Both the metaphase spreads and through acquired tumor promoter hypermethylation (23). Alternatively, it interphase nucleus staining confirmed the single-site location of each probe on could belong to the class of haploinsufficient TSGs. This novel class of 3p21.31, establishing the following order: centromere-cosmids LUCA1- TSGs is predicted to predispose to cancer in a hemizygous (ϩ/Ϫ) state LUCA20-telomere. Pulsed-field gel electrophoresis analysis was performed as but does not show a second hit in the remaining wild-type allele in follows. High molecular weight DNA was prepared in agarose plugs as tumors. Further functional experimental analysis such as growth suppres- described (30). Slices containing ϳ106 cells were digested for 16 h with 50 sion studies and gene knockout strategies will be required to reveal the units of (NotI, Nru1, and Mlu1; Boehringer Mannheim) and resolved identity of the putative 3p21.3 TSG(s). In addition, our study shows that on 1% agarose gels using a Bio-Rad CHEF Mapper (Hercules, CA) and electrophoresis profiles, allowing separation in the range 50–1000 kb. For genomic DNA sequencing in combination with high-quality gene anno- expression analyses, Northern blot hybridization was performed with cDNA tation is an effective method of gene discovery. probes using commercial MTN poly(A)ϩ RNA blots ClonTech (Palo Alto, CA) from a variety of adult human tissues and tumor cell lines and in-house blots with total or poly(A)ϩ RNA were prepared from lung cancer cell lines. MATERIALS AND METHODS Radioactive DNA probes were prepared by random priming Rediprime II (Amersham, Arlington Heights, IL). Hybridization was performed in Ex- Cell Lines and DNA Samples pressHyb hybridization solution according to manufacturer’s instructions Lung cancer cell lines were started and maintained by us using published (ClonTech Laboratories, Palo Alto, CA). In addition, the presence of gene methods, and information on the lines are summarized in the NCI-Navy transcripts was monitored in silico by BLAST homology searches (31) in collection (American Type Culture Collection; Ref. 24). DNA from the SCLC public EST databases. Mutational analyses were performed by RT-PCR-SSCP line, GLC20 (17), was a gift of C. H. C. Buys (University of Groningen, or exon-PCR-SSCP, followed by sequencing of shifted bands as described Groningen, the Netherlands); the SCLC cell line, U2020 (25), was a gift of P. previously (32, 33). Experimental gene discovery by using conserved and Rabbitts (MRC, Cambridge United Kingdom). Lung cancer normal/tumor transcribed genomic fragments was performed as detailed previously (18). paired DNA samples were from the NCI-Navy collection established by B. Johnson (26). Computational and Bioinformatics Procedures

World Wide Web-based Servers and Databases. World Wide Web- Commercial Reagents based servers and databases (34) were used to analyze genomic, cDNA, and The following materials were purchased from the vendors indicated: PCR predicted protein sequences. In addition, the Wisconsin Genetics Computer tool kits, Perkin-Elmer Cetus; DNA sequencing tool kits, Applied Biosystems Group, package 10 (35), and the GENSCAN (27) programs were run at the (Foster City, CA); fluorescent in situ hybridization reagents, Boehringer Advanced Biomedical Computing Center (Frederick Cancer Research and Mannheim (Mannheim, Germany); cDNA and cosmid libraries, ClonTech Development Center), whereas the University of Texas Southwestern Medical Laboratories (Palo Alto CA) and Stratagene (La Jolla, CA); EST clones from Center integrated gene analysis software PANORAMA was run at the Uni- public databases, the I.M.A.G.E consortium7 or Research Genetics (Rockville, versity of Texas Southwestern Medical Center. MD); SSCP tool kits and blotting nylon membranes, Amersham (Arlington DNA and Protein Sequence Analyses. Global sequence alignments were 9 Heights, IL); oligonucleotide primers, Life Technologies, Inc. (Rockville, done using BLAST (31) and Advanced BLAST (36) programs as provided by 10 11 MD); restriction , Life Technologies, Inc., New England Biolabs National Center for Biotechnology Institute, and BLAST2/WU-BLAST. (Beverly, MA), and Amersham (Arlington Heights, IL); MTN poly(A)ϩ RNA Multiple sequence alignments, global and local, were done using the 12 blots, ClonTech Laboratories (Palo Alto, CA); buffers, blotting solutions, and CLUSTAL version W program as provided by EMBL, Baylor Computing RNase free water, Quality Biologicals (Gaithersburg, MD); chemicals, Sigma Chemical Co. (St. Louis, MO); and cell culture media, Life Technologies, Inc. 8 Internet address: http://pompous.swmed.edu/panorama.htm. (Rockville, MD). 9 Internet address:; http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast. 10 Internet address: http://www.ncbi.nlm.nih.gov/. 11 Internet addresses: http://blast.wustl.edu/ and http://www2.ebi.ac.uk/blast2/. 7 Internet address: http://www-bio.llnl.gov/bbrp/image/image.html. 12 Internet address: http://www.bork.embl-heidelberg.de/Alignment/. 6117

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Center,13 and the Wisconsin Genetics Computer Group, package 10 program multiple and often large, making it difficult to define a small consen- (35). Protein structural features were delineated using the EXPASY proteomics sus region that would facilitate a realistic positional cloning effort. tools.14 Protein domains were discovered using Pfam (37) and SMART (38) Fortunately, three overlapping homozygous deletions in lung cancer 15 programs as provided by EMBL. Protein subcellular localization was pre- cell lines (NCI-H740, NCI-H1450, and GLC20; Refs. 9, 17–19, 51) 16 dicted using PSORT (39). Signal peptides, transmembrane helices, and and one in a breast tumor specimen (HCC1500; Ref. 10), in conjunc- membrane topologies were predicted by SPLIT17 (40), TMHMM18 (41), and tion with functional evidence of suppressor activity (9, 52–54), PSORT (39) programs. Protein motifs were found by visually inspecting local alignments or using the protein motifs (42), ProfileScan and Prosite (43) strongly support the existence of a putative TSG(s) in this location and programs. In addition, we used the INTERPRO server (44).19 successively narrowed the critical gene region, first to 370 kb (18), Discovery of Orthologous Genes in Model Organisms. Stringent criteria and more recently, to ϳ120 kb (Ref. 10; Fig. 1). This latest reduction for identification of candidate orthologous genes were applied as suggested of the critical segment by a nesting deletion in a breast (45, 46). In the mouse, orthologous pairs were Ͼ90% identical on the protein assumes that the same gene(s) was targeted in both lung and breast level with Ͼ90% alignment of their entire lengths (47). In the fly, worm, and malignancies (10). However, if the targeted gene(s) were different, yeast, candidates were identified with 20–50% identity over at least 80% of the lung cancer gene(s) could lie in either the ϳ120-kb region or in the their lengths. The TBLASTN program was used to search nonredundant more telomeric ϳ250-kb segment of the 370-kb region. nucleotides, Unigene, and EST databases of the model organisms. EST clusters The genomic DNA cosmid/P1 clone contig (Fig. 1) covering the were then built by the EST assembly machine server20 or the EST Assembler21 overlapping homozygous deletions in 3p21.3 is now sequenced almost at Max Delbru¨ck Center.22 The advanced BLAST2 and Orthologue program ϳ 26 (48) at EMBL was used to confirm the putative orthologous relationships and to completion and is 630 kb long. Fig. 1 summarizes the genetic obtain and ascertain phylogenetic trees. and physical map of the 3p21.3 region on which we have searched for In Silico Gene Discovery Was Performed following Two Different a lung cancer TSG, including contig structure, and successive steps Protocols. Genome-wide repeats and low complexity regions in the genomic leading to the definition of the critical regions of ϳ370 kb, ϳ250 kb, DNA sequences were identified and masked using the program RepeatMas- and ϳ120 kb. We have systematically proceeded to identify and test ker.23 They were then used in BLASTN searches against EST, Unigene, and all of the genes in the region for their candidacy as a TSG (Fig. 1; nonredundant nucleotide databases to identify potential transcripts (ESTs and Table 1). In addition to the genes we detected by manual methods cDNAs) and build EST clusters. Next, genomic sequences assembled from the (such as screening cDNA libraries with cosmid DNAs and using the individual cosmid sequences were subjected to gene prediction programs, i.e., cosmids for exon capture), the genomic sequence allowed us to detect GENSCAN (27) and XGRAIL (49), with default settings to identify coding DNA sequences and corresponding protein sequences. These were then used in other gene candidates in silico. We used BLAST searches for ESTs, BLASTN and TBLASTN searches, respectively, against nonredundant nucle- and gene prediction programs such as GRAIL, GENQUEST, and otide and EST databases to identify ESTs and cDNAs. The ESTs were then GENSCAN (27, 49) including our own integrated informatics tool, 27 assembled into clusters (see above). Genomic information (repetitive elements, PANORAMA. The total number of verified resident genes in the coding exons, ESTs, and known and predicted genes) was also obtained and 630-kb region currently is 25, 6 of which were in the centromeric analyzed by first-pass automatic genome annotation programs, PANORAMA portion of the 630-kb region and outside of the overlap of SCLC or (18), for individual cosmid sequences and by the Rummage package24 for the breast cancer homozygous deletions (Fig. 1; Table 1). For these 25 ϳ whole assembled 630-kb contig sequence. The Rummage analysis was genes, we have reported mutational and expression analysis on 3pK, kindly performed by Drs. A. Rosenthal and R. Schattevoy, both at the Genome IFRD2/SM15, and SEMA3B and laid out the rough outline of some of Sequencing Center (Jena, Austria). Recently, The Genome Annotation Chan- the gene locations in the contig (18, 55–57). The gene prediction nel25 made available their first-pass annotations for most of the contig se- quences. programs, particularly GENSCAN, identified all of the 25 verified Ͼ Gene Annotations. Annotations for the for all of the genes dis- genes and fairly accurately ( 90%) predicted their ORFs and intron/ covered in the contig sequence were compiled from computational predictions, exon structure (although in some cases distinct genes were combined experimental observations, and by transfer of information from the yeast, into one gene). worm, and fly orthologue pairs. Functional conservation between human In addition to the 25 genes, by an intensive informatics effort using proteins and their orthologous counterparts was repeatedly demonstrated ex- the genomic DNA sequence, we found several potential genes (Table perimentally (46, 50). 1). These included two gene candidates (arbitrary names Gene29, cosmid LUCA2, and Gene19, cosmid LUCA10; Table 1), identified by one or two EST hits that did not have intron/exon structure, were RESULTS not detected by the GENSCAN prediction program, did not have a Overlapping and Nested Homozygous Deletions Define 120-kb significant ORF, and did not have a detectable mRNA signal on MTN and 250-kb Regions for a TSG Search and Identification of 25 blots. We believe these may represent genomic DNA contamination Resident Genes in the Overall ϳ630-kb Region. LOH (allele loss) of the EST database. Another set of gene candidates were detected by is an important sign of somatically acquired genetic events in the GENSCAN (29) and GRAIL-EXP (49) gene prediction programs natural history of many tumors and is a useful tool to discover the analysis of genomic DNA (arbitrary names Gene22, Gene23, Gene24, location of new TSGs. However, the regions of allelic losses involv- Gene25, and Gene27; Table 1). Although programs provide a pre- ing 3p in lung cancer (6, 9) and premalignant lesions (12, 14–16) are dicted cDNA sequence, ORF, and intron/exon structure, thus far we have found no EST hits in GenBank, no mRNA signal on MTN blots, 13 Internet address: http://www.hgsc.bcm.tmc.edu/. and no homology of the predicted protein to proteins in GenBank. 14 Internet address: http://www.expasy.ch/tools/. Thus, at the present time we cannot verify these GENSCAN predicted 15 Internet address: http://www.embl-heidelberg.de/. 16 Internet address: http://www.imcb.osaka-u.ac.jp/nakai/psort.html. candidates as actual genes, and they are noted for future reference. 17 Internet address: http://drava.etfos.hr/ϳzucic/split.html. These observations suggest that we have found all, or nearly all, 18 Internet address: http://www.cbs.dtu.dk/services/TMHMM-1.0/. ϳ 19 resident genes in the 630-kb sequence. This contention is likely true Internet address: http://www.ebi.ac.uk/interpro/. ϳ 20 Internet address: http://gcg.tigem.it/cgi-bin/uniestass.pl. for the 120-kb sequence (Fig. 1) that contains all of seven complete 21 Internet address: http://gcg.hgmp.mrc.ac.uk/cgi-bin/Est_blast/Est_blast/ESTblast.pl. genes and a large part of the CACNA2D2 gene. In this critical region, 22 Internet address: http://www.bioinf.mdc-berlin.de/home.html.new. 23 Internet address: http://ftp.genome.washington.edu/cgi-bin/RepeatMasker. 24 Internet address: http://genome.imb-jena.de/ϳschattev/rummage/index.html. 26 Internet address: http://www.ncbi.nlm.nih.gov/genome/guide/HsChr3.shtml. 25 Internet address: http://genome.ornl.gov/. 27 Internet address: http://pompous.swmed.edu/panorama.htm. 6118

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Fig. 1. Genetic and physical characterization of the human tumor nested homozygous deletion region on 3p21.3 showing the steps leading to the identification of the cloned resident candidate tumor suppressor genes. Top, ideogram of the banding pattern of chromosome 3p with some genetic framework markers found in and flanking the deleted region. Next is shown a physical pulsed-field gel electrophoresis map of the homozygously deleted region and its flanking sites. The positions of the rare cutter restriction sites are indicated above the line and the sizes (in kb) of the corresponding NotI restriction fragments are given below the line. The physical map is followed by a diagrammatic representation of the overlapping homozygous deletions discovered in SCLC cell lines (NCI H1450 in blue, NCI H740 in magenta, and GLC20 in green) and a nested deletion in a breast cancer tumor sample and corresponding cell line (H1500 in brick). The sizes of the homozygous deletions, deletion overlaps, and the precisely mapped breakpoints are indicated. The minimum tiling cosmid contig covering the deleted region is shown below the deletions. The position of the framework genetic markers is shown by downward arrows; the GenBank accession nos. for cosmid sequences are given under the lines, and the abbreviated cosmid/P1 clone numbering system given to the Genome Centers (e.g., Luca01, Luca02 . . . Luca22, and P1 clone p3938) is given above the lines. The sequencing was done jointly by The Washington University (Luca12 to P3938) and The Sanger (Luca01 to Luca11) Genome Sequencing Centers using the high-redundancy shotgun procedure and is available from GenBank or the centers ftp sites (see: ftp.sanger.ac.uk/pub/human/sequences/Chr_3/ and ftp://genome.wustl.edu/pub/gsc1/ sequence/st.louis/human/). Finally, a line representing the ϳ630-kb assembled genomic sequence with the positioned resident gene and breakpoints defining critical regions is shown at the bottom. The genes are represented by pointed rectangles, indicating the orientations of transcription. The gene names are given above and the GenBank accession nos. below with downward arrows indicates the position of two genes (temporary names LUCA1.2 and LUCA2.3) on cosmids Luca01 and Luca02, respectively, whose ء the rectangles. The sequencing is not yet completed. The 6 genes outside of the critical region in the centromeric portion are in magenta, the 8 genes in the combined breast and lung 120-kb critical region are in blue, whereas the remaining 11 genes still within the rest of the lung cancer nested homozygous deletions (and additional 250 kb) are all in green. The 6 genes exhibiting at least some point or small mutations have an “M” in their box. Probable pseudogenes and other in silico predicted genes for which we currently cannot confirm their status as a bona fide gene are not shown. the genes reside in the following order with the indicated sizes and addition, in situ hybridization [FISH and two-color FISH with DNA intergenic distances: CACNA2D2 (80 kb), 5-kb intron; PL6 (4.5 kb), from cosmids LUCA1 (Z74618), LUCA8 (Z84495), and LUCA20 1.5-kb intron; 101F6 (2.3 kb), 161-bp intron; NPRL2/g21 (3.3 kb), (AC004693)] located the contig to 3p21.3 and proved the orientation 1.8-kb intron; BLU (4.7 kb), 4.4-kb intron; RASSF1/123F2 (7.6 kb), as shown on Fig. 1 (data not shown). We also performed radiation 1.6-kb intron; FUS1 (3.3 kb), 4.5-kb intron; and HYAL2 (2.7 kb). With hybrid mapping with the TNG panel with selected markers for finer a total of ϳ18 kb of intergenic and ϳ17 kb of intronic sequence, this resolution of the location (not shown). In addition, using the contig region is extremely gene dense and contains 20–25% of coding sequence, we have determined intergenic distances, exon-intron struc- sequence. tures, the intron sizes of the resident genes, and ascertained the The location of the contig on 3p21.3 and its position relative to direction of transcription. These are all features that are immediately other 3p genes were determined by the presence in the ϳ630-kb available by performing a BLAST analysis with our deposited cDNA sequence of several framework genetic markers mapped to this loca- sequences (Table 1) against the individual cosmid or assembled tion (e.g., D3S1621 and D3S1568; Fig. 1), multiple radiation hybrid genomic sequences.29 mapped sequence tagged sites (SHGC-11855 shown), and other In total, the 25 genes occupy ϳ630-kb of genomic DNA, resulting linked genetic markers (see detailed marker information on genomic in an average size of ϳ25 kb/gene, which agrees well with the size of sequence contigs NT_002322, NT_000067, and NT-000069).28 In an average gene (ϳ30 kb) estimated for the whole human genome.

28 Internet address: http://www.ncbi.nlm.nih.gov/genome/guide/HsChr3.shtml. 29 Internet address: http://www.ncbi.nlm.nih.gov/genome/guide/HsChr3.shtml. 6119

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Table 1 Candidate and verified genes identified in the ϳ630-kb 3p21.3 homozygous deletion region and status of their mutation analysis Genea GenBank no. cDNA bp Mutation analysisb (No. found/tested) 3pk/MAPKAP3 U09578 2481 bp (0/20) Gene28 (LUCA1.2) None yet In progress Not done CISH (Gene18) AF132297 2198 bp None foundb Gene29 (LUCA2.2)c H03298/H03299 EST 805 bp Not done (no mRNA) Gene30 (LUCA2.3) None yet In progress Not done (2.4, 10 kb mRNA) HEMK Form I AF131220 1472 bp Not done HEMK Form II AF172244 5737 bp Not done Gene20 AF188706 2366 bp Not done Gene22 (LUCA4)c (GENSCAN predicted) 309 aa Not done CACNA2D2 (Gene26)a AF040709, AF042792, 5482 bp (0/100) Gene19c R00809 EST 1089 bp Not done (no mRNA) PL6 U09584 1860 bp (0/78) 101F6 AF040704 1117 bp (0/78) NPRL2/Gene21a AF040707, AF040708 1351 bp (3/38) 1 stop, 2 missense BLUa U70880, U70824 1739 bp (4/61) 4 missense RASSF1C/123F2SF AF040703, AF061836 1680 bp (0/77) RASSF1A/123F2LFa AF102770 (lung), 1860 bp (0/37) FUS1 AF055479 1691 bp (3/79) 2 stop, 1 Del HYAL2 U09577 1783 bp (1/40) 1 Del HYAL1 U03056, AF173154 2565 bp (3/40) 2 missense, 1 Del HYAL3 AF040710 1861 bp (0/40) FUS2a AF040705, AF040706 1272 bp (4/78) 4 missense SKMC15 U09585 1784 bp (0/63) Gene23 (LUCA14)c (GENSCAN predicted) 139 aa (0/20) SEMA3B/(SEMA-A(V)) U28369 2919 bp (3/39) 3 missense Gene24 (LUCA15)c (GENSCAN predicted) 110 aa Not done GNAI-2 X04828 2596 bp (0/34) Gene17 U49082 2431 bp (0/38) (2 silent mutations found) GNAT-1 X15088 1292 bp (0/35) SEMA3F/SEMA (IV)a U38276, U33920 2719 bp (0/30)d Gene25 (LUCA20)c (GENSCAN predicted) 195 aa Not done Gene27 LUCA19–22)c (GENSCAN predicted) 536 aa (0/20) Gene15/RBM5 U23946 2575 bp (0/18) Gene16/RBM6a U50839 3588 bp (1/39) 1 missense a Several genes have alternatively spliced forms including CACNA2D2 [at least three forms including AF042793 (isoform II)], NPRL2/Gene21 (four forms), BLU (two forms, one in lung and one in testis RNA), RASSF1/123F2 (two forms RASSF1A/123F2LF and RASSF1C/123F2SF and several tissue-associated forms including lung RASSF1A, heart RASSF1D, and pancreas RASSF1E), FUS2 (two forms), SEMA3F/Sem E (IV) (at two forms), and Gene16 (three forms). b Only mutations altering the amino acid sequence are shown. Polymorphisms (silent or aa altering) found in more than one tumor or that did not alter the amino acid sequence are not given. CISH mutation analysis by Uchida et al. (62). Del, deletion of Ͼ1000 bp. c Several predicted genes, Gene22 (LUCA2.2) and Gene19 (LUCA10), have ESTs but appear to be nonexpressed, intronless with no good ORF, and thus probable pseudogenes. Several candidates were predicted by GENSCAN but have no EST hits, no detectable mRNA on multiple tissue Northern blots, and no obvious protein homologies. These include Gene22 (cosmid LUCA4), Gene23 (cosmid LUCA14), Gene25 (cosmid LUCA20), and Gene27 (cosmids LUCA19–22). No EST/cDNAs were found, no MTN blot mRNA signals are seen, and the predicted amino acid sequences have no known protein homologies. d We tested 456 of 753 amino acids of the SEMA3F open reading frame and found no abnormalities. Xiang et al. (63) reported studying the entire ORF in 28 small cell lung cancers and found no abnormalities.

However, distribution of these genes along the sequence is rather The three lung cancer homozygous deletions identified an ϳ370-kb uneven, with the highest gene density (genes in green in Fig. 1) in the region extending from cosmid LUCA10 (Z75742; defined by the ϳ120-kb region (average gene size, ϳ15-kb). Gene sizes varied centromeric end of the GLC20 homozygous deletion) to P3938 dramatically, with the smallest gene size of 2.3 kb (g101F6) and the (AC004814; defined by the telomeric end of the NCI-H740 homozy- largest of ϳ140 kb (CACNA2D2). Actually, this large gene is inter- gous deletion (Fig. 1). The nested homozygous deletion in breast rupted by the centromeric breakpoints of both the HCC1500 breast cancer HCC1500 (10), which covered part of cosmid LUCA06 cancer homozygous deletion (in cosmid LUCA6) and in the SCLC (Z84493) to part of cosmid LUCA13 (AC002455; Fig. 1), divided the GLC20 homozygous deletion in LUCA10 (Fig. 1). The intergenic 19 genes in the 370-kb region into two critical gene sets: 8 genes in distances also differed enormously, from 161 bp (between g101F6 a 120-kb segment extending from part of cosmid LUCA10 to part of and NPRL2/g21)toϳ60 kb (between genes g20 and CACNA2D2). cosmid LUCA13, and 11 genes in the telomeric portion (ϳ250 kb, The intron sizes also varied dramatically, the smaller ones ranging extending from cosmid LUCA13 to P1 clone P3938, AC004814). from 50–1000 bp and the largest being 40–50 kb (in CACNA2D2). These deletions eliminated the 3pk/MAPKAP3 (U09578), CISH It is worthwhile to examine what type of genes (if any) would have (AF132297, temporarily called Gene18 in our GenBank deposit), been missed by our manual and informatics prediction analyses. These HEMK isoforms I and II (AF131220 and AF172244), Gene20 omissions could include genes that lack GenBank matches, genes (AF188706), and two partially characterized genes [Gene28(Luca1.2) whose sequences would not be recognized by the available gene and Gene30(Luca2.3)], all lying in the centromeric portion of the prediction programs, such as non-protein coding genes, and genes contig (ϳ260 kb of genomic DNA located in cosmids LUCA1, whose mRNAs have a very restricted pattern of tissue expression. LUCA2, LUCA3, and LUCA4), from further consideration. In addi- Another approach to this gene saturation problem will be to compare tion, expression and mutation analysis by us of 3pk (U09578) and by the human sequence with the corresponding mouse genomic se- Sithanandam et al. (55) and Uchida et al. (62) of CISH (AF132297) quence. Because functional sequences such as transcriptional enhanc- provided other evidence excluding these genes as well. ers and mRNA-like noncoding RNAs are highly conserved in mam- Expression Analysis of the Candidate Tumor Suppressor malian genomes, they might be readily detected in this comparison Genes. All of the remaining19 genes were analyzed extensively by (58, 59). The effectiveness of this approach has been demonstrated manual and computational methods. Northern analyses with cDNA recently (60, 61). The mouse BAC clone (#AC025353) covering this probes for each gene using commercial poly(A)ϩ RNA MTN blots region was used for this alignment (data not shown). (Clontech) and a panel of lung cancer cell line RNAs revealed the 6120

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Fig. 2. mRNA expression in normal human tissues using MTN blots of 15 of the 630-kb 3p21.3 homozy- gous deletion region resident genes. The RNA filters (#7759, #7760 from Clontech, Palo Alto, CA) contain 2 ␮g of poly(A)ϩ mRNA per lane for each tissue indicated: Lane 1, heart; Lane 2, brain; Lane 3, pla- centa; Lane 4, lung; Lane 5, liver; Lane 6, skeletal muscle; Lane 7, kidney; and Lane 8, pancreas. cDNA probes for the genes were labeled and hybridized as described in “Materials and Methods.” Published ex- amples of MTN expression for some of the other genes are given for 3pk/MAPKAP3 (55), CACNA2D2 (68), SEMA3B and SEMA3F (57), IFRD2/SM15 (56), and HYAL3 (79).

sizes of the respective transcripts and patterns of expression in normal islands associated with the genes showing reduced or absent expres- human tissues and lung cancer samples (Figs. 2 and 3; Table 2). sion is currently under investigation. Expression in Northern blots prepared from total or poly(A)ϩ RNA of Mutation Analysis of the Candidate Tumor Suppressor Genes. 20–30 RNA samples from lung cancer cell lines, representing both Initially, we performed Southern blot analysis on ϳ100 genomic SCLC and NSCLC, revealed, for many of the genes, levels of expres- DNAs from our large panel of lung cancer cell lines representing both sion similar to normal lung. No abnormal transcript sizes suggestive SCLC and NSCLCs (24) using cDNA or genomic probes representing of mutations were found. However, several genes showed reduced each of the 25 genes in our search for other homozygous deletions or expression in the lung cancer lines, i.e., the CACNA2D2 gene not genomic DNA rearrangements (data not shown). We found only the expressed in 50% of the lines, the BLU gene expressed only in 30%, homozygous deletions listed in Fig. 1 and ϳ30-kb homozygous de- the HYAL1 gene expressed in Ͻ30% (Fig. 3), and SEMA3B and letion in SCLC NCI-H524 involving most of the genomic sequence in SEMA3F (19, 57, 63) expressed in Ͻ50%. Thus, expression analysis cosmid LUCA13-interrupting gene FUS1 to gene HYAL1 (data not in lung cancers identified these five genes as potential TSG candidates shown, and see discussion below). Mutational analyses of the resident based on loss of expression in a sizable number (but not all lung genes (summarized in Tables 1 and 3) were performed on lung cancer cancers). Because a possible mutation mechanism is tumor-acquired cDNAs or genomic DNAs by RT-PCR-SSCP or PCR-SSCP, respec- promoter hypermethylation (23), the methylation status of the CpG tively, followed by DNA sequencing of any altered bands detected. A

Table 2 mRNA expression of 3p21.3 genes in Multiple Tissue Northern (MTN) blotsa Gene locus (mRNA kb) GenBank no. Heart Brain Placenta Lung Liver SkMus Kidney Pancreas MAPKAP3 (2.5) U09578 3ϩ Traceb Trace 2ϩ 1ϩ 3ϩ 3ϩ 2ϩ CISH/Gene18 (2.4) AF132297 3ϩ Neg 3ϩ 3ϩ 3ϩ 3ϩ 3ϩ 3ϩ HEMK (1.7, 7, 7.5) AF131220 3ϩ Trace Trace Trace 3ϩ 2ϩ 2ϩ 3ϩ Gene20 (2.4, 2.6) AF188706 3ϩ 2ϩ Neg Neg Trace 2ϩ Trace Trace CACNA2D2 (5.5–5.7) AF040709 2ϩ 2ϩ 1ϩ 3ϩ Neg 2ϩ 1ϩ 2ϩ Gene19 (none)c Neg Neg Neg Neg Neg Neg Neg Neg PL6 (2.2) U09584.1 1ϩ Trace 2ϩ 2ϩ 2ϩ 3ϩ 3ϩ 2ϩ 101F6 (1.5) AF040704.1 1ϩ Trace 2ϩ 2ϩ 3ϩ Trace 1ϩ 1ϩ NPRL2/Gene21 (1.5) AF040707 ϩ 2ϩ 1ϩ 1ϩ 2ϩ 3ϩ 1ϩ 2ϩ BLU (2.0) U70824, U70880 Trace Trace 2ϩ Trace Trace RASSF1/123F2 (2.0) AF040703 3ϩ 1ϩ 2ϩ 1ϩ 1ϩ 2ϩ 1ϩ 3ϩ FUS1 (1.8) AF055479.1 3ϩ 2ϩ 1ϩ 3ϩ 2ϩ 3ϩ 3ϩ 3ϩ HYAL2 (2.0) U09577 2ϩ Trace 3ϩ 3ϩ 3ϩ 2ϩ 3ϩ 1ϩ HYAL1 (2.6) U03056, AF173154 2ϩ Neg Neg 1ϩ 3ϩ 1ϩ 3ϩ Trace FUS2 (1.9) AF040705 3ϩ 2ϩ Neg 1ϩ 1ϩ 3ϩ 1ϩ 2ϩ HYAL3 (2) AF040710 Neg 1ϩ Neg Neg Neg 1ϩ Neg Neg IFRD2/SKMC15 (4) U09585 3ϩ Trace 1ϩ 1ϩ 3ϩ 3ϩ 1ϩ 3ϩ SEMA3B (3.4) U28369 1ϩ 1ϩ 3ϩ Trace Neg 1ϩ 1ϩ 1ϩ G17 (3.0) U49082.1 2ϩ 2ϩ Neg Neg 3ϩ 3ϩ 1ϩ 3ϩ SEMA3F (3.9, 2.9) U38276, U33920 2ϩ 1ϩ 3ϩ 2ϩ Trace 2ϩ 2ϩ 2ϩ G15/RBM5 (4, 2, 1.5) U23946 3ϩ 1ϩ 1ϩ 1ϩ Neg 3ϩ 1ϩ 3ϩ G16/RBM6 (4.0) U50839 1ϩ Trace Trace 2ϩ 1ϩ 2ϩ 2ϩ 3ϩ a Northern blot data for the following genes available in these references: MAPKAP3, Sithanandam et al. (55); IFRD2/Skmc15, Latif et al. (56); SEMA3B, SEMA3F, Sekido et al. (57); and HYAL1,2,3, this report and Csoka et al. (79). The size of the most prominent transcripts in kb are given in parentheses next to the gene locus. b Neg, negative. c Gene19 is given as an example of one of the six genes detected by either a few ESTs or predicted by GENSCAN, which do not give mRNA signals on MTN blots (see Table 2 and “Discussion”). 6121

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Fig. 3. mRNA expression in lung cancer cell lines of 14 of the genes resident in the 630-kb 3p21.3 homozygous deletion region. Replicate Northern blots were made using 20 ␮g of total RNA per lane for each of the samples; probes were labeled and hybridized as described in “Materials and Methods.” Each of the blots was used for two to three different probes with stripping of label between hybridization. The lung cancer cell lines are shown above the panel as well as one B lymphoblastoid cell line (BL5). As positive controls, the various rep- licate blots showed approximately equivalent loading by rRNA amounts and for various positive control probes (not shown). Note that for genes PL6 and G15/RBM5, there is an approximately equal expres- sion in most of the samples for these two genes. As a negative control, note that NCI-H740 RNA homozygously deleted for the entire region provides a negative background signal. The size of the mRNA species for each gene is given on the right of the panel. For published examples of expression of several of the other genes in lung cancer cell lines, see 3pk/MAPKAP3 (55), CACNA2D2 (68), SEMA3B and SEMA3F (57), and IFRD2/SM15 (56). The tumor histologies of the lung cancer lines are: SCLC (H82, H146, H249, H524, H740, H1514, H1618, H2141, H2171, and H2227); adenocarcinoma (H358 [bronchioloalveolar] H838, H1742, and H2077); large cell (H460, H1155, and H1299); and mesothelioma (H290 and H2052; Ref. 24).

large number of both SCLC and NSCLC cell lines and for some Table 1) did not pinpoint a strong candidate gene with a high fre- genes, paired normal and tumor DNA samples, were used. In addition, quency of mutation among either of the critical gene sets. This finding the coding sequences of the FUS1 and the BLU genes were sequenced was unexpected and disappointing. Because it is possible that genes completely in a large number of lung cancer samples, including paired not involved in tumorigenesis may show a low frequency of mutations tumor and normal tissue samples. The results for the eight-gene set in in common tumors related to a “mutator phenotype” expressed by the 120-kb region show that the genes either had no mutations at all tumors (64, 65), the finding of a low mutation rate in a candidate (CACNA2D2, PL6, 101F6, 123F2, and HYAL2), or the mutation rate TSG(s) must be regarded with caution. Accordingly, other lines of was in the range of 5% (NPRL2/g21, BLU, and FUS1; Table 1). The evidence besides finding frequent mutations are needed to rule out same absence or low frequency of mutations was detected in the 11 positionally defined candidate TSGs. These include analysis for tu- genes lying in the more telomeric ϳ250-kb portion of the 630-kb mor-acquired promoter hypermethylation, gene transfer into tumor contig with HYAL1, FUS2, and SEMA3B, each exhibiting a few cells with tests for suppression of the malignant phenotype, and mutations (Table 1). Many examples of the mutations detected in lung disruption of the candidate genes in “knock-out” mice. In addition, we cancer cell lines are given in Table 3. Thus, this extensive mutational now also have to consider the newly recognized class of haploinsuf- analysis (involving 1102 separate tumor sample/gene mutation tests; ficient TSGs, where the presence of only one wild-type allele facili- tates tumorigenesis (see “Discussion”). Table 3 Examples of tumor cell lines bearing homozygous amino acid sequence Informatics Analysis for Predicted Protein Functions and altering mutations of candidate 3p21.3 TSGs Orthologue Identification in Model Organisms. Computational Tumor predictions of biochemical functions could also provide clues as to a b cell line Histology Gene Mutation (codon) whether a particular gene could serve a tumor suppressor function by H1514 SCLC NPR2/G21 CAA to TAA (Stop codon 261) H209 SCLC NPR2/G21 CCT to CTT (Pro28Leu) affecting cell growth and/or survival. Therefore, we next studied the H748 SCLC NPR2/G21 GGC to GAC (Gly86Asp) biochemical functions and subcellular localization of the proteins H125 NSCLC BLU GAC to GAA (Asp198Gln) encoded by the genes using a variety of computational tools. Infor- H157 NSCLC BLU CGA to CAA (Arg407Gln) H524 SCLC FUS1-HYAL1 ϳ30 kb Homozygous deletion mation about protein function is a continuum that begins with the H322 NSCLC FUS1 28 bp deletion (Stop codon 81) finding of homology between proteins within and between species, H1334 NSCLC FUS1 28 bp deletion (Stop codon 81) H1622 SCLC HYAL1 GGC to CGC (Gly196Arg) domain composition, functional motifs, and subcellular localization H2126 NSCLC HYAL1 GCC to ACC (Ala227Ser) signals, extending ultimately to demonstration of function by bio- H460 NSCLC FUS2 CGG to TGG (Arg145Trp) chemical analysis. Finding orthologous genes in model organisms H1373 NSCLC FUS2 ACC to AGC (Thr207Ser) H1648 NSCLC SEMA3B ACT to ATT (Thr415Ile) (mouse, fly, worm, and yeast) permits the transfer of any functional H1155 NSCLC SEMA3B CGC to TGC (Arg348Cys) annotation to the human gene in question; therefore, we performed an H358 NSCLC SEMA3B GAT to CAT (Asp397His) H711 SCLC Gene16/RBM6 TCT to TTT (Ser353Phe) extensive search for orthologues of the resident candidate TSGs in a All tumor cell line names have the NCI- prefix. these model organisms. The results of these computations are sum- b SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer. marized and discussed in the annotations for each of the genes (see 6122

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. 3p21.3 TUMOR SUPPRESSOR GENE POSITIONAL CLONING below). As expected, we found that all 19 genes have true murine experimental confirmation of this predicted function, through injec- orthologues discovered in mouse EST databases. These genes showed tion of CACNA2D2 cRNA into Xenopus oocytes, we have confirmed nearly 90–100% identity/similarity on the protein sequence level and recently that CACNA2D2 acts as a regulatory subunit of voltage-gated about 80–90% on the mRNA sequence level (GenBank accession nos. calcium channels able to augment the function of all three pore- are provided in the annotations). In the worm, highly likely ortholo- forming units (68). BLAST (36) searches in the mouse EST database gous pairs were identified for 14 of the 19 genes using stringent detected two different nonoverlapping EST clones (accession nos. criteria of orthology, i.e., 30–50% amino acid sequence identity or AA000341 and AA008996), which showed 91 and 85% cDNA se- similarity with Ͼ80% alignment of their entire amino acid lengths quence identity with CACNA2D2 (residues 2925–3421 and 4989– (45, 46). In the fly, highly likely orthologues were found for 10 of the 5391), respectively. These EST sequences showed only limited ho- 19 genes among the complete set (ϳ14,000) of fly genes30 (66). We mologies to the murine ␣2␦-1 gene splice forms, indicating that they noticed that orthologous pairs fall into three categories: common for represent true orthologous sequences of the human CACNA2D2 gene. both worm and fly, only present in the worm, and only present in the This was further corroborated by protein alignment of the 86-amino fly. Yeast genes sharing ϳ50% similarity in common domains/ acid ORF encoded by mouse EST AA000341, which was 96% iden- features were found for two of the genes, i.e., PL6 and NPRL2/ tical to the CACNA2D2 isoform I protein (amino acid residues 922- Gene21, probably only the Schizosaccharomyces pombe counterpart 1005). The worm genome also contains two ␣2␦ genes; by stringent (NPRL2)ofNPRL2/g21 should be considered a candidate orthologous criteria of orthology (i.e., ϳ50% identity/similarity with Ͼ80% align- gene. The availability of the complete DNA sequences of yeast (50), ment of their entire amino acid sequence) the worm gene, T24F1.6, worm31, and fly (66) genomes makes it unlikely that we have missed appears to be the orthologue of CACNA2D2, whereas the second any of the orthologous gene pairs for our TSG candidates in these worm ␣2␦ gene, UNC-36 (accession no. P34374), is the orthologue of three model genomes. We now provide annotations for each of the 19 the ␣2␦-1 gene (67). The UNC-36 phenotypes do not affect growth, genes found in the ϳ370-kb segment starting with the genes in the and no phenotypes were yet reported for the T24F1.6 locus. The fly smaller ϳ120-kb sequence (Fig. 1). proteome (ϳ14,000 proteins; Ref. 66) contains three ␣2␦ proteins The ␣2␦-2 calcium channel subunit gene, CACNA2D2, was discov- (accession nos. AAF53505, AAF53476, and AAF58335) of which the ered in silico by both finding EST matches with fragments of genomic first contains a likely orthologue of ␣2␦-1 (44), the second is a likely sequence and by exons predicted by GENSCAN. The gene occupies orthologue of the ␣2␦-2 gene, and the third of a still-not-cloned ϳ140 kb of genomic space and is composed of at least 40 exons. It is human gene; all three have the VWA_DOMAIN. The yeast proteome expressed as a 5.5–5.7 kb mRNA. Three mRNA splice forms have (50) contains only one ion channel gene and appears to have no been detected that code for two protein isoforms in several normal orthologues for either of the ␣2␦ genes. Despite the lack of mutations, tissues. GenBank deposits AF040709 (mRNA isoform 3) and the absence of CACNA2D2 expression in many but not all NSCLCs AF042792 (mRNA isoform 1) differ in the 5Ј untranslated region and with high CACNA2D2 expression in normal lung makes CACNA2D2 encode the same amino acid sequence (protein isoform I), whereas an excellent candidate TSG with the need for testing of function in AF042793 (mRNA isoform 2) differs in the 5Ј translated region and tumor cells and study of acquired promoter hypermethylation as a has a slightly different amino terminal amino acid sequence (protein method of inactivation of . isoform II). The expression of CACNA2D2 is reduced or absent in The PL6 gene was discovered manually by probing Northern blots Ͼ50% of lung cancer cell lines, particularly NSCLCs. However, no with genomic fragments. The gene occupies 4.5 kb of genomic space, mutations were detected in analysis of 60 lung cancer cell lines and 40 is composed of two exons, and expressed as a 2.2-kb mRNA in many paired normal/SCLC tumor samples. The nucleotide sequence sug- normal human tissues including lung. The expression of PL6 is gests that the gene encodes an auxiliary regulatory ␣2-␦ subunit of slightly reduced in some SCLC lines and abundantly represented in calcium channels and joins the ␣2-␦-1 (previously A subunit) gene the human and mouse EST databases. No mutations were detected in (67) as a new and second member of the ␣2-␦ gene family. Three 38 cell lines and 40 paired normal/SCLC tumor samples. By sequence putative transmembrane helices predicted previously in the ␣2␦-1 analysis, PL6 encodes an integral plasma membrane protein [PSORT protein (67) were also predicted in both protein isoforms of the program (39)] with six [SPLIT program (40)] or seven to eight CACNA2D2 gene with the SPLIT 35 program (40). In addition, [TMHMM program (41)] transmembrane helices. The predicted cy- protein isoform I of the CACNA2D2 gene has another membrane helix toplasmic portion of the protein (the last 103 amino acids, residues at the very amino terminus. Using the TMHMM program (41), all 249–351) contains an OMPdecase domain [residues 274–298; three ␣2␦ proteins were predicted to span the membrane only once at PFAM, (39)] that may involve PL6 in protein-protein interactions and the amino (␣2␦-2 isoform I) or at the carboxy termini (␣2␦-1 and a bipartite NLS (residues 282–299; Ref. 39) that may guide it to the ␣2␦-2 isoform II), favoring the single-transmembrane model for the nucleus. The mouse orthologue was discovered in an EST (accession ␣2␦ subunit proteins, which was verified experimentally for the ␣2␦-1 no. W96860), sequenced (our accession no. AF134238), and shown to protein (67). A protein binding a VWA-like domain was discovered be 92% identical on protein and 87% on cDNA levels. The worm by the PFAM (37) program in the extracellular part at similar posi- F11A10.3 gene encoding a multidomain protein that aligns with PL6 tions in all three ␣2␦ subunit proteins (amino acid residues: 291–469 and the aligned region contains the NLS and the OMPdecase domains, suggesting that it is the orthologue of PL6. The fly gene CG9536 and 222–400 for ␣2␦-2 protein isoforms I and II, respectively, and product (450 residues; accession no. AAF52388) is a likely ortho- residues 253–430 for the ␣2␦-1 protein). The VWA-like domain may logue of PL6 (48% similarity over the first 306 residues of PL6) and facilitate the binding of the ␣2␦ complex with the calcium channel is also an integral membrane protein with seven to eight transmem- ␣-1 pore forming subunit protein (67). The almost identical membrane brane helices. It has two HMW kininogen domains (residues 325–349 topologies, similar domain structures, and posttranslational modifica- and 353-3760) but no NLS and OMPdecase domains. The yeast gene tions of all three ␣2␦ subunit proteins strongly support the identity of Yol107w product has substantial homology with PL6 but does not the new ␣2␦-2 gene as a member of the ␣2␦ gene family. To provide have the NLS and the OMPdecase domain. The absence of mutations and robust expression of PL6 in most lung cancers suggest that PL6 30 Internet address: http://morgan.harvard.edu/. 31 Internet addresses: http://www.sanger.ac.uk/Projects/C_elegans/, http://genome. is an unlikely candidate TSG. wustl.edu/gsc/C_elegans/elegans.shtml, and http://www.ncbi.nlm.nih.gov/. The 101F6 gene was discovered manually by screening arrayed 6123

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. 3p21.3 TUMOR SUPPRESSOR GENE POSITIONAL CLONING cDNA libraries with cosmid LUCA12 DNA. The gene space of 3.2 kb The BLU protein is likely a soluble cytoplasmic protein and shares contains four exons encoding a 1.5-kb mRNA. The gene is expressed 30–32% identity over a stretch of 100–112 amino acids (residues in many normal tissues including lung, is highly expressed in SCLC 334–437 or 318–430) with proteins of the MTG/ETO family of and NSCLC cell lines, and is abundantly represented in the EST data transcription factors (69) and the suppressins (70) that may regulate bases. No mutations were detected in 40 cell lines and 40 paired entry into the cell cycle and suppress growth of colon carcinoma cells. normal/SCLC tumor samples. By sequence analysis, 101F6 encodes The “Zn knuckle” motif involved in specific protein-protein interac- an integral plasma membrane protein [PSORT program (39)] with six tions is part of this domain and is present in many proteins.32 No [SPLIT program (40) and TMHMM program (41)] transmembrane orthologous pairs were found in the worm and yeast proteomes (50). helices with both termini in the cytoplasm. No other known domains However, the fly genome (66) contains a true orthologue of BLU: the or significant motifs were detected. The mouse orthologue was dis- CG11253 gene product (accession no. AAF49850) is of similar size covered in the mouse EST database (accession nos. AA285935, (451 residues), has 49% amino acid sequence similarity over the AA198541, and AA198960), sequenced (our accession no. whole length of BLU, and also has a MYND finger domain (residues AF131206), and shown to be 95% identical on the protein and 85% on 412–448). Several other fly, worm, and S. pombe proteins share 35% the cDNA sequence level. No orthologous pairs were detected in the identity with the MTG/ETO domain. The mouse orthologue was fly (66), worm, and yeast proteomes (50). The absence of mutations discovered in mouse EST databases (accession nos. AI595515 and and robust expression of 101F6 in most lung cancers suggest that AI429164), sequenced (our accession no. AF123386), and shown to 101F6 is an unlikely candidate TSG. be 89% identical on protein and 87% on cDNA levels. The loss of The NPRL2/Gene21 gene was discovered in silico by finding both expression in most lung cancers and the occurrence of a few mutations ESTs matches and GENSCAN predicted exons. The gene space of 3.3 make BLU an attractive TSG candidate requiring further functional kb contains 11 exons coding for a 1.5-kb mRNA with multiple splice and promoter methylation status studies. isoforms that are expressed in many normal tissues including lung and The RASSF1/123F2 gene was discovered manually by screening testis and is abundantly represented in the EST databases. NPRL2/ gridded cDNA libraries with cosmid LUCA12 DNA. The gene space Gene21 is well expressed in SCLC and NSCLC lines except for the of 7.6 kb contains 5 exons coding for 2-kb, alternatively spliced SCLC line NCI H1514. A frameshift mutation producing a stop codon mRNAs (“short” and “long” forms, 123F2SF and 123F2LF, that was detected in 1 of 40 lung cancer cell lines. Sequence analysis should now be referred to by Human Genome Organization-approved shows NPRL2/Gene21 encodes a soluble protein that has a bipartite nomenclature as RASSF1C and RASSF1A, respectively) that are well NLS (residues 62–79) and a protein binding domain, granulin (resi- expressed in all analyzed human tissues including lung. The dues 86–98), predicted by PFAM (35, 37. The mouse orthologue was RASSF1C/123FSF but not the RASSF1A/123F2LF mRNAs are well discovered in mouse EST databases (accession nos. AI037102, expressed in most lung cancer cell lines. The mRNA is well repre- AA764527, AA709972, and W64225), sequenced (our accession no. sented in EST databases from normal and tumor tissues. Using GEN- AF131206), and shown to be 97% identical on protein and 90% on SCAN prediction programs, the RASSF1A/123F2LF splicing form cDNA sequence levels. True orthologues were identified: in yeast (the was discovered using RT-PCR on mRNA with a difference in amino NPR2 gene in Saccharomyces cerevisiae, GenBank accession no. acid sequence in the NH terminus, giving a total amino acid sequence P39923, and the hypothetical M 47,000 protein in S. pombe, acces- 2 r of 340 amino acids compared with 270 amino acids for the RASSF1C/ sion no. Z99163); in the fly (66), the CG9104 gene product (accession 123F2SF. The amino acid sequence of RASSF1A/123F2LF contains a no. AAF48677) with 65% similarity over the whole length of the predicted DAG binding domain also found in the related gene NORE1 NPRL2/g21; and in the worm (accession no. U61949) proteome but not found in the RASSF1C/123F2SF cDNA sequence. RASSF1A/ databases. However, only the mouse orthologue contains the bipartite 123F2LF mRNAs also come in multiple tissue-related splicing forms, NLS (residues 62–79) and the granulin domain (residues 86–98). with slight differences in amino acid sequence, including forms for NPRL2/Gene21 mRNA is expressed in most lung cancers. The mu- tations in NPRL2/Gene21, particularly the stop mutations, indicate the lung (RASSF1A, AF102770), heart (RASSF1D, AF102771, and pan- need for further study of this gene as a candidate TSG. creas (RASSF1E, AF102772). No mutations were detected in 40 The BLU gene was discovered manually (and serendipitously) paired normal tumor (SCLC/NSCLC) DNA samples (studied for using PCR primers (kindly provided by B. Vogelstein, Johns Hopkins, RASSF1C/123F2SF and RASSF1/123F2 common region) and in 38 Baltimore, MD) to screen for the presence of the ␤-catenin gene (at lung cancer cell lines (RASSF1C/123F2SF and RASSF1A/123F2LF, the time recently assigned to chromosome region 3p21) in our cosmid all regions). The RASSF1/123F2 protein is a soluble cytoplasmic contig. Although a PCR product was identified, DNA sequence anal- protein that contains a Ras association domain (residues 124–218) ysis showed no sequence relationship of the product to ␤-catenin. This discovered by the SMART (38) and PFAM (37) programs. Although PCR product was used as a probe that identified a mRNA on Northern not all Ras association domains bind RasGTP, the Ras association blot analysis, which then led to the subsequent isolation of the full domain in the mouse paralogue of RASSF1/123F2, NORE1 was BLU cDNA by library screening. The gene space of ϳ4.5 kb contains found to bind RasGTP (71). The NORE1 protein also contains the 11 (testis version) or 12 (lung version) exons coding for a 2-kb, PKC-C1 and DAG/PE domains, which are found in the RASSF1A/ alternatively spliced mRNA, well expressed in lung and testis but not 123F2LF predicted protein but not in the RASSF1C/123F2SF protein. expressed in all other tested human tissues. The EST databases con- Recently, the Kastan group has identified RASSF1/123F2 amino acid tain a moderate number of hits, mostly from lung and testis cDNA sequence (common to both the RASSF1C/123F2SF and RASSF1A/ libraries. The testis isoform contains 11 exons because of a complex 123F2LF proteins) as a potential phosphorylation target for ataxia selection of an alternative acceptor site. The testis-specific protein telangiectasia mutated (72). The mouse orthologue of RASSF1/123F2 isoform contains a different amino acid sequence between residues was discovered in mouse EST databases (accession nos. AA543890, 199 and 234 as compared with the lung-specific isoform; this change AA161846, and AA466998), sequenced (our accession no. results in the loss of one of three PKC phosphorylation sites (residues AF132851), and shown to be 97% identical on protein and 88% on 229–231). The expression in SCLC and NSCLC cell lines is reduced cDNA sequence levels. In contrast, the human orthologue of the or virtually undetectable in 70% of tested lines. Three missense mutations were discovered in a sample of 61 lung cancer cell lines. 32 E. Koonin, personal communication. 6124

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. 3p21.3 TUMOR SUPPRESSOR GENE POSITIONAL CLONING mouse NORE1 and the rat MAXP1 (accession no. AF002251) genes is because of a small (ϳ30 kb) homozygous deletion/rearrangement.34 present in a single human EST (accession no. AA362184). Thus, HYAL2 is abundantly represented in EST databases from normal and RASSF1/123F2 is part of the same gene family as (but not the tumor tissues. No mutations were detected in 40 lung cancer cell lines orthologue of) NORE1 and the rat gene MAXP1. The worm gene, tested. The HYAL2 protein is a member of a large family of hyalu- T24F1.3 (accession no. Z49912), encodes a 615-amino acid hypothet- ronidases (EC3.2.1.35), and in fact the expressed recombinant protein ical protein that shares 33% identity and 53% similarity over 95% of was shown to have enzymatic activity (73). PSORT predicts a signal the length of the RASSF1/123F2 protein. The T24F1.3 protein con- peptide and cell surface and lysosomal sublocalizations (37, 39). ϩ tains in the shared portion with RASSF1/123F2 the Ras association Similarly, SMART predicts a signal peptide and a Ca2 binding domain (residues 396–496), and in addition a PH domain (residues epidermal growth factor-like domain (residues 365–440; Ref. 38). 1–53), and PKC-C1, DAG/PE binding domains (residues:164–214), Recently, the mouse orthologue (AJ000059) was cloned and mapped which are found in the RASSF1A/123F2LF predicted protein. The fly to the syntenic region of mouse chromosome 9 between the micro- (66) and yeast (50) proteomes do not contain a gene with substantial satellite markers D9Mit183 and D9Mit17 (74). The worm gene, homology to 123F2. The absence of expression of RASSF1A/ T22C8.2, encodes a similar size protein of 458 amino acids and shows 123F2LF in many lung cancers makes this isoform an attractive 32% global identity and 50% similarity with the HYAL2 protein and candidate for further promoter hypermethylation and tumor-suppress- is predicted to be an orthologous protein by the Orthologue program ing functional studies. In fact, recent studies by us have shown that (48). The fly (66) and yeast (50) proteome databases do not have any RASSF1A/123F2LF promoter region CpG islands undergo tumor- members with homology to the family of proteins. acquired hypermethylation associated with loss of expression, and that HYAL1 was discovered along with HYAL2, manually by screening forced re-expression of RASSF1A/123F2LF leads to suppression of cDNA libraries with a conserved genomic fragment from cosmid the malignant phenotype.33 LUCA13. It is another hyaluronidase with amino acid sequence ho- The FUS1 gene was discovered manually by screening cDNA mology to HYAL2 and HYAL3 (see below). The gene space of ϳ3.5 libraries with a genomic fragment from the area of cosmids LUCA12 kb contains three exons coding for a 2.6-kb mRNA well expressed in and LUCA13 showing sequence conservation by Southern blot hy- all analyzed human tissues, including lung, and is abundantly repre- bridization and isolated as the fusion (FUS ϭ “fusion”) junction of the sented in EST databases from normal and tumor tissues. However, it ends part of a ϳ30-kb homozygous deletion in SCLC NCI-H524 is not expressed in 18 of 20 lung cancer cell lines. Two missense linking LUCA12 with LUCA13 sequences. The gene space of 3.3 kb mutations were detected in 40 lung cancer cell lines. The HYAL1 contains three exons coding for a 1.8-kb mRNA that is well expressed protein is a member of a large family of (EC3.2.1.35) in all analyzed human tissues including lung and in 20 lung cancer cell and in fact was shown to have enzymatic activity (accession nos. lines. The mRNA is well represented in EST databases from normal U03056 and U96078.1). Triggs-Raine et al. (75) identified two mu- and tumor cells. Three mutations were discovered in 79 lung cancer tations in the HYAL1 alleles of a patient with newly described lyso- cell line DNAs leading to truncated products. The FUS1 protein (110 somal disorder, IX, a mutation that introduces amino acids) is probably a soluble cytoplasmic protein with a high pI a nonconservative amino acid substitution (Glu268Lys) in a putative of 9.69; no domains or known motifs were detected by SMART (38) active site residue and a complex intragenic rearrangement, or PFAM (37) programs. The mouse orthologue was discovered in 1361del37ins14, which results in a premature termination codon. mouse EST databases (AA867009, AA473614, and AA672013), se- They reasoned that the mild phenotype engendered by these mutations quenced (our accession no. AF123387), and shown to be 93% iden- was the result of redundancy resulting from the three tandemly located tical on the protein level and 87% on the cDNA level. The fly hyaluronidases HYAL1, HYAL2, and HYAL3 (discussed below). Thus proteome (66) does not contain a gene with substantial homology to far, no increased incidence of cancer has been reported in these FUS1. However, the worm gene, C09E9.1, shows 41% identity on kindreds. PSORT predicts a signal peptide and a cell surface and lysosomal sublocalizations (39). Similarly, SMART predicts a signal global alignment and 43% identity over 83% of the FUS1 protein 2ϩ length and should be considered a candidate orthologue of FUS1. This peptide and a visible Ca binding epidermal growth factor-like small worm protein (123 amino acids) is predicted to have a bipartite domain (residues 357–430; Ref. 38). Recently, the mouse orthologue was cloned and shown to map to the syntenic mouse chromosome 9 NLS (residues 84–101) and weak similarity with DNA-directed RNA region (accession no. AF011567; Ref. 76). The worm gene, T22C8.2, polymerase subunit AЈ (accession no. P31813). The mutations found encodes a similar size protein of 458 amino acids and shows 31% in FUS1 make it an attractive candidate for further functional TSG global identity and 46% similarity with the HYAL1 protein and studies. contains the same domains. It is predicted to be an orthologous protein The HYAL2 gene along with HYAL1 was discovered manually by by the Orthologue program (48). The fly (66) and yeast (50) proteome screening cDNA libraries with a genomic fragment from LUCA13 databases do not have a member homologous to the hyaluronidase conserved across species in Southern blotting. Because these were the family of proteins. The absent expression and occurrence of mutations first two genes we isolated in our positional cloning effort, they were make HYAL1 an attractive candidate for future promoter methylation initially given the working names LUCA1 and LUCA2 (see GenBank and TSG functional studies. deposits). With the discovery of their function as hyaluronidases, they The FUS2 gene was discovered manually by screening cDNA should now be referred to as HYAL1 and HYAL2 and the LUCA1, 2 libraries with a genomic fragment occurring in cosmid LUCA14 that names reserved for future discovery of a lung cancer functional TSG. showed conservation in Southern blot cross species hybridizations. The gene space of 2.8 kb contains three exons that encode a 2-kb FUS2 also was present in the fusion junction genomic DNA clone mRNA well expressed in all analyzed human tissues including lung, isolated from the NCI-H524 30-kb homozygous deletion but was not well expressed in lung cancer cell lines except SCLC line NCI-H524 involved or rearranged in this deletion but was given the “FUS” working name at the time of its isolation. The gene space of ϳ3.5 kb 33 D. G. Burbee, E. Forgacs, S. Zo¨chbauer-Mu¨ller, L. Shivakumar, K. Fong, B. Gao, D. Randle, A. Virmani, S. Bader, Y. Sekido, F. Latif, S. Milchgrub, A. F. Gazdar, M. I. Lerman, E. Zabarovsky, M. White, and J. D. Minna. RASSF1A in the 3p21.3 homozygous 34 S. Bader, F. Latif, Y. Sekido, M-H. Wei, F-M. Duh, J-Y. Chen, K. Tartoff, C-C. Lee, deletion region: epigenetic inactivation in lung and breast cancer and suppression of the V. Kashuba, R. Gizatullin, E. Zabarovsky, G. Klein, B. Zbar, A. F. Gazdar, M. I. Lerman, malignant phenotype, manuscript in preparation. and J. D. Minna, unpublished data. 6125

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. 3p21.3 TUMOR SUPPRESSOR GENE POSITIONAL CLONING contains an intronless, single-copy gene (accession no. AF040705) The IFRD2/SKMC15/SM15 gene was discovered experimentally by coding for a 1.9-kb mRNA expressed in normal human tissues in- screening cDNA libraries with conserved genomic fragments (56). cluding lung. However, an alternatively spliced form (accession no. GenBank refers to SKMC15/SM15 as IFRD2, and thus we will use the AF040706) with one intron exists that results in the same predicted terminology IFRD2/SM15. The gene space is ϳ6 kb and codes for a amino acid sequence. The alternatively spliced form contains an ϳ4-kb mRNA composed of 12 exons, expressed in several human intron in the 5Ј untranslated region, whereas the other form is intron- tissues including lung (56). It is well represented in EST databases less. The mRNA is well represented in EST databases from normal from normal and tumor cells. The IFRD2/SM15 protein is a soluble and tumor tissues. Four FUS2 missense mutations were detected in 78 nuclear protein as predicted by PSORT (39) and contains a bipartite lung cancer cell lines. The FUS2 protein was predicted to be a soluble NLS at residues 115–132 predicted by ProfileScan (43). PFAM (37) nuclear protein [predicted by PSORT (39)] with interesting domains discovered one Armadillo-␤ catenin-like repeat (residues 249–288), and motifs. SMART (38) and PFAM (37) programs predicted an which suggests the possibility of involvement in APC signaling. No acetyltransferase (GNAT) domain (residues 66–189) and a proline- mutations were found in 63 lung cancer cell lines (56). The mouse rich domain (residues 239–262) that overlaps (residues 234–249) orthologue was discovered in ESTs (accession no. W65790), se- with the Wilms’ tumor protein signature. A Src homology 2 domain quenced and shown to be 93% identical on the protein level and 87% (residues 240–250) was detected by the BLOKS (77) program, on the cDNA level. This true mouse orthologue of IFRD2/SM15 is whereas the EMOTIF (42) program detected a ZP motif (residues different from the mouse gene mIFRD1/PC4, which has its own 25–32) and an eukaryotic thiol (cysteine) protease signature (residues human orthologue located on chromosome 7q22–31 (80). Interest- 180–188), which may explain the suggested weak similarity to furin- ingly, mIFRD1/PC4 and its human orthologue, IFRD1, are not local- like proteases (accession no. AAC02732). The presence of these ized in the nucleus and probably are membrane proteins. IFRD2/SM15 domains raises the intriguing possibility that FUS2 may be directly and IFRD1/PC4 have different patterns of expression during mouse involved in nuclear activities. However, Zegerman et al. (78) dem- development (47, 80). The relation of IFRD2/SM15 and probably onstrated recently that FUS2 functions as an N-acetyltransferase using other members of the family (PC4 and IFRD1) to the IFNs is not a ping-pong mechanism with a specificity for substrates and is a really supported. The slightly shorter worm protein, F58B3.6 (acces- soluble cytoplasmic protein. The worm protein, C56G2.15, shows sion no. Z73427), shows on global alignment 36% identity and 52% 32% identity and 65% similarity on global alignment and should be similarity to the SM15 protein and should be considered a potential considered a true orthologue of the FUS2 gene. As expected, it also orthologue. The fly gene CG3098 product (accession no. AAF51186) contains all but the ZP predicted protein domains and is predicted to is shorter (324 residues) and has 43% similarity to 277 residues of be a nuclear protein. Interestingly, the worm gene contains three small SM15, has a NLS signal (residues 93–110), and should be considered introns in contrast to the one or no intron forms of the human FUS2 a potential orthologue. The expression of IFRD2/SM15 and lack of gene. The mouse orthologue was discovered in mouse EST databases mutations make it a less attractive TSG candidate. (accession nos. AA051756, AA051686, AI425576, and AA833145), The SEMA3B/SEMA A(V) gene was discovered experimentally by sequenced and shown to be 69% identical on protein level and 87% on using DNA fragments from cosmid LUCA14 to screen cDNA librar- cDNA level (accession no. AF172275). The mouse mFUS2 protein ies and for capture of exons (57). The correct nomenclature for this contains an additional 28-amino acid stretch, and similar to the human member of the semaphorin family is SEMA3B [previously referred to protein, is predicted to be a nuclear protein by the PSORT program as SEMA-A(V)]. It is composed of 17 exons spread over 8–10 kb of (39). PFAM (37) and ProfileScan (43) both predict an acetyltrans- genomic space coding for a 3.4-kb mRNA expressed in several ferase (GNAT) domain (residues 92–217) and a proline-rich domain normal tissues including lung and testis and not expressed at all in 12 (residues 267–291). The Flybase (66) contains several ESTs (acces- SCLC lines (Fig. 2; Ref. 57). It is well represented in EST databases sion nos. AI064351, AI109425, and AI404849), which could be from normal and tumor tissues. Three missense mutations were found assembled into a partial cDNA coding for a 168-residue protein that in 39 lung cancer cell lines; all mutations were in NSCLCs. The is 39% identical and 60% similar to the human FUS2 protein and is mouse semaphorin A gene (accession no. X85990) is most likely the predicted to have an acetyltransferase (GNAT) domain (residues mouse orthologue of the SEMA3B gene (86% identity and 89% 13–138). However, the fly proteome (66) does not have a true ortho- similarity on the protein level on global alignment; Ref. 48). Several logue of FUS2, only several proteins with a GNAT domain. The mouse EST clones (accession nos. AI553114, AA518074, and occurrence of mutations and the demonstration of its biochemical AA466386) when translated show 80–94% identity. The worm ge- activity make FUS2 and attractive candidate for future TSG functional nome contains three semaphorin genes, of which the CeSema gene studies. (accession no. U15667) shows 33% identity and 49% similarity over The HYAL3 gene was discovered in silico by finding EST matches the whole length of the CeSema protein and could be considered an and sequence relationship to the HYAL1 and HYAL2 genes. It occupies orthologous gene (48). The fly proteome (66) contains seven sema- 5.5 kb of genomic space and codes for a ϳ2.0-kb mRNA composed phorin proteins, of which the product of the Sema-2a gene (accession of two or three coding exons, expressed in several human tissues no. AAF57990) is of similar size, predicted to be secreted, has a including lung and testis and well represented in EST databases. The similar domain structure, and should be considered a potential candi- protein belongs to the hyaluronidase family of enzymes (EC3.2.1.35) date orthologue of SEMA3B. The SEMA3B protein is predicted by and thus represents the third member of this family in the region. PSORT (39) to be an extracellular secreted protein. SMART (38) and Phylogenetically, it is closer to the worm hyaluronidase gene, PFAM (37) programs identify a signal peptide (residues 1–25), a T22C8.2, than the other members of the human family. No mutations PFAM:SEMA domain (residues 55–497), and one IGc2 domain (res- were found in 40 lung cancer cell lines. No mouse orthologous idues 587–646). Interestingly, the PFAM:SEMA domain is also pres- sequences were found in the databases as of April 2000. HYAL3 RNA ent in the extracellular part of the MET and RON oncoproteins was not expressed in any lung cancer cell lines (data not shown); belonging to the MET family of receptor tyrosine kinases, as discov- however, it has a very restricted pattern of expression in normal ered by the PFAM program (37). Thus, it will be reasonable to test the tissues and was not found by Csoka et al. to be expressed in normal hypothesis that interaction of SEMA3B and SEMA3F (see below) lung (79). The lack of mutations and absent expression in normal lung proteins with these oncogenes may disrupt the activation of MET and make HYAL3 a less attractive TSG candidate. RON and therefore convey a negative growth signal. The lack of 6126

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. 3p21.3 TUMOR SUPPRESSOR GENE POSITIONAL CLONING expression and mutation make SEMA3B an attractive candidate for pendently by several groups (Refs. 19, 57, and 63; accession nos. methylation and TSG functional analysis. U38276 and U33920). The current correct nomenclature for this gene The GNAI2 gene was discovered and cloned 12 years ago as part of is SEMA3F (previously referred to as SEMAIV and SEM IIIF). The studies on G proteins (21). GNAI2 was mapped to 3p21.3 by us and gene space of ϳ28 kb encodes a 3- and/or 4-kb mRNA composed of others and located to the central part of the 370-kb region (Fig. 1; 18 exons. The gene is well expressed in several normal tissues Refs. 9, 17, 18, and 20). It is composed of 8 exons spread over ϳ22 including lung (19, 57, 63) and is represented in EST databases from kb of genomic space. The ϳ2.5-kb mRNA is well expressed in normal normal and tumor tissues and cell lines. Comparison of the cDNA tissues and lung cancer cell lines and is well represented in EST sequences of Xiang et al. (63) and Roche et al. (63; U38276 and databases. No mutations were found in 34 lung cancer cell lines. The U33920) indicate that there are alternatively spliced forms of product is a G protein localized to the endoplasmic reticulum. PFAM SEMA3F. SEMA3F was expressed in several but not all lung cancer (37) predicts a G-␣ domain (residues 6–354) and an arf domain cell lines (15 of 19; Ref. 57). No mutations were found in 30 lung (residues 157–307). The ␣ GTPase function was established experi- cancer lines by Sekido et al. (57) testing 456 of the total 753 amino mentally. The mouse orthologue (accession nos. RGMSI2 and acids for mutations and in tests of the full ORF in 28 SCLC cell lines P08752) is 98% identical and 99% similar on protein and 96% by Xiang et al. (63). ProfileScan (43) predicts an extracellular (se- identical on cDNA levels. The worm orthologue (accession no. creted) protein containing several recognized domains. SMART (38) P51875) is 67% identical, and the fly orthologue (accession no. and PFAM (37) predict a signal peptide (residues 1–18), a single P20353) is 76% identical on the protein level. The newly predicted fly immunoglobulin-like domain (residues 619–680), two PFAM:SEMA gene product G-o␣47A (accession no. AAF58790) is identical in size, domains (residues 57–153 and 184–529), and a proline-rich region 72% similar in amino acid sequence, and should be considered a (residues 715–726). As discussed for SEMA3B, the SEMA domains potential orthologue of GNAI2. The lack of mutations and continued raise the possibility that the SEMA3F protein interrupts signaling by expression of GNAI2 in lung cancers suggest it is an unlikely TSG the MET and RON receptor tyrosine kinase receptors. The mouse candidate. orthologue isoforms a and b were cloned (accession nos. AF80090 The G17 gene was discovered experimentally by cDNA selection and AF080091) and also exist in several EST clones (accession nos. onto cosmid LUCA17, which was then used to screen cDNA libraries. AA216900, W75532, and AA216899). The fly proteome (66) con- The gene space of 17 kb encodes a 3-kb mRNA composed of 18 tains seven semaphorin proteins, of which the product of the CG4383 exons. The mRNA is expressed in several human tissues including gene (accession no. AAF57999) is predicted to be secreted, has a lung, well represented in EST databases from normal and tumor similar domain structure, and could be considered a potential candi- tissues. No mutations were found in 38 lung cancer cell lines, and date orthologue of SEMA3F. This fly protein is similar to the worm Gene17 was expressed in many lung cancers. The product is predicted gene CeSema (accession no. U15667), which is 29% identical and to function as a plasma membrane amino acid transporter by homol- 48% similar to SEMA3F, and both are predicted to be secreted ogy to ABC transporters. It contains 10–11 transmembrane helices proteins. The loss of expression of SEMA3F and results from the [predicted by SPLIT (40) and TMHMM (41) programs] and an Naylor laboratory (52), showing suppression of tumorigenicity of aromatic amino acid permease-2/xan_ur_permease domain (residues mouse A9 fibrosarcoma cells by a P1 clone in the SEMA3F region 67–455) predicted by ProfileScan and PFAM servers. The mouse means this gene has to be considered in functional and methylation orthologue was discovered in several ESTs clones (accession nos. analysis. AI098786, AI048261, and AI466351), sequenced (our accession no. The G15/RBM5 gene was discovered experimentally by screening AI098786), and shown to be 90% identical on cDNA and 97% on arrayed cDNA libraries with cosmid LUCA22 (U73168) DNA. The protein levels. The yeast (50), worm, and fly (66) proteomes contain same gene (called RBM5) was cloned by others (Ref. 81; accession several amino acid transporter genes of similar size. The lack of no. AAD04159). The gene space of 18.5 kb encodes 18 exons ex- mutations and continued expression make Gene17 an unlikely TSG pressed as a main 4-kb mRNA in several human tissues (a minor candidate. 7.5-kb species, and in some tissues, 1.5-kb and 2-kb species are also The GNAT1 gene was cloned 10 years ago (Ref. 22; accession no. expressed). The mRNA is well expressed in several lung cancer cell X15088) and encodes the transducin protein isolated from the eye. We lines and is represented in human and mouse EST databases from and others positioned the gene in 3p21.3, i.e., in the homozygous normal and tumor tissues. No mutations were found in 18 lung cancer deletion overlap region close to the GNAI2 gene (Fig. 1; Refs. 18 and cell lines. According to ProfileScan (43), PFAM (37) and SMART 20). The gene space of ϳ3.5 kb contains seven exons and encodes a (38), the G15/RBM5 protein is a nuclear RNA binding protein fea- 1.5-kb mRNA expressed abundantly in the retina and fetal heart turing two bipartite NLSs (residues 34–51 and 708–725), two RNA- tissues and T-cell lines. GNAT1 was not expressed in lung and lung binding domains (residues 98–178 and 231–315), two zinc finger cancer cell lines. No mutations were found in analysis of genomic motifs, i.e., ZF RANBP (residues 181–210), and ZINC FINGER 2H2 DNA in 35 lung cancer cell lines. The mouse orthologue (48) was 2 (residues 647–677), and a recently recognized D111/G-patch cloned (accession no. P20612) and is 100% identical on global (residues 749–786). G15/RBM5 has amino acid sequence alignment. The worm (accession no. P51875) and fly (accession no. homology with its immediate telomeric neighbor, G16/RBM6/NY-LU- P20353) proteomes contain similarly sized G-proteins with 50 and 12/DEF-3 (see below), indicating that they are part of the same gene 60% identity, respectively, with yet unknown function. The newly family and arose through gene duplication. Studies by Drabkin et al. predicted fly gene product G-o␣65A (accession no. AAF50626) is (82) have shown that both G15/RBM5 and G16/RBM6/NY-LU-12/ identical in size, 86% similar, and should be considered a true ortho- DEF-3 recombinant proteins can specifically bind poly(G) RNA ho- logue of GNAT1. The transducin protein is an ␣1 G-protein subunit mopolymers in vitro. The mouse orthologue was discovered in several localized in the endoplasmic reticulum. PFAM (37) predicts a G-␣ EST clones (accession nos. AA574979, AA139814, and AI197332) domain (residues 2–349) and an arf domain (residues 161–342). The that are 90% identical on cDNA and 97% on the protein level. The restricted tissue distribution of expression of GNAT1 and lack of worm gene, T08B2.5 (accession no. AF000263), is 28% identical and mutations make GNAT1 an unlikely TSG candidate. 45% similar on global protein alignment and is probably a true The SEMA3F/SEMA-IV/SEM IIIF gene, the second semaphorin orthologue (48). The fly proteome (66) contains three similar proteins, gene in the region, was identified experimentally and cloned inde- of which the CG4887 gene product (accession no. AAF51398) shows 6127

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43% similarity over 90% of the G15 length, has a similar domain homozygous deletions were precisely mapped at 3p12 (U2020 region; structure, and is a likely orthologue of G15. The rat RNA-binding Refs. 9, 11, and 96–98), 3p14.2 (FHIT region; Refs. 93 and 95), protein S1–1 (accession no. P70501) has a similar domain structure 3p21.3 (6, 9, 10, 17–19, 51), and 3p21.3–3p22 (94, 99). Allelotyping and is close in amino acid sequence to G15/RBM5; however, a rat EST of histological subtypes of lung cancer showed differences in allele clone (accession no. AI112056) on translation showed 97% identity to loss patterns at several chromosomal sites in addition to 3p between the last 37 amino acids of G15/RBM5 (outside the recognized do- SCLC versus NSCLC histological types (8, 14). In addition, both mains) and probably represents the true rat orthologue of G15/RBM5. SCLCs and squamous cell lung cancers usually undergo allele loss of The lack of mutations and continued expression in most lung cancers a large part of the 3p arm. In contrast, adenocarcinomas of the lung means that G15/RBM5 is an unlikely TSG candidate. tend to have much smaller regions of 3p allele loss (6, 8, 9, 14, The G16/RBM6/DEF-3/NYLU12 gene, the second gene encoding 16, 91). an RNA-binding protein in the homozygous deletion region, was The 3p21.3 location was chosen for the current effort based on discovered experimentally by screening cDNA libraries with a con- allele loss mapping studies showing that this is perhaps the first served breakpoint genomic fragment from P1 clone 3938. Others have chromosomal region with allele loss in preneoplastic lesions and even also cloned this gene (referred to as RBM6, NY-LU-12, and DEF-3). in the histologically normal bronchial epithelium of current and for- The identification of the NY-LU-12 clone through expression cloning mer smokers (12, 14, 16, 100). Thus, TSG(s) residing in this region using antibodies to detect the protein from a patient with lung cancer are likely to play a causative role in the earliest steps of lung cancer by means of the SERAX technology, and the def-3 clone as a differ- pathogenesis. In addition, the presence of overlapping/nested ho- entially expressed gene during myelopoiesis, are of particular interest mozygous deletions in lung and breast cancer and functional studies (81–83). The gene spans the distal breakpoint of the NCI-740 deletion demonstrating the presence of a tumor phenotype-suppressing func- (Ref. 18; Fig. 1), occupies more than 60 kb of genomic DNA, and tion gave us both positional and functional reasons to pursue this encodes more than 16 exons expressed as a 4-kb mRNA in several search (6, 9, 10, 17, 19, 20, 52–54). This exact region has also been human and mouse tissues, including lung. Alternatively spliced forms identified as a site frequently undergoing loss in peripheral blood have been described (83). This mRNA is well expressed in several lymphocytes of lung cancer patients treated in vitro with the tobacco lung cancer cell lines and is represented in human and mouse EST smoke carcinogen benzo(a)pyrene diol epoxide (101). We first ob- databases from normal and tumor tissues. One mutation was found in tained a physical map of the homozygously deleted area, which we 39 lung cancer cell lines. According to ProfileScan (43), PFAM (37), then covered with a cosmid and P1 clone contig (Fig. 1 and Ref. 18). and SMART (38), the G16/RBM6 protein is a nuclear RNA binding Through the auspices of the NCI, we were able to have this ϳ630-kb protein. The following motifs and domains were predicted: a bipartite clone contig sequenced by the Sanger Genome Center (Hinxton, NLS (residues 1016–1033), one RNA-biding domain (residues 456– United Kingdom; cosmids LUCA1-LUCA11) and the St. Louis Ge- 536), two zinc fingers, ZF-RANBP (residues 535–565), and ZF-C2H2 nome Center (LUCA12-LUCA22, P3938). The cosmid DNA se- (residues 955–980, and a D111 DOMAIN (residues 1057–1094). quence accession nos. are given in Fig. 1, and the DNA sequences are Recombinant proteins containing the RNA recognition motifs of available from GenBank. These DNA sequence have been condensed DEF-3(g16/NY- LU-12) and LUCA15 specifically bound poly(G) to several contigs (NT_002322, NT_000067, and NT_000069).29 RNA homopolymers in vitro (82). The mouse orthologue was cloned We identified genes in the 630-kb region by experimental and (accession no. AJ006486) and also exists in several EST clones informatics methods (discussed above), bringing the total number of (accession nos. AA607276 and AA549397) with 90% identity on the verified resident genes to 25. In addition, several candidate “genes” cDNA level and 97% on the protein level. The fly proteome (66) were predicted by BLAST EST hits, GENSCAN and XGRAIL gene contains three similar proteins, of which the CG4887 gene product prediction software that we could not yet confirm as being real genes (accession no. AAF51400) shows 43% similarity over 85% of the G16 as defined by either detection of a mRNA on Northern blots or the length, has a similar domain structure, and is a potential orthologue of presence of a cDNA with exon/intron structure and a reasonable ORF. G16. The lack of mutations and continued expression in lung cancers With the complete genomic DNA sequence, all of the genes could, in make G16/RBM6 an unlikely TSG candidate. retrospect, be identified by informatics methods “in silico.” The 19 genes residing in the largest homozygous deletion overlap region DISCUSSION (from cosmid LUCA6 to part of P1 clone P3938) were analyzed by experimental (expression and mutational) analyses and computational We initiated these studies to gain insight into the molecular mech- methods (for predicted function using web-based servers) for infor- anisms of lung cancer pathogenesis with the goal of identifying and mation supporting their candidacy as a TSG. The extensive search for cloning a lung cancer TSG(s) residing in chromosome region 3p21.3. mutations in the 19-gene set did not identify a single gene with a high Our interest in this region began with our initial cytogenetic descrip- frequency of mutation in the analyzed lung tumors but did identify tion of a chromosome 3p deletion abnormality in SCLC and subse- NPRL2/Gene21, BLU, FUS1, HYAL1, FUS2, and SEMA3B as candi- quently in NSCLCs Ͼ17 years ago (84–86). This was followed by dates for further study because of the occurrence of a few mutations. our discovery of 3p allele loss in SCLC (87–90). During this period, In addition, a significant frequency of absent mRNA expression in many groups also described 3p allele loss in lung cancer and other lung cancer cell lines was found for the CACNA2D2, RASSF1A, common cancers and found 3p cytogenetic abnormalities (9). Karyo- HYAL1, BLU, and SEMA3B genes, also indicating their possible typing, comparative genomic hybridization, interphase FISH (summa- involvement in lung carcinogenesis as candidate TSGs with homozy- rized in Ref. 91), and identification of homozygous deletions, in gotic inactivation in tumors. The combination of mutations and absent conjunction with numerous molecular allelotyping studies, have iden- expression in BLU, HYAL1, SEMA3B, and for the RASSF1A/123F2LF tified a minimum of four separate consensus regions of 3p allele loss, mRNA isoform suggests special attention be paid to these genes. With i.e., 3p25-p26, 3p21-p22, 3p14.2 (FHIT region), and 3p12 (U2020 the recognition of tumor-acquired promoter hypermethylation as a homozygous deletion region; Refs. 6, 8, 9, 11, 17, 19, 51, and 92–95). frequent mechanism of gene inactivation, those genes with loss of A more recent study with very high density allelotyping, as well as expression need to be studied for such acquired promoter changes results from multiple groups, have identified at least eight different 3p (23). In addition, functional information documenting tumor suppres- LOH sites (14). To aid in this TSG positional cloning effort, the sor or other relevant activity (e.g., DNA repair) will be required to 6128

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. 3p21.3 TUMOR SUPPRESSOR GENE POSITIONAL CLONING provide a convincing argument for TSG identification. Computational not confirm as real genes (i.e., that did not give positive signals on analyses predicting function for the encoded proteins among the Northern blots or show several matches in searches of the EST 19-gene set did not reveal obvious homology to a known TSG, nor did databases). In contrast, GENSCAN accurately predicted the location it exclude genes from having a possible TSG function. and nearly all of the intron/exon structures of the genes we did There are several obvious features of this 3p21.3 region that pro- identify. Related to the gene detection question is the possibility that vide information on the evolution of this part of the genome. There are there is a tissue-specific splicing isoform that we have not detected, four examples of gene duplication: the G proteins GNAI2 and GNAT1; and it is this isoform that is mutated or has altered expression. Because the hyaluronidases HYAL1, HYAL2, and HYAL3; the semaphorins, of this possibility, we have particularly scrutinized GENSCAN exon SEMA3B and SEMA3F; and the RNA binding proteins encoded by predictions, ESTs mapping to intragenic regions not covered by our Gene15/RBM5 and Gene16/RBM6/DEF-3/Ny-Lu-12 genes. Each of current cDNA sequences, and poly(A) Northern blots containing these duplications (or triplications in the case of the hyaluronidase normal lung RNA for other possible mRNA forms. In fact, this genes) occurred within 50–100-kb regions. In the case of the G- methodology has allowed us to detect many different mRNA splicing protein and semaphorin gene regions, a complex or sequential dupli- variants, including those for at least 8 of the 19 genes including cation must have occurred to account for the orientation of these four CACNA2D2, NPRL2/Gene21, BLU, RASSF1/123F2, HYAL1, FUS2, genes. In the case of Gene16 and Gene15 and the hyaluronidase SEMA3F, and Gene16/RBM6/DEF-3/Ny-Lu-12. Our detailed gene genes, it appears tandem duplications occurred. Other genes (e.g., analysis, coupled with an ever-enlarging EST database including FUS2 and Gene17) then arose between these gene clusters. These information from many different tissues, will facilitate further mRNA duplicated genes pairs (or triplets) all have about the same amino acid splicing form detection. However, our analysis was not able to rule ϳ ( 40–50%) to their duplicated partners, indicat- out the possibility that this 3p21.3 region encodes one or more ing that these duplications arose at approximately the same time. Also mRNA-like noncoding RNA type genes that may function as TSG(s). with the exception of the G-protein genes (showing 67% homology), We do not know of any currently available informatics approach that the hyaluronidase, semaphorin, and RNA binding protein encoding can identify such genes. Alignment of the human genomic sequence ϳ genes all have 30% amino acid sequence homology with their with the syntenic mouse sequence may provide clues to the location of orthologous/homologous worm or fly genes. In each case, the worm additional genes such as these by detecting conserved sequences in the or fly genomes have only one copy of the gene and not two, as are intergenic/intronic regions where these genes could reside (59).35 present in the human and mouse genomes. Nevertheless, continued searches of the ever-growing EST database as The absence of frequent mutations in the resident candidate TSGs well as comparing the sequence of the homologous region in the suggests we have to consider several other possibilities: mouse (when the whole sequence is available) would provide addi- (a) We need to consider the possibility that the homozygous dele- tional support to the assumption that we have identified all of the tions only represent random genetic damage from tobacco smoke genes in this region. carcinogen exposure or the presence of a previously unknown fragile Another possibility is that we have identified the gene and that it is site, and thus, this specific 3p21.3 region does not contain a TSG. inactivated by a combination of mutation (uncommon) and loss of There are no obvious ways to prove or disprove that the three expression (frequent) mechanisms. Recently, methylation of the pro- overlapping homozygous deletions in the SCLCs are random dele- moter region and allele loss leading to loss of expression of p16INK4A tions. However, the recurrent presence of allele loss in this very have been identified as a combination of mechanisms leading to the specific region in multiple lung cancers and respiratory preneoplastic inactivation of this accepted TSG in lung cancer (108). In addition, lesions and the occurrence of a homozygous deletion in a breast several other TSGs, such as RB, VHL, and BRCA1, as well as a series cancer not exposed to tobacco smoke carcinogens, as well as the of genes related to tumorigenesis such as E-cadherin (E-CAD), death- microcell hybrid data indicating a tumor suppressor function in this associated protein kinase (DAPK), glutathione S-transferase P1 region, all argue against this possibility (10, 14, 16, 19, 52–54, 100). Homozygous deletions have also been crucial in the identification of (GSTP1), O-6-methylguanine-DNA-methyltransferase (MGMT), and several TSGs in other tumor types such as p16 (102), SMAD4 (103), CACNA1G, a T-type calcium channel gene, also have been found to BRCA2 (104), and p53 (105). Also, homozygous deletions have been have their expression turned off by tumor-acquired promoter hyper- found in lung cancer for these and other TSGs including RB, p53, p16, methylation, some of which occur in lung cancers (6, 23, 108, 110– FHIT, and PTEN (33, 93, 95, 106–108). Finally, in recently com- 112). The definition of the precise structure for each of the 19 genes pleted genome-wide scans of lung cancers using Ͼ600 polymorphic and their associated CpG islands in putative promoter regions identi- markers, we have found only a few chromosomal sites with recurrent fied by the current work will greatly aid in our current search for homozygous deletions that include 3p12 (U2020 region), 3p14.2 promoter hypermethylation as a mechanism of inactivation of expres- (FHIT region), 3p21.3 (the current region under study), 9p21 (p16/ sion of genes in this region. Recent evidence indicates this to be the INK4 region), 10q (PTEN region), and a few other regions [Girard et mechanism for loss of expression of RASSF1A. In this regard, 5 of the al. (109) and Refs. 8, 11, 14, 33, and 95]. These studies include over 19 genes showed loss of expression in a large fraction of at least one 40 markers distributed over chromosome arm 3p. Thus, in our expe- histological type of lung cancer (CACNA2D2, RASSF1A, SEMA3B, rience, the occurrence of recurring homozygous deletions in the same BLU, and HYAL1). A more complex version of this possibility is that chromosomal region are very uncommon and appear to target TSGs. more than one gene in this 3p213 region can serve as a TSG. Because (b) It is possible that we have not identified all of the genes residing Ͼ15 different mutations occurred independently of one another (in 6 in the critical region. Although this remains a possibility, the combi- genes, BLU, NPRL2/Gene21, FUS1, HYAL1, FUS2, and SEMA3B) nation of extensive computational and experimental approaches and independently of the loss of expression (in 5 genes) in the 40–80 (screening multiple cDNA libraries with cosmid clones and using the lung cancers tested, it is possible that nearly all of the lung tumor lines cosmid clones for exon capture) would argue against this possibility. had either a mutation or loss of expression for one of these 8 genes. No matches in EST databases suggestive of other genes have been Thus, these 8 genes in particular need to undergo detailed functional detected in regular searches using the intergenic and intronic se- tests to rule out their potential tumor suppressor function. quences. In addition, the gene identification programs GENSCAN (27) and XGRAIL (49) predicted only five candidates that we could 35 Internet address: http://bio.cse.psu.edu/pipmaker/. 6129

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A final possibility is that the TSG in this 3p21.3 region is 1 (or carcinoma in situ lesions are evident (12, 15, 16, 100). Lung embry- more) of the 19 cloned genes we have identified but does not conform ology identifies lung stem cells as the primitive columnar epithelia to the classical two-mutation model for TSGs requiring homozygous derived from the primordial upper gut at days 40–45 of development inactivation in the tumor (113, 114). Instead, the gene may represent (127). These columnar cells first differentiate into multipotent neu- an example of the emerging class of haploinsufficient TSGs inferred roendocrine cells (probably, the cells of origin of SCLC) and a variety from mouse models of human cancer (115–118). Recently it has been of multipotent bronchial epithelia cells (probable the cells of origin of discovered that mice hemizygous (ϩ/Ϫ) for p27/Kip1 or TGF␤ show NSCLC). In fact, some lung cancers, within the same tumor, exhibit ϩ ϩ a higher spontaneous tumor rate compared with mice / at these histological features of several lung cancer types, indicating a possible loci and rapidly develop multiple tumors when challenged with car- common stem cell of origin (128, 129). The embryology of the lung cinogens. Analysis of these tumors demonstrated that the remaining would therefore suggest that the same genes could be involved in both wild-type alleles for these genes remained intact and expressed in the SCLC and NSCLC carcinogenesis. tumors (115, 116). Mice hemizygous for the Ptch gene frequently In the light of these observations, the following model of lung develop the rare tumor medulloblastoma with continued expression of cancer pathogenesis is proposed. Consequent to smoking damage, one wild-type Ptch allele. This provides evidence that haploinsuffi- 3p21.3 allele loss occurs in thousands of different sites throughout the ciency of Ptch leads to medulloblastoma in mice, and a similar respiratory epithelium, leaving the putative 3p21.3 TSG haploinsuf- circumstance appears to occur in humans as well (117). Similarly, ficient. These initiated cell(s) grow to form clonal patches of ϳ50– haploid loss of the tumor suppressor Smad4/Dpc4 initiated gastric 100,000 cells (ϳ15–17 doublings) throughout the lung. The next hit polyposis and cancer in mice, suggesting that haploinsufficiency of could occur in another cancer-causing gene (such as RB, p53, p16, or Smad4 is sufficient for tumor initiation (118). In addition, the auto- somal dominant syndrome of familial platelet disorder with predispo- “gene X”), leading to the next stages of invasive cancer. The RB and sition to acute myelogenous leukemia (FPD/AML and MIM 601399) p53 genes are mutated in nearly 100% of SCLC samples, whereas in humans was found to be related to haploinsufficiency of the CBFA2 mutations of p53 occur in 50% of NSCLC and some form of p16 gene (119). Thus, we need to consider the possibility that the TSG in inactivation (also inactivating the “RB pathway”) occurs in a very the 3p21.3 region may be an example of an acquired abnormality in large fraction of NSCLCs (6). Alternatively, it is possible that the such a haploinsufficient gene. 3p21.3 TSG undergoes allele loss and then a second inactivating event This brings us to the discussion of lung cancer gene model(s). (either an uncommon mutation or loss of expression through promoter Inherited (120), initiating (114), gate-keeper (121), and cancer-caus- hypermethylation), which is required to allow the clonal outgrowth of ing (our term) genes follow the two-hit proposition for cancer causa- these initiated cells. In any event, the nearly universal presence of tion (113, 114, 120–123). These genes (TSG or oncogenes) are 3p21.3 allele loss in SCLC and squamous cell of the lung usually discovered in an inherited family cancer syndrome through and its occurrence in Ͼ50% of adenocarcinomas of the lung suggest genetic mapping and positional cloning (113, 114). Consistent with that this alteration is an obligate rate-limiting step in the pathogenesis the two-hit model, the corresponding tumors show loss of one allele of many lung cancers (6, 8, 14, 16). In this regard, it will be very and inactivating mutations of the remaining wild-type allele (in the interesting to study SCLCs arising in the rare families segregating a case of a TSG). In the case of an inherited activated oncogene (such mutant RB allele in the germ-line in which the majority of affected as MET), the tumors may show loss of the wild-type allele with a individuals surviving to adulthood have developed SCLC to see simultaneous duplication of the mutant allele or a second activating whether tumors arising under these conditions also have to undergo mutation in the remaining wild-type allele (124). The net result of 3p21.3 allele loss (130). It should also be noted that allele loss that these rate-limiting genetic changes (122, 123) is a homozygous status includes 3p21.3 and immediately surrounding 3p21 regions would for these genes in tumors. In contrast, the haploinsufficient TSGs lose lead to the hemizygotic state for several other possible cancer-predis- one copy in the germ-line or somatic tissues, predispose as a result of posing genes residing in the area, including, MLH1, TGF␤RII, ␤-cate- this loss to cancer but remain hemizygous (and express the wild-type nin, RON, and Wnt5, which also could contribute to malignant trans- allele) in tumors. Because the acquisition of other genetic alterations formation. Functional testing by gene transfer into lung cancer cells is still rate-limiting in developing cancer (122, 123), another mutation and gene disruption strategies in mice are now necessary to test this in a different gene would appear to be required in the haploinsufficient model and identify the putative 3p21.3 TSG(s). cancer model. It is tempting to assume that it could be a mutation in Studies of familial lung cancer risk, including data from lung cancer a second known TSG such as p53, RB,orp16, all of which are occurring in young nonsmoking individuals, are compatible with frequently mutated in many common cancers with 3p21.3 allele loss Mendelian codominant inheritance of a rare major autosomal gene including lung cancers (6). There is no reason to exclude the possi- that produces earlier age of onset of lung and probably other cancers bility that classical TSGs and the haploinsufficient cancer-causing (7, 131, 132). A new national consortium (Genetic Epidemiology of genes both could be involved in the development of sporadic malig- nancies arising from a common stem cell. In addition, it is likely that Lung Cancer) has been formed that has accumulated enough families 3p21.3 allele loss is involved in the development of all histological with informative cases with an apparently inherited predisposition to types of lung cancer. Premalignant lesions in the lung have 3p21.3 lung cancer to begin genetic linkage analysis. In due time, a classical LOH that is also found in a large fraction of SCLCs and NSCLCs (12, TSG(s) (with homozygous inactivation in tumors) segregating in these 15, 16, 100, 125). These include clones with 3p21.3 allele loss in lung cancer pedigrees could also be discovered. A causative role for histologically normal-appearing epithelium found in the lungs of this inherited lung cancer gene in the origin of sporadic lung cancers current and former smokers (16, 100). Recently, we have been able to would also have to be ascertained. Linkage to markers in this 3p21.3 estimate the size of these clonal patches with 3p21.3 and other sites of region can be readily tested, and if found, the genes we have reported LOH to be ϳ90,000 cells, as well as determine that the smoking- in this study would need to be screened for germ-line mutations. In damaged lung contains thousands of such patches (126). We also any event, the early involvement of chromosome 3p21.3 allele loss in know from studies of multiple markers that allele loss at classic TSG premalignant lesions and sporadic lung cancers argues that one or loci, such as at RB (13q14) and p53 (17p13), occurs after 3p allele loss more of the genes we have found should play a critical role in the at a later stage of carcinogenesis when histologically dysplastic or development of common, sporadic lung tumors. 6130

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2000 American Association for Cancer Research. The 630-kb Lung Cancer Homozygous Deletion Region on Human Chromosome 3p21.3: Identification and Evaluation of the Resident Candidate Tumor Suppressor Genes

Michael I. Lerman and John D. Minna

Cancer Res 2000;60:6116-6133.

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