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Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein.

Koo J, Cabarcas-Petroski S, Petrie JL, Diette N, White RJ, Schramm L - BMC Cancer (2015)

Bottom Line: In addition, expression was compared between mice fed diets enriched or deprived of isoflavones.Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter.In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, St. John's University, Queens, New York, 11439, USA.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Azacytidine induces demethylation and expression of BRF2 selectively in MCF-7 cells. a, b, c MCF-7 and (d, e) MDA-MB-231 cells were treated with 5 μM 5-Azacytidine for 24, 48 and 72 h. Post-treatments, total RNA was isolated and analyzed by qRT-PCRfor expression of (a, d) BRF2 and (c, e) BRF1 mRNAs. The ΔΔCt method with GAPDH and RPS13 expression levels was used as a reference for normalization. Meta-analysis of two independent experiments performed in triplicate was completed 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. (b) Genomic methylation of BRF2 promoter DNA in MCF-7 cells was analyzed with methylation-sensitive restriction enzymes, as in Fig. 4a
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Fig5: Azacytidine induces demethylation and expression of BRF2 selectively in MCF-7 cells. a, b, c MCF-7 and (d, e) MDA-MB-231 cells were treated with 5 μM 5-Azacytidine for 24, 48 and 72 h. Post-treatments, total RNA was isolated and analyzed by qRT-PCRfor expression of (a, d) BRF2 and (c, e) BRF1 mRNAs. The ΔΔCt method with GAPDH and RPS13 expression levels was used as a reference for normalization. Meta-analysis of two independent experiments performed in triplicate was completed 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. (b) Genomic methylation of BRF2 promoter DNA in MCF-7 cells was analyzed with methylation-sensitive restriction enzymes, as in Fig. 4a

Mentions: If demethylation of promoter DNA can influence transcription of the BRF2 gene, then treatment with the methylation inhibitor 5-azacytidine might be predicted to induce expression. Indeed, BRF2 mRNA levels were elevated within 48 h of adding 5-azacytidine to MCF-7 cells, along with promoter demethylation (Figs. 5a and b). This response was specific, as it was not shown by BRF1 mRNA when assayed in parallel (Fig. 5c). Furthermore, the same treatment failed to induce either BRF1 or BRF2 in MDA-MB-231 cells (Figs. 5d and e). These data suggest that BRF2 promoter activity may be sensitive to DNA methylation, which responds to daidzein in MCF-7 cells.Fig. 5


Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein.

Koo J, Cabarcas-Petroski S, Petrie JL, Diette N, White RJ, Schramm L - BMC Cancer (2015)

Azacytidine induces demethylation and expression of BRF2 selectively in MCF-7 cells. a, b, c MCF-7 and (d, e) MDA-MB-231 cells were treated with 5 μM 5-Azacytidine for 24, 48 and 72 h. Post-treatments, total RNA was isolated and analyzed by qRT-PCRfor expression of (a, d) BRF2 and (c, e) BRF1 mRNAs. The ΔΔCt method with GAPDH and RPS13 expression levels was used as a reference for normalization. Meta-analysis of two independent experiments performed in triplicate was completed 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. (b) Genomic methylation of BRF2 promoter DNA in MCF-7 cells was analyzed with methylation-sensitive restriction enzymes, as in Fig. 4a
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4647806&req=5

Fig5: Azacytidine induces demethylation and expression of BRF2 selectively in MCF-7 cells. a, b, c MCF-7 and (d, e) MDA-MB-231 cells were treated with 5 μM 5-Azacytidine for 24, 48 and 72 h. Post-treatments, total RNA was isolated and analyzed by qRT-PCRfor expression of (a, d) BRF2 and (c, e) BRF1 mRNAs. The ΔΔCt method with GAPDH and RPS13 expression levels was used as a reference for normalization. Meta-analysis of two independent experiments performed in triplicate was completed 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. (b) Genomic methylation of BRF2 promoter DNA in MCF-7 cells was analyzed with methylation-sensitive restriction enzymes, as in Fig. 4a
Mentions: If demethylation of promoter DNA can influence transcription of the BRF2 gene, then treatment with the methylation inhibitor 5-azacytidine might be predicted to induce expression. Indeed, BRF2 mRNA levels were elevated within 48 h of adding 5-azacytidine to MCF-7 cells, along with promoter demethylation (Figs. 5a and b). This response was specific, as it was not shown by BRF1 mRNA when assayed in parallel (Fig. 5c). Furthermore, the same treatment failed to induce either BRF1 or BRF2 in MDA-MB-231 cells (Figs. 5d and e). These data suggest that BRF2 promoter activity may be sensitive to DNA methylation, which responds to daidzein in MCF-7 cells.Fig. 5

Bottom Line: In addition, expression was compared between mice fed diets enriched or deprived of isoflavones.Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter.In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, St. John's University, Queens, New York, 11439, USA.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus