4-hydroxy estradiol-induced oxidant-mediated signaling is involved in the development of breast cancer

Breast cancer is a disease associated with excess exposures to estrogens. While the mode of cancer causation is unknown, others have shown that oxidative stress induced by prolonged exposure to estrogens mediates renal, liver, endometrial and mammary tumorigenesis though the mechanism(s) underling this process is unknown. In this study, we show that 4-hydroxyl 17β-estradiol (4-OHE2), a catechol metabolite of estrogen, induces mammary tumorigenesis in a redox dependent manner. We found that the mechanism of tumorigenesis involves redox activations of nuclear respiratory factor-1 (NRF1); a transcriptions factor associated with regulation of mitochondria biogenesis and oxidative phosphorylation (OXPHOS), as well as mediation of cell survival and growth of cells during periods of oxidative stress. Key findings from our study are as follows: (i) Prolonged treatments of normal mammary epithelial cells with 4-OHE2, increased the formation of intracellular reactive oxygen species (ROS). (ii) Estrogen-induced ROS activates redox sensitive transcription factors NRF1. (iii) 4-OHE2 through activation of serine-threonine kinase and histone acetyl transferase, phosphorylates and acetylate NRF1 respectively. (iv) Redox mediated epigenetic modifications of NRF1 facilitates mammary tumorigenesis and invasive phenotypes of breast cancer cells via modulations of genes involved in proliferation, growth and metastasis of exposed cells. (v) Animal engraftment of transformed clones formed invasive tumors. (vi) Treatment of cells or tumors with biological or chemical antioxidants, as well as silencing of NRF1 expressions, prevented 4-OHE2 induced mammary tumorigenesis and invasive phenotypes of MCF-10A cells. Based on these observations, we hypothesize that 4-OHE2 induced ROS epigenetically activate NRF1 through its phosphorylation and acylation. This, in turn, through NRF1-mediated transcriptional activation of the cell cycle genes, controls 4-OHE2 induced cell transformation and tumorigenesis.^

Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

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.