Journal: Cancer Research

Article Title: Supraphysiological Androgens Promote the Tumor Suppressive Activity of the Androgen Receptor through cMYC Repression and Recruitment of the DREAM Complex

doi: 10.1158/0008-5472.CAN-22-2613

Figure Lengend Snippet: SPA repression of E2F target genes and prostate cancer proliferation depends on RBL1 and RBL2. A, E2F1 ChIP-seq volcano plot showing differentially bound E2F1 binding sites between EtOH- and SPA-treated LNCaP DKO RB1 −/− ;TP53 −/− cells. B, Overlap between RB1-bound sites in LNCaP and E2F1-bound sites in LNCaP DKO cells. These E2F1:RB1 bound sites were mapped to promoters of genes, which were overlapped with genes transcriptionally repressed by SPA. C, Graph of over-represented GO categories in overlapping RB1/E2F-bound and SPA-repressed genes. D, Growth assay results for knockout and parental cells treated with 10 μmol/L ENZ or SPA for 5 days. E–G , Cell-cycle profile changes induced by SPA on LNCaP ( E ), LNCaP DKO ( F ), or LNCaP DKO;RBL1/2 ( G ) cells using flow cytometry to measure EdU incorporation (S-phase) and FxCycle violet staining (DNA-content). H, R1881 dose–response assay comparing LNCaP DKO cells with LNCaP DKO;RBL1/2 cells with and without ectopic expression of RBL2. I, R1881 dose–response assay on VCAP and the VCAP RB1 –14 RB1 -knockout clonal cell line transfected with siRNA to RBL1/2 or a nontargeting control (NTC). *, P < 0.05, by one-way ANOVA with Dunnett multiple-comparison test. Data represent the mean ± SD.

Article Snippet: The following antibodies were used for Western blots: GAPDH (Cell Signaling Technology, no. 2118L), RB1 (Cell Signaling Technology, no. 9309S), RBL1 (Cell Signaling Technology, no. 89798S), RBL2 (Cell Signaling Technology, no. 13610S), TP53 (Santa Cruz Biotechnology, no. sc-126), AR (Cell Signaling Technology, no. 5153S), and p27 (Abcam, ab32034).

Techniques: ChIP-sequencing, Binding Assay, Growth Assay, Knock-Out, Flow Cytometry, Staining, Expressing, Transfection, Control, Comparison

Journal: Cancer Research

Article Title: Supraphysiological Androgens Promote the Tumor Suppressive Activity of the Androgen Receptor through cMYC Repression and Recruitment of the DREAM Complex

doi: 10.1158/0008-5472.CAN-22-2613

Figure Lengend Snippet: Androgen-repressed cell-cycle genes bound by the DREAM complex. A, RB-family, E2F1, and LIN37 genomic binding sites determined by ChIP-seq from LNCaP cells and mapped to annotated regions. B, Boxplots of GIGGLE similarity scores for RBL1 ChIP-seq from LNCaP cells. C, Boxplots of GIGGLE similarity scores for LIN37 ChIP-seq from LNCaP cells. D, Overlapping CUT&RUN peaks bound by RB1 LNCaP cells, RBL1 in LNCaP DKO-RBL2 cells, and RBL2 LNCaP DKO-RBL1 cells. E, RB-family co-bound sites mapped to gene promoters and overlapped with SPA-repressed genes in LNCaP DKO cells (FDR < 0.05). F, Heatmap of mean-centered log 2 (FPKM) RNA-seq values for the top 10 androgen-repressed genes bound by all three RB-family members in LNCaP and isogenic gene knockout sublines. G, GO enrichment analysis on androgen repressed genes co-bound by RB-family members. H and I , Chromatin peaks for ChIP-seq datasets for E2F1 (LNCaP DKO ), LIN37 (LNCaP), LIN9 (LNCaP), and IgG (LNCaP DKO ); and for CUT&RUN peaks for RB1 (LNCaP), RBL1 (LNCaP DKO-RBL2 ), RBL2 (LNCaP DKO-RBL1 ), and LNCaP DKO-RBL1/2 (PRL1L2) as a negative control for each antibody on the promoters for SPA-repressed genes CDK1 ( H ) and CCNA2 ( I ).

Article Snippet: The following antibodies were used for Western blots: GAPDH (Cell Signaling Technology, no. 2118L), RB1 (Cell Signaling Technology, no. 9309S), RBL1 (Cell Signaling Technology, no. 89798S), RBL2 (Cell Signaling Technology, no. 13610S), TP53 (Santa Cruz Biotechnology, no. sc-126), AR (Cell Signaling Technology, no. 5153S), and p27 (Abcam, ab32034).

Techniques: Binding Assay, ChIP-sequencing, RNA Sequencing, Gene Knockout, Negative Control

Journal: Cancer Research

Article Title: Supraphysiological Androgens Promote the Tumor Suppressive Activity of the Androgen Receptor through cMYC Repression and Recruitment of the DREAM Complex

doi: 10.1158/0008-5472.CAN-22-2613

Figure Lengend Snippet: Androgen-mediated repression of cell-cycle genes is dependent on RBL1 and RBL2. A, GSVA signature score heatmaps of gene expression signatures on RNA-seq data generated from LNCaP models treated with SPA (10 nmol/L R1881) for 48 hours. B, Heatmap of mean-centered log 2 (FPKM) RNA-seq values for the androgen-upregulated AR Activity signature genes. LNCaP and LNCaP-DKO data are replotted from . C, Plot of GSEA enrichment scores of MSigDB Hallmark gene sets representing SPA-induced gene expression changes in LNCaP DKO and LNCaP DKO-RBL1/2 cells. D, Heatmap of mean-centered log 2 (FPKM) RNA-seq values for the cell-cycle progression (CCP) signature genes. LNCaP and LNCaP-DKO data are replotted from . E, Heatmap of mean-centered log 2 (FPKM) RNA-seq values for the top 10 SPA-repressed genes in the LNCaP DKO-RBL1/2 cells. LNCaP and LNCaP-DKO data are replotted from .

Article Snippet: The following antibodies were used for Western blots: GAPDH (Cell Signaling Technology, no. 2118L), RB1 (Cell Signaling Technology, no. 9309S), RBL1 (Cell Signaling Technology, no. 89798S), RBL2 (Cell Signaling Technology, no. 13610S), TP53 (Santa Cruz Biotechnology, no. sc-126), AR (Cell Signaling Technology, no. 5153S), and p27 (Abcam, ab32034).

Techniques: Gene Expression, RNA Sequencing, Generated, Activity Assay