Fig 2: Cell cycle arrest effects of MOX in C6 and U251 cells. Cells were treated with various concentrations of MOX (0, 10, 15 and 20 µmol/l) for 48 h. Following staining with propidium iodide, the DNA content was analyzed using flow cytometric analysis. (A) Representative histograms for the changes in the cell cycle for C6 and U251 cells. (B) Graphs represent the percentage cell cycle distributions at the G0/G1, S and G2/M phases in C6 and U251 cells. (C) Effect of MOX on the protein expression levels of CDK2, CDK4, CDK6, cyclin D1 and cyclin E. C6 and U251 cells were treated with indicated MOX concentrations for 48 h and then subjected to western blotting analysis. Data are presented as the mean ± SD. *P<0.05 and ***P<0.001 vs. the control. MOX, moxidectin; CDK, cyclin-dependent kinase.

Fig 3: Drug responses show reciprocal relationships based on cell-cycle states(A) Heatmap showing drugs with reciprocal response relationships between groups of cell lines with high and low expression in CCND1/RB1 and CCNE1/CDKN2A (determined using upper and lower expression quantiles; N = 71 in each quantile group, N = 140 in the intermediate group). Values shown are the ratio between mean drug response of cell lines with high gene expression compared to mean drug response of cell lines with low gene expression (color bar is the log fold-change [logFC] in the comparison groups).(B) Volcano plots with trend lines showing the correlation between drug response log fold-changes and p values across each gene for specific drug families.(C) Correlation plot showing drug family response trendline correlation coefficients and p values (*p < 0.05, **p < 0.01, ***p < 0.001).(D) Correlation between CCND1, CCNE1, RB1, and CDKN2A gene expression, with drug sensitivity in PDX models. PDX models with gene expression greater than the 75th percentile are marked in red and models with gene expression less than the 25th percentile are marked in blue. PDX models that have no information regarding drug sensitivity were removed. The correlation coefficient and related p valuate are shown.(E) Representative multispectral staining for PDX models that are RB proficient (HCI-009) and RB deficient (HCI-012) (scale bar, 100 mm).(F) The indicated PDX models were treated with CDK4/6 (palbociclib) and/or mTOR (AZD8186) inhibitor, and the effect on tumor growth was measured by calipers. The number of mice for each treatment group is shown, and the means and SEMs are plotted.(G) The effect of the indicated drugs and combinations were evaluated in isogenic MCF7 and MB231 cells harboring RB deletion. Cell viability was determined using the CellTiter-Glo (CTG) assay. The column represents means and SDs from triplicates (**p < 0.01, ***p < 0.001 as determined by Student’s t test).

Fig 4: Overexpression of cyclin D-CDK complex components rescues AR-mediated growth suppression of HPr-1AR.(A) Transient overexpression of cyclin D1 and cyclin D2 was validated by immunoblot analysis. (B) Transient overexpression of either cyclin D1 or cyclin D2 decreased the proportion of HPr-1AR cells in G0/G1 phase of the cell cycle and increased the proportion of cells in S phase. However, the increases in cell cycle progression due to cyclin D1 and cyclin D2 overexpression remained completely sensitive to androgen, such that DHT treatment increased the G0/G1 populations by the same amount (6–7%) before and after transient overexpression of cyclin D1 or D2. The expression vectors used in these experiments also express green fluorescent protein, which allowed for gating and analysis of transfected cells among a background of untransfected cells. (C) Stable overexpression of CDK4 and CDK6 was validated by immunoblot analysis. (D) Stable overexpression of either CDK4 or CDK6 decreased the proportion of HPr-1AR cells in G0/G1 phase of the cell cycle and increased the proportion of cells in S phase. Moreover, the cell cycle progression after CDK4 and CDK6 overexpression became completely resistant to androgen, such that the proportions of cells in G0/G1, S, and G2/M phases of the cell cycle were unaffected by DHT treatment. The integrated proviral vectors used in these experiments also express red fluorescent protein, which allowed for sorting, gating and analysis of transduced cells among a background of uninfected cells. (E) Stable overexpression of either CDK4 or CDK6 increased the proliferation of HPr-1AR cells. In addition, the increases in proliferation due to CDK4 and CDK6 overexpression became completely resistant to DHT treatment. * P < 0.05.

Fig 5: Attenuation of CDK expression and cyclin D-CDK4/6 activity by androgen in PC3-Lenti-AR.PC3-Lenti-AR cells were treated with 10 nM DHT or vehicle control for 24 hours, and (A) cyclin mRNA levels and (B) CDK and CDK inhibitor mRNA levels were determined using QPCR. The cyclin, CDK2, and CDKN1B mRNAs were unaffected by androgen treatment, whereas CDK4 and CDK6 mRNAs were androgen-repressed 1.8 fold or more. CDKN1A mRNA was DHT-induced 2.3 fold or more at 24 hours. Data represent the mean ± SEM, n = 3. (C) PC3-Lenti-AR cells were treated with several DHT doses (10-10, 10-9, 10-8 M) or vehicle control for 24 hours, and significant decreases in CDK4 and CDK6 mRNAs occurred at DHT doses ranging from 0.1–10 nM, n = 3. These DHT doses also increased CDKN1A mRNA. (D) AR antagonist 2-hydroxyflutamide (OHF, 10 µM) suppressed the down-regulation of CDK mRNAs and the up-regulation of CDKN1A mRNA by 1 nM DHT treatment at 24 hours, n = 3. (E) In comparison to control samples, DHT treatment for 24 hours significantly decreased CDK4 and CDK6 mRNAs and pre-mRNAs 1.5 fold or more and increased CDKN1A mRNA and pre-mRNA 2 fold or more, suggesting AR-mediated regulation of transcription at these genes, n = 3. (F) PC3-Lenti-AR cells were treated with 10 nM DHT or vehicle control for 24–48 hours (h), and immunoblot analysis of cell lysates reveals that the relative levels of the CDK proteins and various phosphorylated forms of RB (S780 and S807/811) decreased and CDKN1A expression increased over time with androgen treatment compared to vehicle. (G) CDK4 and CDK6 proteins were reduced 50% or more at 24 hours and 65% or more at 48 hours, and phosphorylated CDK2 as well as total CDK2 protein decreased 50% with androgen treatment at 48 hours. In addition, the phosphorylated forms of RB declined 50% or more at 24 hours and 55% or more at 48 hours. Relative Cyclin D/GAPDH, CDK/GAPDH and AR/GAPDH and p-RB/total RB protein ratios are shown using the mean ± SEM, n = 4. * P < 0.05.