ar negative prostate cancer cell line pc3  (ATCC)

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: A CHRM4 and AR protein levels of LNCaP, 22Rv1, C4-2, C4-2-MDVR, PC3, and LASCPC01 cells, measured by a western blot analysis. B , C CHRM4 mRNA abundances in LNCaP and C4-2 cells during 1, 2, 3, 4, and 5 months of 20 μM MDV3100 treatment, measured by an RT-qPCR analysis. * vs. parental LNCaP or C4-2 cells, by a one-way ANOVA. D Relative CHRM4 mRNA levels of C4-2 cells cultured in charcoal-stripped serum (CSS)-containing medium for 5 and 10 days, followed by treatment with 10 nM dihydrotestosterone (DHT) for 24 h. Quantification of relative mRNA levels is presented as the mean ± SEM of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.0001. E CHRM4, androgen-responsive markers (KLK3 and NKX3-1), and AR protein levels in C4-2 cells cultured in CSS-containing medium for 5 and 10 days, followed by treatment with 10 nM DHT for 24 h. F CHRM4, KLK3, NKX3-1, and AR protein levels of LNCaP and C4-2 cells cultured in 20 μM MDV3100 for 1 week. G Relative mean expressions of the AR, KLK3, NKX3-1, and CHRM4 in LNCaP cells from 3 weeks to 11 months of androgen withdrawal (ADT) in the GDS3358 database. * vs. the control, by a one-way ANOVA. H GSEAs of TCGA prostate dataset revealing negative associations between high CHRM4 expression in prostate tissues with gene signatures representing androgen-responsive signaling (GO, Nelson, Wang, PID, and Hallmark). NES normalized enrichment score, FDR false discovery rate.

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: Western Blot, Quantitative RT-PCR, Cell Culture, Control, Expressing

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: A ChIP-sequencing analysis of the detected DNA-binding sites for the AR of the CHRM4 gene in cells in response to 0.5 or 4 h of AR-ligand R1881 treatment labeled as black boxes in the tracks. ChIP-sequencing data were downloaded from the Gene Expression Omnibus (GEO) (GSE84432) and analyzed by Genome Brower (Genomics Institute). B Schematic of the predicted AR resonse elements (AREs) and an introduced single- or double-binding site mutant in regulatory sequence reporter constructs of human CHRM4 (GRCh38:11). ChIP assay showing binding of the AR and acetyl-H3 to predicted AREs of the CHRM4 gene regulatory sequence following treatment of C4-2 cells with 10 nM dihydrotestosterone (DHT) ( C ) or 20 μM MDV3100 ( D ) for 24 h. Sheared chromatin from nuclear extracts was precipitated with antibodies to the AR and acetyl-H3, and predictive primers ( B , black arrows) were used to quantify the precipitated DNA by a qPCR. Enrichment of each protein to each site is given as a percentage of the total input and then normalized to IgG. * vs. the vehicle (Veh) ( C ) or DMSO ( D ), by a one-way ANOVA. E , F ChIP assay showing binding of the AR and acetyl-H3 to predicted AREs of the CHRM4 gene regulatory sequence in PC3 cells following stable transfection with an empty vector (EV) or AR cDNA vector ( E ) or in C4-2 cells with a non-targeting control (NC) or AR siRNA transfection ( F ). * vs. the EV ( E ) or NC ( F ), by a one-way ANOVA. G Relative CHRM4 and AR mRNA levels of C4-2 cells transfected with the NC or AR siRNA, measured by an RT-qPCR analysis, * vs. the NC. H , I Relative mean florescence intensity (MFI) of the GFP reporter gene containing a wild-type (WT)- or mutant (M)-ARE from the CHRM4 regulatory sequence in C4-2 cells following treatment with 10 nM DHT ( H ) or 20 μM MDV3100 ( I ) for 48 h. * vs. WT; # vs. the vehicle (Veh) ( H ) or DMSO ( I ), by a two-way ANOVA. J , K Relative MFI of the GFP reporter gene containing a WT- or M-ARE from the CHRM4 regulatory sequence in PC3 cells following stable transfection with the EV or AR cDNA vector ( J ) or in C4-2 cells following NC or AR siRNA transfection ( K ). * vs. the EV ( J ) or NC ( K ), by a two-way ANOVA. Quantification of the ChIP assay, relative MFI values, and mRNA levels are presented as the mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: ChIP-sequencing, Binding Assay, Labeling, Gene Expression, Mutagenesis, Sequencing, Construct, Stable Transfection, Plasmid Preparation, Control, Transfection, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: A , B CHRM4 and NE marker (CHGA, SYP, and ENO2) mRNA levels in C4-2 cells stably transfected with an empty vector (EV) or a CHRM4-expressing vector ( A ) or in PC3 cells stably transfected with the non-targeting control (NC) or CHRM4 shRNA vector (B) , measured by an RT-qPCR analysis, * vs. the EV ( A ) or NC ( B ), by a one-way ANOVA. C Western blot showing CHRM4 and NE marker protein levels in CHRM4-modified C4-2 and PC3 cells. D GSEA analysis of TCGA prostate cancer dataset revealed positive correlations between higher CHRM4 expression in prostate tissues and gene profiles reflecting NEPC-responsiveness. NES, normalized enrichment score; FDR, false discovery rate. E – H Relative cell migration and invasion through Matrigel (E , F) and proliferation (G , H) of CHRM4-overexpressing C4-2 ( E , G ) or CHRM4-knockdown (KD) PC3 cells ( F , H ). n = 5 per group. * vs. the EV ( E , G ) or NC ( F , H ), by a one-way ANOVA. I – K Tumor growth analysis ( I , J ) and tumor weights ( K ) of CHRM4-KD PC3 cells subcutaneously inoculated into male nude mice for 8 weeks. Tumor weights were measured on the day tumors were collected. Tumor sizes were measured once a week and analyzed by a one-way ANOVA. IHC staining ( L ) and representative intensities ( M ) of CHRM4, ENO2, CHGA, and KI67 in subcutaneous tumors from J . * vs. NC-bearing tumors, by a two-tailed Student’s t -test. Quantification of relative mRNA levels, and migration, invasion, and proliferation are presented as the mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: Marker, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, shRNA, Quantitative RT-PCR, Western Blot, Modification, Migration, Knockdown, Immunohistochemistry, Two Tailed Test

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: A GSEAs of TCCA prostate cancer dataset showing that high abundance of CHRM4 mRNA in prostate cancer samples was positively linked to a wide range of cytokine-responsive gene signatures (GO, KEGG, and BIOCARTA). NES, normalized enrichment score; FDR, false discovery rate. B Venn diagram showing the number of overlapping genes identified in the top four cytokine-responsive gene sets. C The list of five overlapping gene candidates includes INHBC, IFNA17, IFNG, IL1RN , and TNFSF8 from ( B ). Pearson correlations among the five candidate genes and CHRM4 were analyzed in TCGA prostate cancer dataset by XY correlation analyses in GraphPad Prism. Relative mRNA levels of INHBC, IFNA17, IFNG , and IL1RN in C4-2 cells stably transfected with an empty vector (EV) or a CHRM4-expressing vector ( D ) or in PC3 cells stably transfected with a non-targeting control (NC) or CHRM4 shRNA vector, examined by an RT-qPCR. * vs. the EV ( D ) or NC ( E ), by a one-way ANOVA. F Kaplan–Meier analyses of IFNA17 alterations in the GSE21032 dataset. A log-rank (Mantel–Cox) test was used for the survival curve analysis. Hazard ratio = 0.3806, p = 0.0378. G Mean expression levels of IFNA17 in normal prostate ( n = 28), primary prostate cancer ( n = 111), and metastatic prostate cancer ( n = 13) samples in the GSE21032 dataset. * vs. normal prostate; # vs. Primary, *p < 0.05, ***p < 0.001, by a two-way ANOVA. H Mean expression levels of IFNA17 in prostate cancer patient samples in the GSE21032 dataset by pathologic Gleason scores (GSs). * vs. GS6. * p < 0.05, by a one-way ANOVA. I IFNA17 protein levels in LNCaP, VCaP, C4-2, PC3, LASCPC01, and C4-2-MDVR cells, measured by a Western blot analysis. IFNA17 cytokine concentrations in supernatants of cultured medium derived from LNCaP, VCaP, C4-2, PC3, LASCPC01, and C4-2-MDVR cells ( J ) or C4-2 and PC3 cells expressing the EV and CHRM4 cDNA or the NC and CHRM4 shRNA vectors ( K ), measured with an ELISA kit. * vs. LNCaP cells ( J ) or the EV ( K ); # vs. the NC ( K ), by a two-way ANOVA. Quantification of relative mRNA levels and IFNA17 contents is presented as the mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. L GSEAs of TCGA prostate dataset revealing negative associations between high IFNA17 expression in prostate tissues with gene signatures representing androgen-responsive signaling (GO, Nelson, Wang, PID, and Hallmark). NES normalized enrichment score, FDR false discovery rate. M IFNA17 cytokine concentrations in patient sera derived from benign prostatic hyperplasia (BHP; n = 23), primary prostate cancer ( n = 16), and castration-resistant prostate cancer (CRPC) samples ( n = 8). * vs. BPH; # vs. primary prostate cancer, by a two-way ANOVA. N Representative images of IHC staining of CHRM4 and CHGA in selected tissue sections from patients diagnosed with BHP, primary prostate cancer, and CRPC from ( M ).

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, shRNA, Quantitative RT-PCR, Western Blot, Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: Various prostate cancer cells were treated with 0, 1, 5, 10, 25, 50, and 100 μM of the small-molecule drugs, ceritinib ( A ), PD102807 ( B ), and LY2033298 ( C ) for 24 h, and cell viability was assessed by an MTT colorimetric assay. * vs. the control (0 μM). n = 8 per group. D , E Sphere-formation assay of PC3 and LASCPC01 cells treated with DMSO or 5 μM ceritinib during 1 week. * vs. DMSO. n = 5 per group by a t -test. F , G Relative CHRM4, MYCN, IFNA17, NE marker (CHGA, ENO2, and SYP), and immune checkpoint (PDL1 and CTLA4) mRNA levels in PC3 and LASCPC01 cells treated with DMSO or ceritinib at 10 and 25 μM for 24 h, as measured by an RT-qPCR analysis. * vs. DMSO, by a one-way ANOVA. Quantification of relative mRNA expressions is presented as the mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. H , I Tumor growth monitoring of LASCPC01 cells subcutaneously injected into male nude mice. One month after injection, DMSO or ceritinib (25 mg/kg) was intraperitoneally inoculated into mice once a week for 4 weeks. The tumor volume was measured every week, and tumor tissues were collected on the last day of the experiment. DMSO-injected mice ( n = 13); ceritinib-injected mice ( n = 14). * vs. DMSO, *** p < 0.001, by a t -test. IHC staining ( J ) and representa t ive intensities ( K ) of CHRM4, IFNA17, MYCN, KI67, ENO2, and PDL1 in subcutaneous tumors from I . * vs. DMSO. ** p < 0.01, *** p < 0.001. Significance was examined by a two-tailed Student’s t -test. L A schematic summary of this study. Our study focused on androgen-deprivation therapy (ADT)-induced NE prostate cancer (NEPC) to determine the mechanism by which androgen receptor (AR) loss of function might promote CHRM4-driven AKT/MYCN signaling leading to increased IFNA17 and PDL1 expressions. Increased abundances of IFNA17 and PDL1 may be regulated by the MYCN transcription factor through a positive feedback mechanism. Serum IFNA17 levels can be considered a prognostic biomarker in NEPC-like prostate cancer, and targeting CHRM4 may have the potential to inhibit NEPC progression.

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: Colorimetric Assay, Control, Tube Formation Assay, Marker, Quantitative RT-PCR, Injection, Immunohistochemistry, Two Tailed Test, Biomarker Discovery