Abstract

Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via upregulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER-) metastatic breast cancer cells, equol induced elevated levels of eIF4G which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNA’s translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of cMyc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant downregulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer promoting effects of equol via upregulation of eIF4GI and selective initiation of translation on mRNAs that utilize noncanonical initiation, including certain oncogenes. http://www.jbc.org/cgi/doi/10.1074/jbc.M114.617415 The latest version is at JBC Papers in Press. Published on January 15, 2015 as Manuscript M114.617415 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Equol increases cancer potential via c-Myc upregulation 2 INTRODUCTION Isoflavones are found in nutritionally relevant amounts in soybeans comprising ~3.5 mg/gm soy protein in traditional soy foods. Studies have reported a range of 1-25 μM soy isoflavones in the human circulation following consumption of soy products, which is sufficient for physiological activity (1-4). Due to the structural similarity to 17β estradiol, these phytoestrogens have been extensively studied for their potential estrogenic or antiestrogenic effects in breast cancer (5;6). Moreover, soy isoflavones may have additional estrogen-independent effects in aggressive ERbreast cancers (5;7). Daidzein is the second most prominent isoflavone in soy and approximately 70% of daidzein can be metabolized by the intestinal microflora to the metabolite equol (8). Only ~3050% of humans have the gut microflora necessary to convert daidzein to equol (8;9); therefore, not all humans are affected by equol. Equol is also chemically similar to estrogen and has 80 times more ERβ affinity than daidzein (10-12). The effects of equol, specifically in ERbreast cancers, or established aggressive breast cancers, remains unclear and requires additional study (7;8;13;14). At high concentrations (50-350 μM), equol has been implicated in inhibition of cancer cell growth, invasion, tumor progression, and cancer risk (15-19), while low physiological concentrations of equol reportedly increases cancer cell proliferation (14;20). In ER+ human breast cancer cells, equol increases estrogenic activity and cell proliferation, but does not affect tumor growth in mice (20-23). Dietary daidzein also failed to reduce mammary tumor growth in rats when metabolized to ~1μM equol in serum (24), although others have shown inhibitory effects of daidzein and equol on ER+ breast cancer cells and tumors (25;26). Thus, whether daidzein and equol prevents or promotes breast cancer in experimental models is not established, and may be concentration and ER status dependent. Accordingly, the molecular mechanisms by which equol acts on breast cancer cells need to be better understood, enabling recommendations for soy consumption in breast cancer patients. To this end, we previously tested the effect of individual and combined soy isoflavones on immune impaired (nude) mice with mammary tumors, established from ERcancer cells. We reported that dietary daidzein increases mammary tumor growth and metastasis (27). These findings support our recent in vitro studies which demonstrated that equol is the active metabolite of daidzein and increases breast cancer cell malignancy, primarily via upregulation of the eukaryotic initiation factor eIF4GI and its translation of mRNA encoding oncogenic proteins (28). Translational control has received increased attention in recent years due to its emerging significance in cancer development and progression (29). Translation initiation is typically the rate-limiting step and therefore, a primary site for regulation. Accordingly, the levels of two eukaryotic initiation factors that are members of the cap-initiation eIF4F complex, consisting of eIF4E (Cap binding protein) and eIF4G (initiation complex molecular scaffold), are frequently elevated in human cancers, and have been associated with poor prognosis and outcome (2931). Overexpression of eIF4GI is critical for the modes of translation initiation in eukaryotic cells that bypass or have a reduced requirement for eIF4E, including IRES-dependent mechanisms that allow the 40S ribosome to be directly recruited to the mRNA (32;33). It is thought that under the physiological stress conditions that exist in large tumors (i.e., growth arrest, amino acid starvation, hypoxia), cancer cells rely on noncanonical, eIF4E deficient, IRES-dependent translation of a subset of mRNAs encoding progrowth, pro-angiogenic and pro-survival proteins, such as, BCL2, Bcl -Xl, c-Myc, p120 catenin, and vascular endothelial growth factor A (VEGF) A, among others (29;34-36). In our recent study using MDA-MB 435 ERmetastatic breast cancer cells treated with equol, we showed that the transcription factor c-Myc was also elevated in addition to upregulated levels of eIF4GI and the increased translation of IREScontaining mRNAs that control cell survival and cell proliferation (28). This result is relevant because c-Myc is overexpressed in a variety of human cancers and plays an important role in multiple signaling pathways including cell growth, cell proliferation, metabolism, ribosome biogenesis, microRNA regulation, cell death, and cell survival (37-39). With the objective of determining whether the equol-mediated upregulation of eIF4G promotes by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Equol increases cancer potential via c-Myc upregulation 3 preferential synthesis of c-Myc, which has an IRES element (40), or whether c-Myc upregulation by equol leads to eIF4GI transcription, as shown in (41), we investigated the effects of silencing eIF4GI or c-Myc in equoltreated metastatic breast cancer cells. Here, we show that reducing eIF4GI levels results in a marked reduction in IRES dependent mRNA translation, decreased polysomal association of those mRNAs, and corresponding oncogenic protein levels of specific IRES-containing mRNAs. The increase in cell viability and mammosphere formation (an indicator of tumorigenic potential (42;43)) in response to equol was not affected until c-Myc was also targeted by siRNA, which in addition, significantly downregulated eIF4GI levels. Taken together, these data implicate c-Myc, and the consequent increase in eIF4GI followed by selective translation of oncogene mRNAs, in the pro-transforming effects of equol on breast cancer progression. EXPERIMENTAL PROCEDURES Cell culture – Metastatic variant of MDA-MB-435 (ER−) (gift of Dr. Danny Welch, The University of Kansas Cancer Center) and Hs578t (ER−) metastatic human breast cancer cells (American Type Culture Collection (ATCCC), Manassas, VA) were maintained in complete culture medium: Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Houston, TX) supplemented with 10% fetal bovine serum (Invitrogen, Houston, TX) at 37°C in 5% CO2. The MCF-10A mammary epithelial cells (ATCCC) were maintained in DMEM supplemented with 10% horse serum, EGF, hydrocortisone, cholera toxin, and insulin, as described previously (44). Cell lines were authenticated and found to be mycoplasma free. Cell treatment – Quiescent mammary epithelial cells or metastatic cancer cells were treated with 0 (vehicle, 0.1% DMSO) or 25 μM of (R,S) Equol (LC Laboratories, Woburn, MA) in DMEM and 5% FBS media for 24 h. Western blotting – Cells were lysed and western blotted, as described in (27). Primary antibodies to eIF4E, eIF4GI, c-Myc, p120 catenin, Bcl-Xl, Cyclin-D1, GAPDH, JunB and β-actin proteins (Epitomics, Burlingame, CA, Cell Signaling, Danvers, MA, Sigma-Aldrich Comp., St Louis, MO) were used. The integrated density of positive bands was quantified using Image J software, as described in (27). Cell viability assay – Cell viability was determined by the CellTiter 96 Non-Radioactive Cell Proliferation kit according to manufacturer instructions (Promega, Madison, WI). Briefly, quiescent 1 x 10 MDA-MB-435 cells were added to each well of a 96-well plate and treated for 24 h with vehicle or 25 μM equol. Following equilibration, 15 μL/well of MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent was added and the plates incubated at 37°C for 4 h. Stop solution (100 μL) was added to each well, and the plates incubated to facilitate solubilization of newly formed formazan salts. The absorbance at 570 nm was measured using an ELISA plate reader. Real-Time reverse transcriptase polymerase reaction (RT-PCR) analysis – Real-time quantitative RT-PCR analysis was performed as described (28). Briefly, total RNA was extracted using