Journal of Gastroenterology

, Volume 44, Issue 9, pp 897–911 | Cite as

Differential gene expression in normal esophagus and Barrett’s esophagus

  • Jacob Wang
  • Rong Qin
  • Yan Ma
  • Huiyun Wu
  • Heiko Peters
  • Matthew Tyska
  • Nicholas J. Shaheen
  • Xiaoxin Chen
Original Article—Alimentary Tract

Abstract

Purpose

As the premalignant lesion of human esophageal adenocarcinoma (EAC), Barrett’s esophagus (BE) is characterized by intestinal metaplasia in the normal esophagus (NE). Gene expression profiling with microarray and serial analysis of gene expression (SAGE) may help us understand the potential molecular mechanism of human BE.

Methods

We analyzed three microarray datasets (two cDNA arrays and one oligonucleotide array) and one SAGE dataset with statistical tools, significance analysis of microarrays (SAM) and SAGE(Poisson), to identify individual genes differentially expressed in BE. Gene set enrichment analysis (GSEA) was used to identify a priori defined sets of genes that were differentially expressed. These gene sets were grouped according to either certain signaling pathways (GSEA curated), or the presence of consensus binding sequences of known transcription factors (GSEA motif). Immunohistochemical staining (IHC) was used to validate differential gene expression.

Results

Both SAM and SAGE(Poisson) identified 68 differentially expressed genes (55 BE genes and 13 NE genes) with an arbitrary cutoff ratio (≥4-fold). With IHC on matched pairs of NE and BE tissues from 6 patients, these genes were grouped into 6 categories: category I (25 genes only expressed in BE), category II (5 genes only expressed in NE), category III (8 genes expressed more in BE than in NE), and category IV (2 genes expressed more in NE than in BE). Differential expression of the remaining genes was not confirmed by IHC either due to false discovery (category V), or lack of proper antibodies (category VI). Besides individual genes, the TGFβ pathway and several transcription factors (CDX2, HNF1, and HNF4) were identified by GSEA as enriched pathways and motifs in BE. Apart from 9 target genes known to be up-regulated in BE, IHC staining confirmed up-regulation of 19 additional CDX1 and CDX2 target genes in BE.

Conclusion

Our data suggested an important role of CDX1 and CDX2 in the development of BE. The IHC-confirmed gene list will lead to future studies on the molecular mechanism of BE.

Keywords

Barrett’s esophagus Intestinal metaplasia Expression profile SAM GSEA 

Abbreviations

BE

Barrett’s esophagus

EAC

Esophageal adenocarcinoma

FDR

False discovery rate

GenMapp

Gene Map Annotator and Pathway Profiler

GO

Gene ontology

GSEA

Gene set enrichment analysis

IHC

Immunohistochemical staining

IM

Intestinal metaplasia

NE

Normal esophagus

SAGE

Serial analysis of gene expression

SAM

Significance analysis of microarrays

Supplementary material

535_2009_82_MOESM1_ESM.doc (496 kb)
Supplementary material (DOC 495 kb)
535_2009_82_MOESM2_ESM.xls (9.9 mb)
Supplementary files (XLS 10110 kb)
535_2009_82_MOESM3_ESM.ppt (260 kb)
Supplementary figures (PPT 260 kb)

References

  1. 1.
    Chen X, Yang CS. Esophageal adenocarcinoma: a review and perspectives on the mechanism of carcinogenesis and chemoprevention. Carcinogenesis. 2001;22:1119–29.PubMedCrossRefGoogle Scholar
  2. 2.
    Jankowski JA, Harrison RF, Perry I, Balkwill F, Tselepis C. Barrett’s metaplasia. Lancet. 2000;356:2079–85.PubMedCrossRefGoogle Scholar
  3. 3.
    Fitzgerald RC. Barrett’s oesophagus and oesophageal adenocarcinoma: how does acid interfere with cell proliferation and differentiation? Gut. 2005;54(Suppl 1):i21–6.PubMedCrossRefGoogle Scholar
  4. 4.
    Krishnadath KK. Novel findings in the pathogenesis of esophageal columnar metaplasia or Barrett’s esophagus. Curr Opin Gastroenterol. 2007;23:440–5.PubMedCrossRefGoogle Scholar
  5. 5.
    van Baal JW, Krishnadath KK. High throughput techniques for characterizing the expression profile of Barrett’s esophagus. Dis Esophagus. 2008;21:634–40.PubMedCrossRefGoogle Scholar
  6. 6.
    Barrett MT, Yeung KY, Ruzzo WL, Hsu L, Blount PL, Sullivan R, et al. Transcriptional analyses of Barrett’s metaplasia and normal upper GI mucosae. Neoplasia. 2002;4:121–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Fox CA, Sapinoso LM, Zhang H, Zhang W, McLeod HL, Petroni GR, et al. Altered expression of TFF-1 and CES-2 in Barrett’s esophagus and associated adenocarcinomas. Neoplasia. 2005;7:407–16.PubMedCrossRefGoogle Scholar
  8. 8.
    Gomes LI, Esteves GH, Carvalho AF, Cristo EB, Hirata R Jr, Martins WK, et al. Expression profile of malignant and nonmalignant lesions of esophagus and stomach: differential activity of functional modules related to inflammation and lipid metabolism. Cancer Res. 2005;65:7127–36.PubMedCrossRefGoogle Scholar
  9. 9.
    Greenawalt DM, Duong C, Smyth GK, Ciavarella ML, Thompson NJ, Tiang T, et al. Gene expression profiling of esophageal cancer: comparative analysis of Barrett’s esophagus, adenocarcinoma, and squamous cell carcinoma. Int J Cancer. 2007;120:1914–21.PubMedCrossRefGoogle Scholar
  10. 10.
    Hao Y, Triadafilopoulos G, Sahbaie P, Young HS, Omary MB, Lowe AW. Gene expression profiling reveals stromal genes expressed in common between Barrett’s esophagus and adenocarcinoma. Gastroenterology. 2006;131:925–33.PubMedCrossRefGoogle Scholar
  11. 11.
    Helm J, Enkemann SA, Coppola D, Barthel JS, Kelley ST, Yeatman TJ. Dedifferentiation precedes invasion in the progression from Barrett’s metaplasia to esophageal adenocarcinoma. Clin Cancer Res. 2005;11:2478–85.PubMedCrossRefGoogle Scholar
  12. 12.
    Kimchi ET, Posner MC, Park JO, Darga TE, Kocherginsky M, Karrison T, et al. Progression of Barrett’s metaplasia to adenocarcinoma is associated with the suppression of the transcriptional programs of epidermal differentiation. Cancer Res. 2005;65:3146–54.PubMedGoogle Scholar
  13. 13.
    Ostrowski J, Mikula M, Karczmarski J, Rubel T, Wyrwicz LS, Bragoszewski P, et al. Molecular defense mechanisms of Barrett’s metaplasia estimated by an integrative genomics. J Mol Med. 2007;85:733–43.PubMedCrossRefGoogle Scholar
  14. 14.
    Ostrowski J, Rubel T, Wyrwicz LS, Mikula M, Bielasik A, Butruk E, et al. Three clinical variants of gastroesophageal reflux disease form two distinct gene expression signatures. J Mol Med. 2006;84:872–82.PubMedCrossRefGoogle Scholar
  15. 15.
    Pohler E, Craig AL, Cotton J, Lawrie L, Dillon JF, Ross P, et al. The Barrett’s antigen anterior gradient-2 silences the p53 transcriptional response to DNA damage. Mol Cell Proteomics. 2004;3:534–47.PubMedCrossRefGoogle Scholar
  16. 16.
    Selaru FM, Zou T, Xu Y, Shustova V, Yin J, Mori Y, et al. Global gene expression profiling in Barrett’s esophagus and esophageal cancer: a comparative analysis using cDNA microarrays. Oncogene. 2002;21:475–8.PubMedCrossRefGoogle Scholar
  17. 17.
    van Baal JW, Milano F, Rygiel AM, Bergman JJ, Rosmolen WD, van Deventer SJ, et al. A comparative analysis by SAGE of gene expression profiles of Barrett’s esophagus, normal squamous esophagus, and gastric cardia. Gastroenterology. 2005;129:1274–81.PubMedCrossRefGoogle Scholar
  18. 18.
    Wang S, Zhan M, Yin J, Abraham JM, Mori Y, Sato F, et al. Transcriptional profiling suggests that Barrett’s metaplasia is an early intermediate stage in esophageal adenocarcinogenesis. Oncogene. 2006;25:3346–56.PubMedCrossRefGoogle Scholar
  19. 19.
    Xu Y, Selaru FM, Yin J, Zou TT, Shustova V, Mori Y, et al. Artificial neural networks and gene filtering distinguish between global gene expression profiles of Barrett’s esophagus and esophageal cancer. Cancer Res. 2002;62:3493–7.PubMedGoogle Scholar
  20. 20.
    van Baal JW, Diks SH, Wanders RJ, Rygiel AM, Milano F, Joore J, et al. Comparison of kinome profiles of Barrett’s esophagus with normal squamous esophagus and normal gastric cardia. Cancer Res. 2006;66:11605–12.PubMedCrossRefGoogle Scholar
  21. 21.
    Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001;98:5116–21.PubMedCrossRefGoogle Scholar
  22. 22.
    Cai L, Huang H, Blackshaw S, Liu JS, Cepko C, Wong WH. Clustering analysis of SAGE data using a Poisson approach. Genome Biol. 2004;5:R51.PubMedCrossRefGoogle Scholar
  23. 23.
    Lewin K, Appelman H. Barrett’s esophagus, columnar dysplasia, and adenocarcinoma of the esophagus. In: Appelman KLaH, editor. Tumors of the Esophagus and Stomach. Washington: AFIP; 1995. p. 99–144.Google Scholar
  24. 24.
    Liu T, Zhang X, So CK, Wang S, Wang P, Yan L, et al. Regulation of Cdx2 expression by promoter methylation, and effects of Cdx2 transfection on morphology and gene expression of human esophageal epithelial cells. Carcinogenesis. 2007;28:488–96.PubMedCrossRefGoogle Scholar
  25. 25.
    Wong NA, Wilding J, Bartlett S, Liu Y, Warren BF, Piris J, et al. CDX1 is an important molecular mediator of Barrett’s metaplasia. Proc Natl Acad Sci USA. 2005;102:7565–70.PubMedCrossRefGoogle Scholar
  26. 26.
    Yang Q, Bermingham NA, Finegold MJ, Zoghbi HY. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science. 2001;294:2155–8.PubMedCrossRefGoogle Scholar
  27. 27.
    Eda A, Osawa H, Satoh K, Yanaka I, Kihira K, Ishino Y, et al. Aberrant expression of CDX2 in Barrett’s epithelium and inflammatory esophageal mucosa. J Gastroenterol. 2003;38:14–22.PubMedCrossRefGoogle Scholar
  28. 28.
    Silberg DG, Furth EE, Taylor JK, Schuck T, Chiou T, Traber PG. CDX1 protein expression in normal, metaplastic, and neoplastic human alimentary tract epithelium. Gastroenterology. 1997;113:478–86.PubMedCrossRefGoogle Scholar
  29. 29.
    Chen X, Qin R, Liu B, Ma Y, Su Y, Yang CS, et al. Multilayered epithelium in a rat model and human Barrett’s esophagus: similar expression patterns of transcription factors and differentiation markers. BMC Gastroenterol. 2008;8:1.PubMedCrossRefGoogle Scholar
  30. 30.
    Milano F, van Baal JW, Buttar NS, Rygiel AM, de Kort F, DeMars CJ, et al. Bone morphogenetic protein 4 expressed in esophagitis induces a columnar phenotype in esophageal squamous cells. Gastroenterology. 2007;132:2412–21.PubMedCrossRefGoogle Scholar
  31. 31.
    Slack JM, Tosh D. Transdifferentiation and metaplasia—switching cell types. Curr Opin Genet Dev. 2001;11:581–6.PubMedCrossRefGoogle Scholar
  32. 32.
    Brabender J, Marjoram P, Salonga D, Metzger R, Schneider PM, Park JM, et al. A multigene expression panel for the molecular diagnosis of Barrett’s esophagus and Barrett’s adenocarcinoma of the esophagus. Oncogene. 2004;23:4780–8.PubMedCrossRefGoogle Scholar
  33. 33.
    Glickman JN, Blount PL, Sanchez CA, Cowan DS, Wongsurawat VJ, Reid BJ, et al. Mucin core polypeptide expression in the progression of neoplasia in Barrett’s esophagus. Hum Pathol. 2006;37:1304–15.PubMedCrossRefGoogle Scholar
  34. 34.
    Jovov B, Van Itallie CM, Shaheen NJ, Carson JL, Gambling TM, Anderson JM, et al. Claudin-18: a dominant tight junction protein in Barrett’s esophagus and likely contributor to its acid resistance. Am J Physiol Gastrointest Liver Physiol. 2007;293:G1106–13.PubMedCrossRefGoogle Scholar
  35. 35.
    Kumble S, Omary MB, Fajardo LF, Triadafilopoulos G. Multifocal heterogeneity in villin and Ep-CAM expression in Barrett’s esophagus. Int J Cancer. 1996;66:48–54.PubMedCrossRefGoogle Scholar
  36. 36.
    Madsen J, Nielsen O, Tornoe I, Thim L, Holmskov U. Tissue localization of human trefoil factors 1, 2, and 3. J Histochem Cytochem. 2007;55:505–13.PubMedCrossRefGoogle Scholar
  37. 37.
    Mitas M, Almeida JS, Mikhitarian K, Gillanders WE, Lewin DN, Spyropoulos DD, et al. Accurate discrimination of Barrett’s esophagus and esophageal adenocarcinoma using a quantitative three-tiered algorithm and multimarker real-time reverse transcription-PCR. Clin Cancer Res. 2005;11:2205–14.PubMedCrossRefGoogle Scholar
  38. 38.
    van Baal JW, Bozikas A, Pronk R, Ten Kate FJ, Milano F, Rygiel AM, et al. Cytokeratin and CDX-2 expression in Barrett’s esophagus. Scand J Gastroenterol. 2008;43:132–40.PubMedCrossRefGoogle Scholar
  39. 39.
    Wong NA, Warren BF, Piris J, Maynard N, Marshall R, Bodmer WF. EpCAM and gpA33 are markers of Barrett’s metaplasia. J Clin Pathol. 2006;59:260–3.PubMedCrossRefGoogle Scholar
  40. 40.
    Christie KN, Thomson C. The distribution of carbonic anhydrase II in human, pig and rat oesophageal epithelium. Histochem J. 2000;32:753–7.PubMedCrossRefGoogle Scholar
  41. 41.
    Xia SH, Hu LP, Hu H, Ying WT, Xu X, Cai Y, et al. Three isoforms of annexin I are preferentially expressed in normal esophageal epithelia but down-regulated in esophageal squamous cell carcinomas. Oncogene. 2002;21:6641–8.PubMedCrossRefGoogle Scholar
  42. 42.
    Mobasheri A, Wray S, Marples D. Distribution of AQP2 and AQP3 water channels in human tissue microarrays. J Mol Histol. 2005;36:1–14.PubMedCrossRefGoogle Scholar
  43. 43.
    Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. Histopathology. 2002;40:403–39.PubMedCrossRefGoogle Scholar
  44. 44.
    Gerber JK, Richter T, Kremmer E, Adamski J, Hofler H, Balling R, et al. Progressive loss of PAX9 expression correlates with increasing malignancy of dysplastic and cancerous epithelium of the human oesophagus. J Pathol. 2002;197:293–7.PubMedCrossRefGoogle Scholar
  45. 45.
    Ihrie RA, Marques MR, Nguyen BT, Horner JS, Papazoglu C, Bronson RT, et al. Perp is a p63-regulated gene essential for epithelial integrity. Cell. 2005;120:843–56.PubMedCrossRefGoogle Scholar
  46. 46.
    South AP. Plakophilin 1: an important stabilizer of desmosomes. Clin Exp Dermatol. 2004;29:161–7.PubMedCrossRefGoogle Scholar
  47. 47.
    Nishimori T, Tomonaga T, Matsushita K, Oh-Ishi M, Kodera Y, Maeda T, et al. Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin. Proteomics. 2006;6:1011–8.PubMedCrossRefGoogle Scholar
  48. 48.
    Mori-Akiyama Y, van den Born M, van Es JH, Hamilton SR, Adams HP, Zhang J, et al. SOX9 is required for the differentiation of paneth cells in the intestinal epithelium. Gastroenterology. 2007;133:539–46.PubMedCrossRefGoogle Scholar
  49. 49.
    Niimi T, Nagashima K, Ward JM, Minoo P, Zimonjic DB, Popescu NC. Claudin-18, a novel downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing. Mol Cell Biol. 2001;21:7380–90.PubMedCrossRefGoogle Scholar
  50. 50.
    Haveri H, Westerholm-Ormio M, Lindfors K, Maki M, Savilahti E, Andersson LC, et al. Transcription factors GATA-4 and GATA-6 in normal and neoplastic human gastrointestinal mucosa. BMC Gastroenterol. 2008;8:9.PubMedCrossRefGoogle Scholar
  51. 51.
    Calon A, Gross I, Lhermitte B, Martin E, Beck F, Duclos B, et al. Different effects of the Cdx1 and Cdx2 homeobox genes in a murine model of intestinal inflammation. Gut. 2007;56:1688–95.PubMedCrossRefGoogle Scholar
  52. 52.
    Barros R, Pereira B, Duluc I, Azevedo M, Mendes N, Camilo V, et al. Key elements of the BMP/SMAD pathway co-localize with CDX2 in intestinal metaplasia and regulate CDX2 expression in human gastric cell lines. J Pathol. 2008.Google Scholar

Copyright information

© Springer 2009

Authors and Affiliations

  • Jacob Wang
    • 1
    • 2
  • Rong Qin
    • 1
  • Yan Ma
    • 1
  • Huiyun Wu
    • 3
  • Heiko Peters
    • 4
  • Matthew Tyska
    • 5
  • Nicholas J. Shaheen
    • 2
  • Xiaoxin Chen
    • 1
    • 2
  1. 1.Cancer Research Program, Julius L. Chambers Biomedical/Biotechnology Research InstituteNorth Carolina Central UniversityDurhamUSA
  2. 2.Division of Gastroenterology and Hepatology, Department of Medicine, Center for Gastrointestinal Biology and DiseaseUniversity of North Carolina at Chapel HillChapel HillUSA
  3. 3.Department of Biostatistics, Vanderbilt University School of MedicineVanderbilt-Ingram Cancer Center/Biostatistics Shared ResourceNashvilleUSA
  4. 4.Institute of Human Genetics, International Centre for LifeUniversity of Newcastle upon TyneNewcastle upon TyneUK
  5. 5.Department of Cell and Developmental BiologyVanderbilt University Medical CenterNashvilleUSA

Personalised recommendations