Abstract
The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.
Highlights
Elevated lifetime estrogen exposure is a well-known major risk factor for breast cancer
Tamoxifen and other antiestrogens are thought to prevent cancer through their actions at the estrogen receptor (ER), other mechanisms cannot be ruled out as these compounds block metabolism and redox cycling of estrogen and are free radical scavengers [26]. 4-OH-E2 induces an estrogenic response in the uterus of ERa null mice, and this response is not inhibited by the antiestrogen ICI182780 [27]
When manganese superoxide dismutase (MnSOD) were over-expressed in these cells, reactive oxygen species (ROS) levels increased significantly compared to cells treated with estrogen alone (Fig. 1C)
Summary
Elevated lifetime estrogen exposure is a well-known major risk factor for breast cancer. A large body of epidemiological and experimental evidence points to a role for estrogen in the etiology of human breast cancer [1,2,3,4,5,6,7,8,9]. Estrogens are complete breast carcinogens, as they are capable of initiating and triggering growth and selection to generate palpable malignancy [8,9,10,11,12,13,14]. The signaling mechanisms by which estrogen contributes in the initiation of breast cancer remain the subject of a long-standing controversy. This is due, in part, to the inability to resolve whether estrogen or estrogen metabolites are procarcinogenic. We have previously shown that E2-induced renal tumor formation is decreased in animals exposed to inhibitors of estrogen metabolism or to hormonally potent estrogens undergoing reduced metabolic conversion to catechol metabolites compared to E2 [10,11,12,15]
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