| Breast cancer is a common malignancy affecting women in western countries. The cellular processes leading to mammary carcinogenesis have yet to be elucidated. Oxidative stress has come under scrutiny as a contributing factor, with 17beta-estradiol (E2) and epidermal growth factor (EGF), agents that can increase oxidative stress, being linked to breast cancer development. MCF-10A cells, categorized as ER-alpha-negative, were used to test our hypothesis that non-transformed human breast epithelial cells can be transformed by increased oxidant stress. To accomplish this goal, we exposed cells to treatments and culture conditions that modulate oxidative stress. These included treatments with E2, 2-fluoroestradiol, Ro 41-0960, ICI 182,780 (ICI), Eukarion-134 (EUK), and caffeic acid phenethyl ester (CAPE) or their combinations. Our studies led to the development of a protocol in which the effects of chronic, physiologically relevant microenvironmental alterations on cellular transformation can be examined. Results indicated that both oxidative stress and ER-alpha-mediated events were operational in MCF-10A neoplastic transformation. Increased oxidative stress due to manipulation of EGF and hydrocortisone in culture medium induced transformation. Chronic administration of 1 nM E2 resulted in significant increases in transformation rates and oxidative stress markers. These E2-induced elevations were significantly suppressed by EUK and CAPE. ER-alpha-mediated events were also necessary for cell transformation, since both the antiestrogen ICI and weakly estrogenic phenol red abrogated it. Unexpectedly, gene expression array and western blotting analyses provided the first documentation of detectable ER-alpha and ER-beta and their mRNAs in MCF-10A cells. This analysis also indicated for the first time a possible direct association of EGF receptor (EGFR) and ER-alpha in cells, as well as high induction of a novel ternary complex that also includes ER-beta (ERalpha/ERbeta/EGFR) in cells most prone to transformation. ER/EGFR crosstalk may provide the proliferative capacity needed for transformation, explaining observed ER-mediated events and EGF hypersensitivity. Overall, our studies indicated that oxidative stress-mediated DNA damage and ER-alpha-mediated proliferative pathways act in concert to effect MCF-10A neoplastic transformation. |
