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Onc2011554.Pdf Oncogene (2012) 31, 3939–3948 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Identification of novel CHD1-associated collaborative alterations of genomic structure and functional assessment of CHD1 in prostate cancer W Liu1,2, J Lindberg3, G Sui4, J Luo5, L Egevad6,TLi1,2, C Xie1,2, M Wan4, S-T Kim1,2, Z Wang1,2, AR Turner1,2, Z Zhang1,2, J Feng1,2, Y Yan7, J Sun1,2, GS Bova8, CM Ewing5, G Yan5, M Gielzak5, SD Cramer4, RL Vessella9, SL Zheng1,2, H Gro¨nberg3, WB Isaacs5 and J Xu1,2,7 1Center for Cancer Genomics, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 2Center for Human Genomics and Personalized Medicine Research, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 3Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden; 4Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 5Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 6Department of Pathology and Cytology, Karolinska University Hospital, Stockholm, Sweden; 7Center for Genetic Epidemiology and Prevention, Van Andel Research Institute, Grand Rapids, MI, USA; 8Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA and 9Department of Urology, University of Washington, Seattle, WA, USA A clearer definition of the molecular determinants that drive Introduction the development and progression of prostate cancer (PCa) is urgently needed. Efforts to map recurrent somatic deletions Although many prostate cancer (PCa) tumors are in the tumor genome, especially homozygous deletions indolent and pose little health hazard, an important (HODs), have provided important positional information in subset are aggressive and progress to disseminated the search for cancer-causing genes. Analyzing HODs in the disease, resulting in B34 000 deaths in the United States tumors of 244 patients from two independent cohorts and 22 every year (Siegel et al., 2011). Recent studies have PCa xenografts using high-resolution single-nucleotide highlighted the collaborative nature of multiple genomic polymorphism arrays, herein we report the identification of alterations underlying the critical process of PCa CHD1, a chromatin remodeler, as one of the most frequently progression (Carver et al., 2009; King et al., 2009; Ding homozygously deleted genes in PCa, second only to PTEN et al., 2011). in this regard. The HODs observed in CHD1, including Indeed, recent deep sequencing of the exome and deletions affecting only internal exons of CHD1, were found the whole genome of tumor cells has revealed the to completely extinguish the expression of mRNA of this extraordinary complexity of genomic alterations that gene in PCa xenografts. Loss of this chromatin remodeler in characterize human cancers (Berger et al., 2011; clinical specimens is significantly associated with an Robbins et al., 2011). This complexity consists of increased number of additional chromosomal deletions, both various combinations of base substitution, transloca- hemi- and homozygous, especially on 2q, 5q and 6q. tions, gene fusion and copy-number alterations (CNAs). Together with the deletions observed in HEK293 cells stably It is becoming increasingly clear that CNAs are a major transfected with CHD1 small hairpin RNA, these data component of the landscape of the PCa tumor genome suggest a causal relationship. Downregulation of Chd1 in (Taylor et al., 2010; Robbins et al., 2011; Kan et al., mouse prostate epithelial cells caused dramatic morphologi- 2010). To assess the significance of these alterations in cal changes indicative of increased invasiveness, but did not the development of PCa, it is necessary to distinguish result in transformation. Indicating a new role of CHD1, alterations driving proliferation of cancer cells versus these findings collectively suggest that distinct CHD1- random changes or passengers. Beroukhim et al. (2007) associated alterations of genomic structure evolve during have developed an important tool (Genomic Identifica- and are required for the development of PCa. tion of Significant Targets in Cancer (GISTIC)) that has Oncogene (2012) 31, 3939–3948; doi:10.1038/onc.2011.554; been used in the identification of a number of cancer published online 5 December 2011 genes (Beroukhim et al., 2010; Taylor et al., 2010). After candidate regions of CNAs are identified, a Keywords: CHD1; homozygous deletion; prostate cancer major challenge is how to then further identify candidate cancer genes, as the size of deletions and amplifications are usually very large, covering many genes, especially in high-grade and metastatic PCa. Correspondence: Dr WB Isaacs, Brady Urological Institute, Johns Hopkins Medical Institutions, Marburg 115, 600 North Wolfe Street, Using GISTIC with stringent criteria can help to narrow Baltimore, MD 21287, USA. the search to a target region with fewer genes. Even so, a E-mail: [email protected] or Dr J Xu, Center for Cancer Genomics, majority of such regions in the tumor genome are Wake Forest University School of Medicine, Medical Center Boulevard, hemizygous, with their in vivo biological effect on the Winston-Salem, NC 27157, USA. growth advantage of cancer cells being difficult to infer E-mail: [email protected] Received 5 July 2011; revised 14 September 2011; accepted 9 October because a second, unaltered allele is still present, 2011; published online 5 December 2011 notwithstanding established haploinsufficient or CHD1 and genomic alterations in prostate cancer WLiuet al 3940 dominant cancer genes. The unequivocal loss-of-func- Table 1 Screening for HOD in significant CNAs regions of deletion tion associated with homozygous gene deletions (HODs) identified by GISTICa can simplify this process. As a result, identifying and Representative JHH Sweden Combined characterizing HODs have led to the discovery of gene and CNA-region multiple recessive human cancer genes with PTEN (cytoband) being an important example in PCa. Number % Number % Number % Herein we report the assessment of HOD in the tumor PTEN(10q23.31) 18 12.77 16 15.53 34 13.93 genomes of 244 patients with primary PCa from two CHD1(5q21.1) 10 7.09 11 10.68 21 8.61 independent cohorts and 22 xenografts via genome-wide BNIP3L(8p21.2) 3 2.13 2 1.94 5 2.05 analysis of CNAs. While uncovering a number of new LRP1B(2q22.1) 2 1.42 3 2.91 5 2.05 RB1(13q14.2) 3 2.13 1 0.97 4 1.64 recurrent HODs using allele-specific analysis, we dis- USP10(16q24.1) 1 0.71 2 1.94 3 1.23 cover that CHD1 is the second, only to PTEN, most TMPRSS2- 3 2.13 0 0.00 3 1.23 frequent homozygously deleted gene in PCa. We find ERG(21q22) that loss of both the alleles of CHD1 is significantly HTR3A(11q23.2) 2 1.42 NA NA 2 0.82 associated with additional, potentially targeted HOD in RYPB(3p13) 2 1.42 0 0.00 2 0.82 MAP3K7(6q15) 1 0.71 1 0.97 2 0.82 the tumor genome. Accordingly, chromosomal deletions TP53(17p13.1) 0 0.00 1 0.97 1 0.41 associated with experimental knockdown of CHD1 CDKN1B(12p13.1) 1 0.71 0 0.00 1 0.41 provide evidence that CHD1 may have a causal role in SERPINB5(18q21.33- 0 0.00 0 0.00 0 0.00 prevention of deletion events. These data revealed a 22.1) PDE4D(5q11.2) 0 0.00 0 0.00 0 0.00 novel CHD1-associated coordinative network of altera- DISC1(1q42.2) 0 0.00 0 0.00 0 0.00 tions, and suggest a new role for this chromatin remodeler in the development of PCa. Abbreviations: CNAs, copy-number alterations; GISTIC, Genomic Identification of Significant Targets in Cancer; HOD, homozygous deletions; JHH, the Johns Hopkins Hospital. Results NA, not tested due to in significant in GISTIC analysis. aGISTIC was used to distinguish the regions of CNAs that drive cancer growth from numerous random CNAs that accumulate during cancer CHD1 is the second most frequent homozygously deleted development. gene in PCa Recurrent somatic deletions in the tumor genome, especially HODs, have been informative targets in the (Supplementary Figures 2–5) with informative samples search for tumor-suppressor genes. To uncover the full harboring deletions in these regions and confirmed our spectrum of HODs in primary PCa tumors, we findings from the GISTIC analysis as described above. performed a comprehensive analysis of DNA CNAs in Importantly, genome-wide allele-specific analysis re- the tumor genomes of surgical specimens of cancer vealed multiple occurrences of CHD1 HOD in both of tissues from 244 PCa patients using Affymetrix high- the cohorts from JHH and Sweden, with frequencies of resolution single-nucleotide polymorphism arrays 7.1% and 10.7%, respectively (Table 1 and Supplemen- (Affymetrix, Santa Clara, CA, USA). To identify CNAs tary Figures 6 and 7). In comparison, HOD frequency at that likely drive cancer growth, we first used GISTIC PTEN was observed in B13% and 16%, respectively, of with a q ¼ 0.01 (false discovery rate (FDR)) and a join- the sample in these two cohorts. Thus, at this resolution, segment-size of 80 probes to identify the significant CHD1 is second only to PTEN as the most frequent regions of deletions. The data revealed 20 significant homozygously deleted gene in the tumor genome of CNA regions, including 15 and 13 deletions (Supple- PCa. mentary Figure 1) in the Johns Hopkins Hospital (JHH) In primary PCa tumors, the size of HODs affecting and Swedish cohorts, respectively, as well as 5 and 7 CHD1 ranged from B138 kb to 2898 kb, in some amplifications. Although distinct CNAs unique to a instances covering RGMG, FAM174A and ST8SIA4, specific cohort were observed, the overall patterns were in addition to CHD1 (Supplementary Table 1). Three of remarkably similar. For example, all the 13 regions of the HODs eliminated the 50 region of CHD1 (Figure 1a), deletion identified in the Swedish cohort overlapped while the majority removed the whole gene (Figure 1b).
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