Functional & Integrative Genomics

, Volume 11, Issue 2, pp 215–224 | Cite as

Integrating proteomic and transcriptomic high-throughput surveys for search of new biomarkers of colon tumors

  • Michal Mikula
  • Tymon Rubel
  • Jakub Karczmarski
  • Krzysztof Goryca
  • Michal Dadlez
  • Jerzy Ostrowski
Original Paper

Abstract

To the search of new colon tumor biomarkers in the transition from normal colon (NC) mucosa to adenoma (AD) and adenocarcinoma (AC), we integrated microarray data with the results of a high-throughput proteomic workflow. In proteomic study, we used a modified isoelectric focusing protocol on strips with an immobilized pH gradient to separate peptides labeled with iTRAQ (isobaric tags for relative and absolute quantitation) tags followed by liquid chromatography–tandem mass spectrometry analysis. Gene expression measurements were done using Affymetrix GeneChip HG-U133plus2 microarrays and quantitative reverse transcriptase PCR (q-RT-PCR). We identified 3,886 proteins with at least two peptides. Of them, 1,061 proteins were differentially expressed [FC ≥ 1.5; FDR ≤ 0.01] in two pair-wise comparisons: AD vs. NC and AC vs. AD while 15 and 23 proteins were progressively up-regulated and down-regulated in the NC/AD/AC sequence, respectively. The quantitative proteomic information was subsequently correlated with microarray data. For a collection of genes with the same direction of changes of both mRNA and protein levels, we obtained 785/853/795 genes in AD vs. NC/AC vs. NC/AC vs. AD comparison, respectively. Further evaluation of sequentially altered gene expression by q-RT-PCR on individual samples of 24 NCs, 42 ADs, and 26 ACs confirmed progressive expression of six genes: biglycan, calumenin, collagen type XII, alpha 1 (COL12A1), monoamine oxidase A (MAOA), ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), and MOCO sulphurase C-terminal domain-containing 2 (MOSC2). Among them, three continuously down-regulated (MAOA, ENTPD5, and MOSC2) and one continuously overexpressed (COL12A1) are reported, to our best knowledge, for the first time in a connection to colon cancer onset.

Keywords

Gene expression Colorectal cancer Microarrays Mass spectrometry Data integration 

Notes

Acknowledgments

This work was supported by grants from the Polish Ministry of Science and Higher Education: PBZ-MNiI-2/1/2005 and R13 010 03 (MS software development). We thank for technical assistance to Magdalena Skrzypczak.

Supplementary material

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References

  1. Bairoch ABL, Altairac S, Amendolia V, Auchincloss A, Argoud-Puy G, Axelsen K, Baratin D, Blatter MC, Boeckmann B, Bolleman J, Bollondi L, Boutet E, Quintaje SB, Breuza L, Bridge A, deCastro E, Ciapina L, Coral D, Coudert E, Cusin I, Delbard G, Dornevil D, Roggli PD, Duvaud S, Estreicher A, Famiglietti L, Feuermann M, Gehant S, Farriol-Mathis N, Ferro S, Gasteiger E, Gateau A, Gerritsen V, Gos A, Gruaz-Gumowski N, Hinz U, Hulo C, Hulo N, James J, Jimenez S, Jungo F, Junker V, Kappler T, Keller G, Lachaize C, Lane-Guermonprez L, Langendijk-Genevaux P, Lara V, Lemercier P, Le Saux V, Lieberherr D, Lima Tde O, Mangold V, Martin X, Masson P, Michoud K, Moinat M, Morgat A, Mottaz A, Paesano S, Pedruzzi I, Phan I, Pilbout S, Pillet V, Poux S, Pozzato M, Redaschi N, Reynaud S, Rivoire C, Roechert B, Schneider M, Sigrist C, Sonesson K, Staehli S, Stutz A, Sundaram S, Tognolli M, Verbregue L, Veuthey AL, Yip L, Zuletta L, Apweiler R, Alam-Faruque Y, Antunes R, Barrell D, Binns D, Bower L, Browne P, Chan WM, Dimmer E, Eberhardt R, Fedotov A, Foulger R, Garavelli J, Golin R, Horne A, Huntley R, Jacobsen J, Kleen M, Kersey P, Laiho K, Leinonen R, Legge D, Lin Q, Magrane M, Martin MJ, O’Donovan C, Orchard S, O’Rourke J, Patient S, Pruess M, Sitnov A, Stanley E, Corbett M, di Martino G, Donnelly M, Luo J, van Rensburg P, Wu C, Arighi C, Arminski L, Barker W, Chen Y, Hu ZZ, Hua HK, Huang H, Mazumder R, McGarvey P, Natale DA, Nikolskaya A, Petrova N, Suzek BE, Vasudevan S, Vinayaka CR, Yeh LS, Zhang J (2009) The Universal Protein Resource (UniProt) 2009. Nucleic Acids Res 37:D169–174CrossRefGoogle Scholar
  2. Barderas R, Babel I, Casal JI (2010) Colorectal cancer proteomics, molecular characterization and biomarker discovery. Proteomics: Clin Appl 4:159–178CrossRefGoogle Scholar
  3. Becker JC, Fukui H, Imai Y, Sekikawa A, Kimura T, Yamagishi H, Yoshitake N, Pohle T, Domschke W, Fujimori T (2007) Colonic expression of heme oxygenase-1 is associated with a better long-term survival in patients with colorectal cancer. Scand J Gastroenterol 42:852–858PubMedCrossRefGoogle Scholar
  4. Blanquez MJ, Regadera J, Marino J, Newman RE, Notario V (2002) Gradual deregulation and loss of PCPH expression in the progression of human laryngeal neoplasia. Mol Carcinog 35:186–195PubMedCrossRefGoogle Scholar
  5. Blanquez MJ, Arenas MI, Conde I, Tirado OM, Paniagua R, Notario V (2004) Deregulated expression of the PCPH proto-oncogene in human breast cancers. Int J Oncol 25:821–830PubMedGoogle Scholar
  6. Bustin SA, Li SR, Dorudi S (2001) Expression of the Ca2 + -activated chloride channel genes CLCA1 and CLCA2 is downregulated in human colorectal cancer. DNA Cell Biol 20:331–338PubMedCrossRefGoogle Scholar
  7. Cargile BJ, Sevinsky JR, Essader AS, Stephenson JL Jr, Bundy JL (2005) Immobilized pH gradient isoelectric focusing as a first-dimension separation in shotgun proteomics. J Biomol Tech 16:181–189PubMedGoogle Scholar
  8. Chen G, Gharib TG, Huang CC, Taylor JM, Misek DE, Kardia SL, Giordano TJ, Iannettoni MD, Orringer MB, Hanash SM, Beer DG (2002) Discordant protein and mRNA expression in lung adenocarcinomas. Mol Cell Proteomics 1:304–313PubMedCrossRefGoogle Scholar
  9. Choe L, D’Ascenzo M, Relkin NR, Pappin D, Ross P, Williamson B, Guertin S, Pribil P, Lee KH (2007) 8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer’s disease. Proteomics 7:3651–3660PubMedCrossRefGoogle Scholar
  10. Dowell JA, Frost DC, Zhang J, Li L (2008) Comparison of two-dimensional fractionation techniques for shotgun proteomics. Anal Chem 80:6715–6723PubMedCrossRefGoogle Scholar
  11. Galamb O, Gyorffy B, Sipos F, Spisak S, Nemeth AM, Miheller P, Tulassay Z, Dinya E, Molnar B (2008) Inflammation, adenoma and cancer: objective classification of colon biopsy specimens with gene expression signature. Dis Markers 25:1–16PubMedGoogle Scholar
  12. Galamb O, Sipos F, Spisak S, Galamb B, Krenacs T, Valcz G, Tulassay Z, Molnar B (2009) Potential biomarkers of colorectal adenoma-dysplasia-carcinoma progression: mRNA expression profiling and in situ protein detection on TMAs reveal 15 sequentially upregulated and 2 downregulated genes. Cell Oncol 31:19–29PubMedGoogle Scholar
  13. Honore B, Vorum H (2000) The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells. FEBS Lett 466:11–18PubMedCrossRefGoogle Scholar
  14. John R, El-Rouby NM, Tomasetto C, Rio MC, Karam SM (2007) Expression of TFF3 during multistep colon carcinogenesis. Histol Histopathol 22:743–751PubMedGoogle Scholar
  15. Kaiser S, Park YK, Franklin JL, Halberg RB, Yu M, Jessen WJ, Freudenberg J, Chen X, Haigis K, Jegga AG, Kong S, Sakthivel B, Xu H, Reichling T, Azhar M, Boivin GP, Roberts RB, Bissahoyo AC, Gonzales F, Bloom GC, Eschrich S, Carter SL, Aronow JE, Kleimeyer J, Kleimeyer M, Ramaswamy V, Settle SH, Boone B, Levy S, Graff JM, Doetschman T, Groden J, Dove WF, Threadgill DW, Yeatman TJ, Coffey RJ Jr, Aronow BJ (2007) Transcriptional recapitulation and subversion of embryonic colon development by mouse colon tumor models and human colon cancer. Genome Biol 8:R131PubMedCrossRefGoogle Scholar
  16. Kwong KY, Bloom GC, Yang I, Boulware D, Coppola D, Haseman J, Chen E, McGrath A, Makusky AJ, Taylor J, Steiner S, Zhou J, Yeatman TJ, Quackenbush J (2005) Synchronous global assessment of gene and protein expression in colorectal cancer progression. Genomics 86:142–158PubMedCrossRefGoogle Scholar
  17. Lee S, Bang S, Song K, Lee I (2006) Differential expression in normal-adenoma-carcinoma sequence suggests complex molecular carcinogenesis in colon. Oncol Rep 16:747–754PubMedGoogle Scholar
  18. Lengqvist J, Uhlen K, Lehtio J (2007) iTRAQ compatibility of peptide immobilized pH gradient isoelectric focusing. Proteomics 7:1746–1752PubMedCrossRefGoogle Scholar
  19. Liu X, Wang X, Zhang J, Lam EK, Shin VY, Cheng AS, Yu J, Chan FK, Sung JJ, Jin HC (2010) Warburg effect revisited: an epigenetic link between glycolysis and gastric carcinogenesis. Oncogene 29:442–450PubMedCrossRefGoogle Scholar
  20. Mendel RR, Bittner F (2006) Cell biology of molybdenum. Biochim Biophys Acta 1763:621–635PubMedCrossRefGoogle Scholar
  21. Ostergaard M, Hansen GA, Vorum H, Honore B (2006) Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton. Proteomics 6:3509–3519PubMedCrossRefGoogle Scholar
  22. Ostrowski J, Wyrwicz LS (2009) Integrating genomics, proteomics and bioinformatics in translational studies of molecular medicine. Expert Rev Mol Diagn 9:623–630PubMedCrossRefGoogle Scholar
  23. Read R, Hansen G, Kramer J, Finch R, Li L, Vogel P (2009) Ectonucleoside triphosphate diphosphohydrolase type 5 (ENTPD5)-deficient mice develop progressive hepatopathy, hepatocellular tumors, and spermatogenic arrest. Vet Pathol 46:491–504PubMedCrossRefGoogle Scholar
  24. Robson SC, Sevigny J, Zimmermann H (2006) The E-NTPDase family of ectonucleotidases: structure function relationships and pathophysiological significance. Purinergic Signal 2:409–430PubMedCrossRefGoogle Scholar
  25. Rouzaut A, Recio JA, Notario V (2001) Expression of the protein product of the PCPH proto-oncogene in human tumor cell lines. Radiat Res 155:181–187PubMedCrossRefGoogle Scholar
  26. Rybaczyk LA, Bashaw MJ, Pathak DR, Huang K (2008) An indicator of cancer: downregulation of monoamine oxidase-A in multiple organs and species. BMC Genomics 9:134PubMedCrossRefGoogle Scholar
  27. Skrzypczak M, Goryca K, Rubel T, Paziewska A, Mikula M, Jarosz D, Pachlewski J, Oledzki J, Ostrowski J (2010) Modeling oncogenic signaling in colon tumors by multidirectional analyses of microarray data directed for maximization of analytical reliability. PLoS ONE 5:e13091PubMedCrossRefGoogle Scholar
  28. Stella J, Bavaresco L, Braganhol E, Rockenbach L, Farias PF, Wink MR, Azambuja AA, Barrios CH, Morrone FB, Oliveira Battastini AM (2010) Differential ectonucleotidase expression in human bladder cancer cell lines. Urol Oncol 28:260–267PubMedCrossRefGoogle Scholar
  29. Uwaje NC, Mueller NS, Maccarrone G, Turck CW (2007) Interrogation of MS/MS search data with an pI Filter algorithm to increase protein identification success. Electrophoresis 28:1867–1874PubMedCrossRefGoogle Scholar
  30. Wang Y, Ma Y, Lu B, Xu E, Huang Q, Lai M (2007) Differential expression of mimecan and thioredoxin domain-containing protein 5 in colorectal adenoma and cancer: a proteomic study. Exp Biol Med 232:1152–1159, Maywood, NJCrossRefGoogle Scholar
  31. Weber CK, Sommer G, Michl P, Fensterer H, Weimer M, Gansauge F, Leder G, Adler G, Gress TM (2001) Biglycan is overexpressed in pancreatic cancer and induces G1-arrest in pancreatic cancer cell lines. Gastroenterology 121:657–667PubMedCrossRefGoogle Scholar
  32. Yates JR 3rd (2004) Mass spectral analysis in proteomics. Annu Rev Biophys Biomol Struct 33:297–316PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Michal Mikula
    • 1
  • Tymon Rubel
    • 2
    • 5
  • Jakub Karczmarski
    • 1
  • Krzysztof Goryca
    • 3
  • Michal Dadlez
    • 4
  • Jerzy Ostrowski
    • 1
    • 3
    • 6
  1. 1.Department of Oncological GeneticsMaria Sklodowska-Curie Memorial Cancer Center and Institute of OncologyWarsawPoland
  2. 2.Laboratory of Bioinformatics and Systems BiologyMaria Sklodowska-Curie Memorial Cancer Center and Institute of OncologyWarsawPoland
  3. 3.Department of Gastroenterology and HepatologyMedical Center for Postgraduate Education WarsawWarsawPoland
  4. 4.Institute of Biochemistry and BiophysicsPolish Academy of SciencesWarsawPoland
  5. 5.Institute of RadioelectronicsWarsaw University of TechnologyWarsawPoland
  6. 6.Cancer Center-InstituteWarsawPoland

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