Biomarker Discovery for Breast Cancer
Estrogens contribute to development of breast cancer by acting as a growth hormone. There is also evidence for a second mechanism whereby metabolites of estrogens react to damage directly DNA, leading to nucleobase removal and abasic-site formation. Estrogens are metabolized to catechol estrogens (CEs), which are further oxidized to catechol-estrogen quinones (CEQs). These latter compounds are nucleophiles that damage DNA via a Michael addition. Fortunately, buildup of the reactive CEs is impeded in most women by catechol-O-methyltransferase (COMT), and the CEQs can be removed by conjugation with glutathione (GSH), via an S-transferase, or reduced to reform CEs via a reductase.
If these biosynthetic/protection mechanisms are not in balance, however, the accumulating CEQs react with Ade and Gua of DNA, causing them to depurinate and release modified nucleobases, 4-OH-E1(E2)-Ade and 4-OH-E1(E2)-Gua. These molecules indicate the imbalance and serve as biomarkers for cancer risk.
Our approach to testing these biomarkers is to synthesize reference materials, develop a method of analysis, and test breast-tissue specimens. Dr. Rebecca Aft, an oncology surgeon of the Washington University School of Medicine, provides specimens.
Recently, we implemented a synthetic procedure to prepare reference materials and isotopically labeled reference materials to serve as internal standards. We also implemented a new extraction procedure to remove the putative biomarkers from breast tissue specimens and implemented capillary HPLC with a LTQ-Fourier transform mass spectrometer for analysis. An advantage is the use of accurate mass as another criterion for specificity. The overall procedure requires that the analyte have the correct retention time, the correct nominal m/z, the correct accurate m/z, and a product-ion spectrum that is nearly identical with that of authentic materials. All these implementations have been successful. We wish to analyze samples of breast tissue taken in surgery of women with cancer and to compare the levels of the modified DNA bases with those in tissue of women who are having cosmetic breast surgery.
1. Q. Zhang, R.L. Aft and M.L. Gross, Estrogen Carcinogenesis: Specific Identification of Estrogen-Modified Nucleobase in Breast Tissue from Women, Chem. Res. Tox., 21, 1509 (2008).
2. Qiang Zhang and Michael L. Gross, Efficient Synthesis, Liquid-Chromatography Purification, and Tandem Mass Spectrometric Characterization of Estrogen-Modified DNA Bases, Chem. Res. Toxicol., 21, 1244-1252 (2008).