Figure 1.
AR and c-Myc levels are positively correlated in CRPC specimens.
A) Z-scores (to normal prostate specimens) of AR versus c-Myc mRNA expression across 140 human CRPC metastases. The Pearson correlation coefficient, linear regression, and F test for significantly non-zero slope were performed for each pair of genes. B) Fisher’s exact test and odds ratio on the contingency table analyzing the co-occurrence of tumors with AR or c-Myc z-scores greater than 2.
Figure 2.
AR and c-Myc promote ligand-independent prostate cancer cell growth.
A) LNCaP, abl and 22RV1 cells were transfected with 50 nM of non-targeted control (NTC) or AR RNAi oligonucleotides. Cells were switched to charcoal-stripped serum on the day of transfection. Cell growth was determined 5 days later for LNCaP and 6 days later for abl and CRPC 22RV1 with the trypan blue exclusion method. B) Immunoblot for AR expression. The lower bands in the AR immunoblot in 22RV1 cells reflect the presence of an AR transcript variant [7]. B) LNCaP, abl and 22RV1 cells were transfected with 50 nM of NTC or c-Myc RNAi oligonucleotides. Cells were switched to charcoal-stripped serum on the day of transfection. Cell growth was determined 5 days later for LNCaP cells and 6 days later for abl and 22RV1 with the trypan blue exclusion method. Immunoblot for c-Myc protein expression. C) LNCaP cells with stable overexpression of empty vector (EV) or c-Myc were generated. These cells were transfected with 50 nM of non-targeted control (NTC) or AR siRNA oligonucleotides. Cell growth was determined 6 days later with the trypan blue exclusion method. Immunoblot for AR and c-Myc protein expression. The higher bands on the c-Myc immunoblot in the c-Myc-overexpressing cells represent the ectopically-expressed c-Myc. *denotes p<0.05 compared to NTC.
Figure 3.
c-Myc expression is not activated by androgen ligands.
LNCaP, abl, and 22RV1 cells were grown in charcoal-stripped serum for 72 hours and then treated with 10 nM R1881 (or ethanol vehicle) for 4 hours. A) Chromatin immunoprecipitation was performed to determine the enrichment of AR and histone H3 acetylation (AcH3) at the c-Myc and KLK3 enhancer elements. B) QRT-PCR was performed to determine the mRNA levels of KLK3 and c-Myc relative to actin. C) Immunoblotting was performed to determine the protein levels of AR, c-Myc, and actin. *denotes p<0.05 compared to vehicle.
Figure 4.
AR promotes ligand-independent expression of c-Myc.
LNCaP, abl and 22RV1 cells were transfected with 50 nM of non-targeted control (NTC) or AR RNAi oligonucleotides. Cells were then grown in charcoal-stripped serum for 96 hours. At the end of the treatment, cells were harvested to extract mRNA and protein. A) QRT-PCR was performed to determine the levels of c-Myc relative to actin. B) Immunoblotting was performed to determine the levels of AR, c-Myc and actin. C) Parallel treatments were performed and cells were cross-linked and processed for ChIP to determine AR and histone H3 acetylation (AcH3) enrichment at the c-Myc enhancer. *denotes p<0.05 compared to NTC.
Figure 5.
AR suppression recapitulates the effect of c-Myc suppression on c-Myc target gene expression.
LNCaP and abl cells were transfected with 50 nM of non-targeted control (NTC) and either A) AR or B) c-Myc RNAi oligonucleotides. Cells were switched to charcoal-stripped serum on the day of transfection and harvested 96 hours later. QRT-PCR was performed to determine the levels of the indicated c-Myc target genes relative to actin. *denotes p<0.05 compared to NTC.
Figure 6.
JQ1 treatment suppresses c-Myc expression and function and reduces ligand-independent prostate cancer cell survival.
LNCaP, abl, and 22RV1 cells were grown in charcoal-stripped serum and treated with vehicle, 50 nM, 250 nM or 500 nM JQ1 every 24 hours for 72 hours. A) Immunoblotting was performed to determine the protein levels of c-Myc. B) QRT-PCR was performed to determine the mRNA level of c-Myc and c-Myc targets genes KIF11, CDKN1A, TPX2, and AURKB relative to actin. *denotes p<0.05 compared to vehicle. C) Cell viability was determined at the end of treatment with the trypan blue exclusion method. p<0.01 for the 250 nM and 500 nM doses vs. vehicle in all three cell lines.
Figure 7.
AR and c-Myc are critical drivers of ligand-independent mechanisms of prostate cancer progression.
Currently, androgen deprivation therapies that interfere with androgen ligand activation of the AR are primarily used to treat this disease. These therapies suppress AR’s androgen ligand-dependent function and suppress expression of androgen ligand-dependent (AD) AR target genes. However, despite these treatments prostate cancer progression is inevitable. The AR also promotes the expression of androgen ligand-independent pathways such as c-Myc. The c-Myc gene is commonly upregulated in prostate cancer and contributes to androgen ligand-independent prostate cancer progression. This model strongly suggests that AR or c-Myc-directed therapies would complement current androgen deprivation strategies.