Journal: Cancer Research Communications

Article Title: Potent Stimulation of the Androgen Receptor Instigates a Viral Mimicry Response in Prostate Cancer

doi: 10.1158/2767-9764.CRC-21-0139

Figure Lengend Snippet: MeT has potent androgenic and growth suppressive activity in prostate cancer cells. A, MeT potently suppresses growth of LNCaP cells (left graph), as determined by Trypan blue growth assay. The response of cells to DHT is shown on the right. Error bars are ± SEM. P values (using day 7 data) were determined using ANOVA and Dunnett multiple comparisons tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). NS, not significant. B, MeT inhibits the growth of CRPC models of prostate cancer (C4-2B, MR49F, and V16D), as determined by Trypan blue growth assay. Statistical analysis was as for A . C, Activation of AR transcriptional activity by MeT in LNCaP cells (top) and PC3 cells (bottom). PC-3 cells were transfected with plasmids expressing AR and a probasin-luciferase reporter for 4 hours prior to 20 hours treatment with MeT or DHT; LNCaP cells were transfected only with the probasin-luciferase reporter. Transcriptional activity values represent the mean of six technical replicates; results are representative of three independent experiments. Error bars are SEM. Unpaired t tests were used to compare MeT and DHT (***, P < 0.001; ****, P < 0.0001). D, Venn diagram showing the overlap of AR cistromes in LNCaP cells treated with DHT or MeT (1 nmol/L each). E, Read density plots (top) and heatmaps (bottom) representing AR ChIP-seq peak sets from D . Far right heatmaps (“MeT/DHT”) represent MeT ChIP-seq signals corrected to DHT ChIP-seq signals. F, Most highly enriched motifs in the MeT (top) and DHT (bottom) cistromes. Motifs were identified using a de novo Gibbs motif sampling approach. P values represent enrichment over genomic background, calculated using Fisher exact tests. G, Heatmap of RNA-seq data for genes differentially expressed by 24 hours of MeT treatment (compared with Vehicle; FDR < 0.05). The heatmap was generated using ClustVis after applying unit variance scaling to each gene.

Article Snippet: Statistical analyses for grouped quantitative data were carried out using two-tailed unpaired t test or ANOVA (GraphPad Prism 9).

Techniques: Activity Assay, Growth Assay, Activation Assay, Transfection, Expressing, Luciferase, ChIP-sequencing, Sampling, RNA Sequencing, Generated

Journal: Cancer Research Communications

Article Title: Potent Stimulation of the Androgen Receptor Instigates a Viral Mimicry Response in Prostate Cancer

doi: 10.1158/2767-9764.CRC-21-0139

Figure Lengend Snippet: DNA replication and repair pathways are repressed by potent androgenic stimulation of prostate cancer cells. A, Normalized enrichment scores (NES) for Hallmark gene sets representing RNA-seq data from LNCaP cells treated with 1 nmol/L MeT or DHT for 24 hours. B and C, Heatmap of RNA-seq data for androgen-regulated genes associated with DNA repair and DNA replication in LNCaP cells treated with 1 nmol/L MeT or 1 nmol/L DHT for 24 hours. Heatmaps were generated using ClustVis after applying unit variance scaling to each gene. D, Average read density plots for AR chromatin binding proximal (<100 kb) to DNA repair/replication genes in LNCaP cells treated with 1 nmol/L MeT or 1 nmol/L DHT for 4 hours. E, Cell-cycle analysis by DAPI labeling and flow cytometry after 72 hours of treatment with 1 nmol/L MeT or 1 nmol/L DHT. Unpaired t tests were used to compare data at different cell-cycle phases (i.e., G 1 , S, and G 2 –M) between treatment groups (**, P < 0.01; ****, P < 0.0001). F, Flow cytometry–based Annexin V/7-AAD apoptosis assays after 96 hours of treatment with MeT or DHT. Data represent the mean ± SEM of triplicate samples and are representative of three independent experiments. Dead and dying cell proportions were compared with vehicle using ANOVA and Tukey multiple comparison tests (**, P < 0.01; ****, P < 0.0001; NS, not significant). G, Assessment of DNA DSBs after androgen treatment. γH2AX foci were quantitated in LNCaP cells 6 hours after treatment with indicated doses of MeT, DHT, or a positive control (H 2 O 2 ). Error bars are SEM. P values (day 7) were determined using ANOVA and Dunnett multiple comparisons tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significant).

Article Snippet: Statistical analyses for grouped quantitative data were carried out using two-tailed unpaired t test or ANOVA (GraphPad Prism 9).

Techniques: RNA Sequencing, Generated, Binding Assay, Cell Cycle Assay, Labeling, Flow Cytometry, Comparison, Positive Control

Journal: Cancer Research Communications

Article Title: Potent Stimulation of the Androgen Receptor Instigates a Viral Mimicry Response in Prostate Cancer

doi: 10.1158/2767-9764.CRC-21-0139

Figure Lengend Snippet: MeT elicits viral mimicry and enhances interplay with T cells in a mouse model of prostate cancer. A, Expression of HLA genes and B2M , as determined by qRT-PCR, following 3 or 6 days of treatment of LNCaP cells with the indicated doses of MeT or DHT or a vehicle control. Gene expression was normalized to GAPDH . Error bars are SEM; P values (treatment compared with vehicle at each timepoint) were determined using ANOVA and Dunnett multiple comparisons tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). B, AR activity is associated with the Reactome “class I MHC-mediated antigen processing and presentation” gene set in TCGA (left) and SU2C (right) datasets. Activity scores were calculated using ssGSEA. P and r values were determined using Pearson correlation tests. C, Western blot analysis showing AR protein expression in RM1 cells following treatment with the indicated doses of MeT or vehicle control in both full (androgen replete) and charcoal-stripped (androgen depleted) media. GAPDH is shown as a loading control. D, MeT suppresses growth of RM1 cells, as determined by sulforhodamine B colorimetric assay mean absorbance (550 nm) is shown at the indicated timepoints; error bars are ± SEM. P values were determined using unpaired t tests at day 5 (***, P < 0.001; ****, P < 0.0001). E, Expression of ERVs ( Rltr1B , Rltr45, Erv-L, Erv3 Mta ) , Line-1 , Rig-I Irf7, and Psmb9 in RM1 cells as determined by qRT-PCR following 3 days of treatment with the indicated doses of MeT. Gene expression was normalized to Hprt. Vehicle for each gene was set to 1. Error bars are SEM; P values (treatment compared to vehicle at each timepoint) were determined using ANOVA and Dunnett multiple comparisons tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). F, ICS assay demonstrating IFNγ + in CD8 + T cells. T cells were cocultured with RM1 cells treated with indicated doses of MeT or DHT for 3 days. Vehicle control for each AR ligand was set to 1. Error bars are SEM; P values (treatment compared with vehicle at each timepoint) were determined using ANOVA and Dunnett multiple comparisons tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Statistical analyses for grouped quantitative data were carried out using two-tailed unpaired t test or ANOVA (GraphPad Prism 9).

Techniques: Expressing, Quantitative RT-PCR, Control, Gene Expression, Activity Assay, Western Blot, Colorimetric Assay