AR expression and activity within the prostate epithelial hierarchy of differentiation.

<p>A) Representative images of expression of CD133 and AR counterstained with DRAQ5™ within prostate EpCAM^+VE α₂β₁^HI CD133^+VE (Left panel), α₂β₁^HI CD133^–VE (central panel) and α₂β₁^LOW CD133^–VE cells (right panel). <b>B)</b> Expression of the AR regulated genes PSA, KLK2 and TMPRSS2 normalised to GAPDH (n = 10) (p<0.001 comparing α₂β₁^HI and α₂β₁^LOW). Error bars represent standard error of the mean.</p

Expression of the AR within the prostate epithelial hierarchy of differentiation.

<p>Error bars represent standard error of the mean. <b>A)</b> Expression of AR transcript relative to GAPDH. Error bars represent standard error of the mean for n = 10. <b>B)</b> Upper dotplot representative of CD133 staining for progenitor α₂β₁^HI cells. Lower left dotplot representative of AR expression in CD133^–VE gated α₂β₁^HI cells. Lower right dotplot representative of AR expression in CD133^+VE gated α₂β₁^HI cells. Gates were set according to appropriate isotype controls. <b>C)</b> Mean percentage of cells expressing the AR in CD133^+VE and CD133^–VE α₂β₁^HI cells (n = 6). <b>D)</b> Representative histograms for fluorescence of α₂β₁^HI and α₂β₁^LOW isotype controls and AR detection. <b>E)</b> Mean fold change in median staining relative to isotype control for AR stained α₂β₁^HI cells and α₂β₁^LOW cells (n = 6).</p

Strategy of enrichment for required cell types.

<p><b>A)</b> Schematic work flow for enrichment of epithelial cells for assessment of AR expression. <b>B)</b> Purity of selection by expression of the lineage specific markers CD24 (epithelial), CD146 (endothelial) and CD45 (haematopoietic) normalised to GAPDH following real-time PCR for unsorted prostate epithelia and EpCAM/HEA sorted epithelia, error bars represent standard error of the mean for n = 3. <b>C)</b> CD133/1 Sorted samples were assessed for purity by co-expression of the CD133/2 epitope, confirming that these two epitopes are co-expressed in the prostate and that our CD133 selection efficiently enriches for CD133^+VE cells: Upper dotplot representative of CD133/2 staining for unsorted α₂β₁^HI epithelial cells; lower left dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^–VE cells; lower right dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^+VE cells. Gates are set according to appropriate isotype controls. <b>D)</b> Confirmation of CD133 enrichment with real-time PCR. CD133 expression is shown normalised to GAPDH, error bars represent standard error of the mean n = 10.</p

Validation of AR detection with flow cytometry.

<p><b>A)</b> Percentage of cells staining above a no antibody control for either isotype antibody (hollow points) or PG-21 AR antibody (solid points) in LNCaP (circles) or PC3 (triangles) across a dilution series. <b>B)</b> Representative staining patterns for PC3 (upper dotplots) and LNCaP (lower dotplots) for either 1∶200 isotype antibody (left dotplots) or 1∶200 PG-21 AR antibody (right dotplots) of equivalent concentrations. Gates set according to isotype control. <b>C)</b> Left dotplot representative of staining of LNCaP with isotype control, right dotplots representative of AR staining in non-transfected LNCaP (upper), LNCaP transfected with scrambled siRNA (middle dotplot) and LNCaP transfected with AR siRNA (lower). Gates were set according to an appropriate isotype control. <b>D)</b> Percentage of cells staining positive for AR relative to an isotype control in non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA. Error bars represent standard error of the mean for n = 3. <b>E)</b> Western blots of AR expression for non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA are shown using a different AR antibody (G122-434, BD Pharmingen).</p

Characterisation of a Tip60 Specific Inhibitor, NU9056, in Prostate Cancer

<div><p>Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an <em>in vitro</em> HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC₅₀ 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8–27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer.</p> </div

NU9056 reduces PSA and p53 protein levels.

<p>To confirm the effects of Tip60 on androgen receptor activity we used 2.5 nM siRNA specifically targeted against Tip60 in LNCaP cells, or non-silencing control. Knockdown was achieved after 48 hours in steroid depleted medium after which time 10 nM DHT was applied to induce androgen receptor activity and PSA expression. RNA was collected after 24 hours DHT stimulation, reverse transcription and real-time PCR performed. Expression of (A) PSA and (B) Tip60 was normalised relative to HPRT1 expression. (C) LNCaP cells were treated with 24 µM NU9056 over 48 hours and protein samples were collected in SDS sample buffer. Protein analysis was carried out by SDS PAGE and Western blotting for p53, p21, AR, PSA and alpha tubulin. (D) Densitometry was performed on Western blots. All experiments were performed twice and the mean ± standard deviation is shown.</p

IC₅₀ values of 1,2-bis(isothiazol-5-yl)disulfane (7) and related compounds towards HATs.

<p>To assess the activity against Tip60 HAT, <i>in vitro</i> HAT assays using ³H acetyl-CoA were carried out using histones as substrates. Assays were performed in quadruplicate and repeated twice. Individual IC₅₀ values are presented.</p

Table of Tip60 IC₅₀ values of isothiazoles and related compounds.

<p>To assess the activity against Tip60 HAT, <i>in vitro</i> HAT assays using ³H acetyl-CoA were carried out using histones as substrates. Assays were performed in quadruplicate and repeated twice. For compounds producing >50% inhibition at 100 μM, IC₅₀ values were calculated. Individual IC₅₀ values are presented. For other compounds the % inhibition at 100 μM is presented.</p

Chemical synthesis of Tip60 inhibitors.

<p>Part 1 - Synthesis of compounds <b>4–7</b>. Part 2 - Synthesis of Compounds <b>1</b> and <b>11</b>.</p

NU9056 inhibits protein acetylation in prostate cancer cell lines.

<p>LNCaP cells were treated with increasing concentrations of NU9056 or the control compound, 1,2 bis(4-pyridyl)-ethane for 24 hours. (A) Levels of Tip60 were assessed by Western blotting. (B) LNCaP cells were treated with 2 µM TSA for 6 hours and levels of histone H4 acetylated-lysine 16, histone H4 acetylated-lysine 8 and histone H3 acetylated lysine 14 were assessed by Western blotting. LNCaP cells were treated with increasing concentrations of NU9056 or the control compound for 2 hours, then treated with the HDAC inhibitor TSA (2 µM) for a further 4 hours. (C) Levels of histone H4 acetylated-lysine 16, histone H4 acetylated-lysine 8 and histone H3 acetylated lysine 14 were assessed by Western blotting. (D) LNCaP cells were treated with 24 µM NU9056 over 4 days and the levels of acetylated tubulin were assessed by Western blotting. (E) LNCaP cells were treated with increasing concentrations of NU9056 or the control compound for 2 hours, then treated with the HDAC inhibitor TSA (2 µM) for a further 4 hours. Levels of acetylated tubulin were assessed by Western blotting. Alpha-tubulin was used as a loading control. Representative blots are shown for duplicate experiments.</p