There is a clear association between excessive exposure to estrogens and the development of cancer in hormone sensitive tissues (breast, endometrium). The central hypothesis of this project is that the formation of electrophilic/redox active quinoids is an important mechanism of carcinogenesis for estrogens. o-Quinones are known metabolites of estrogens. Our data strongly suggests that estrogen receptors (ERs) play a major role in estrogen o-quinone-induced DNA damage. Selective alkylation of ERs by the o- quinones generates a highly redox active """"""""Trojan horse"""""""" which selectively targets estrogen sensitive genes.
The specific aims are: 1. What is the role of ERs in catechol estrogen induced DNA damage? We will first investigate modification of purified ERs by estrogen o-quinones with MALDI-TOF and LC-MS-MS experiments. The influence of o-quinone/catechol binding/alkylation of ER on binding to the ERE or on oxidative DNA strand cleavage of ERE oligonucleotide sequences will be studied by gel shift assays. A possible estrogenic effect or functional perturbations between ER and ERE will be examined by ERE-luciferase assays in ER positive MCF-7 cells. Finally, we will conduct chromatin immuno-precipitation assays with ER antibodies in breast cancer cells treated with estrogen o-quinones to analyze DNA damage of estrogen sensitive genes compared to the whole genomic DNA. The isolated estrogen sensitive genes will be investigated for oxidative/alkylation by LC-MS-MS or for point mutation by amplification with PCR and subsequent DNA sequencing. 2. What are the protein targets of catechol estrogens? Selectivity for ERs, ER coregulators, histone 3 and/or redox sensitive detoxification enzymes? We plan to employ novel COATag (covert oxidized activated tag) methodology (i.e., estrogens or catechol metabolites linked to biotin) and the """"""""click chemistry"""""""" or modified Staudinger ligation approaches to examine potential protein covalent modification in rat mammary subcellular fractions and MCF-7 cells. The targeted proteins will be isolated using avidin affinity chromatography, separated by 2D electrophoresis, digested, and analyzed by MALDI-TOF and LC-MS-MS. 3. What is the role of ERs in cellular transformation and induction of DNA damage in vivo? Transformation studies will be performed in MCF-10A cells that are either ER negative, ERa, or ER? positive. The transformed clones will be implanted into athymic nude mice to investigate their ability to induce tumor formation. The role of ERs on catechol estrogen-induced DNA damage will be assessed in the mouse mammary organ culture (MMOC) model and in vivo using wild type and ER knockout mice. These data will be correlated with the DNA damage experiments described in Aim 1 and the protein targets identified in Aim 2 in order to give an overall picture of the involvement of ERs in estrogen carcinogenesis.
