Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer.

The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.


RNA isolation and processing for microarrays
Total RNA was isolated using the Qiagen RNeasy kit (Qiagen, 74106) following the manufacturer's recommendations. RNA concentration and purity was measured using a NanoDrop instrument (Thermo Scientific).
For microRNA-profiling, total RNA was isolated using Trizol reagent (Life technologies, 15596026) following the manufacturer's recommendations.
Resuspended cRNA samples were hybridized onto Human . Missing probes were imputed using Illumina's GenomeStudio Gene Expression Module.

Microrarray analysis
The imputed probe datasets were analyzed using the freely available J-Express 2012 software (http://jexpress. bioinfo.no/site/). The raw data was quantile normalized and log2 transformed prior to analysis. Differential expression analysis was performed using the grouped triplicate experiments and Rank product analysis. Probes with a q-value of < 0.05 were considered significantly up-or downregulated. For hierarchical clustering using complete linkage and Pearson correlation, differentially expressed probes were merged and high level mean and variance normalized.
Gene expression data published in Taylor et al., were downloaded from the NCBI GEO data repository (accession number: GSE21034) and analyzed using J-Express.

Reverse transcription and quantitative real-time PCR (qRT-PCR)
500 ng to 1 μg total RNA was reverse transcribed using the SuperScript VILO kit (Applied Biosystems, 11754) following the manufacturer's recommendations. qRT-PCR was performed using SYBR green master mix (Applied Biosystems, 4385612). Amplification was performed in duplicate series using the ABI 7900HT FAST Sequence Detection System (Applied Biosystems) with the following cycling conditions, 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15s and 60°C for 60s. Transcript levels were normalized to vehicle controls and the expression levels of beta-actin using the 2^ddCt method.

Identification of putative MYC binding sites and primer design
To identify candidate consensus MYC binding sites associated with purine biosynthesis enzymes, we downloaded ENCODE datasets from the National Center for Biotechnology Information (NCBI) data repository (http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi) for six cell lines: lymphoblastoid cells -GM12878 (GSM822290); embryonic stem cells -H1-hESC (GSM822274); hepatocytes -HepG2 (GSM822291); cervical cancer cells -HeLa-S3 (GSM935320); endothelial cells -HUVEC (GSM822298) and myeloid leukemia cells -K562 (GSM935516). We identified overlapping MYC consensus binding sites in a minimum of three cancer cell lines lying at the transcription start sites (TSS) of every gene in the pathway. Approximate primer sites are shown in Figure S2A (black rectangle) and sequences can be found in Figure S2B.

Chromatin immunoprecipitation (ChIP)
ChIP was performed using the Human MYC ExactaChIP Chromatin IP kit (R&D, ECP3696) with slight modifications to the manufacturer's protocol. Briefly, LNCaP or VCaP cells were seeded in charcoal-stripped medium in 150 mm dishes for 72 h prior to 4 h stimulation with 1 nM R1881 or EtOH control and crosslinking with 1% formaldehyde (Sigma, F8775). After scraping and cell lysis, chromatin was sheared to an average fragment size of 200-500 bp using a Bioruptor NextGen (Diagenode), cleared and diluted. Prior to overnight incubation with 5 μg MYC/control IgG antibody, 1% of total chromatin was taken as input control. After harvesting the antibody-DNA complexes on a 4°C rotator for 1 h using 50 μl magnetic Streptavidin beads (R&D, MAG999), the DNA was eluted and reverse crosslinked for 16-20 h shaking at 65°C using 200 μl of a 1% SDS in NaHCO3 solution. Subsequently, the DNA was purified using phenolchloroform-isoamylalcohol extraction and reverse phaselock tubes (5Prime, 2302830), precipitated with EtOH abs., washed with 80% EtOH and finally resuspended in 60 μl Tris-HCl (pH 8.0). ChIP qPCR was performed using the SYBR green master mix and same amplification conditions as mentioned above. Results are being displayed as '% of input' using the formula 2^(ct(Input)ct(antibody)). For a detailed list of primer pairs used, see Figure S2.

Recursive partitioning
To identify statistically significant biochemical recurrence courses, recursive partitioning was performed on a single gene expression profile using the 'party' package from CRAN (Han et al, 2012) with accompanying biochemical recurrence data taken from (Huang et al, 2008). Kaplan Meier plots of the risk of biochemical recurrence were produced using the 'survival' package and p-values from the log rank test were corrected using the Bonferroni correction method.

Fluorescent-based caspase cleavage assay
Appropriate amounts of LNCaP or VCaP cells were seeded in 384-well plates and allowed to attach for 48 h at which point they received drug treatment (12 wells per condition). Induction of apoptosis was monitored using the CellPlayer 96-well Caspase-3/7 reagent (Essen Bioscience, 4440) at a final concentration of 1:5,000 on the Incucyte FLR instrument (Essen Bioscience). Phase contrast and fluorescence pictures were taken every two hours for a total of 96 h. Analysis was performed using the inbuilt object counting algorithm.

NTP measurements
LNCaP and VCaP cells were seeded in 100 mm dishes and allowed to attach for 48 h at which point the received fresh drug-containing medium. For every condition, a duplicate plate was included for protein normalization. Cells were washed once with a 150 mM NaCl solution, scraped and immersed in 600 μl of ice-cold extraction solution (15% w/v trichloroacetic acid, 15 mM MgCl 2 ) and frozen in liquid nitrogen.
The samples were vortexed for 30s and incubated for 10 min at 4°C. Supernatants were collected after centrifugation at 20,000 g for 1 min, and added to 800 μl of ice-cold extraction mixture consisting of 10 ml of Freon (1, 1, 2-trichlorotrifluoroethane, Aldrich, Sigma-Aldrich Sweden AB > 99%) and 2.8 ml of trioctylamine (Sigma-Aldrich Sweden AB, 98%). The samples were vortexed and centrifuged for 1 min at 20,000 g. The aqueous phase was collected and added to 700 μl of the same ice-cold extraction mixture. The samples were vortexed as above. 70 μl of the aqueous phase containing nucleoside triphosphates (NTP) was adjusted to pH 3.4 with 6 M HCl, separated on a Partisphere SAX HPLC column (125 mm × 4.6 mm, Hichrome, United Kingdom) under isocratic elution with 0.35 M potassium phosphate buffer (pH 3.4, 2.5% v/v acetonitrile) and quantified using a LaChrom Elite ® HPLC system (VWR International).    G TCTTCTCTTCGACTACCCAGTGG  CAAATTGAGCTGATCCTTCTGGA  ADSL  CGGCCAAGGAAGTCTTAATG  C TTGGTTCGCTTCTTTGACC   ADSL  TTGCTAAGGAACGAGCCAGT  ATCTCGGACACGCTTCAAGT  A TIC  TTTTCAGCATGGGAGCTTCT  GGGCAAATGCCTGTTATTCT   ATIC  AACCCTCACACCCATCTCAG  ACATCGGACAATGCAACAAA  ADSS  GTGTATTCTGGGGCCACTGT  CCCTGTCCCCATCTCTGTTA   ADSS  GGATTTACTGCGTTGGCACT  TGTTCCATCCTGGGAGAGTC  A DSSL1  C CGCGTGTTTATTTCAGGAT  GGGACTTCAGTCCAGACGAG   ADSSL1  CGGATCCTTAACACCTGGAG  GCAGCAGGTGGAAGTCGTA  I MPDH1  G CCGCTGATCAGGTAGTCC  G TCAGCAGTAGCAGCAGCAG   IMPDH1  ATTGGACCTCGCTACACACC  CAGGTAGTCCGCCATGCTA  I MPDH2  T TGAACTGGCCGAGGTCCT  GCACATAGTGGCCTTGTCACTG   IMPDH2  AGTGGCTCCATCTGCATTACG  A CCTTGTACACTGCTGTTGCTTG  nega ve  AACTCCACATTTCCTAAGTGACC  CCAACCCACACCAAGTACC   KLK3  GCAGCATTGAACCAGAGGAG  A GAACTGGGGAGGCTTGAGT   MYC  T ACCCTCTCAACGACAGCAG  T  were transfected with 25 nM (IMPDH2 ambion) or 50 nM (IMPDH2 pool/PAICS) or equivalent amounts of non-targeting control siRNA (siCTRL) for 72 h. Protein lysates were harvested, separated by SDS-PAGE and blotted for the indicated proteins. B. Cell viability results of siRNA and Abiraterone/MDV3100 treated cells. Cells were transfected with 25 nM IMPDH1 or equal amounts of siCTRL for 48 h. Following treatment with the indicated drugs for another 72 h, viability relative to DMSO and siCTRL was assessed using a MTS-based assay. Doses for LNCaP were 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 1 μM Abiraterone and 100nM MDV3100. n = 3-4 C. Caspase cleavage assay using the fluorescent-based CellPlayer system. LNCaP (top) and VCaP (bottom) cells were allowed to attach for 48 h prior to treatment with the indicated drug combinations. Activation of caspase as determined by fluorescence was monitored for a total of 96 h using an automated imaging system (Incucyte). 2-Deoxyglucose and Metformin (LNCaP) or Hydroxyurea (VCaP) have been shown to induce apoptosis in the respective cell lines and were used as positive controls. D. Western Blot analysis of LNCaP treated with mycophenolic acid (10 μM), hydroxyurea (1 mM) or guanosine (100 μM) for the indicated timepoints. Protein lysates were harvested, separated by SDS-PAGE and blotted for the indicated proteins. Protein levels were normalized to 6 h DMSO control and GAPDH.