Abstract
Chronic cigarette smoking exposes airway epithelial cells to thousands of carcinogens, oxidants and DNA-damaging agents, creating a field of molecular injury in the airway and altering gene expression. Studies of cytologically normal bronchial epithelial cells from smokers have identified transcription-based biomarkers that may prove useful in early diagnosis of lung cancer, including a number of p53-regulated genes. The ability of p53 to regulate transcription is critical for tumor suppression, and this suggests that single-nucleotide polymorphisms (SNPs) in functional p53 binding sites (p53 response elements, or p53REs) that affect gene expression could influence susceptibility to cancer. To connect p53RE SNP genotype with gene expression and cancer risk, we identified a set of 204 SNPs in putative p53REs, and performed cis expression quantitative trait loci (eQTL) analysis, assessing associations between SNP genotypes and mRNA levels of adjacent genes in bronchial epithelial cells obtained from 44 cigarette smokers. To further test and validate these genotype–expression associations, we searched published eQTL studies from independent populations and determined that 53 % (39/74) of the bronchial epithelial eQTLs were observed in at least one of other studies. SNPs in p53REs were also evaluated for effects on p53-DNA binding using a quantitative in vitro protein–DNA binding assay. Last, based on linkage disequilibrium, we found 6 p53RE SNPs associated with gene expression were identified as cancer risk SNPs by either genome-wide association studies or candidate gene studies. We provide an approach for identifying and evaluating potentially functional SNPs that may modulate the airway gene expression response to smoking and may influence susceptibility to cancers.
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References
Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, Kang HM, Marth GT, McVean GA (2012) An integrated map of genetic variation from 1,092 human genomes. Nature 491:56–65. doi:10.1038/nature11632
Ahrendt SA, Chow JT, Xu LH, Yang SC, Eisenberger CF, Esteller M, Herman JG, Wu L, Decker PA, Jen J, Sidransky D (1999) Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer. J Natl Cancer Inst 91:332–339
Bandele OJ, Wang X, Campbell MR, Pittman GS, Bell DA (2011) Human single-nucleotide polymorphisms alter p53 sequence-specific binding at gene regulatory elements. Nucleic Acids Res 39:178–189. doi:10.1093/nar/gkq764
Cheung VG, Spielman RS, Ewens KG, Weber TM, Morley M, Burdick JT (2005) Map** determinants of human gene expression by regional and genome-wide association. Nature 437:1365–1369
Dunham I, Kundaje A, Aldred SF, Collins PJ, Davis C, Doyle F, Epstein CB, Frietze S, Harrow J, Kaul R, Khatun J, Lajoie BR, Landt SG, Lee BK, Pauli F, Rosenbloom KR, Sabo P, Safi A, Sanyal A, Shoresh N, Simon JM, Song L, Trinklein ND, Altshuler RC, Birney E, Brown JB, Cheng C, Djebali S, Dong XJ, Dunham I, Ernst J, Furey TS, Gerstein M, Giardine B, Greven M, Hardison RC, Harris RS, Herrero J, Hoffman MM, Iyer S, Kellis M, Khatun J, Kheradpour P, Kundaje A, Lassmann T, Li QH, Lin X, Marinov GK, Merkel A, Mortazavi A, Parker SCJ, Reddy TE, Rozowsky J, Schlesinger F, Thurman RE, Wang J, Ward LD, Whitfield TW, Wilder SP, Wu W, ** HLS, Yip KY, Zhuang JL, Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, Snyder M, Pazin MJ, Lowdon RF, Dillon LAL, Adams LB, Kelly CJ, Zhang J, Wexler JR, Green ED, Good PJ, Feingold EA, Bernstein BE, Birney E, Crawford GE, Dekker J, Elnitski L, Farnham PJ, Gerstein M, Giddings MC, Gingeras TR, Green ED, Guigo R, Hardison RC, Hubbard TJ, Kellis M, Kent WJ, Lieb JD, Margulies EH, Myers RM, Snyder M, Stamatoyannopoulos JA, Tenenbaum SA et al (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489:57–74. doi:10.1038/Nature11247
Franklin WA, Gazdar AF, Haney J, Wistuba II, La Rosa FG, Kennedy T, Ritchey DM, Miller YE (1997) Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis. J Clin Invest 100:2133–2137
Grundberg E, Small KS, Hedman AK, Nica AC, Buil A, Keildson S, Bell JT, Yang TP, Meduri E, Barrett A, Nisbett J, Sekowska M, Wilk A, Shin SY, Glass D, Travers M, Min JL, Ring S, Ho K, Thorleifsson G, Kong A, Thorsteindottir U, Ainali C, Dimas AS, Hassanali N, Ingle C, Knowles D, Krestyaninova M, Lowe CE, Di Meglio P, Montgomery SB, Parts L, Potter S, Surdulescu G, Tsaprouni L, Tsoka S, Bataille V, Durbin R, Nestle FO, O’Rahilly S, Soranzo N, Lindgren CM, Zondervan KT, Ahmadi KR, Schadt EE, Stefansson K, Smith GD, McCarthy MI, Deloukas P, Dermitzakis ET, Spector TD (2012) Map** cis- and trans-regulatory effects across multiple tissues in twins. Nat Genet 44:1084–1089. doi:10.1038/ng.2394
Harden SV, Thomas DC, Benoit N, Minhas K, Westra WH, Califano JA, Koch W, Sidransky D (2004) Real-time gap ligase chain reaction: a rapid semiquantitative assay for detecting p53 mutation at low levels in surgical margins and lymph nodes from resected lung and head and neck tumors. Clin Cancer Res 10:2379–2385
Herbst RS, Heymach JV, Lippman SM (2008) Lung cancer. N Engl J Med 359:1367–1380
Hindorff LA, Sethupathy P, Junkins HA, Ramos EM, Mehta JP, Collins FS, Manolio TA (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci 106:9362–9367. doi:10.1073/pnas.0903103106
Lampe JW, Stepaniants SB, Mao M, Radich JP, Dai H, Linsley PS, Friend SH, Potter JD (2004) Signatures of environmental exposures using peripheral leukocyte gene expression: tobacco smoke. Cancer Epidemiol Biomark Prev 13:445–453
Liang L, Morar N, Dixon AL, Lathrop GM, Abecasis GR, Moffatt MF, Cookson WO (2013) A cross-platform analysis of 14,177 expression quantitative trait loci derived from lymphoblastoid cell lines. Genome Res 23:716–726. doi:10.1101/gr.142521.112
Morley M, Molony CM, Weber TM, Devlin JL, Ewens KG, Spielman RS, Cheung VG (2004) Genetic analysis of genome-wide variation in human gene expression. Nature 430:743–747
Nikulenkov F, Spinnler C, Li H, Tonelli C, Shi Y, Turunen M, Kivioja T, Ignatiev I, Kel A, Taipale J, Selivanova G (2012) Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis. Cell Death Differ 19:1992–2002. doi:10.1038/cdd.2012.89
Noureddine MA, Menendez D, Campbell MR, Bandele OJ, Horvath MM, Wang X, Pittman GS, Chorley BN, Resnick MA, Bell DA (2009) Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts. PLoS Genet 5:e1000462
Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55:74–108
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81:559–575
Riley T, Sontag E, Chen P, Levine A (2008) Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 9:402–412
Schadt EE, Molony C, Chudin E, Hao K, Yang X, Lum PY, Kasarskis A, Zhang B, Wang S, Suver C, Zhu J, Millstein J, Sieberts S, Lamb J, GuhaThakurta D, Derry J, Storey JD, Avila-Campillo I, Kruger MJ, Johnson JM, Rohl CA, van Nas A, Mehrabian M, Drake TA, Lusis AJ, Smith RC, Guengerich FP, Strom SC, Schuetz E, Rushmore TH, Ulrich R (2008) Map** the genetic architecture of gene expression in human liver. PLoS Biol 6:e107
Shields PG (1999) Molecular epidemiology of lung cancer. Ann Oncol 10(Suppl 5):S7–S11
Slaughter DP, Southwick HW, Smejkal W (1953) Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 6:963–968
Spira A, Beane J, Shah V, Liu G, Schembri F, Yang X, Palma J, Brody JS (2004) Effects of cigarette smoke on the human airway epithelial cell transcriptome. Proc Natl Acad Sci 101:10143–10148
Spira A, Beane JE, Shah V, Steiling K, Liu G, Schembri F, Gilman S, Dumas YM, Calner P, Sebastiani P, Sridhar S, Beamis J, Lamb C, Anderson T, Gerry N, Keane J, Lenburg ME, Brody JS (2007) Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer. Nat Med 13:361–366
Steiling K, Ryan J, Brody JS, Spira A (2008) The field of tissue injury in the lung and airway. Cancer Prev Res (Phila Pa) 1:396–403
Stranger BE, Forrest MS, Clark AG, Minichiello MJ, Deutsch S, Lyle R, Hunt S, Kahl B, Antonarakis SE, Tavare S, Deloukas P, Dermitzakis ET (2005) Genome-wide associations of gene expression variation in humans. PLoS Genet 1:e78
Stranger BE, Nica AC, Forrest MS, Dimas A, Bird CP, Beazley C, Ingle CE, Dunning M, Flicek P, Koller D, Montgomery S, Tavare S, Deloukas P, Dermitzakis ET (2007) Population genomics of human gene expression. Nat Genet 39:1217–1224
Tomso DJ, Inga A, Menendez D, Pittman GS, Campbell MR, Storici F, Bell DA, Resnick MA (2005) Functionally distinct polymorphic sequences in the human genome that are targets for p53 transactivation. Proc Natl Acad Sci 102:6431–6436
Toyooka S, Tsuda T, Gazdar AF (2003) The TP53 gene, tobacco exposure, and lung cancer. Hum Mutat 21:229–239
Veyrieras JB, Kudaravalli S, Kim SY, Dermitzakis ET, Gilad Y, Stephens M, Pritchard JK (2008) High-resolution map** of expression-QTLs yields insight into human gene regulation. PLoS Genet 4:e1000214. doi:10.1371/journal.pgen.1000214
Wang X, Tomso DJ, Chorley BN, Cho HY, Cheung VG, Kleeberger SR, Bell DA (2007) Identification of polymorphic antioxidant response elements in the human genome. Hum Mol Genet 16:1188–1200
Wang X, Chorley BN, Pittman GS, Kleeberger SR, Brothers J 2nd, Liu G, Spira A, Bell DA (2010) Genetic variation and antioxidant response gene expression in the bronchial airway epithelium of smokers at risk for lung cancer. PLoS One 5:e11934. doi:10.1371/journal.pone.0011934
Westra HJ, Peters MJ, Esko T, Yaghootkar H, Schurmann C, Kettunen J, Christiansen MW, Fairfax BP, Schramm K, Powell JE, Zhernakova A, Zhernakova DV, Veldink JH, Van den Berg LH, Karjalainen J, Withoff S, Uitterlinden AG, Hofman A, Rivadeneira F, t Hoen PA, Reinmaa E, Fischer K, Nelis M, Milani L, Melzer D, Ferrucci L, Singleton AB, Hernandez DG, Nalls MA, Homuth G, Nauck M, Radke D, Volker U, Perola M, Salomaa V, Brody J, Suchy-Dicey A, Gharib SA, Enquobahrie DA, Lumley T, Montgomery GW, Makino S, Prokisch H, Herder C, Roden M, Grallert H, Meitinger T, Strauch K, Li Y, Jansen RC, Visscher PM, Knight JC, Psaty BM, Ripatti S, Teumer A, Frayling TM, Metspalu A, van Meurs JB, Franke L (2013) Systematic identification of trans eQTLs as putative drivers of known disease associations. Nat Genet 45:1238–1243. doi:10.1038/ng.2756
Yu W, Gwinn M, Clyne M, Yesupriya A, Khoury MJ (2008) A navigator for human genome epidemiology. Nat Genet 40:124–125. doi:10.1038/ng0208-124
Zeller T, Wild P, Szymczak S, Rotival M, Schillert A, Castagne R, Maouche S, Germain M, Lackner K, Rossmann H, Eleftheriadis M, Sinning CR, Schnabel RB, Lubos E, Mennerich D, Rust W, Perret C, Proust C, Nicaud V, Loscalzo J, Hubner N, Tregouet D, Munzel T, Ziegler A, Tiret L, Blankenberg S, Cambien F (2010) Genetics and beyond–the transcriptome of human monocytes and disease susceptibility. PLoS One 5:e10693. doi:10.1371/journal.pone.0010693
Zeron-Medina J, Wang X, Repapi E, Campbell MR, Su D, Castro-Giner F, Davies B, Peterse EF, Sacilotto N, Walker GJ, Terzian T, Tomlinson IP, Box NF, Meinshausen N, De Val S, Bell DA, Bond GL (2013) A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection. Cell 155:410–422. doi:10.1016/j.cell.2013.09.017
Acknowledgments
This work was funded in part by the Intramural Research Program (projects: ZO1-ES100475 and Z01 ES046008) of the National Institute of Environmental Health Sciences, National Institutes of Health and grants to Avrum Spira from the National Institute of Health (U01ES016035, R01CA124640). We are grateful for comments from Drs. Daniel Menendez and Jack Taylor, NIEHS.
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Conceived and designed the experiments: AS DAB. Performed the experiments: XW GSP OJB JB JBII GL. Analyzed the data: XW DAB. Wrote the paper: XW DAB.
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Wang, X., Pittman, G.S., Bandele, O.J. et al. Linking polymorphic p53 response elements with gene expression in airway epithelial cells of smokers and cancer risk. Hum Genet 133, 1467–1476 (2014). https://doi.org/10.1007/s00439-014-1483-8
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DOI: https://doi.org/10.1007/s00439-014-1483-8