Sociedade Brasileira de Espectrometria de Massas – BrMASS
Proteins & Peptides
MRM Analysis of expression and protein secretion from Mammary
Epithelial Cells Induced to EMT by TGF-2
Daniele Albuquerque1,2, Camila de Souza Palma1,2, Carolina Hassibe Thomé1,2, Fernanda
Ursoli Ferreira Melo2, Valéria Cerqueira César1, Gabriela Canchaya1; Vera Lúcia
Aparecida Aguillar Epifânio1; Simone Haddad2; Eduardo Brant de Oliveira1;
Dimas Covas2 Vitor Marcel Faça1,3
[email protected]
1
Department of Biochemistry and Immunology – FMRP/USP; 2National Center for Cell
Based Therapy – Hemotherapy Center of Ribeirão Preto – FMRP/USP; 3Center for
Integrative Systems Biology (CISBi NAP) – FMRP/USP, SP, Brazil
Breast cancer is one of the most common type of invasive cancer in females worldwide, although
presents relatively good prognosis if diagnosed and treated early. Breast cancer is caused by
several genetic and epigenetic alterations. Genetic mutations in BRCA-1 and BRCA-2 genes as well
as in TP53, PTEN, ATM, CDH1, CHEK2 e STK11 (LKB1) are often detected [1]. In addition to these
known frequent mutations, growth factors such as TGB-β play important roles in breast cancer
progression and metastasis. The TGF-β plays a dichotomous role during normal development of the
mammary gland and carcinoma. In the normal breast epithelium, autocrine signaling of TGF-β
maintains homeostasis, controls cell proliferation, induces apoptosis and senescence. However,
changes that occur during the carcinogenic process induced resistance to the effects of TGF-β
homeostatic thereby allow the activation of favorable responses to tumor growth, metastasis and
cell survival [2]. Interestingly, this growth factor also induces Epithelial-Mesenchymal Transition
(EMT), where normal epithelial cells during embryogenesis, or malignant cells during tumor
progression and metastasis, lose their intracellular contacts and acquire a migratory character [3]. In
this study, MCF10A human mammary epithelial cells were treated with TGB‐β2 in order to induce
migratory and invasive characteristics achieved during EMT. After 72 hours, the total extract and
conditioned medium were monitored through a Multiple Reaction Monitoring (MRM) method, on a
XevoTQS-Triple quadrupole coupled to Ultra Performance Liquid Chromatography (UPLC-MS/MS) for
detection and quantification of 12 proteins relevant to the process of EMT. MRM results showed that
there was increased expression of the transcription factor TWIST and other proteins relevant to
EMT such as N-cadherin in the total extract. In addition, there was increased secretion
metallopeptidase inhibitor 1 (TIMP1), mesothelin (MSLN) and matrix metalloproteinase 2 (MMP2)
after treatment with the growth factor. With the development of MRM methods and expansion of
the biomarkers panel for EMT we expect to evaluate novel proteins relevant to breast cancer
progression and metastasis in clinical samples and correlate quantitative changes with disease stage
as well validate potential diagnostic or therapeutic targets for metastatic disease.
[1] Bradbury, A. R.; Olopade, O.I. Rev Endocr Metab Disord. 2007, 8(3), 255 ‐ 267.
[2] Elliott, R. L.; Blobe, G.C. J Clin Oncol. 2005, 23(9), 2078 ‐ 2093.
[3] Kalluri, R.; Weinberg, R. A. J Clin Invest. 2009, 119(6), 1420 – 1428.
SUPPORT: FAPESP grants 2011/09740-1 and 2012/02518-4; Center for Integrative Systems Biology - CISBi, NAP/USP
grant 12.1.17598.1.3, Research Support Centers - University of São Paulo