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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 and epigenetic regulation of ST3Gal I in LNCap cells.(A) LNCap 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 for ST3Gal I 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 I 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 I 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. (D) LNCap cells were treated with 5-aza-2′-deoxycytidine (5-azadC) (0–50 µM) for 120 h, by refeeding with fresh medium with or without 5-azadC at 72 h. The quantitative real-time PCR analyses for ST3Gal I 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. (E) LNCap cells were treated with 5 µM trichostatin A (TSA) for 48 h. The quantitative real-time PCR analyses for ST3Gal I 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.
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pone-0031234-g005: Androgen-dependent and epigenetic regulation of ST3Gal I in LNCap cells.(A) LNCap 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 for ST3Gal I 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 I 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 I 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. (D) LNCap cells were treated with 5-aza-2′-deoxycytidine (5-azadC) (0–50 µM) for 120 h, by refeeding with fresh medium with or without 5-azadC at 72 h. The quantitative real-time PCR analyses for ST3Gal I 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. (E) LNCap cells were treated with 5 µM trichostatin A (TSA) for 48 h. The quantitative real-time PCR analyses for ST3Gal I 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.

Mentions: Although GD1a is synthesized from GM1 mainly by ST3Gal II, ST3Gal I may also contribute to the synthesis of GD1a [6], [21]–[24]. We previously reported that ST3Gal I was expressed in LNCap cells, while the expression of ST3Gal II was silenced [20]. Therefore, the regulation of ST3Gal I transcription may be different from that of ST3Gal II. We thereafter examined whether the expression of ST3Gal I, like ST3Gal II, was controlled by testosterone in LNCap cells. In this experiment, the LNCap cells were treated with testosterone and incubated for 120 h. The quantitative real-time PCR analyses showed that testosterone treatment resulted in increased expression of ST3Gal I in LNCap cells (Fig. 5A), although the expression of ST3Gal VI, the sialyltransferase required for the synthesis of sialyl paragloboside, was not induced by testosterone treatment (Figure S4). Furthermore, the testosterone-mediated induction of ST3Gal I in LNCap cells was suppressed by bicalutamide (Fig. 5B). To ensure that there were no androgens present in the cell culture media, LNCap cells were incubated in charcoal-stripped serum for 48 h. The basal level of ST3Gal I was not significantly different between the LNCap cells cultured with 10% FBS and charcoal-stripped serum (Fig. 5C). Then, the LNCap cells were treated with 100 nM testosterone, and the time-course of the changes following testosterone treatment was evaluated. The expression of ST3Gal I increased 72 h after testosterone treatment and remained elevated for more than 120 h in LNCap cells (Fig. 5C). In PC3 and PNT2 cells, no significant increase in the expression of ST3Gal I was detected after testosterone treatment (Figure S5).


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 and epigenetic regulation of ST3Gal I in LNCap cells.(A) LNCap 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 for ST3Gal I 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 I 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 I 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. (D) LNCap cells were treated with 5-aza-2′-deoxycytidine (5-azadC) (0–50 µM) for 120 h, by refeeding with fresh medium with or without 5-azadC at 72 h. The quantitative real-time PCR analyses for ST3Gal I 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. (E) LNCap cells were treated with 5 µM trichostatin A (TSA) for 48 h. The quantitative real-time PCR analyses for ST3Gal I 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275626&req=5

pone-0031234-g005: Androgen-dependent and epigenetic regulation of ST3Gal I in LNCap cells.(A) LNCap 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 for ST3Gal I 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 I 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 I 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. (D) LNCap cells were treated with 5-aza-2′-deoxycytidine (5-azadC) (0–50 µM) for 120 h, by refeeding with fresh medium with or without 5-azadC at 72 h. The quantitative real-time PCR analyses for ST3Gal I 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. (E) LNCap cells were treated with 5 µM trichostatin A (TSA) for 48 h. The quantitative real-time PCR analyses for ST3Gal I 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.
Mentions: Although GD1a is synthesized from GM1 mainly by ST3Gal II, ST3Gal I may also contribute to the synthesis of GD1a [6], [21]–[24]. We previously reported that ST3Gal I was expressed in LNCap cells, while the expression of ST3Gal II was silenced [20]. Therefore, the regulation of ST3Gal I transcription may be different from that of ST3Gal II. We thereafter examined whether the expression of ST3Gal I, like ST3Gal II, was controlled by testosterone in LNCap cells. In this experiment, the LNCap cells were treated with testosterone and incubated for 120 h. The quantitative real-time PCR analyses showed that testosterone treatment resulted in increased expression of ST3Gal I in LNCap cells (Fig. 5A), although the expression of ST3Gal VI, the sialyltransferase required for the synthesis of sialyl paragloboside, was not induced by testosterone treatment (Figure S4). Furthermore, the testosterone-mediated induction of ST3Gal I in LNCap cells was suppressed by bicalutamide (Fig. 5B). To ensure that there were no androgens present in the cell culture media, LNCap cells were incubated in charcoal-stripped serum for 48 h. The basal level of ST3Gal I was not significantly different between the LNCap cells cultured with 10% FBS and charcoal-stripped serum (Fig. 5C). Then, the LNCap cells were treated with 100 nM testosterone, and the time-course of the changes following testosterone treatment was evaluated. The expression of ST3Gal I increased 72 h after testosterone treatment and remained elevated for more than 120 h in LNCap cells (Fig. 5C). In PC3 and PNT2 cells, no significant increase in the expression of ST3Gal I was detected after testosterone treatment (Figure S5).

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