Modeling Truncated AR Expression in a Natural Androgen Responsive Environment and Identification of RHOB as a Direct Transcriptional Target

<div><p>Recent studies identifying putative truncated androgen receptor isoforms with ligand-independent activity have shed new light on the acquisition of androgen depletion independent (ADI) growth of prostate cancer. In this study, we present a model system in which a C-terminally truncated variant of androgen receptor (TC-AR) is inducibly expressed in LNCaP, an androgen-dependent cell line, which expresses little truncated receptor. We observed that when TC-AR is overexpressed, the endogenous full length receptor (FL-AR) is transcriptionally downmodulated. This in essence allows us to “replace” FL-AR with TC-AR and compare their individual properties in exactly the same genetic and cellular background, which has not been performed before. We show that the TC-AR translocates to the nucleus, activates transcription of AR target genes in the absence of DHT and is sufficient to confer ADI growth to the normally androgen dependent LNCaP line. We also show that while there is significant overlap in the genes regulated by FL- and TC-AR there are also differences in the respective suites of target genes with each AR form regulating genes that the other does not. Among the genes uniquely activated by TC-AR is RHOB which is shown to be involved in the increased migration and morphological changes observed in LN/TC-AR, suggesting a role of RHOB in the regulation of androgen-independent behavior of prostate cancer cells.</p> </div

Knockdown of RHOB affects cell shape and cell migration following overexpression of TC-AR in LN/TC-AR/shR-RHOB.

<p><b>A</b> LN/TC-AR cells were pre-cultured in hormone-depleted media for 48 hours then treated with Low Dox, High Dox, 1 nM DHT or vehicle for 24 hours. Whole cell lysates were harvested and subjected to western blot analysis. Membranes were probed with α-RHOB or α-actin. <b>B</b> Graphic representation of TC-AR binding sites 100 kb within the region of adjacent to RHOB gene. Arrows above show TC-AR binding sites (+3.8 kb and +47.5 kb downstream of TES). <b>C</b> Androgen-deprived LN/TC-AR/shR-empty and LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle only. Whole cell lysates were harvested 48-hours post-treatment and subjected to western blot analysis. Membranes were probe with either α-RHOB (top) or α-actin (bottom). <b>D</b> Androgen-deprived LN/TC-AR/shR-RHOB cells were treated with 1 nM DHT, Low Dox, High Dox or vehicle. Images were acquired 48-hours post-treatment. <b>E</b> LN/TC-AR/shR-RHOB cells were pre-cultured in serum free media (SFM) for 24 hours then seeded to migration chambers with various treatments in the presence of SFM for an additional 48 hours after which time fluorescence was detected. Fold induction is relative to untreated control. Prior results for LN/TC-AR (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049887#pone-0049887-g003" target="_blank">Figure 3B</a>) are included for comparison. <b>F</b> MTT assay of LN/TC-AR/shR-RHOB in hormone depleted media following treatment with 1 nM DHT, Low Dox, High Dox or vehicle as control showing that DHT independent growth previously shown for LN/TC-AR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049887#pone-0049887-g002" target="_blank">Figure 2D</a>) is not inhibited by knockdown of RHOB. Bright field images were acquired with an Olympus microscope using 20× magnification.</p

Expression of FLAG-tagged TC-AR in LN/TC-AR cell line suppresses endogenous AR at both transcriptional and translational levels.

<p><b>A</b> Schematic structure of prototypic AR truncated variants. (i) Two truncated AR variants used in our study. (ii) AR splice variants from Guo et al 2009 and Hu et al 2009. (iii) AR splice variants from Dehm et al 2008. (iv) AR splice variant from Sun et al 2010. Numbers represent exon numbers. 2b, 3b, CE1, and CE3 represent cryptic exons. The length of translated sequence by cryptic exons is shown at the right. <b>B</b> Membrane was probed with α-FLAG M2. Right lane contains 100 µg whole cell lysate harvested from LN/TC-AR cells induced with 100 ng/mL doxycycline for 48 hours. Left lane contains 100 µg whole cell lysate harvested from uninduced LN/TC-AR. <b>C</b> LNCaP (LN) and CWR22R<i>v1</i> (CWR) lysates serve as control for LN/TC-AR cells which were treated with various concentrations of doxycycline. All lanes contain 100 µg whole cell lysate per lane and the membrane was probed with α-AR(PG-21). <b>D</b> LN/TC-AR cells were treated with either Low Dox or High Dox. At designated time points, RNA was isolated and quantitative RT-PCR was performed to determine mRNA levels of endogenous AR and TC-AR. Fold induction is relative to uninduced LN/TC-AR control. <b>E</b> Co-immunoprecipitation assay reveals that in LN/TC-AR cells induced with 4 ng/mL doxycycline (Lanes 3&4), TC-AR, but not FL-AR, is precipitated by IP with α-FLAG agarose beads (Lanes 4) indicating that the two AR forms do not heterodimerize. As controls, identically prepared/immunoprecipitated uninduced LN/TC-AR cell lysate is shown in Lanes 1 (WCL) and 2 (IP).</p

TC-AR is transcriptionally active in the absence of DHT and confers ADI growth <i>in vitro</i>.

<p><b>A</b> Luciferase assay showing DHT-independent transcription of a transiently transfected AR regulated promoter following induction of TC-AR in LN/TC-AR. LN/ TC-AR cells were co-transfected with pPSA6.0-luc and pH 48-ren in hormone depleted media and treated with either low concentrations of doxycycline, DHT 1 nM or vehicle as control for 24 hours. Fold induction is relative to untreated control. <b>B</b> Immunostaining of LN/TC-AR shows androgen independent nuclear localization of TC-AR (right). Cells were counterstained with DAPI to identify nuclei (left) and images were acquired with an Olympus fluorescent microscope using 20× magnification. <b>C</b> Chromatin Immunoprecipitation (ChIP) showed the recruitment of AR and RNA polymerase II to the <i>KLK3</i> promoter. LN/TC-AR cells were pre-cultured in androgen depleted medium for 72 hours then treated with Low Dox for 24 hours. Anti-FLAG M2 (for FLAG-tagged TC-AR) and α-RNAP2 antibody were used in separate aliquots to immunoprecipitate cross-linked protein and DNA. <b>D</b> Cell count assay showing the growth of LN/TC-AR in hormone depleted media following treatment with 1 nM DHT, Low Dox, High Dox or vehicle as control.</p

Cell shape and motility change of LN/TC-AR under different dox treatments.

<p><b>A</b> LN/TC-AR cells were grown in the presence of hormone depleted media and treated with various concentrations of doxycycline or 1 nM DHT. CWR22R<i>v1</i> cells were grown in RPMI supplemented with 10% FBS. At 48-hours post-treatment representative images of each sample group were acquired. <b>B</b> LN/TC-AR cells were pre-cultured in serum free media (SFM) for 24 hours then seeded to migration chambers with various treatments in the presence of SFM for an additional 48 hours after which time fluorescence was detected. Fold induction is relative to untreated control.</p

STUbL-like function is important for K-Rta transactivation activity.

<p>Reporter assays were performed in 293 cells using the indicated K-Rta target gene reporters. Reporter plasmids were cotransfected with K-Rta Wt or mutants, and luciferase activity was measured at 48 hours post-transfection. Luciferase activity of reporter with empty expression plasmids was normalized to a value of 1. Fold activation over control is shown.</p

K-Rta Wt but not Ring-finger domain mutant can degrade SUMO-modified proteins.

<p>(<b>A</b>) Flag-SUMO and K-Rta wild type or mutant were cotransfected into 293T cells, and probed with indicated antibodies. The K-Rta Ring-like domain (C141, H145) is important for SUMO degradation. (<b>B</b>) MG132 recovered SUMO proteins from degradation. 24 hours after the K-Rta transfection, MG132 or DMSO (vehicle) was added into the culture media and cells were harvested after 12 hours of treatment. SUMO-modified proteins were probed with anti-Flag antibody. (<b>C</b>) SUMO degradation during KSHV reactivation in BCBL-1. After induction of K-Rta expression in TREx-K-Rta-BCBL-1 with doxycycline (Dox), SUMO-modified proteins were probed with anti-Flag antibody. K-Rta induction was confirmed with an anti-K-Rta antibody and GAPDH was served as the loading control. (<b>D</b>) Recovery of SUMO-modified proteins with MG132 in BCBL-1. KSHV reactivation was triggered by induction of K-Rta in BCBL-1 cells in either the presence or absence of MG132. The amount of SUMO-modified proteins was examined by immunoblotting with an anti-Flag antibody. The accumulation of SUMO-modified conjugates was evident by increments of higher molecular weight entities.</p

Immunofluorescence analysis (IFA).

<p><b> (A)</b> IFA was performed with anti-PML (Green) and anti-K-Rta (Red) antibodies. Arrows indicate cells showing overexpression of K-Rta. <b>(B)</b> K-Rta degrades PML. PML wild type or PML del-SUMO mutant was cotransfected with K-Rta or K-Rta mutants. K-Rta wild type preferentially degrades PML wild type, which can be modified by SUMO in vivo. K-Rta Ring mutant (H145L) or SUMO-binding mutant (<i>Δ</i>SIM) was not able to degrade PML. <b>(C)</b> Endogenous SUMO-modified proteins. K-Rta expression was induced by addition of doxycycline (Dox). Cell lysates were prepared 48 hours post-induction. Indicated proteins were probed with specific antibodies and 25 µg of total protein was loaded in each lane. GAPDH was used as a loading control.</p

K-Rta physically associates with SUMO.

<p>(<b>A</b>) (a) Recombinant GST and GST-SUMO were purified from an <i>E. coli</i> expression system, and GST-SUMO chains were generated <i>in vitro</i>. Flag-K-Rta protein was purified from recombinant baculovirus infected Sf9 cells (right panel). (b)The GST-Pull down assay was performed by incubating GST, GST-SUMO-1, -SUMO-2, -SUMO-3, -SUMO-2 chain or –SUMO-3 chain with purified Flag-K-Rta. Association was detected by immunoblotting with anti-Flag antibody. (<b>B</b>) Mapping of SUMO-binding domain. The indicated K-Rta deletion protein was incubated with SUMO-2 chains, and the interaction was probed with an anti-SUMO monoclonal antibody (upper panel). The membrane was also stained with Ponceau to show amount of GST-K-Rta deletion protein on the membrane (bottom panel). Asterisks showed non-specific interaction between C-terminal K-Rta and IgG. (<b>C</b>) Generation of K-RtaΔSIM. (a) Multiple alignments between KSHV Rta and RRV Rta. Conserved hydrophobic clusters (putative SIM) are marked in bold letters. Mutations that showed decreased SUMO degradation are underlined. (b) Mutations introduced in this study are shown. (c) SUMO degradation by K-Rta mutants. Immunoblotting was performed after the transfection of indicated K-Rta mutant plasmids along with SUMO expression vector in 293T cells. Mutations at SIM-3, -4, or -5 impaired K-Rta SUMO degradation function. (d) K-Rta Wt but not K-RtaΔSIM degrades SUMO modified proteins. Increasing amounts of K-Rta Wt or K-RtaΔSIM3&5 (0.25, 0.5, or 1.0 ug) were cotransfected with SUMO. The SIM mutation completely abolished the SUMO degradation function of K-Rta. (<b>D</b>) (a) GST pull-down analyses with purified K-Rta Wt or K-Rta ΔSIM mutants are shown. The interaction was probed with anti-Flag antibody. (b) Ponceau staining shows the amount and purity of GST- or GST-SUMO proteins used in the assay.</p

Sumo inhibits KSHV gene expression and reactivation.

<p><b>(A)</b> A dual-inducible cell line was generated with the TREx FRT recombination system. Indicated protein was probed with specific antibodies <b>(B)</b>, and gene expression was examined by northern blotting with probes to the coding regions of K-bZIP or ORF65 <b>(C)</b>. Ubc9 wild type decreased gene expression; however, the Ubc9 mutant increased viral gene expression compared to control cells. <b>(D)</b> SUMO inhibits viral reactivation. Indicated plasmids were cotransfected into recombinant KSHV infected Vero cells. (a) RFP positive cells were counted 72 hours after transfection in five randomly selected fields. (b) Average of RFP positive cells are shown. The total amount of plasmid/well was kept at 2 micrograms by adding control vector DNA. (c) Viral copy number after transfection of K-Rta or mutants into recombinant KSHV infected Vero cells is shown. Supernatants were collected 5 days post-transfection and encapsidated viral DNA was measured by qt-PCR (left panel). K-Rta expression was confirmed by immunoblotting (right panel).</p