Supplement
Patient samples
Clinical samples for RNA sequencing (RNA-seq) were prospectively collected from
Newcastle upon Tyne Hospitals National Health Service (NHS) Trust as part of the GenTax
(tumour profiling in an open-labelled, two-arm study investigating the tolerability and
efficacy of taxotere in patients with hormone-naïve high-risk prostate cancer [PCa]) study
[11], and ethical approval was granted by the local research and ethics committee
(Northumberland, Tyne and Wear NHS Strategic Health Authority Local Research Ethics
Committee Ref: 2003/11). Informed consent was obtained from all subjects who had a clinical
suspicion of advanced PCa. Tissue for diagnosis was obtained by transrectal ultrasound
(TRUS)–guided prostatic biopsy (BK Medical, Herlev, Denmark; 8818).
Routine histopathologic assessment of tumour biopsies was undertaking by hematoxylin and
eosin staining to confirm the diagnosis and determine the Gleason sum score [16]. Routine
radiologic staging investigations (computed tomography [CT], magnetic resonance imaging,
and radioisotope bone scintillography) were performed accordingly to national guidelines
[40]. Patient eligibility for study inclusion was cT3/T4 disease (TNM classification, 2009), a
prostate-specific antigen (PSA) level ≥50 ng/ml or Gleason score ≥8, or metastatic disease to
be commenced on standard androgen-deprivation therapy (ADT) consisting luteinising
hormone-releasing hormone agonists with antiandrogen “flare” protection. Further material
for RNA-seq was taken by TRUS biopsy from eight eligible patients prior to commencement
of ADT and again at approximately 22 wk following initiation of ADT. Biopsies were
specifically taken from tumour-rich areas of the prostate, where typically more than 60% of
the initial diagnostic cores taken were occupied by tumour. All tissue was stored at −80°C.
Patient demographics and clinicopathologic parameters were recorded. Patients were also
assessed using the Karnofsky performance status scoring system [41] to determine suitability
for further intervention. Serum PSA was measured approximately every 3 wk until
approximately 22 wk, and then every 3 mo; repeat staging CT scanning was undertaken
approximately 6 mo after diagnosis for patients with N+ or M+ disease to assess the
radiologic treatment response. PSA progression was defined as two consecutive rises in PSA
above nadir at least 2 wk apart, although it is not known whether patients subsequently
fulfilled the European Association of Urology (EAU) criteria for castration-resistant PCa
(CRPC) disease [42].
For immunohistochemistry validation experiments, we used a completely separate,
prospectively collected cohort of matched prostate tumours from patients prior to ADT and
after the development of CRPC, as previously described [43,44]. Patients were selected for
analysis if they initially responded to ADT but subsequently relapsed (defined as two
consecutive rises in PSA >10%). As this cohort is historical, it is not known whether patients
fulfilled recent EAU criteria for castration-resistant disease [42]. All material was used in
accordance with approval granted by the Multicentre Research Ethics Committee for Scotland
(MREC/01/0/36) and Local Research and Ethical Committee.
RNA extraction, library preparation, and sequencing
Total RNA was extracted from pre- and post-ADT samples using the RNeasy Mini Kit
(Qiagen, Venlo, The Netherlands; 74104) according to the manufacturer’s instructions. RNA
quantity and quality were evaluated by spectrophotometry using the NanoDrop 2000
spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and 2100 Bioanalyzer
(Agilent Technologies, Santa Clara CA, USA) RNA electropherograms, with calculation of
RNA integrity number (RIN) [45]. Samples were included for RNA sequencing if RIN > 6
and total RNA > 500 ng. Samples were processed using the Illumina RNA-seq protocol
(Illumina, San Diego, CA, USA) according to the manufacturer’s instructions without poly(A)
messenger RNA (mRNA) selection. The complementary DNA (cDNA) sample library was
subsequently normalised using the Illumina duplex-specific nuclease protocol according to
the manufacturer’s instructions. Cluster generation was performed using the Illumina Cluster
Generation Kit according to the manufacturer’s instructions. The amplified library was
sequenced on the HiSeq 2000 (Illumina) with a paired-end sequencing strategy. The read
length was set at 90 nt, with an expected library size of 200 base pairs.
Bioinformatics
The quality of raw reads was first assessed using the FastQC package
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and mapped to human genome
assembly hg19 using TopHat v.1.4.1 [46], with a junctions library derived from Ensembl
version 68. Samples were subjected to quality control by examining the percentage of reads
uniquely mapping to the genome, the percentage of reads mapping to known protein coding
sequences, the number of exon junctions identified, the percentage of spliced reads, and the
number of genes with 90% base coverage (Supplemental Table 1). On the basis of these
measures, one sample was discarded from further analysis. Gene fusions were identified by
mapping reads to the human genome using TopHat-Fusion version 0.1.0 [47]. Differentially
expressed genes were identified by counting the number of reads mapping to each gene from
Ensembl 68 using HTSeq v.0.5.3 (“HTSeq: Analysing high-throughput sequencing data with
Python,” www.huber.embl.de/users/anders/HTSeq). Read counts were normalised using the
trimmed mean of M values method and differential expression tested for using a paired
generalised linear model design with the Bioconductor 2.11 edgeR package [48]. Enriched
pathways were identified by downloading gene pathway associations from the Kyoto
Encyclopaedia of Genes and Genomes database [19] and testing each pathway for enrichment
in significantly up- and downregulated genes (false discovery rage [FDR] <0.05) with a
hypergeometric test using a custom script. Pathways are said to be enriched if the enrichment
over background was at least twofold and the FDR <0.05. Associations between androgen
receptor (AR) binding and differential expression were examined by overlapping previously
published AR binding peaks [18], with gene promoter regions. Briefly, each gene was
assigned a promoter region extending 2 kb upstream and 1 kb downstream of the transcription
start site binding peaks [18] and were converted to human reference genome hg19 coordinates
using the LiftOver program (http://genome.ucsc.edu) and overlapped with the promoter
regions. The significance of enrichments was tested using a hypergeometric test. Raw data
have been deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under
accession number GSE48403, and all details are Minimum Information About a Microarray
Experiment (MIAME) compliant.
Immunohistochemistry
Immunohistochemistry using an antibody to β-catenin (C19220, BD Transduction Labs) and
AR (M3562, Dako) was performed as previously described [49,50], and immunoreactivity
was scored using the weighted histoscore method [51]. Histoscores were calculated from the
sum of (1 × % cells staining weakly positive) + (2 × % cells staining moderately positive) +
(3 × % cells staining strongly positive), with a maximum of 300. The mean of the four
histoscores obtained from assessment of immunoreactivity of four technical replicates was
used for statistical analysis. The one-sample Kolmogorov-Smirnov test was used for
assessment of normality of data. Wilcoxon signed-rank tests were employed to identify
differences between groups, and correlations were identified using the Pearson product
moment correlation coefficient (Pearson r). All tests were undertaken using SPSS version
19.0 computer software (IBM Corp., Armonk, NY, USA). A p value <0.05 was taken to
indicate statistical significance. All images were captured using a ScanScope CS scanner
(Aperio, Vista, CA, USA) and viewed using the Slidepath Gateway viewer (Leica
Microsystems, Wetzlar, Germany).
Reagents and cell lines
All cells were grown at 37°C in 5% CO2. LNCaP (CRL-1740, ATCC) and CWR22 (CRL2505, ATCC) cells were maintained in RPMI-1640 medium (Life Technologies, Carlsbad,
CA, USA; 31870-025) with 20 mM L-glutamine (Life Technologies, 25030-024)
supplemented with 10% foetal bovine serum (FBS; PAA Laboratories, Yeovil Somerset, UK;
A15-101). LNCaP-AI cells were derived from LNCaP parental cells and maintained as
previously described [12]. Where indicated, LNCaP and CWR22 cells were cultured for 72 h
in medium supplemented with 10% dextran charcoal stripped FBS (Lonza, Basel,
Switzerland; DE14-820F) to produce a steroid-deplete medium (DCC), following which 10
nM dihydrotestosterone (PerkinElmer, Waltham, MA, USA; NET453001MC) in 0.1%
ethanol or vehicle was added for 24 h. All cells were harvested and total RNA extracted using
the RNeasy Mini Kit (74104) according to the manufacturer’s instructions.
Quantitative reverse transcription-polymerase chain reaction
We generated cDNA by reverse transcription of 1 µg (untreated cells grown in steady-state
conditions) or 200 ng (drug-treated cells) of total RNA using the High-Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA; 4368814) according
to the manufacturer’s instructions. Quantitative PCR was performed on the 7500 Fast RealTime PCR machine (Applied Biosystems, 4351106) using triplicate cDNA templates with the
TaqMan Universal PCR Master mix (Roche Diagnostics, Indianapolis, IN, USA; 4304437)
and Universal Probe Library set (Roche, 04683633001) per the manufacturer’s instructions.
All primer sequences are listed in Supplemental Table 6. All experimental results shown are
the means of at least three independent experiments plus or minus standard deviation (SD).
The one-tailed independent sample t test was used to identify statistically significant
differences in total transcript expression, with p < 0.05 taken to indicate statistical
significance.
Proliferation assays
Assays were carried out using the WST-1 cell proliferation reagent (Roche, 05015944001) per
the manufacturer’s instructions. Briefly, cells were seeded into flat-bottomed 96-well plates at
a density of 4000 to 10 000 cells per well and grown to approximately 20–30% confluence.
Medium was then replaced with fresh medium containing either 10 μm XAV939 (Novartis
Pharmaceuticals, Plantation, FL, USA) in 0.1% dimethyl sulfoxide (DMSO) [13] or or
vehicle. After 72 h, 10 µl WST-1 reagent was added to each well, mixed, and left to develop
for 1 h before reading the absorbance at 450 nm (with reference wavelength at 650 nm) using
the SpectraMax Plus384 Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA,
USA). Results were corrected for cell-only controls. All experimental results shown are the
means of at least three independent experiments plus or minus SD. The one-tailed
independent sample t test was used to identify statistically significant differences in
proliferation rates, with p < 0.05 taken to indicate statistical significance.
Cell cycle analysis
Cells were synchronised in the G0/G1 phase by serum withdrawal for 24 h prior to
resuspension in serum-containing medium and treatment with 10 μm XAV939 in 0.1%
DMSO [13] or vehicle. After 72 h, cellular DNA was labelled with 10 uM
Bromodeoxyuridine (BrdU; GE Healthcare, Cleveland, OH, USA; RPN201) for 1 h. Cells
were then fixed in 70% ethanol and incubated sequentially with anti-BrdU primary antibody
(BD Biosciences, Franklin Lakes, NJ, USA; 555627) and Alexa Fluor 488 (Life
Technologies, A11001) secondary antisera. Finally, cells were stained with 10 μg/ml
propidium iodine (Fluka, St Louis, MO, USA; 81845) in the presence of 5 μg of RNaseA
(Qiagen). Data were collected and analysed using CellQuest Pro software (BD Biosciences,
337452) on a FACScan cytometer (BD Biosciences, 337452). At least 10,000 cells were
evaluated for each sample, and isotope controls were used as negative controls where
appropriate. Percentages of cells in the different stages in the cell cycle were estimated from
their DNA content as read by propidium iodine (sub-G1 [DNA <2N], G2-M [DNA 4N], and
polyploid [DNA >4N]) and BrdU incorporation (S phase). All experimental results shown are
the means of at least three independent experiments plus or minus SD. The one-tailed
independent sample t test was used to identify statistically significant differences in the
percentage of cells with BrdU incorporation, with p < 0.05 taken to indicate statistical
significance.
Supplemental references
[40] Graham J, Baker M, Macbeth F, Titshall V. Diagnosis and treatment of prostate cancer:
summary of NICE guidance. BMJ 2008;336:610–2.
[41] Karnofsky DA, Burchenal JH. The clinical evaluation of chemotherapeutic agents in
cancer. In: MacLeod CM, editor. Evaluation of chemotherapeutic agents. New York, NY:
Columbia University Press; 1949.
[42] Mottet N, Bellmunt J, Bolla M, et al. EAU guidelines on prostate cancer. Part II:
treatment of advanced, relapsing, and castration-resistant prostate cancer. Eur Urol
2011;59:572–83.
[43] Armstrong K, Ahmad I, Kalna G, et al. Upregulated FGFR1 expression is associated
with the transition of hormone-naive to castrate-resistant prostate cancer. Br J Cancer
2011;105:1362–9.
[44] McCall P, Gemmell LK, Mukherjee R, Bartlett JM, Edwards J. Phosphorylation of the
androgen receptor is associated with reduced survival in hormone-refractory prostate cancer
patients. Br J Cancer 2008;98:1094-101.
[45] Schroeder A, Mueller O, Stocker S, et al. The RIN: an RNA integrity number for
assigning integrity values to RNA measurements. BMC Mol Biol 2006;7:3.
[46] Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-seq.
Bioinformatics 2009;25:1105–11.
[47] Kim D, Salzberg SL. TopHat-Fusion: an algorithm for discovery of novel fusion
transcripts. Genome Biol 2011;12:R72.
[48] Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential
expression analysis of digital gene expression data. Bioinformatics 2010;26:139–40.
[49] Ahmad I, Morton JP, Singh LB, et al. β-Catenin activation synergizes with PTEN loss to
cause bladder cancer formation. Oncogene 2011;30:178–89.
[50] Willder JM, Heng SJ, McCall P, et al. Androgen receptor phosphorylation at serine 515
by Cdk1 predicts biochemical relapse in prostate cancer patients. Br J Cancer 2013;108:139–
48.
[51] Fraser JA, Reeves JR, Stanton PD, et al. A role for BRCA1 in sporadic breast cancer. Br
J Cancer 2003;88:1263–70.
[52] Ngan S, Stronach EA, Photiou A, Waxman J, Ali S, Buluwela L. Microarray coupled to
quantitative RT-PCR analysis of androgen-regulated genes in human LNCaP prostate cancer
cells. Oncogene 2009;28:2051–63.