Article Figures & Data
Figures
- Figure 1.
YM155 synergizes with high-androgen therapy to suppress prostate cancer viability. A, Design of high-throughput screen in LNCaP grown in 10% FBS media supplemented with either 1 nmol/L R1881, 10 μmol/L enzalutamide (ENZ), or DMSO. B, Average CellTiter-Glo luciferase signal (viability) for all control wells on drug screen 96 hours after cell seeding. C and D, Inhibitory concentration for 50% viability (IC50) plotted for various doses of R1881 (C) and (D) enzalutamide.
- Figure 2.
High-dose androgens suppress BIRC5 levels and increase YM155-mediated DNA damage. A, Western blots for BIRC5 (survivin), γH2AX, Beclin, ATG5, caspase-3, and PARP1 on LNCaP treated for 24 hours with 0.5 or 1 μmol/L YM155 with and without a 48-hour preincubation with 10 nmol/L R1881. B, LNCaP viability (CellTiter-Glo) in response to YM155 with or without 2.5 nmol/L testosterone (T; error bars = SD; n = 4). C, YM155 IC50 values of LNCaP preincubated 24 hours with 10 nmol/L R1881 or vehicle control then exposed to a dose range of YM155 for 24, 48, and 72 hours (error bars = 95% confidence interval, n = 4).
- Figure 3.
SLC35F2 is regulated by AR signaling. A, qRT-PCR showing relative fold change of SLC35F2 in LNCaP and VCaP exposed to 10 μmol/L enzalutamide (ENZ), vehicle (veh), 10 nmol/L R1881 (error bars = SD, n = 6). B, RNA-seq CPM of prostate cancer cell lines cultured with 1 nmol/L R1881 or vehicle for 24 hours, each in duplicate. C, Western blot analysis of SLC35F2 on LNCaP exposed to 10 μmol/L enzalutamide, 10 nmol/L R1881, or ethanol vehicle. D, Western blots of SLC35F2 and BIRC5 in response to a dose range of R1881 in LNCaP. E, Western blot analysis of SLC35F2 with or without a blocking peptide of SLC35F2 and GAPDH on VCaP and LNCaP cultured with 10 nmol/L R1881 for 48 hours. F, Western blot of SLC35F2 on VCaP cultured with 10 nmol/L R1881 or vehicle for 48 hours and 5 μg/mL cycloheximide (CHX) or 5 μmol/L MG132 for 24 hours (*, P < 0.05).
- Figure 4.
The membrane transport protein SLC35F2 is regulated by AR activation. A, ChIP-seq histograms of AR binding to the SLC35F2 locus from published datasets. Black arrows, peaks selected for validation. B, ChIP-qPCR of peaks “AR1-3” in LNCaPs cultured in CSS with DHT, enzalutamide (ENZ), or ethanol vehicle control (EtOH).
- Figure 5.
SLC35F2 and ABCB1 expression determine response to YM155. A, YM155 dose–response curve for LNCaPs transduced with SLC35F2 or GFP overexpression vectors. B, IC50 values plotted for curves in A. C, Same as B for VCaPs. D, YM155 IC50 values of LNCaPs transduced with shRNA vectors to SLC35F2 (shSLC) compared with a nontargeting control (NTC) vector. E, Same as D for VCaP. F, YM155 dose–response curves are plotted for LNCaPs overexpressing ABCB1 with and without 10 nmol/L R1881 [error bars, SD (A) and 95% confidence interval (B–E); *, P < 0.05, n = 4].
- Figure 6.
SLC35F2 expression correlates with AR activity and androgen levels in CRPC. A, Pearson correlation plot comparing AR activity and SLC35F2 expression in 171 CRPC samples. B, Waterfall plot of microarray signal intensities for SLC35F2 in primary tumors (n = 11), and CRPC (n = 171). C, Intratumoral testosterone and DHT levels in the LuCaP 96 PDX model in intact mice, at 7 and 21 days postcastration (Cx), and after castration-resistant (CR) regrowth. D, qRT-PCR for tumoral SLC35F2 expression in LuCaP 96 xenograft samples shown in C. E, Spearman correlation comparing intratumoral testosterone levels with normalized SLC35F2 mRNA expression in LuCaP 96 tumors. F, Intratumoral androgen levels in LuCaP 23. G, qRT-PCR for tumoral SLC35F2 expression in LuCaP 23. H, Spearman correlation comparing normalized SLC35F2 levels and intratumoral DHT in LuCaP 23. Student t tests comparing SLC35F2 levels in intact and castrate mice.
Additional Files
Supplementary Data
- Fig S1 - Fig S1. Androgen Mediated Growth Suppression in LnCaP
- Fig S2 - Fig S2. SLC35F2 Expression Determines Sensitivity to YM155
- Fig S3 - Fig S3. Tumor Androgen Levels Correlate with SLC35F2 Expression
- Supplementary Table - Correlation of tumor androgen levels with SLC35F2 transcript expression in PDX models
Volume 15, Issue 5
