A Genome-Wide Screen for Microdeletions Reveals Disruption of Polarity Complex Genes in Diverse Human Cancers
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Published OnlineFirst March 9, 2010; DOI: 10.1158/0008-5472.CAN-09-3458 Published Online First on March 9, 2010 as 10.1158/0008-5472.CAN-09-3458 Priority Report Cancer Research A Genome-Wide Screen for Microdeletions Reveals Disruption of Polarity Complex Genes in Diverse Human Cancers S. Michael Rothenberg1,3,4, Gayatry Mohapatra1,5, Miguel N. Rivera1,3,5, Daniel Winokur1, Patricia Greninger1,2, Mai Nitta1,5, Peter M. Sadow5, Gaya Sooriyakumar1, Brian W. Brannigan1, Matthew J. Ulman1, Rushika M. Perera1, Rui Wang6, Angela Tam1,2, Xiao-Jun Ma7, Mark Erlander7, Dennis C. Sgroi1,5, James W. Rocco1, Mark W. Lingen8, Ezra E.W. Cohen9, David N. Louis1,5, Jeffrey Settleman1,2, and Daniel A. Haber1,3 Abstract In a genome-wide screen of 684 cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contact-dependent proliferation. Searching specifically for small intragenic microdeletions using high-resolution genomic arrays may be com- plementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes. Cancer Res; 70(6); 2158–64. ©2010 AACR. Introduction efforts used genome-wide PCR-based methods to detect rare homozygous deletions (2, 3), array-based hybridization tech- Classic tumor suppressor gene discovery involved loss-of- nologies (4, 5), and, most recently, whole-genome exon rese- heterozygosity screens, followed by detailed mapping of quencing (6, 7). However, the complexity of these efforts has tumor-dependent allelic losses (1), whereas subsequent limited the number of tumors that can be screened by each method, a critical consideration because the recurrence of a Authors' Affiliations: 1Massachusetts General Hospital Cancer Center and gene-targeting event across different cancers is key to distin- 2Center for Molecular Therapeutics, Harvard Medical School, Charlestown, Massachusetts; 3Howard Hughes Medical Institute, 4Center for Head and guishing driver from passenger mutations. Neck Cancers, 5Department of Pathology, and 6Biostatistics Center, Here, we performed a high-resolution array-based gene Massachusetts General Hospital and Harvard Medical School, Boston, copy number analysis of 684 cancer cell lines, with the goal Massachusetts; 7Biotheranostics, San Diego, California; and Departments of 8Pathology and 9Medicine, University of Chicago, Chicago, Illinois of identifying rare homozygous deletions across a broad pan- Note: Supplementary data for this article are available at Cancer el of diverse cancer types. Although initial arrays based on Research Online (http://cancerres.aacrjournals.org/). BACs or cDNAs contained relatively sparse genomic probes, The data discussed in this publication have been deposited in NCBI Gene the advent of oligonucleotide arrays with every increasing Expression Omnibus (21) and are accessible through GEO Series probe density has paved the way for simultaneously interro- accession no. GSE20306 (http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE20306). gating large numbers of tumors at increased resolution for discrete regions of genomic loss (8, 9). However, genomic in- Author Contributions: S.M. Rothenberg, G. Mohapatra, and M.N. Rivera directed the study, performed functional work, and interpreted the data. stability in epithelial cancers has complicated attempts at D. Winokur performed PCR, RT-PCR, and sequencing analysis. P. Greninger finding recurrent deletions using copy number screens, lead- performed copy number analysis. M. Nitta performed FISH analysis. G. ing to algorithms that readily reveal large, frequent amplifi- Sooriyakumar, M.J. Ulman, and B.W. Brannigan performed sequencing analysis. P.M. Sadow, D.N. Louis, and M.W. Lingen contributed sam- cations and deletions, possibly at the expense of focal or less ples and performed pathologic analysis. R.M. Perera and A. Tam per- frequent variation in allelic dosage (10, 11). In contrast, we formed functional work. X-J. Ma and M. Erlander performed oligonucleotide array hybridization. D.C. Sgroi performed laser capture reasoned that the use of a very large cancer cell line panel microdissection. J.W. Rocco, E.E.W. Cohen, and D.N. Louis contrib- would enable a focused screen for small intragenic deletions, uted samples and gave comments on the manuscript. S.M. Rothenberg, and that recurrence of such focal deletions across different G. Mohapatra, M.N. Rivera, D.N. Louis, J. Settleman, and D.A. Haber conceived the study. S.M. Rothenberg and D.A. Haber wrote the man- cancers would serve as evidence of functional significance. uscript. S. Michael Rothenberg, G. Mohapatra, and M.N. Rivera contrib- In addition, the primary screen provides the key target cells uted equally to this work. in which to test reconstitution of the deleted gene. Corresponding Authors: Daniel A. Haber and Jeffrey Settleman, Massa- chusetts General Hospital Cancer Center, CNY7 Building 149, 13th Street, Charlestown, MA 02129. Phone: 617-726-5628; Fax: 617-726-5637; E-mail: Materials and Methods [email protected] and [email protected]. doi: 10.1158/0008-5472.CAN-09-3458 Human cancer cell lines. Sources for human cancer cell ©2010 American Association for Cancer Research. lines are listed in Supplementary Table S1. To minimize the 2158 Cancer Res; 70(6) March 15, 2010 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 9, 2010; DOI: 10.1158/0008-5472.CAN-09-3458 Microdeletions Disrupting Polarity Complex Genes chance of cross-contamination or misidentification, genomic PCR-basedexonresequencingandreversetranscrip- DNA for array hybridization was prepared from each cell line tion-PCR. See Supplementary Data. within 6 mo of receipt and as soon as possible after initial cul- Fluorescence in situ hybridization. All BAC probes were ture (always within 1 mo). Methods of cell line verification per- obtained from BAC/PAC Resources (Children's Hospital, formed by the individual repositories include STR DNA typing, Oakland, CA). PARD3 gene copy number was determined cytogenetic analysis, and immunophenotyping. Additional de- by two-color fluorescence in situ hybridization (FISH) on 5- tails of verification steps are described in Supplementary Data. μm-thick sections following published protocols (13). Images Primary tumors. Frozen and paraffin-embedded tumor were acquired using an Olympus BX61 fluorescent micro- samples were obtained from the MGH and University of Chi- scope equipped with a charge-coupled device camera, and cago Departments of Pathology (SCCHN) and the MGH Brain analysis was done with Genus software (Applied Imaging). Tumor Bank (GBM). The presence of tumor within each tumor At least 100 nuclei were scored for each sample. Nontumor tissue was confirmed by a pathologist (P.M.S. and M.W.L. for tissue samples were used as controls (Cybrdi, Inc.). SCCHN; D.N.L. for GBM) by H&E staining of representative tis- DNA and shRNA constructs and lentivirus expression. sue sections. In cases where tumor cells composed <70% of the The cDNA for PARD3 was generously provided by Ian Macara tissue, microdissection was used to isolate a homogeneous tu- (University of Virginia, Charlotsville, VA). See Supplementary mor cell population. Data for additional details on cDNA and shRNA vector con- Array hybridization. Sample processing for genomic struction and expression. DNA, complexity reduction, amplification, purification, label- Immunoblotting and immunofluorescence. See Supple- ing, and hybridization to GeneChip Human Mapping 250K mentary Data. Sty Arrays10 were done as described in the Affymetrix Gene- Growth assays. Cells were plated at 50,000 to 100,000 per Chip Human Mapping 500K assay manual (P/N 701930 Rev. 2). well in triplicate in six-well plates. At each time point, cells Genomic DNA samples from six EBV-transformed B-lympho- were detached with trypsin, stained with 1:10 dilution of 0.1% cyte cell lines were included as normal controls for copy num- trypan blue, and counted using a Cellometer Auto T4 (Nex- ber calculations (Coriell Institute for Medical Research, celom Bioscience LLC). Medium was refreshed on the re- Camden, NJ). Array hybridization, data acquisition, and anal- maining wells every 3 d. ysis for Agilent Human 105K oligonucleotide arrays were based on published methods (12). Additional methods de- Results scribing preparation of nucleic acid and data acquisition and initial processing are available in Supplementary Data. Analysis of 684 specimens with 250,000 SNP probes iden- Copy number analysis and identification of candidate tified 11,328 regions with a reduction in copy number consis- tumor suppressor loci. The method for identifying homozy- tent with a homozygous deletion (<0.3; Supplementary Fig. gous deletions is outlined in Supplementary Fig. S1B and S1; Supplementary Table S1). Filtering for overlap with exons described in detail in Supplementary Data. Briefly, after calcu- of protein-coding genes and involvement of the candidate lation of copy number for each SNP probe using dCHIP,11 a gene by at least one other deletion in an unrelated cancer cell copy number threshold equal to <0.3 was applied to select can- line produced 211 genes identified as being inactivated by fo- didate deleted regions