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SUPPLEMENTAL METHODS
Gene Expression Profiling
For gene expression profiling of Abl cells expressing, under a tetracycline-inducible promoter,
SPOP-WT or SPOP mutants F102C, F133V, F133L (or control vector), the cells were treated
with doxycycline (200 ng/ml) for 48 hours. Total RNA was extracted using Trizol (Life
Technologies) and purified with the RNeasy Mini Kit (Qiagen) following the manufacturer’s
instructions. RNA was reverse transcribed and the microarray hybridization was performed using
the Illumina Gene Expression Sentrix BeadChip HumanHT-12_V4 (Illumina) at the Laboratory
for Translational Genomics at Baylor College of Medicine. Microarray scanned images were
imported to Illumina® GenomeStudio for data quality control and the transcriptome profile data
was quantile-normalized by the Bioconductor lumi package (1). Differences in gene expression
of these samples (SPOP-F102C, SPOP-F133L or SPOP-F133V vs SPOP-WT or control vector)
were inferred utilizing the Bioconductor limma package (2) (p<0.05) and imposing a fold change
exceeding 4/3x. For hierarchical clustering, gene expression of all SPOP-regulated genes was
transformed to z-scores (with respect to the Control Vector values), using the R statistical
system.
Genesets of AR- or SRC3-dependent genes in PC were determined by treating LNCaP cells with
Silencer Select predesigned siRNA (Life Technologies) for AR or SRC-3 or non-target (NT)
control for 48 hours, respectively, at a final concentration of 50nM, using Lipofectamine
RNAiMAX (Life Technologies) according to the manufacturer’s instructions. RNA was
extracted as above and analyzed on Affymetrix Human Exon 1.0 ST Array platform at the
Genomic and RNA Profiling (G.A.R.P.) Shared Resource at Baylor College of Medicine. Gene
expression differences were inferred utilizing the t-test and imposing a fold change exceeding
4/3x (p<0.05), using the R statistical system.
Androgen- and Drug-induced signatures in PC cells
Previously published signatures of PC cells transcriptional response to several agents were
derived from the Gene Expression Omnibus (GEO): androgen (3-4); direct AR antagonists:
enzalutamide (GSE50661 (5), GSE44924 (6), GSE44905 (7)), ARN-509 (GSE51873 (8)),
bicalutamide (GSE7708 (9) and GSE8533); other agents reported to have anti-AR activity:
compound 30 (GSE32892 (10)), celastrol and gedunin (GSE5505 (3)); HDAC inhibitor (TSA:
GSE20433 (11), GSE9000 (12)); EZH2 inhibition via siRNA (GSE20433 (11)); roscovitine
(GSE20433 (11)); peruvoside, digoxin, strophanthidin (GSE35126 (13)); HTI-286 and docetaxel
(GSE8325 (14)); N-butylidenephthalide (BP, GSE33883); zebularine (GSE51629); and
clorgyline (GSE19822 (15)). Each drug signature was partitioned into up- and down-regulated
genes.
Gene Set Enrichment Analysis
Gene Set Enrichment Analysis (GSEA) was carried out using the GSEA software package (16)
to assess the degree of similarity among the studied gene signatures. To define the signature of
each mutant SPOP (F102C, F133V, F133L, and for all three mutants combined), genes were
ranked by the fold change between the mutant and the WT SPOP samples. We examined the
SPOP mutant signatures in an unbiased fashion against the entire Molecular Signatures Database
(MSigDB, http://www.broadinstitute.org/gsea/msigdb/), containing 10,295 gene signatures (as of
6/8/2014); we used adjusted q<0.05 as our filtering criteria. For the androgen response signatures
(3-4), we utilized the up-regulated genes (induced by androgen). For the gene signature derived
from AR and SRC-3 siRNAs, we utilized the down-regulated genes (positively regulated by AR
and SRC-3, respectively). Normalized Enrichment Score (NES) and adjusted q-values were
computed utilizing the GSEA method, based on 1000 random permutations of the ranked genes.
Comparison of AR activity scores in PC cells in vitro
Utilizing the gene expression profiles obtained from Abl cells transfected with control vector
(CV), wt-SPOP, and mutant-SPOPs and treated with doxycycline (200 ng/ml) for 48 hours, we
calculated the expression of each gene as a respective z-score for each sample, relative to the
average expression of that gene in the cells transfected with the control vector. For each of the
genes in two published androgen-induced signatures (Nelson et al. (4) and Hieronymus et al.
(3)), we computed the sum z-score for each sample (the z-scores of downregulated genes were
subtracted from the z-scores of upregulated genes), as described previously (17), which
represents the AR activity scores for that sample.
Comparison of SPOP signature score vs AR activity score in PC patient cohorts
We downloaded gene expression datasets from multiple previously reported human PC specimen
cohorts: Cai et al. (18), Taylor et al. (17), Grasso et al. (19), and the Cancer Genome Atlas
(TCGA) (https://tcga-data.nci.nih.gov/tcga/). Within each dataset, we utilized the expression of
each gene to calculate its respective z-score for each sample, relative to the normal prostate gland
specimens available in that cohort. For each SPOP gene signature and for each of two published
androgen-induced signatures (Nelson et al. (4) and Hieronymus et al. (3)), we computed the sum
z-score for each sample (the z-scores of downregulated genes were subtracted from the z-scores
of upregulated genes), as described previously (17). Finally, for each pair of signatures we
plotted the cumulative z-scores on the x and y axis, and computed the Pearson Correlation
Coefficient R and associated p-value using the R statistical system.
Plasmid Constructs
AR constructs: To construct a pcDNA3-AR-Flag expression vector, the AR coding cDNA
sequence (with 2xFlag in-frame fusion at the C-terminal of the AR protein, introduced by PCR
primers) was amplified by PCR from the previously reported pCMV5-AR plasmid (20). The
primer sequences are shown in Suppl. Table 1. The PCR product was digested with restriction
enzyme BglII and XbaI, resolved on 1% agarose gel, recovered and ligated into pcDNA3 vector
that was digested by BamHI/XbaI. The resulting construct was confirmed by DNA sequencing.
The pcDNA3-ARv7-Flag vector was constructed by replacing the Bsu36i/XbaI sequence of
pcDNA3-AR with Bsu36i/XbaI digested, PCR amplified Flag-tagged C-terminal coding
sequence of ARv7.
To generate expression vectors for mutated AR protein variants at the SPOP Binding Consensus
(SBC) motif (AR-A646D, AR-S647F, AR-S648N and AR-STT648/649/650AAA), in vitro
mutagenesis was performed as previously described (20). The PCR-amplified AR mutant cDNAs
were digested with Bsu36i/EcoR1 and inserted into pcDNA3-AR-Flag vector (digested by the
same restriction enzyme pair) to generate pcDNA3-AR-A646D-Flag, -AR-S647F-Flag, -ARS648N-Flag and –AR-S648A/T649A/T650A-Flag expression vectors.
SPOP constructs: The pcDNA3.1-HA-SPOPwt construct was digested by MscI/EcoRV. The
sequence corresponding to HA-SPOP(aa1-aa172) was religated to give pcDNA3.1-HASPOPaa1-aa172 (SPOP N-terminal MATH domain expression vector). A PCR primer containing
a translation start codon (ATG) in-frame with a HA-tag and the SPOP coding sequence after
aa172 was designed to amplify the HA-SPOP(aa173-aa374) coding sequence (containing the
BTB domain). The PCR-amplified fragment was digested by BamHI/XhoI, and inserted into
pcDNA3.1 vector digested by the same pair of enzymes to generate pcDNA3.1-HASPOP(aa173-aa374). The sequences of PCR primers used are listed in Suppl. Table 1. The
resulting plasmid constructs were confirmed by DNA sequencing. The expression vectors,
human SPOP (SPOPwt) and its mutation variants inserted in pcDNA3.1 vector (pcDNA3.1-HASPOP-Y87C, -SPOP-Y87N, -SPOP-F102C, -SPOP-S119N, -SPOP-F125V, -SPOP-W131G, SPOP-F133V and –SPOP-F133L), were described previously (20). The pSG5-HA-Ub (HAtagged human ubiquitin expression vector) and the mammalian expression vector for dominantnegative inhibitor of cullin 3 (pcDNA3-Flag-CUL3 DN), were obtained from Addgene
(Cambridge, MA).
Protein half-life determination analysis
The intracellular stability of AR protein in Abl PC cells was estimated by protein half-life assay.
Briefly, the cells were treated with 100 μg/ml cycloheximide (CHX) and collected at different
time points, as indicated. The collected cells (normalized to same number of cells) were washed
by PBS and lysed in 1x RIPA buffer (Cell Signaling Technology, Danvers, MA) containing
phosphatase inhibitors and protease inhibitors (F. Hoffmann-La Roche Ltd). The same volume of
the lysate (corresponding to same cell number) from each time point was mixed with SDS-PAGE
loading buffer and separated by SDS-PAGE. Immunoblot analyses were conducted for the
expression levels of AR protein in the cell lysates, as previously described (20). The volume
density of protein bands on the membrane were imaged and quantified by QuantitiOne software
(Bio-Rad) on a Molecular Imager® VersaDoc™ MP 4000 System (Bio-Rad). The results for
each timepoint were plotted as % of the corresponding control (0 timepoint).
Transient Transfection of Expression Vectors in Mammalian Cells
293T cells or HeLa cells were transfected with expression vectors using Superfect transfection
reagent (Qiagen) following the manufacturer’s instructions. Typically, for a 6-well plate, 3 μg of
DNA/well was transfected; and for a 10-cm plate 20 μg of DNA were utilized. Forty-eight hours
post-transfection, the cells were harvested and washed with PBS. Total RNA or total cell lysates
were prepared as previously described (20), and used for immunoprecipitation, qRT-PCR, or
immunoblot analyses.
Protein Co-Immunoprecipitation (Co-IP) Analysis
To conduct co-IP experiments in PC cell lines, the cells were plated with or without treatment, as
needed in the corresponding experiments. The cells were collected, washed by PBS and lysed in
NP-40 lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The
cell lysates were used in co-IP analysis as we previously described (20). For in vitro analysis of
protein-protein interactions in 293T and HeLa cells, the cells were co-transfected with combined
mix of SPOP (WT or mutant) expression vectors (or control vector) and expression vectors
containing the cDNA of interest, respectively. In select experiments, the proteasome inhibitor
bortezomib (PS341, 250 nM) was added for the last 8 hours of transfection, as indicated. The cell
lysates were incubated with antibodies for co-IP assay. The immuno-complexes were
precipitated using protein G-Dynabeads, eluted by 1xSDS loading buffer and separated by SDSPAGE.
Immunoblotting analyses
After SDS-PAGE, the proteins were transferred to nitrocellulose membrane and detected by
immunoblotting using monoclonal antibodies or specific anti-sera, as indicated. Blots were
washed with 1X PBST and incubated with Anti-mouse or anti-rabbit HRP-conjugated secondary
antibodies for 1 hour. Protein signals were detected with SuperSignal West chemiluminescent
substrate (Thermo Scientific, inc., Waltham, MA) and developed with x-ray films according to
the manufacturer’s instructions (Thermo Scientific, Inc) followed by densitometric analysis
using NIH ImageJ image analysis software (21), or on a Molecular Imager® VersaDoc™ MP
4000 System (Bio-Rad) using QuantitiOne software (Bio-Rad) for quantitative analysis of the
band intensity.
Real-time Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA was prepared from cultured cells using RNeasy Mini Kit (Qiagen) following the
manufacturer’s instructions. Total RNA was quantified and reverse transcribed. The resulting
cDNA was combined with sensiFAST SYBR No ROX kit (Bioline, Taunton, MA) and primer
pairs to detect specific transcripts (AR-dependent genes: SGK1, CAMKK2, ABCC4, HOMER2,
SEPP1 and NKX3.1, Suppl. Table 1) by Sybr Green Realtime PCR. For RT-qPCR analysis of
18S and AR transcripts, a TaqMan® One-Step RT-PCR Master Mix Kit (Life Technologies) was
used. Taqman assays for eukaryotic 18S rRNA (endogenous control) and for AR mRNA
quantification (hs0090244) were obtained from Life Technologies.
The RT-qPCRs were performed on a StepOne Plus Realtime PCR System (Life Technologies),
and amplification data were processed as previously described (20). Values were normalized to
total RNA or 18S rRNA, as indicated. Each experiment was repeated at least three times, and the
results were analyzed for statistical significance. Differences in transcript levels between samples
(e.g. with vs without doxycycline treatment) were analyzed using t test and considered
significant if P value <0.05.
Xenograft studies of human PC cells
We examined the impact of mt-SPOP on the growth of PC cells, using Abl cells engineered to
express, under a tetracycline-inducible promoter, wt-SPOP or SPOP-F102C. Two million cells
per mouse (mixed with Matrigel at a volume ratio of 1:1) were injected sc in the flank of SCIDBeige male mice (n=5 per group). The mice were fed with water containing doxycycline (200
µg/ml) starting on the next day after cell injection, and kept on doxycycline afterwards, in order
to induce expression of WT and mt-SPOPs, respectively. The size of the tumors was compared
between wt-SPOP and SPOP-F102C tumors by unpaired t-test. All animal studies were
conducted in accordance with institutional guidelines. All procedures were approved by the
BCM Institutional Animal Care and Use Committee (IACUC) that is accredited by the
Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
SPOP knockout model
Spop Knockout-First-Reporter Tagged Insertion mice (Spoptm1a(KOMP)Wtsi) were obtained
from the International Knockout Mouse Phenotyping Consortium (KOMP, (22)). In this mutant
mouse line, a Knockout-First-Reporter Tagged Insertion containing the β-galactosidase (β-gal,
LacZ) gene has been engineered into the Spop locus after exon 3, interrupting the expression of
the full-length SPOP and resulting in a non-expressive allele. For confirmation, we documented
that the prostates of the male hemizygotes express approximately ~50% the amount of Spop
mRNA and protein compared to wild-type mice. As SPOP mRNA levels in metastatic human
PCs are, on average, ~50% of normal prostate levels, these hemizygote male mice already
recapitulate the SPOP status of many metastatic SPOP WT human PCs. Moreover, this model is
also relevant to human SPOP-mt PC, because human SPOP mutations always occur in
heterozygote fashion. Immunoblot analyses were conducted for SPOP, AR and β-actin in the
prostates of nine-month old wild-type and Spop hemizygous mice.
PC Cell Proliferation Analysis in vitro
In vitro analysis of PC cells proliferation was conducted by counting viable cells. Briefly, 50,000
cells per well were plated in 6-well plates in culture media supplemented with regular Fetal
Bovine Serum (FBS) or Charcoal-Stripped Fetal Bovine Serum (CSS, to determine PC cell
proliferation in the absence of hormone). All media were supplemented with doxycycline (50
ng/ml in all wells) and replenished every 48 hrs with fresh medium, serum and antibiotics. The
cells were collected and counted for the number of total viable cells (per well) via trypan blue
dye exclusion using a Countess Automated Cell Counter (Life Technologies, Carlsbad, CA) on
days 4, 8 and 13. Experimental conditions were repeated in triplicate, and average±SD was
calculated. The results were plotted as the fold of initial number of cells plated in each well
(x50,000) versus elapsed course of time (days) that cells were in culture for count. The
experiments were repeated for 4 times, giving comparable results.
We also utilized the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,
as previously described (20).
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