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Androgen-regulated transcriptional control of sialyltransferases in prostate cancer cells.

Hatano K, Miyamoto Y, Mori M, Nimura K, Nakai Y, Nonomura N, Kaneda Y - PLoS ONE (2012)

Bottom Line: The expression of gangliosides is often associated with cancer progression.This testosterone-dependent ST3Gal II expression was suppressed by RelB siRNA, indicating that RelB activated ST3Gal II transcription in the testosterone-induced demethylated promoter.This is the first report indicating that the expression of a sialyltransferase is transcriptionally regulated by androgen-dependent demethylation of the CpG sites in its gene promoter.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan.

ABSTRACT
The expression of gangliosides is often associated with cancer progression. Sialyltransferases have received much attention in terms of their relationship with cancer because they modulate the expression of gangliosides. We previously demonstrated that GD1a production was high in castration-resistant prostate cancer cell lines, PC3 and DU145, mainly due to their high expression of β-galactoside α2,3-sialyltransferase (ST3Gal) II (not ST3Gal I), and the expression of both ST3Gals was regulated by NF-κB, mainly by RelB. We herein demonstrate that GD1a was produced in abundance in cancerous tissue samples from human patients with hormone-sensitive prostate cancers as well as castration-resistant prostate cancers. The expression of ST3Gal II was constitutively activated in castration-resistant prostate cancer cell lines, PC3 and DU145, because of the hypomethylation of CpG island in its promoter. However, in androgen-depleted LNCap cells, a hormone-sensitive prostate cancer cell line, the expression of ST3Gal II was silenced because of the hypermethylation of the promoter region. The expression of ST3Gal II in LNCap cells increased with testosterone treatment because of the demethylation of the CpG sites. This testosterone-dependent ST3Gal II expression was suppressed by RelB siRNA, indicating that RelB activated ST3Gal II transcription in the testosterone-induced demethylated promoter. Therefore, in hormone-sensitive prostate cancers, the production of GD1a may be regulated by androgen. This is the first report indicating that the expression of a sialyltransferase is transcriptionally regulated by androgen-dependent demethylation of the CpG sites in its gene promoter.

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Androgen-dependent regulation of ST3Gal II in LNCap cells.(A) LNCap, PC3, and PNT2 cells were treated with or without testosterone (0–1000 nM) for 120 h, by refeeding with fresh medium with or without testosterone at 72 h. The quantitative real-time PCR analyses of ST3Gal II mRNA were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. **P<0.001. (B) LNCap cells were treated with or without testosterone (0–100 nM) and simultaneously with or without 10 µM bicalutamide for 120 h, by refeeding with fresh medium with or without testosterone and/or bicalutamide at 72 h. The quantitative real-time PCR analyses for ST3Gal II were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. **P<0.001. (C) LNCap cells were incubated in charcoal-stripped serum (CSS) for 48 h and then treated with 100 nM testosterone for the indicated times. The quantitative real-time PCR analyses for ST3Gal II were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. *P<0.05, **P<0.001.
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pone-0031234-g002: Androgen-dependent regulation of ST3Gal II in LNCap cells.(A) LNCap, PC3, and PNT2 cells were treated with or without testosterone (0–1000 nM) for 120 h, by refeeding with fresh medium with or without testosterone at 72 h. The quantitative real-time PCR analyses of ST3Gal II mRNA were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. **P<0.001. (B) LNCap cells were treated with or without testosterone (0–100 nM) and simultaneously with or without 10 µM bicalutamide for 120 h, by refeeding with fresh medium with or without testosterone and/or bicalutamide at 72 h. The quantitative real-time PCR analyses for ST3Gal II were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. **P<0.001. (C) LNCap cells were incubated in charcoal-stripped serum (CSS) for 48 h and then treated with 100 nM testosterone for the indicated times. The quantitative real-time PCR analyses for ST3Gal II were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. *P<0.05, **P<0.001.

Mentions: The LNCap cell culture medium is routinely supplemented with 10% fetal bovine serum (FBS). A recent report showed that media supplemented with 10% FBS contains only castrate levels of testosterone [29]; in contrast, hormone-sensitive prostate cancers of untreated patients usually grow in an environment containing testosterone in vivo. To analyze the transcriptional control of ST3Gal II in hormone-sensitive prostate cancers, we examined whether the expression of ST3Gal II was controlled by testosterone in LNCap cells. LNCap cells were treated with testosterone (0–1000 nM), and were incubated for 120 h. The quantitative real-time PCR analyses showed that the expression of ST3Gal II was higher in LNCap cells treated with testosterone than in the LNCap cells that were not (Fig. 2A). Furthermore, the induction of ST3Gal II after testosterone treatment was suppressed by an anti-androgen, bicalutamide, in LNCap cells (Fig. 2B). To ensure that there were no androgens present in the media, LNCap cells were incubated in charcoal-stripped serum for 48 h. The basal level of ST3Gal II was not significantly different between the 10% FBS- and charcoal stripped serum-supplemented LNCap cells (Fig. 2C). The LNCap cells were subsequently treated with 100 nM testosterone, and the time-course of expression following testosterone treatment was evaluated. The expression of ST3Gal II was increased 48 h after testosterone treatment, and remained elevated for more than 120 h in the LNCap cells (Fig. 2C). To evaluate the NF-κB activity after testosterone treatment, LNCap cells were transfected with an NF-κB luciferase reporter construct and incubated for 120 h with or without testosterone. The NF-κB activity was not significantly different in the testosterone-treated LNCap cells compared to the cells cultured without testosterone (Figure S1). In PC3 and PNT2 cells, no significant increase in the expression of ST3Gal II was detected regardless of whether the cells cultured with or without testosterone (Fig. 2A). The expression of ST3Gal II did not increase after testosterone treatment in the PC3 cells at any time point up to 120 h (Figure S2). Based on these findings, we hypothesized that the media with castrate levels of testosterone led to the epigenetic silencing of ST3Gal, a gene required for the synthesis of GD1a, in LNCap cells.


Androgen-regulated transcriptional control of sialyltransferases in prostate cancer cells.

Hatano K, Miyamoto Y, Mori M, Nimura K, Nakai Y, Nonomura N, Kaneda Y - PLoS ONE (2012)

Androgen-dependent regulation of ST3Gal II in LNCap cells.(A) LNCap, PC3, and PNT2 cells were treated with or without testosterone (0–1000 nM) for 120 h, by refeeding with fresh medium with or without testosterone at 72 h. The quantitative real-time PCR analyses of ST3Gal II mRNA were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. **P<0.001. (B) LNCap cells were treated with or without testosterone (0–100 nM) and simultaneously with or without 10 µM bicalutamide for 120 h, by refeeding with fresh medium with or without testosterone and/or bicalutamide at 72 h. The quantitative real-time PCR analyses for ST3Gal II were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. **P<0.001. (C) LNCap cells were incubated in charcoal-stripped serum (CSS) for 48 h and then treated with 100 nM testosterone for the indicated times. The quantitative real-time PCR analyses for ST3Gal II were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. *P<0.05, **P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275626&req=5

pone-0031234-g002: Androgen-dependent regulation of ST3Gal II in LNCap cells.(A) LNCap, PC3, and PNT2 cells were treated with or without testosterone (0–1000 nM) for 120 h, by refeeding with fresh medium with or without testosterone at 72 h. The quantitative real-time PCR analyses of ST3Gal II mRNA were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. **P<0.001. (B) LNCap cells were treated with or without testosterone (0–100 nM) and simultaneously with or without 10 µM bicalutamide for 120 h, by refeeding with fresh medium with or without testosterone and/or bicalutamide at 72 h. The quantitative real-time PCR analyses for ST3Gal II were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. **P<0.001. (C) LNCap cells were incubated in charcoal-stripped serum (CSS) for 48 h and then treated with 100 nM testosterone for the indicated times. The quantitative real-time PCR analyses for ST3Gal II were performed, and the expression levels are reported as the means ± S.E. (n = 3) of the fold difference in mRNA after normalizing the values to the expression level of untreated cells. *P<0.05, **P<0.001.
Mentions: The LNCap cell culture medium is routinely supplemented with 10% fetal bovine serum (FBS). A recent report showed that media supplemented with 10% FBS contains only castrate levels of testosterone [29]; in contrast, hormone-sensitive prostate cancers of untreated patients usually grow in an environment containing testosterone in vivo. To analyze the transcriptional control of ST3Gal II in hormone-sensitive prostate cancers, we examined whether the expression of ST3Gal II was controlled by testosterone in LNCap cells. LNCap cells were treated with testosterone (0–1000 nM), and were incubated for 120 h. The quantitative real-time PCR analyses showed that the expression of ST3Gal II was higher in LNCap cells treated with testosterone than in the LNCap cells that were not (Fig. 2A). Furthermore, the induction of ST3Gal II after testosterone treatment was suppressed by an anti-androgen, bicalutamide, in LNCap cells (Fig. 2B). To ensure that there were no androgens present in the media, LNCap cells were incubated in charcoal-stripped serum for 48 h. The basal level of ST3Gal II was not significantly different between the 10% FBS- and charcoal stripped serum-supplemented LNCap cells (Fig. 2C). The LNCap cells were subsequently treated with 100 nM testosterone, and the time-course of expression following testosterone treatment was evaluated. The expression of ST3Gal II was increased 48 h after testosterone treatment, and remained elevated for more than 120 h in the LNCap cells (Fig. 2C). To evaluate the NF-κB activity after testosterone treatment, LNCap cells were transfected with an NF-κB luciferase reporter construct and incubated for 120 h with or without testosterone. The NF-κB activity was not significantly different in the testosterone-treated LNCap cells compared to the cells cultured without testosterone (Figure S1). In PC3 and PNT2 cells, no significant increase in the expression of ST3Gal II was detected regardless of whether the cells cultured with or without testosterone (Fig. 2A). The expression of ST3Gal II did not increase after testosterone treatment in the PC3 cells at any time point up to 120 h (Figure S2). Based on these findings, we hypothesized that the media with castrate levels of testosterone led to the epigenetic silencing of ST3Gal, a gene required for the synthesis of GD1a, in LNCap cells.

Bottom Line: The expression of gangliosides is often associated with cancer progression.This testosterone-dependent ST3Gal II expression was suppressed by RelB siRNA, indicating that RelB activated ST3Gal II transcription in the testosterone-induced demethylated promoter.This is the first report indicating that the expression of a sialyltransferase is transcriptionally regulated by androgen-dependent demethylation of the CpG sites in its gene promoter.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan.

ABSTRACT
The expression of gangliosides is often associated with cancer progression. Sialyltransferases have received much attention in terms of their relationship with cancer because they modulate the expression of gangliosides. We previously demonstrated that GD1a production was high in castration-resistant prostate cancer cell lines, PC3 and DU145, mainly due to their high expression of β-galactoside α2,3-sialyltransferase (ST3Gal) II (not ST3Gal I), and the expression of both ST3Gals was regulated by NF-κB, mainly by RelB. We herein demonstrate that GD1a was produced in abundance in cancerous tissue samples from human patients with hormone-sensitive prostate cancers as well as castration-resistant prostate cancers. The expression of ST3Gal II was constitutively activated in castration-resistant prostate cancer cell lines, PC3 and DU145, because of the hypomethylation of CpG island in its promoter. However, in androgen-depleted LNCap cells, a hormone-sensitive prostate cancer cell line, the expression of ST3Gal II was silenced because of the hypermethylation of the promoter region. The expression of ST3Gal II in LNCap cells increased with testosterone treatment because of the demethylation of the CpG sites. This testosterone-dependent ST3Gal II expression was suppressed by RelB siRNA, indicating that RelB activated ST3Gal II transcription in the testosterone-induced demethylated promoter. Therefore, in hormone-sensitive prostate cancers, the production of GD1a may be regulated by androgen. This is the first report indicating that the expression of a sialyltransferase is transcriptionally regulated by androgen-dependent demethylation of the CpG sites in its gene promoter.

Show MeSH
Related in: MedlinePlus