Proteomics of Putative Protein Biomarkers in Human Breast Cancer Tissues

Breast cancer is the second leading death-causing cancer in women, after lung cancer. Breast cells are normally responsive to the estrogen hormone as they have the receptor for it. Breast cancer types that express the estrogen receptor have been targeted by anti-hormonal drugs yet treatment is not always successful due to the resistance of some breast cancer cells. Studies have shown that some breast cancer cells overlying broken breast-duct walls have lost their estrogen receptor (ER) and became more aggressive favoring metastasis more than the ER positive (ER+) cancer cells lying beside them in the duct. Those ER negative (ER-) cells are not affected by anti-hormonal treatments. Therefore more prognostic biomarkers are needed to increase the life expectancy in breast cancer affected women. Post translational modifications, such as glycosylation and phosphorylation, are essential to correct functioning of proteins within the cell. The addition of sugar moieties to a glycoprotein changes its structure and thus its function, which has been frequently correlated with cancerous transformation. Similarly, phosphorylation or dephosphorylation of a protein directly affects the protein's function. Identifying such post translational differences at different stages of breast cancer is essential for recognizing possible targets or specific biomarkers. In this work, proteins extracted from ER+ and ER- human breast cancer tissues were separated by two dimensional gel electrophoresis (2-DE), and stained for glycoproteins and total proteins. Breast cancer tissue lysates were N-deglycosylated in order to identify the N-linked glycoproteins. The 2-DE scans were analyzed and the proteins with significant differential expression among the ER+, ER-, and their N-deglycosylated proteins were identified by means of a reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer (LC-LTQ/FTICR-MS). Spot count results showed that 30% of the total lysate proteins are glycoproteins. Alpha-1-acid, alpha-1- antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor were among the identified glycosylated proteins, further verified by Western blotting in both ER+ and ER- human breast tissues. A possible glycosylation difference in alpha-1-antitrypsin was found to be expressed highest in the ER- samples. This glycosylation difference is a potential tumor-derived biomarker for breast cancer progression.