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Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein
© Koo et al. 2015
Received: 28 August 2015
Accepted: 6 November 2015
Published: 16 November 2015
BRF2 is a transcription factor required for synthesis of a small group of non-coding RNAs by RNA polymerase III. Overexpression of BRF2 can transform human mammary epithelial cells. In both breast and lung cancers, the BRF2 gene is amplified and overexpressed and may serve as an oncogenic driver. Furthermore, elevated BRF2 can be independently prognostic of unfavorable survival. Dietary soy isoflavones increase metastasis to lungs in a model of breast cancer and a recent study reported significantly increased cell proliferation in breast cancer patients who used soy supplementation. The soy isoflavone daidzein is a major food-derived phytoestrogen that is structurally similar to estrogen. The putative estrogenic effect of soy raises concern that high consumption of soy foods by breast cancer patients may increase tumor growth.
Expression of BRF2 RNA and protein was assayed in ER-positive or –negative human breast cancer cells after exposure to daidzein. We also measured mRNA stability, promoter methylation and response to the demethylating agent 5-azacytidine. In addition, expression was compared between mice fed diets enriched or deprived of isoflavones.
We demonstrate that the soy isoflavone daidzein specifically stimulates expression of BRF2 in ER-positive breast cancer cells, as well as the related factor BRF1. Induction is accompanied by increased levels of non-coding RNAs that are regulated by BRF2 and BRF1. Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter. Functional significance of demethylation is supported by induction of BRF2 by the methyltransferase inhibitor 5-azacytidine. None of these effects are observed in an ER-negative breast cancer line, when tested in parallel with ER-positive breast cancer cells. In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet. In striking contrast, BRF2 and BRF1 mRNA levels are suppressed in matched male mice fed the same isoflavone-enriched diet.
The BRF2 gene that is implicated in cancer can be induced in human breast cancer cells by the isoflavone daidzein, through promoter demethylation and/or mRNA stabilization. Dietary isoflavones may also induce BRF2 in female mice, whereas the converse occurs in males.
RNA polymerase (pol) III has the responsibility of synthesizing a variety of short noncoding RNAs such as tRNAs and the spliceosomal U6 snRNA . Initiation by pol III requires TFIIIB , a transcription factor complex with at least two forms in mammalian cells [2, 3]. Gene-internal pol III promoters, such as those found in tRNA genes, require TFIIIB composed of TBP, BDP1 and BRF1 subunits, whereas gene-external pol III promoters, as exemplified by U6 genes, require TFIIIB containing TBP, BDP1 and BRF2 . Aberrant pol III transcription is a feature of many tumor types . This reflects, in part, the fact that TFIIIB is strongly regulated by pathways involving oncogenes and tumor suppressors [4, 5]. For example, MYC  and the MAP kinase ERK  bind to TFIIIB and stimulate its activity, whereas an array of tumor suppressors inhibit TFIIIB activity , either directly or indirectly, including BRCA1 , PTEN [10, 11], p53 , and the RB family .
The BRF2 subunit of TFIIIB is encoded by an oncogene at 8p12 that is frequently amplified and overexpressed in breast cancers and lung squamous cell carcinomas (SqCC) [14–20, 21]. BRF2 drives the 8p12 amplification in SqCC . Its overexpression stimulates proliferation and saturation density of human bronchial epithelial cells, whereas its knockdown specifically suppresses proliferation and anchorage-independent growth of SqCC cells with 8p12 amplification . Copy number increases and overexpression of BRF2 are apparent in most pre-invasive bronchial carcinomas in situ, with minimal staining in benign lesions . BRF2 induction was therefore proposed as an early event in development of lung SqCC, that might serve as a marker and/or therapeutic target . Subsequent independent studies reported elevated BRF2 protein in lung and esophageal SqCC, where high BRF2 was independently prognostic of unfavorable survival for both lung (P = 0.007) and esophageal (P = 0.009) SqCC [23, 24]. BRF2 overexpression may also be an oncogenic driver in some breast cancers and human mammary epithelial cells can be transformed by transfection of the BRF2 gene . Analysis of published datasets, using the Web-based Oncomine platform, reveals that BRF2 was amongst the top 1 % of genes overexpressed in a study  of 154 invasive breast carcinomas (p = 3.53E-10), whilst a larger study of over two thousand breast samples  confirmed BRF2 overexpression in several tumor subgroups, with invasive ductal breast carcinomas the most significant (p = 2.17E-21). The cBioPortal cancer genomics database [27–29] reveals amplification of the BRF2 gene in 12 % of 825 tumors in the Breast Invasive Carcinoma study (TCGA, Nature 2012) .
The chemopreventive polyphenol EGCG, enriched in green tea, specifically decreases TFIIIB activity in cervical cancer cells . The polyphenols genistein and daidzein are isoflavone components of soybeans, a major crop in the United States and globally . These soy isoflavones are major food-derived phytoestrogens that are structurally similar to estrogen with the capacity to weakly bind to estrogen receptors (ERs) . The putative estrogenic effect of soy raises the concern that high consumption of soy foods by breast cancer patients and/or women at high risk for breast cancer may increase estrogen-dependent breast tumor growth . A recent study reported a significant increase in cell proliferation in breast cancer patients who used soy supplementation . Dietary soy isoflavones increase metastasis to lungs in an experimental model of breast cancer . These data prompted us to investigate if the soy isoflavone daidzein regulates TFIIIB. We found that 10 uM daidzein stimulates expression of the TFIIIB subunits BRF1 and BRF2 in ER-positive breast cancer cells, as well as pol III products U6 snRNA and tRNAi Met. Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and raises levels of their protein products. It also triggers selective demethylation of the BRF2 promoter. These effects are not seen in an ER-negative breast cancer line. An isoflavone-enriched diet also induces BRF2 in female mice, but has the opposite effect in males. These in vitro and in vivo data suggest that dietary isoflavones differentially regulate TFIIIB expression, an important observation given the evidence that BRF2 can drive tumorigenesis and is predictive of poor prognosis.
Cell lines and daidzein treatment
MCF-7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in DMEM supplemented with FBS (5 % v/v), nonessential amino acids (100 mM), L-glutamine (5 mM), streptomycin (100 μg/ml), and penicillin (100 units/ml); all from BioWhittaker, Walkersville, MD. Cells were grown at 37 °C in a humidified atmosphere of 95 % air and 5 % CO2 as previously described [37, 38]. Daidzein (Sigma) treatments are as described in figure legends.
Asynchronous MCF-7 and MDA-MB-231 cells were plated at 1 × 104 cell/well in 6-well plates. After 24 h, cells were treated with 5 μM 5-azacytidine (Sigma) for 24, 48 and 72 h. At each time point, total RNA was collected using RNeasy total RNA isolation kit (Qiagen), according to the manufacturer's protocol and cDNA subsequently prepared to be used in qRT-PCRassays.
Quantitative reverse transcription PCR (qRT-PCR)
Total RNA was extracted from cancer cell lines using the RNeasy total RNA isolation kit (Qiagen), according to the manufacturer's protocol and qPCR was performed using diluted cDNA from treated breast cancer cells and SsoAdvanced™ Universal SYBR® Green Supermix (BioRad). Gene specific primers include: BRF2-forward, 5’-CAG AAG TGG AGA CCC GAG AG-3’; BRF2-reverse, 5’-CAG GGA GGG TTA GGG ACA CT-3’; BRF1-forward, 5’-GGC ATT GAT GAC CTG GAG AT-3’; BRF1-reverse, 5’-ACC AGA GGC CTC AAC CTT TT-3’; BDP1-forward, 5’-TGG AAG AAG CTG GAA GGA GA-3’; BDP1-reverse, 5’-TTC CTC AAT GGC ATC AAT CA-3’; TBP-forward, 5’-CGG CTG TTT AAC TTC GCT TC-3’; TBP reverse, 5’-CTG TTG TTG TTG CTG CTG CT-3’; U6-forward, 5’-GGT CGG GCA GGA AAG AGG GC-3’; U6-reverse, 5’- GCTAAT CTT CTC TGT ATC GTT CC-3’; tRNAi Met-forward, 5’- CTG GGC CCA TAA CCC AGA G-3’; tRNAi Met-reverse, 5’-TGG TAG CAG AGG ATG GTT TC-3’; GAPDH-forward, 5’- TCCACCACCCTGTTGCTGTA-3’; GAPDH-reverse, 5’- ACC ACA GTC CAT GCC ATC AC-3’; RPS13-forward, 5’-GTT GCT GTT CGA AAG CAT CTT G-3’; RPS13-reverse, 5’-AAT ATC GAG CCA AAC GGT GAA-3’; actin β-forward, 5’-TAG CGG GGT TCA CCC ACA CTG TGC CCC A-3’; actin β-reverse, 5’- CTA GAA GCA TTT GCG GTG GAC CGA TGG A-3’. Real time quantitative PCR reactions were carried out using the Bio-Rad CFX Connect System. The ΔΔCt method was employed for each gene tested as noted in figures using GAPDH and RPS13 expression levels for normalization. Meta-analysis of data using one-way ANOVA with a Tukey post-test with a 95 % confidence interval (GraphpadPrism3.03, San Diego California USA); * = p <0.05; ** = p < 0.01; *** = p < 0.001.
Western blot analysis
Total cellular protein was extracted using Cytobuster Protein Extraction Reagent (Merck Millipore; 71009). Proteins were fractionated by SDS-PAGE and transferred to nitrocellulose membranes, which were incubated overnight with antibodies against BRF1 (Bethyl Laboratories; A301-228A), BRF2 and actin (Santa Cruz Biotechnology; sc-390312 and sc-1615). Membranes were then incubated with HRP-conjugated anti-goat (Dako; P0449) anti-rabbit and anti-mouse (Cell Signaling; 7074 and 7076) IgG for 1 h. Bands were visualized using the enhanced chemiluminescence method.
Promoters were analyzed using MethPrimer  to identify potential CpG islands. NEBcutter V2.0  was used to identify methylation sensitive restriction enzyme (MSRE) sites sensitive within promoter sequences. Genomic DNA was isolated from MCF-7 and MD-MB-231 cancer cells using DNeasy Blood & Tissue Kit (Qiagen) using the manufacturer’s protocol. Restriction enzymes AciI, AscI, BanI, BfuAI, BsrFI, BsrBI, BseYI, BfuAI, BspEI, Cac81, FspI, NciI, NruI (New England Biolabs) and HpyCH4III/Taal (Fermentas) were used. Restriction digestions were then analyzed by real time PCR using Universal SYBR Green (BioRad) and primers spanning the promoters regions and primers spanning the promoters regions (BRF2-forward, 5’-GGC CTC CAA AAG CGT T-3’; BRF2-reverse, 5’-AGC TGG CTC TGC GAA TAG T-3’; BRF1-forward, 5’-GGG GTT GGG TCC CAG GTC GC-3’; BRF1-reverse, 5’-GTC CTC CAG CAC TGA GCC GC-3’; U6-forward, 5’- AAG TAT TTC GAT TTC TTG GC-3’; U6-reverse, 5’- AAT ATG GAA CGC TTC ACG-3’; tRNAi Met-forward, 5’-TAG ATA GCA GAG TGG CGC A-3’; tRNAi Met-reverse, 5’-AAC TCC GAT AGC AGA GGA TG-3’). Results were quantified using the ΔΔCt method and normalized to RPS13 expression levels. Data presented are average of three independent experiments. Statistical analysis was performed using one-way ANOVA with a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p <0.05; ** = p < 0.01; *** = p < 0.001.
mRNA stability of the BRF1 and BRF2 genes
The mRNA stability of BRF1 and BRF2 mRNA was determined following actinomycin D treatment. Actinomycin D was added to a final concentration of 5 μg/ml to block transcription . At 0, 4, 8, 12 and 24 h post-actinomycin D treatment, the cells were harvested, and mRNA was quantified by qRT-PCRas described above. Data show relative expression values at time points indicated with control untreated sample set to 1 at 0 h post-actinomycin D treatment.
BRF2 protein levels were measured from untreated and daidzein treated MCF-7 and MD-MB-231 cells using a human BRF2 ELISA kit (MyBioSource) as per the manufacturer’s protocol. Data presented are average of four independent experiments. Statistical analysis was performed using one-way ANOVA with a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p <0.05; ** = p < 0.01; *** = p < 0.001.
Animals and isoflavone treatment
Female and male C57BL/6 J mice were purchased from Taconic Farms (Germantown, New York). Mice were housed 4 per cage in a temperature-controlled St. John’s University Animal Care Facility with alternating 12:12 h light–dark cycles, with ad libitum access to water and commercial Purina 5001 or 5 K96 chows (Fig. 7), as approved in its entirety by SJU IACUC (SJU Protocol number 1831.0). Mice were numbered with permanent marker identification, and acclimated to animal penthouse for 72 h prior to start of experiment. Mice were treated for a total of three weeks and monitored daily for signs of stress. Weekly tail bleeds and a terminal cardiac puncture bleed were collected and analyzed for TFIIIB levels via qRT-PCR.
Daidzein induces BRF1, BRF2 and pol III transcript expression in MCF-7 breast cancer cells
Daidzein decreases methylation of the BRF2 promoter specifically in MCF-7 cells
Daidzein increases stability of BRF1 and BRF2 mRNAs in MCF-7 cells
Commercial rodent chow differentially regulates BRF1 and BRF2 in a sex-dependent manner
Breast cancer is the leading cause of cancer-related deaths in females in developing countries and the second leading cause in developed countries [51, 52]. It has been estimated that there will be approximately 1.7 million new cases of breast cancer in 2020, a 26 % increase from current incidence rates, mostly due to the increase in new cases in the developing world . As diet is known to influence the incidence of breast cancer , identifying the impact of specific foods is imperative. Soy has been studied extensively for its anti-cancer properties, but the epidemiological results have been contradictory, in part because soy can regulate gene expression via estrogen- dependent and independent pathways. We therefore investigated the molecular effects of the isoflavone daidzein in both ER-positive and ER-negative cancer cells. We observed clear effects in the former that were not seen in the latter. Specifically, daidzein raises expression of BRF2 and BRF1 in MCF-7 cells, as well as pol III transcripts that depend on these TFIIIB subunits for their synthesis. It is noteworthy that another polyphenol, EGCG that is enriched in green tea, also affects expression of BRF1 and BRF2 selectively, but in this case elicits an inhibitory effect, in keeping with its anti-cancer activity .
Induction of BRF2 and BRF1 by daidzein may be explained by stabilization of their respective mRNAs, although additional effects might also contribute. For example, we also observed demethylation of the BRF2 promoter that might result in a transcriptional response. How the polyphenol produces these effects remains to be established. A clear possibility is that the ER is involved, either directly or indirectly, as this can be weakly bound by daidzein . Indeed, ER has been shown to bind directly to the BRF1 promoter in MCF-7 cells  and 17β-estradiol has been shown to induce tRNA synthesis . Those studies did not investigate BRF2 or its target genes, such as U6. Circumstantial evidence in support of ER involvement is provided by the differential response in ER-positive MCF-7 and ER-negative MDA-MB-231 cells in culture, and the gender-specificity of induction in mice. However, further studies will be required to establish if ER mediates the observed effects. In terms of the current study, we consider the crucial finding to be that physiologically relevant doses of a common dietary compound can stimulate expression of a gene with established oncogenic properties. This raises concerns for women consuming soy.
It remains to be determined how BRF2 exerts oncogenic effects, although the obvious possibility is through transcriptomic changes, given its well-established function as a transcription factor. Genome-wide analyses showed that BRF2 has a highly restricted set of target genes , all encoding short pol III-dependent noncoding RNAs. In addition to U6 snRNA, these include 7SK RNA, which regulates transcription of protein-coding genes by inhibiting P-TEFb [58–60], and hY RNAs, which promote DNA replication . The hY RNAs are commonly overexpressed in tumors [62, 63]. For example, hY1 is overexpressed ~13-fold in lung, prostate and other cancers (P = 9.7 × 10−26) and its knockdown inhibits proliferation of lung and prostate cell lines . As a key component of spliceosomes, U6 snRNA is an essential BRF2-dependent product. It was consistently detected at elevated levels in sera from 140 breast cancer patients, relative to 115 healthy age-matched controls . U6 was also the most strongly upregulated (P < 0.0001; FDR <0.001) non-coding RNA in serum from 75 glioblastoma multiforme patients, relative to matched healthy controls . MicroRNAs are widely believed to have considerable potential as biomarkers, but U6 outperformed all 381 miRNAs tested in this study . It has also been suggested that BRF2 might serve as a biomarker, given its early induction in development of lung SqCC , where high BRF2 is independently prognostic (P = 0.007) of unfavourable survival , as also reported (P = 0.009) for oesophageal SqCC . Our demonstration that BRF2 levels can be monitored in mouse serum provides initial evidence that a non-invasive test is practical for this potential biomarker.
This study demonstrates, both in vitro and in vivo, that a common soy isoflavone, at dietary concentrations, can influence expression of BRF2 and BRF1, essential components of the pol III-specific transcription factor TFIIIB. The response may involve an epigenetic component and gender-dependence in mice. Its importance is suggested by recent evidence that BRF2 can be oncogenic and prognostic of poor survival.
Animal experiments presented were approved by SJU IACUC protocol 1831.0 in its entirety.
Availability of supporting data
Authors declare there is no additional supporting data to be made available. All DNA promoter sequences are readily available in PubMed database.
We thank Helen Scaramell, Director Animal Center at St. John’s University, for helpful discussions.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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