Current prognostic factors are insufficient for precise risk-discrimination in breast cancer patients with low grade breast tumors, which, in disagreement with theoretical prognosis, occasionally form early lymph node metastasis. To identify markers for this group of patients, we employed iTRAQ-2DLC-MS/MS proteomics to 24 lymph node positive and 24 lymph node negative grade 1 luminal A primary breast tumors. Another group of 48 high-grade tumors (luminal B, triple negative, Her-2 subtypes) was also analyzed to investigate marker specificity for grade 1 luminal A tumors. From the total of 4405 proteins identified (FDR < 5%), the top 65 differentially expressed together with 30 previously identified and control markers were analyzed also at transcript level. Increased levels of carboxypeptidase B1 (CPB1), PDZ and LIM domain protein 2 (PDLIM2), and ring finger protein 25 (RNF25) were associated specifically with lymph node positive grade 1 tumors, whereas stathmin 1 (STMN1) and thymosin beta 10 (TMSB10) associated with aggressive tumor phenotype also in high grade tumors at both protein and transcript level. For CPB1, these differences were also observed by immunohistochemical analysis on tissue microarrays. Up-regulation of putative biomarkers in lymph node positive (versus negative) luminal A tumors was validated by gene expression analysis of an independent published data set (n = 343) for CPB1 (p = 0.00155), PDLIM2 (p = 0.02027) and RELA (p = 0.00015). Moreover, statistically significant connections with patient survival were identified in another public data set (n = 1678). Our findings indicate unique pro-metastatic mechanisms in grade 1 tumors that can include up-regulation of CPB1, activation of NF-κB pathway and changes in cell survival and cytoskeleton. These putative biomarkers have potential to identify the specific minor subpopulation of breast cancer patients with low grade tumors who are at higher than expected risk of recurrence and who would benefit from more intensive follow-up and may require more personalized therapy.

‖Masaryk University Faculty of Medicine Institute of Biostatistics and Analyses Brno Czech Republic;

§§University of Southampton School of Medicine Cancer Sciences Division Institute for Life Sciences Center for Proteomic Research Southampton UK

¶Proteomics Mass Spectrometry The Wellcome Trust Sanger Institute Cambridge CB10 1SA UK;

**ThermoFisher Scientific Hemel Hempstead UK;

‡‡Laboratory of Computational Biology Center for Human Genetics University of Leuven Belgium;

From the ‡Masaryk Memorial Cancer Institute Regional Centre for Applied Molecular Oncology Brno Czech Republic;

From the ‡Masaryk Memorial Cancer Institute Regional Centre for Applied Molecular Oncology Brno Czech Republic; ‖Masaryk University Faculty of Medicine Institute of Biostatistics and Analyses Brno Czech Republic;

From the ‡Masaryk Memorial Cancer Institute Regional Centre for Applied Molecular Oncology Brno Czech Republic; §Masaryk University Faculty of Science Department of Biochemistry Brno Czech Republic;

Zobrazit více v PubMed

Ross J. S., Hortobagyi G.N. (Eds) (2005) Molecular Oncology of Breast Cancer, Jones and Barlett Publishers, Sadbury, MA, U.S.A.

Cress A. E., Nagle R.B., (Eds.) (2006) Cell Adhesion and Cytoskeletal Molecules in Metastasis, Springer, Dodrecht, The Netherlands

Mansel R. E., Fodstad O., Jiang W.G., (Eds.) (2007) Metastasis of Breast Cancer, Springer, Dodrecht, The Netherlands

Harris L., Fritsche H., Mennel R., Norton L., Ravdin P., Taube S., Somerfield M. R., Hayes D. F., Bast R. C., Jr. (2007) American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J. Clin. Oncol. 25, 5287–5312 PubMed

Stephens R. W., Brunner N., Janicke F., Schmitt M. (1998) The urokinase plasminogen activator system as a target for prognostic studies in breast cancer. Breast Cancer Res. Treat. 52, 99–111 PubMed

Maryas J., Faktor J., Dvorakova M., Struharova I., Grell P., Bouchal P. (2014) Proteomics in investigation of cancer metastasis: functional and clinical consequences and methodological challenges. Proteomics 14, 426–440 PubMed

Bouchal P., Roumeliotis T., Hrstka R., Nenutil R., Vojtesek B., Garbis S. D. (2009) Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis. J. Proteome Res. 8, 362–373 PubMed

Scigelova M., Hornshaw M., Giannakopulos A., Makarov A. (2011) Fourier Transform Mass Spectrometry. Mol. Cell. Proteomics 10, 1–19 PubMed PMC

R_Development_Core_Team (2008) A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria

Bookout A. L., Mangelsdorf D. J. (2003) Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl. Receptor Signal. 1, e012 PubMed PMC

Haibe-Kains B., Desmedt C., Loi S., Culhane A. C., Bontempi G., Quackenbush J., Sotiriou C. (2012) A three-gene model to robustly identify breast cancer molecular subtypes. J. Natl. Cancer Inst. 104, 311–325 PubMed PMC

Wirapati P., Sotiriou C., Kunkel S., Farmer P., Pradervand S., Haibe-Kains B., Desmedt C., Ignatiadis M., Sengstag T., Schutz F., Goldstein D. R., Piccart M., Delorenzi M. (2008) Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures. Breast Cancer Res. 10, R65. PubMed PMC

Gyorffy B., Lanczky A., Eklund A. C., Denkert C., Budczies J., Li Q., Szallasi Z. (2010) An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res. Treat. 123, 725–731 PubMed

Vizcaino J. A., Deutsch E. W., Wang R., Csordas A., Reisinger F., Rios D., Dianes J. A., Sun Z., Farrah T., Bandeira N., Binz P. A., Xenarios I., Eisenacher M., Mayer G., Gatto L., Campos A., Chalkley R. J., Kraus H. J., Albar J. P., Martinez-Bartolome S., Apweiler R., Omenn G. S., Martens L., Jones A. R., Hermjakob H. (2014) ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat. Biotechnol. 32, 223–226 PubMed PMC

Paik S., Tang G., Shak S., Kim C., Baker J., Kim W., Cronin M., Baehner F. L., Watson D., Bryant J., Costantino J. P., Geyer C. E., Jr., Wickerham D. L., Wolmark N. (2006) Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J. Clin. Oncol. 24, 3726–3734 PubMed

van 't Veer L. J., Dai H., van de Vijver M. J., He Y. D., Hart A. A., Mao M., Peterse H. L., van der Kooy K., Marton M. J., Witteveen A. T., Schreiber G. J., Kerkhoven R. M., Roberts C., Linsley P. S., Bernards R., Friend S. H. (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature 415, 530–536 PubMed

Swaisgood C. M., Schmitt D., Eaton D., Plow E. F. (2002) In vivo regulation of plasminogen function by plasma carboxypeptidase B. J. Clin. Invest. 110, 1275–1282 PubMed PMC

Prasad S., Ravindran J., Aggarwal B. B. (2010) NF-kappaB and cancer: how intimate is this relationship. Mol. Cell. Biochem. 336, 25–37 PubMed PMC

Qu Z., Fu J., Yan P., Hu J., Cheng S. Y., Xiao G. (2010) Epigenetic repression of PDZ-LIM domain-containing protein 2: implications for the biology and treatment of breast cancer. J. Biol. Chem. 285, 11786–11792 PubMed PMC

Bowe R. A., Cox O. T., Ayllon V., Tresse E., Healy N. C., Edmunds S. J., Huigsloot M., O'Connor R. (2014) PDLIM2 regulates transcription factor activity in epithelial-to-mesenchymal transition via the COP9 signalosome. Mol. Biol. Cell 25, 184–195 PubMed PMC

Asamitsu K., Tetsuka T., Kanazawa S., Okamoto T. (2003) RING finger protein AO7 supports NF-kappaB-mediated transcription by interacting with the transactivation domain of the p65 subunit. J. Biol. Chem. 278, 26879–26887 PubMed

Ding W., Li C., Hu T., Graves-Deal R., Fotia A. B., Weissman A. M., Coffey R. J. (2008) EGF receptor-independent action of TGF-alpha protects Naked2 from AO7-mediated ubiquitylation and proteasomal degradation. Proc. Natl. Acad. Sci. U.S.A. 105, 13433–13438 PubMed PMC

Belletti B., Baldassarre G. (2011) Stathmin: a protein with many tasks. New biomarker and potential target in cancer. Expert Opin. Therapeutic Targets 15, 1249–1266 PubMed

Trovik J., Wik E., Stefansson I. M., Marcickiewicz J., Tingulstad S., Staff A. C., Njolstad T. S., MoMaTec Study, G., Vandenput I., Amant F., Akslen L. A., Salvesen H. B. (2011) Stathmin overexpression identifies high-risk patients and lymph node metastasis in endometrial cancer. Clin. Cancer Res. 17, 3368–3377 PubMed

Jeon T. Y., Han M. E., Lee Y. W., Lee Y. S., Kim G. H., Song G. A., Hur G. Y., Kim J. Y., Kim H. J., Yoon S., Baek S. Y., Kim B. S., Kim J. B., Oh S. O. (2010) Overexpression of stathmin1 in the diffuse type of gastric cancer and its roles in proliferation and migration of gastric cancer cells. Br. J. Cancer 102, 710–718 PubMed PMC

Golouh R., Cufer T., Sadikov A., Nussdorfer P., Usher P. A., Brunner N., Schmitt M., Lesche R., Maier S., Timmermans M., Foekens J. A., Martens J. W. (2008) The prognostic value of Stathmin-1, S100A2, and SYK proteins in ER-positive primary breast cancer patients treated with adjuvant tamoxifen monotherapy: an immunohistochemical study. Breast Cancer Res. Treat. 110, 317–326 PubMed

Rana S., Maples P. B., Senzer N., Nemunaitis J. (2008) Stathmin 1: a novel therapeutic target for anticancer activity. Expert Rev. Anticancer Therapy 8, 1461–1470 PubMed

Williams K., Ghosh R., Giridhar P. V., Gu G., Case T., Belcher S. M., Kasper S. (2012) Inhibition of stathmin1 accelerates the metastatic process. Cancer Res. 72, 5407–5417 PubMed PMC

Huang L., Zheng M., Zhou Q. M., Zhang M. Y., Jia W. H., Yun J. P., Wang H. Y. (2011) Identification of a gene-expression signature for predicting lymph node metastasis in patients with early stage cervical carcinoma. Cancer 117, 3363–3373 PubMed

Feher L. Z., Pocsay G., Krenacs L., Zvara A., Bagdi E., Pocsay R., Lukacs G., Gyory F., Gazdag A., Tarko E., Puskas L. G. (2012) Amplification of thymosin beta 10 and AKAP13 genes in metastatic and aggressive papillary thyroid carcinomas. Pathol. Oncol. Res. 18, 449–458 PubMed

Bergamaschi A., Katzenellenbogen B. S. (2012) Tamoxifen downregulation of miR-451 increases 14–3-3zeta and promotes breast cancer cell survival and endocrine resistance. Oncogene 31, 39–47 PubMed PMC

Mizejewski G. J. (1999) Role of integrins in cancer: survey of expression patterns. Proc. Soc. Exp. Biol. Med. 222, 124–138 PubMed

Tuck A. B., O'Malley F. P., Singhal H., Harris J. F., Tonkin K. S., Kerkvliet N., Saad Z., Doig G. S., Chambers A. F. (1998) Osteopontin expression in a group of lymph node negative breast cancer patients. Int. J. Cancer 79, 502–508 PubMed

Tuck A. B., Chambers A. F. (2001) The role of osteopontin in breast cancer: clinical and experimental studies. J. Mammary Gland Biol. Neoplasia 6, 419–429 PubMed

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