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

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

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

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

WST-1 proliferation assay of LNCaP cells (<i>A</i>) and 22R<i>v</i>1 cells (<i>B</i>).

<p>Cells were transfected with 50 nM of miR-125bm or 50 nM of miRNA negative control (miR-NC) for 5 days. Cell proliferation was measured by WST-1 assay. <i>p14^ARF</i> siRNA (sip14) was used as a control. The results are expressed as proliferation relative to that of miR-NC-treated cells, and shown as mean ± SD (n = 4).</p

<i>MiR-125b</i> down-regulates p14^ARF in CaP cells.

<p><i>A</i>) Western blot analysis of expression levels of p14^ARF in LNCaP (<i>top</i>) and 22Rv1 cells (<i>bottom</i>). Cells grown in 10% FBS media were transfected with 50 nM of miR-125bm or anti-<i>miR-125b</i> (anti-125b) for 72 hrs or treated with 5.0 nM of R1881 androgen for 48 hrs. Then, 50 µg of protein per sample was analyzed. Both miR-negative control (miR-NC) and anti-miR negative control (anti-NC) were used as controls, and β-actin was used as a loading control. <i>B</i>) Western blot analysis of expression levels of p14^ARF, mdm2 and p53 in lenti-<i>miR-125b</i>-overexpressed PC-346C xenograft tumor. Both untreated xenograft (untreat.) and lenti-miRNA control vector-infected PC-346C xenograft (vector) were used as controls. In both <i>A</i> and <i>B</i>, the numbers under the gels are the average fold changes of p14^ARF protein from three independent gels relative to the corresponding controls. Fold changes were calculated by scanning the p14^ARF bands and normalizing for β-actin bands. <i>C</i>) Luciferase assay of <i>miR-125b</i> binding to the 3′-UTR of <i>p14^ARF</i> mRNA in LNCaP cells. The assay was repeated three times with each assay being performed in three wells and similar results were obtained each time. The representative results are shown as a mean ±SD (n = 3).</p

Schematic model of <i>miR-125b</i>-controlled oncopathway in CaP cells.

<p>In CaP cancer cells, p14^ARF facilitates apoptosis in a p53-dependent (<i>left</i>) and p53-independent (<i>right</i>) manner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061064#pone.0061064-Muer1" target="_blank">[30]</a>. Since <i>miR-125b</i> directly targets p14^ARF and other pro-apoptotic molecules, deregulation of <i>miR-125b</i> can modulate proliferation and apoptosis in both p53-positive and p53-deficient CaP cells. Black arrows represent upregulated molecules and white arrows represent downregulated molecules. Broken arrow indicates undefined upregulation of Bak1 activity by p14^ARF.</p

<i>MiR-125b</i> regulates the p53 network.

<p><i>A</i>) Western blot analysis of Mdm2 and p53 in miR-125bm-treated LNCaP (<i>top</i>) and 22R<i>v</i>1 cells (<i>bottom</i>). Cells were transfected with 50 nM of miR-125bm or miR-negative control (miR-NC) for 72 hrs. Equal amounts of protein (50 µg) were used to detect the expression levels of Mdm2, p53, p21 and Puma. <i>B</i>) Western blot analysis of p14^ARF, Mdm2 and p53 in <i>p14^ARF</i> siRNA (sip14)-treated LNCaP (<i>top</i>) and 22R<i>v</i>1 cells (<i>bottom</i>). Cells were treated with sip14 and the cellular levels of p14^ARF, p53 and Mdm2 were analyzed. β-actin was used as a loading control. <i>C</i>) Co-immunoprecipitation analysis of protein interaction between p14^ARF and Mdm2 in 22R<i>v</i>1 cells. Cells were transfected with miR-125bm and 1.0 mg protein was immunoprecipitated with anti-p14^ARF antibody or the rabbit IgG. The resultant immunecomplexes were used to detect the level of Mdm2 by Western blot analysis using anti-Mdm2 antibody. Input: 50 µg protein from total cell lysate. IP: immunoprecipitation. IB: immunoblotting.</p

Inactivation of <i>miR-125b</i> induces apoptosis in p53-positive CaP cells.

<p><i>A</i>) Detection of SMAC and activated caspase 3 (Cas-3) in LNCaP (<i>left</i>) and 22R<i>v</i>1 (<i>right</i>) cells. Cells were transfected with 50 nM miR-125bm or 50 nM anti-<i>miR-125b</i> (anti-125b) for 5 days, and the levels of SMAC and Cas-3 were measured by Western blot analysis. β-actin was used as loading control. The numbers under the gels are the average fold changes of SMAC and Cas-3 from three independent gels relative to the corresponding controls. <i>B</i>) Detection of anti-<i>miR-125b</i>-induced apoptosis in 22R<i>v</i>1 cells. Cells were transfected using 50 nM anti-<i>miR-125b</i> for 72 hrs and apoptotic cell death was detected using TUNEL assay. The green nuclear fluorescence indicates the apoptotic cleavage of nuclear DNA (<i>left</i>). For quantitation of apoptotic cell death, 400 cells were counted and apoptosis is expressed as % apoptosis (apoptotic cells/400×100%). Quantitative analysis was performed three times and result was expressed as mean ± SE (n = 3) (<i>right</i>). Cells treated with irradiation (IR, 6 Gy) were used as a positive control. <i>C</i>) TUNEL assay of apoptotic death of 22R<i>v</i>1 cells that were treated with anti-<i>miR-125b</i> followed by <i>p14^ARF</i> antisense (sip14). Result was expressed as mean ± SE (n = 3). <i>D</i>) Western blot analyses of p14^ARF, p53 and Bak1 levels in 22R<i>v</i>1 cells. <i>Left</i>: 22R<i>v</i>1 cells were transfected with anti-<i>miR-125</i>; <i>right</i>: anti-<i>miR-125</i>-transfected 22R<i>v</i>1 cells were treated with sip14. Both anti-miR-NC (anti-NC) and scramble siRNA were used as controls.</p