Limits...
Regulation of the androgen receptor by SET9-mediated methylation.

Gaughan L, Stockley J, Wang N, McCracken SR, Treumann A, Armstrong K, Shaheen F, Watt K, McEwan IJ, Wang C, Pestell RG, Robson CN - Nucleic Acids Res. (2010)

Bottom Line: Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor.Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9.Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes.

View Article: PubMed Central - PubMed

Affiliation: Solid Tumour Target Discovery Group, Northern Institute for Cancer Research, Newcastle University, Paul O'Gorman Building, Newcastle Upon Tyne, NE2 4HH, UK. luke.gaughan@ncl.ac.uk

ABSTRACT
The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.

Show MeSH

Related in: MedlinePlus

SET9 regulates LNCaP cell proliferation, apoptosis and is aberrantly expressed in prostate cancer. (A) WST-1 proliferation assays were conducted in SET9-depleted and non-depleted LNCaP cells grown in steroid-depleted media supplemented with 10 nM DHT for 72 h. Data represents the mean of three independent experiments. (B) LNCaP cells grown in serum-containing media and transiently transfected with non-silencing (N/S), SET9 and/or p53 siRNAs were treated with and without 0.5 µM doxorubicin for 24-h prior to caspase 3 analysis. Data represents the mean of three independent repeats ± standard error (asterisk represents statistical significance <0.05). (C) Representative SET9 staining of prostate tissue using an anti-SET9 antibody in immunohistochemistry (left panel) and table summarizing nuclear and stromal staining pattern of SET9 in cancer and normal tissue (right panel).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045589&req=5

Figure 7: SET9 regulates LNCaP cell proliferation, apoptosis and is aberrantly expressed in prostate cancer. (A) WST-1 proliferation assays were conducted in SET9-depleted and non-depleted LNCaP cells grown in steroid-depleted media supplemented with 10 nM DHT for 72 h. Data represents the mean of three independent experiments. (B) LNCaP cells grown in serum-containing media and transiently transfected with non-silencing (N/S), SET9 and/or p53 siRNAs were treated with and without 0.5 µM doxorubicin for 24-h prior to caspase 3 analysis. Data represents the mean of three independent repeats ± standard error (asterisk represents statistical significance <0.05). (C) Representative SET9 staining of prostate tissue using an anti-SET9 antibody in immunohistochemistry (left panel) and table summarizing nuclear and stromal staining pattern of SET9 in cancer and normal tissue (right panel).

Mentions: Given SET9 is important for AR-mediated PSA expression in LNCaP prostate cancer cells (Figure 4A), we next sought to investigate the role of SET9 in androgen-dependent cell proliferation. Transient transfection of an siRNA against SET9 resulted in a 40% reduction in hormone-dependent LNCaP cell growth (Figure 7A) compared to control cells indicating a pro-proliferative effect of SET9 on the AR signalling cascade. A similar result was achieved using doxycycline-induced SET9 knockdown in LNCaP cells (data not shown). Further analysis revealed that the reduction in LNCaP cell number upon SET9 knockdown was, in part, due to a small, but significant increase in apoptosis as measured by caspase 3 expression (Figure 7B, left panel). This finding was unexpected considering SET9- mouse embryonic fibroblasts (MEFs) demonstrated reduced apoptosis in response to the DNA damaging agent doxorubicin which was found to be the result of attenuated p53 activity (48). To test the effect of DNA damage on cell apoptosis with and without SET9-depletion in our system, LNCaP cells transiently transfected with either SET9 or N/S siRNAs were treated with 0.5 µM doxorubicin for 24-hours prior to caspase 3 expression analysis. As expected, control LNCaP cells showed a robust increase in apoptosis from ∼9–40% in the presence of doxorubicin (Figure 7B, middle panel). Interestingly, doxorubicin-induced apoptosis was enhanced significantly by SET9 knockdown (Figure 7B, middle panel, compare bars 1 and 2) suggesting that SET9 function in LNCaP cells is anti-apoptotic as opposed to pro-apoptotic in MEFs (48) and U2OS cells (Supplementary Figure S9A) [as described in ref. (31)]. Furthermore, although p53 knockdown had no effect on doxorubicin-induced LNCaP cells apoptosis, dual depletion of SET9 and p53 in LNCaP cells abrogated the effects of SET9 knockdown alone (Figure 7B, right panel, compare bars 2 and 4 and Supplementary Figure S9B) indicating that p53 remains functional in the absence of SET9 to facilitate cell death.Figure 7.


Regulation of the androgen receptor by SET9-mediated methylation.

Gaughan L, Stockley J, Wang N, McCracken SR, Treumann A, Armstrong K, Shaheen F, Watt K, McEwan IJ, Wang C, Pestell RG, Robson CN - Nucleic Acids Res. (2010)

SET9 regulates LNCaP cell proliferation, apoptosis and is aberrantly expressed in prostate cancer. (A) WST-1 proliferation assays were conducted in SET9-depleted and non-depleted LNCaP cells grown in steroid-depleted media supplemented with 10 nM DHT for 72 h. Data represents the mean of three independent experiments. (B) LNCaP cells grown in serum-containing media and transiently transfected with non-silencing (N/S), SET9 and/or p53 siRNAs were treated with and without 0.5 µM doxorubicin for 24-h prior to caspase 3 analysis. Data represents the mean of three independent repeats ± standard error (asterisk represents statistical significance <0.05). (C) Representative SET9 staining of prostate tissue using an anti-SET9 antibody in immunohistochemistry (left panel) and table summarizing nuclear and stromal staining pattern of SET9 in cancer and normal tissue (right panel).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045589&req=5

Figure 7: SET9 regulates LNCaP cell proliferation, apoptosis and is aberrantly expressed in prostate cancer. (A) WST-1 proliferation assays were conducted in SET9-depleted and non-depleted LNCaP cells grown in steroid-depleted media supplemented with 10 nM DHT for 72 h. Data represents the mean of three independent experiments. (B) LNCaP cells grown in serum-containing media and transiently transfected with non-silencing (N/S), SET9 and/or p53 siRNAs were treated with and without 0.5 µM doxorubicin for 24-h prior to caspase 3 analysis. Data represents the mean of three independent repeats ± standard error (asterisk represents statistical significance <0.05). (C) Representative SET9 staining of prostate tissue using an anti-SET9 antibody in immunohistochemistry (left panel) and table summarizing nuclear and stromal staining pattern of SET9 in cancer and normal tissue (right panel).
Mentions: Given SET9 is important for AR-mediated PSA expression in LNCaP prostate cancer cells (Figure 4A), we next sought to investigate the role of SET9 in androgen-dependent cell proliferation. Transient transfection of an siRNA against SET9 resulted in a 40% reduction in hormone-dependent LNCaP cell growth (Figure 7A) compared to control cells indicating a pro-proliferative effect of SET9 on the AR signalling cascade. A similar result was achieved using doxycycline-induced SET9 knockdown in LNCaP cells (data not shown). Further analysis revealed that the reduction in LNCaP cell number upon SET9 knockdown was, in part, due to a small, but significant increase in apoptosis as measured by caspase 3 expression (Figure 7B, left panel). This finding was unexpected considering SET9- mouse embryonic fibroblasts (MEFs) demonstrated reduced apoptosis in response to the DNA damaging agent doxorubicin which was found to be the result of attenuated p53 activity (48). To test the effect of DNA damage on cell apoptosis with and without SET9-depletion in our system, LNCaP cells transiently transfected with either SET9 or N/S siRNAs were treated with 0.5 µM doxorubicin for 24-hours prior to caspase 3 expression analysis. As expected, control LNCaP cells showed a robust increase in apoptosis from ∼9–40% in the presence of doxorubicin (Figure 7B, middle panel). Interestingly, doxorubicin-induced apoptosis was enhanced significantly by SET9 knockdown (Figure 7B, middle panel, compare bars 1 and 2) suggesting that SET9 function in LNCaP cells is anti-apoptotic as opposed to pro-apoptotic in MEFs (48) and U2OS cells (Supplementary Figure S9A) [as described in ref. (31)]. Furthermore, although p53 knockdown had no effect on doxorubicin-induced LNCaP cells apoptosis, dual depletion of SET9 and p53 in LNCaP cells abrogated the effects of SET9 knockdown alone (Figure 7B, right panel, compare bars 2 and 4 and Supplementary Figure S9B) indicating that p53 remains functional in the absence of SET9 to facilitate cell death.Figure 7.

Bottom Line: Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor.Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9.Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes.

View Article: PubMed Central - PubMed

Affiliation: Solid Tumour Target Discovery Group, Northern Institute for Cancer Research, Newcastle University, Paul O'Gorman Building, Newcastle Upon Tyne, NE2 4HH, UK. luke.gaughan@ncl.ac.uk

ABSTRACT
The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.

Show MeSH
Related in: MedlinePlus