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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

Daidzein increases stability of BRF1 and BRF2 mRNAs selectively in MCF-7 cells. MCF-7 cells were treated with 5 μg/ml of actinomycin D, either alone (white bars) or with 10 μM daidzein (black bars) for 24 h. The cells were then harvested at 4, 8, 12 and 24 h. The RNA was extracted and analyzed by qRT-PCRfor levels of (a) BRF2, (b) BRF1, (c) RPS13, and actin β mRNAs. ΔΔCt method with RPS13 and actin β expression levels were calculated for assay normalization. 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
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Fig6: Daidzein increases stability of BRF1 and BRF2 mRNAs selectively in MCF-7 cells. MCF-7 cells were treated with 5 μg/ml of actinomycin D, either alone (white bars) or with 10 μM daidzein (black bars) for 24 h. The cells were then harvested at 4, 8, 12 and 24 h. The RNA was extracted and analyzed by qRT-PCRfor levels of (a) BRF2, (b) BRF1, (c) RPS13, and actin β mRNAs. ΔΔCt method with RPS13 and actin β expression levels were calculated for assay normalization. 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

Mentions: The 3’-untranslated regions (3’-UTR) of BRF1 and BRF2 mRNAs are AU-rich, which may influence their stability [42]. Various classes of polyphenols have been shown to post-transcriptionally regulate levels of mRNA containing AU-rich elements by raising expression of RNA binding proteins [46–49]. To test if daidzein stabilizes BRF mRNAs, as a potential mechanism of induction, we treated MCF-7 cells with the transcription inhibitor actinomycin D [41] and monitored mRNA decay using qRT-PCR. Although 10 μM daidzein was unable to prevent the turnover of BRF mRNAs by 24 h, it prevented degradation of both BRF2 (p < 0.01; Fig. 6a) and BRF1 (p < 0.05; Fig. 6b) for 12 h after transcription inhibition. This effect is specific, as mRNA encoding ribosomal protein S13 (RPS13) decayed with similar kinetics in the presence or absence of daidzein, when assayed in parallel (Fig. 6c). The selective stabilization of BRF mRNAs offers a potential explanation for the increased expression seen when MCF-7 cells are exposed to 10 μM daidzein. Additional mechanisms may also be involved, such as demethylation of the BRF2 promoter (Fig. 4b).Fig. 6


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)

Daidzein increases stability of BRF1 and BRF2 mRNAs selectively in MCF-7 cells. MCF-7 cells were treated with 5 μg/ml of actinomycin D, either alone (white bars) or with 10 μM daidzein (black bars) for 24 h. The cells were then harvested at 4, 8, 12 and 24 h. The RNA was extracted and analyzed by qRT-PCRfor levels of (a) BRF2, (b) BRF1, (c) RPS13, and actin β mRNAs. ΔΔCt method with RPS13 and actin β expression levels were calculated for assay normalization. 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
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Related In: Results  -  Collection

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Fig6: Daidzein increases stability of BRF1 and BRF2 mRNAs selectively in MCF-7 cells. MCF-7 cells were treated with 5 μg/ml of actinomycin D, either alone (white bars) or with 10 μM daidzein (black bars) for 24 h. The cells were then harvested at 4, 8, 12 and 24 h. The RNA was extracted and analyzed by qRT-PCRfor levels of (a) BRF2, (b) BRF1, (c) RPS13, and actin β mRNAs. ΔΔCt method with RPS13 and actin β expression levels were calculated for assay normalization. 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
Mentions: The 3’-untranslated regions (3’-UTR) of BRF1 and BRF2 mRNAs are AU-rich, which may influence their stability [42]. Various classes of polyphenols have been shown to post-transcriptionally regulate levels of mRNA containing AU-rich elements by raising expression of RNA binding proteins [46–49]. To test if daidzein stabilizes BRF mRNAs, as a potential mechanism of induction, we treated MCF-7 cells with the transcription inhibitor actinomycin D [41] and monitored mRNA decay using qRT-PCR. Although 10 μM daidzein was unable to prevent the turnover of BRF mRNAs by 24 h, it prevented degradation of both BRF2 (p < 0.01; Fig. 6a) and BRF1 (p < 0.05; Fig. 6b) for 12 h after transcription inhibition. This effect is specific, as mRNA encoding ribosomal protein S13 (RPS13) decayed with similar kinetics in the presence or absence of daidzein, when assayed in parallel (Fig. 6c). The selective stabilization of BRF mRNAs offers a potential explanation for the increased expression seen when MCF-7 cells are exposed to 10 μM daidzein. Additional mechanisms may also be involved, such as demethylation of the BRF2 promoter (Fig. 4b).Fig. 6

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