Journal: Cell reports

Article Title: A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer

doi: 10.1016/j.celrep.2022.110417

Figure Lengend Snippet: (A) Schematic of 22Rv1/AR-V7-GFP reporter cell line. CRISPR/Cas9 editing and homology-directed repair were used to insert a GFP-containing cassette directly upstream of the cryptic exon 3 (CE3) stop codon in 22Rv1 cells. (B) Schematic of the CRISPR/Cas9 screening strategy used to identify regulators of AR/AR-V7 expression in 22Rv1/AR-V7-GFP cells. (C) Screen hits, plotted by STARS score on day 5 or day 12 after library transduction, determined by enrichment of sgRNAs in the sorted GFP-negative population as compared with the starting library pool. Top: Scatterplot of STARS scores for screen hits on day 5 versus day 12. For plotting purposes, hits that scored at only one timepoint were assigned a STARS score of 0.5 for the timepoint that they were not enriched. Bottom: Plots of false discovery rate (FDR) versus STARS score of hits from day 5 (left) or day 12 (right) timepoints. Selected high-scoring hits are labeled. (D) Arrayed validation of screen hits by RT-qPCR in parental 22Rv1 cells. Heatmap shows relative AR-FL and AR-V7 expression in 22Rv1 cells at the indicated timepoints after knockout of selected screen hits. mRNA levels are normalized to a control sgRNA (control98). Data are presented as the mean of n = 4 technical replicates. (E) Enrichment analysis showing gene ontology (GO) terms significantly enriched among screen hits scoring on either day 5 or day 12 with FDR < 0.25. See also Figure S1 and Tables S1 and S2.

Article Snippet: In vivo studies 5 million 22RV1 cells resuspended in 1:2 matrigel and conditioned media were injected subcutaneously into male NCRNU (CrTac:NCr- Foxn1 nu ) mice (Taconic, USA).

Techniques: CRISPR, Expressing, Transduction, Labeling, Biomarker Discovery, Quantitative RT-PCR, Knock-Out, Control

Journal: Cell reports

Article Title: A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer

doi: 10.1016/j.celrep.2022.110417

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: In vivo studies 5 million 22RV1 cells resuspended in 1:2 matrigel and conditioned media were injected subcutaneously into male NCRNU (CrTac:NCr- Foxn1 nu ) mice (Taconic, USA).

Techniques: Western Blot, Immunoprecipitation, Recombinant, Cell Viability Assay, Luciferase, Expressing, Gene Expression, shRNA, Software

Journal: Cell reports

Article Title: A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer

doi: 10.1016/j.celrep.2022.110417

Figure Lengend Snippet: (A) Relative AR-FL, AR-V7, and PRMT1 expression, as assessed by RT-qPCR, with or without PRMT1 knockdown by dox-inducible shRNA in the prostate cancer cell lines 22Rv1, VCaP, and LNCaP. Expression is shown relative to no dox. Error bars represent mean ± SD, n = 3 biological replicates. (B) Relative luciferase activity upon PRMT1 knockdown in LNCaP cells transduced with an androgen-responsive MMTV-Luciferase reporter. Luciferase activity is normalized to cell viability for each condition and shown relative to no dox. Error bars represent mean ± SD, n = 6 biological replicates. (C) Relative AR-FL expression in LNCaP cells after treatment with the PRMT1 inhibitor furamidine at the indicated doses. Expression is shown relative to DMSO. Error bars represent mean ± SD, n = 3 biological replicates. (D) Relative MMTV-Luciferase activity in LNCaP cells upon treatment with furamidine at the indicated doses. Luciferase activity is normalized to cell viability at each concentration and shown relative to DMSO. Error bars represent mean ± SD, n = 3 biological replicates. For A-D, statistical significance was determined by t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S2.

Article Snippet: In vivo studies 5 million 22RV1 cells resuspended in 1:2 matrigel and conditioned media were injected subcutaneously into male NCRNU (CrTac:NCr- Foxn1 nu ) mice (Taconic, USA).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, shRNA, Luciferase, Activity Assay, Transduction, Concentration Assay

Journal: Cell reports

Article Title: A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer

doi: 10.1016/j.celrep.2022.110417

Figure Lengend Snippet: (A) Western blot showing relative AR-FL and AR-V7 expression in parental prostate cancer cell lines. (B) Proliferation of AR-expressing (LNCaP, 22Rv1, VCaP) or non-AR-expressing (PC3) cell lines with or without dox-induced PRMT1 knockdown. Confluence readings were taken using an IncuCyte live-cell imager. Error bars represent mean ± SD of the following numbers of biological replicates: n = 4 (LNCaP), n = 8 (22Rv1), n = 4 (VCaP), n = 3 (PC3). (C) Doubling times of prostate cancer cell lines with or without PRMT1 knockdown, estimated by nonlinear regression of confluence readings shown in (A). Data are presented as mean with 95% CI. Statistical significance was determined by t-test. (D) Relative viability (normalized to DMSO) of prostate cancer cell lines after 5 days of treatment with furamidine at the indicated concentrations. Error bars represent mean ± SD, n = 3 biological replicates. **p < 0.01, ****p < 0.0001. See also Figure S7.

Article Snippet: In vivo studies 5 million 22RV1 cells resuspended in 1:2 matrigel and conditioned media were injected subcutaneously into male NCRNU (CrTac:NCr- Foxn1 nu ) mice (Taconic, USA).

Techniques: Western Blot, Expressing, Knockdown

Journal: Cell reports

Article Title: A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer

doi: 10.1016/j.celrep.2022.110417

Figure Lengend Snippet: (A) Heatmaps showing percent viability of prostate cancer cell lines after 7 days of combination treatment with the indicated doses of furamidine and enzalutamide. LNCaP/AR-Enh cells are derived from the parental LNCaP line and contain knock-in of an additional copy of the AR enhancer (Takeda et al., 2018). Viabilities are shown relative to the DMSO condition. Quantized heatmaps of Bliss synergy index are shown below each cell line. A box is drawn around the dose combination in each cell line that resulted in the maximum Bliss synergy score. Data represent the mean of n = 3 biological replicates. (B) Percent viabilities for single-agent compared to combination treatment at the doses indicated by boxes in (A) for each cell line. Dotted lines indicate predicted additive effect of enzalutamide (E) and furamidine (F), calculated by multiplying the percent viabilities upon single-agent treatment at the respective doses. Error bars represent mean ± SD, n = 3 biological replicates. (C) Western blot showing relative AR protein levels in LNCaP and LNCaP/AR-Enh cells upon furamidine treatment in the context of androgen depletion. Cells were seeded in media supplemented with charcoal stripped serum and treated with DMSO or furamidine (8 μM) for 5 days. (D) Densitometric quantification of western blot in (C). AR protein levels are normalized to actin and shown relative to parental LNCaP treated with DMSO. Error bars represent mean ± SD, n = 3 biological replicates. For A-D, statistical significance was determined by t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Impact of enzalutamide (Enz) or furamidine (Fur) monotherapy, or combination treatment, on 22Rv1 xenograft growth. Error bars represent mean ± SEM for the indicated number of biological replicates. *p < 0.05 for enzalutamide + furamidine vs enzalutamide at the final time point (t-test), **p < 0.01 for enzalutamide + furamidine versus each other condition by 2-way ANOVA and Tukey’s method.

Article Snippet: In vivo studies 5 million 22RV1 cells resuspended in 1:2 matrigel and conditioned media were injected subcutaneously into male NCRNU (CrTac:NCr- Foxn1 nu ) mice (Taconic, USA).

Techniques: Derivative Assay, Knock-In, Western Blot

Journal: Cell reports

Article Title: A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer

doi: 10.1016/j.celrep.2022.110417

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: In vivo studies 5 million 22RV1 cells resuspended in 1:2 matrigel and conditioned media were injected subcutaneously into male NCRNU (CrTac:NCr- Foxn1 nu ) mice (Taconic, USA).

Techniques: Western Blot, Immunoprecipitation, Recombinant, Cell Viability Assay, Luciferase, Expressing, Gene Expression, shRNA, Software

Journal: British Journal of Cancer

Article Title: Insulin-like growth factor binding protein-6 inhibits prostate cancer cell proliferation: implication for anticancer effect of diethylstilbestrol in hormone refractory prostate cancer

doi: 10.1038/sj.bjc.6602520

Figure Lengend Snippet: Inhibition of the proliferation of human prostate cancer cell lines LNCaP and PC-3 by diethylstilbestrol (DES). Cells were incubated for 24 h, and thereafter CM was aspirated away and the cells were incubated with CM containing various concentrations of DES. After 72 h, the number of viable cells was measured by MTT assay. Optical densities (OD) of cell lysates were measured at a wavelength of 540 nm. The values are expressed as means+s.d. ( n =3), and the P -values were <0.05 ( * ) and <0.01 (†).

Article Snippet: The human prostate cancer cell lines LNCaP and PC-3 were purchased from Dainippon Pharmaceutical (Tokyo, Japan) and cultured in RPMI (Sigma, St Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) (Moregate, Bulimba, Australia).

Techniques: Inhibition, Incubation, MTT Assay