Targeting of CYP17A1 Lyase by VT-464 Inhibits Adrenal and Intratumoral Androgen Biosynthesis and Tumor Growth of Castration Resistant Prostate Cancer

Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a validated treatment target for the treatment of metastatic castration-resistant prostate cancer (CRPC). Abiraterone acetate (AA) inhibits both 17α-hydroxylase (hydroxylase) and 17,20-lyase (lyase) reactions catalyzed by CYP17A1 and thus depletes androgen biosynthesis. However, coadministration of prednisone is required to suppress the mineralocorticoid excess and cortisol depletion that result from hydroxylase inhibition. VT-464, a nonsteroidal small molecule, selectively inhibits CYP17A1 lyase and therefore does not require prednisone supplementation. Administration of VT-464 in a metastatic CRPC patient presenting with high tumoral expression of both androgen receptor (AR) and CYP17A1, showed significant reduction in the level of both dehydroepiandrosterone (DHEA) and serum PSA. Treatment of a CRPC patient-derived xenograft, MDA-PCa-133 expressing H874Y AR mutant with VT-464, reduced the increase in tumor volume in castrate male mice more than twice as much as the vehicle (P < 0.05). Mass spectrometry analysis of post-treatment xenograft tumor tissues showed that VT-464 significantly decreased intratumoral androgens but not cortisol. VT-464 also reduced AR signaling more effectively than abiraterone in cultured PCa cells expressing T877A AR mutant. Collectively, this study suggests that VT-464 therapy can effectively treat CRPC and be used in precision medicine based on androgen receptor mutation status.


MDA-PCa-133 Xenograft Tumor Tissue
The subcutaneous xenograft tumors were harvested when the tumor size reached 1.5 cm diameter according to institutional guidelines. The tumor tissue were divided for 1) fixation in 10% aqueous formaldehyde solution for paraffin embedding, 2) snap freezing and storage at -80 0 C, and 3) subsequent implantation. Tissue sections from all formalin-fixed, paraffin-embedded (FFPE) blocks derived from the xenografts were reviewed. Representative blocks were selected and a tissue microarray (TMA) containing 0.6 mm diameter cores was constructed as previously described (1,2)

Immunohistochemistry (IHC) analysis
Tissue sections (4 µm) from the tumors of xenograft, or patient biopsy specimens were subjected to IHC analyses, using an Autostainer Plus (Dako North America, Inc.) as previously published (2). Briefly, serial 4-µm formalin-fixed paraffin-embedded tissue sections of prostate biopsy or xenograft TMA were deparaffinized by incubation at room temperature in xylene for 5 minutes and then rinsed in 100% ethanol. Antigen retrieval was performed by heating the sections in a Target Retrieval Solution (S1699, Dako, Carpentaria, CA, USA).

Construction of AR and mutant expression plasmids
The DNAs of wild-type AR and AR-V7 open reading frames were PCR amplified using cDNAs of MDA-PCa-180-30 xenografts (2). The amplification was done using HotStart hifidelity polymerase kit (QIAGEN), the amplified DNAs were cloned using the pCR8/GW//TOPO TA Cloning Kit (Life Technologies), and then plasmids from several colonies were sequenced to match wild-type AR and AR-V7. Each cDNA was then amplified using an AR or AR-V7 TOPO plasmid and primers that introduced ClaI at the 5'-end and XbaI at the 3'-end in order to clone each cDNA into the p3xFLAG-CMV-14 vector and induce expression of each protein as a fusion with the 3xFlag tag. The T877A and H874Y mutants were PCR amplified using cDNAs of C4-2B cells and the MDA-PCa-133 xenograft, respectively, and also expressed as a fusion with the 3xFlag tag.

Transfection and Reporter Assay
Method used for Figure  collected after the incubation to measure the luciferase activity as described earlier, and to measure expression of AR or AR-mutants proteins by western blot as described below. The relative luciferase activity in each set expressing AR or AR mutants was calculated after subtraction of the basal luciferase activity with the vector plasmid control.

Preparation of Cell or Tissue Protein Extracts and Western Blot Analysis
Whole cell protein extracts were prepared from cultured C4-2B or PC3 cells in a lysis buffer containing 1% Triton x-100, 50 mM HEPES ( pH 7.4), 150 mM NaCl, Frozen tumor tissues were grinded with mortar and pestle, incubated with the extraction buffer (2 ml of buffer per 0.1 g of tissue) in ice for 30 min, sonicated for the supernatant was collected and used for western blot analysis as shown supplemental Figure 6C.
Cell or tissue lysates (40 µg of protein) were separated on 4%-20% Tris-glycine polyacrylamide gels, transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA) and incubated overnight at 4°C with the primary antibodies described above. Next, the mixtures were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling) at a 1:3,000 dilution. Immunoblots were developed by using enhanced chemiluminescence (Amersham GE Healthcare Bio-Sciences Corp. Piscataway, NJ).

RNA and QRT-PCR analysis
Total RNA was extracted from cell lines using the RNeasy Mini Kit (Qiagen). The relative mRNA level for each gene was quantified by using QRT-PCR with SYBR Green (Applied Biosystems, Inc., Foster City, CA) and the primers described above. The mRNA level for each gene was calculated from the values in the linear range of the PCR cycles and normalized to the value for the reference mRNA of GAPDH and ACTB (2) as follows: ΔC T = average C T of reference − average C T of the specific gene, where C T is the threshold cycle. The relative level of expression of each gene with respect to the reference gene (GAPDH or ACTB) was then calculated as 2 ΔCT . For each gene, data from three independent measurements were used to calculate means and standard deviations.

Measurements of PSA and testosterone in mouse serum
Mouse blood was collected by either mandibular venipuncture or cardiac puncture in an Eppendorf centrifuge tube, kept on the lab bench for 30 min at room temperature, and centrifuged at 3,000 rpm for 5 min at room temperature, and then the supernatant (serum) was collected for analysis. Serum PSA and testosterone were measured by ELISA using kits from American Qualex, San Clemente, CA (catalog no. KD4310), and Alpha Diagnostic International, San Antonio, TX (catalog no. 1880), respectively, following the protocols provided in each kit.
Hydroxylamine hydrochloride and ultrapure methanol and water (Chromasolv) were purchased from Sigma-Aldrich. Steroid internal standards progesterone-13 C 3 , dihydrotestosterone-13 C 3 , cortisol-2 H 4 , and testosterone-13 C 3 (0.5 ng each) were added to C4-2B, PC3, and MDA-PCa-133 homogenates. Steroids were extracted using tert-butyl methyl ether, and the separated organic layer was evaporated under nitrogen. The extracts were subsequently derivatized using hydroxylamine hydrochloride in water/methanol (5). An Agilent (Santa Clara, CA) 6490 triple quadrupole mass spectrometer equipped with a Jet Stream electrospray ion source(Agilent), a 1290 Infinity ultrahigh-performance liquid chromatography system (Agilent) and MassHunter Workstation software (Agilent) was used to quantify steroids. Chromatographic separation of steroid oximes was conducted with a Chromolith C 18 reverse phase column (50 x 2 mm) with a matching Chromolith guard column (5 x 2 mm) using a mobile phase gradient from 30% methanol-water with 0.1% formic acid to 95% methanol-water with 0.1% formic acid. Steroid oximes were introduced into the electrospray ion   Maity et al. Supplementary Fig 5