Fig 2: CHRM4/AKT/MYCN upregulates IFNA17 and PDL1 in prostate cancer after androgen-deprivation therapy.A Immunoblots of CHRM4, IFNA17, phosphorylated (p)-AKT, AKT, and MYCN proteins in C4-2 cells cultured in charcoal-stripped serum (CSS)-containing medium for 1 or 2 weeks, followed by treatment with 10 nM dihydrotestosterone (DHT) for 24 h. B Immunoblots showing CHRM4, IFNA17, p-AKT, AKT, and MYCN protein levels in C4-2 cells expressing a non-targeting control (NC) or CHRM4 shRNA vector, then treated with 20 ng/ml IFNA17 protein for 24 h. C Relative mRNA levels of MYCN, IFNA17, neuroendocrine (NE) markers (CHGA, SYP, and ENO2), and immune checkpoints (PDL1 and CTLA4) in LASCPC01 cells with NC or MYCN siRNA transfection, measured by an RT-qPCR analysis. * vs. the NC, by a one-way ANOVA. D Relative mRNA levels of MYCN, IFNA17, PDL1, and CTLA4 in C4-2 cells with NC or MYCN siRNA transfection, followed 20 ng/ml IFNA17 protein treatment for 24 h, as measured by an RT-qPCR analysis. * vs. -IFNA17; # vs. the NC, by a two-way ANOVA. E Schematic of the predicted E-boxes and an introduced single-binding site mutant in regulatory sequence reporter constructs of human IFNA17 (GRCh38:9). F ChIP assay showing binding of MYCN and acetyl-H3 to predicted E-box1 and E-box2 of the IFNA17 gene regulatory sequence in C4-2 cells following treatment with 20 ng/ml of the IFNA17 protein for 24 h. Sheared chromatin from nuclear extracts was precipitated with antibodies to MYCN and acetyl-H3, and predictive primers (E, 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. -IFNA17, by a one-way ANOVA. G ChIP assay showing reduced binding of MYCN and acetyl-H3 to the predicted E-box1 and E-box2 of the IFNA17 gene regulatory sequence in LASCPC01 cells with NC or MYCN siRNA transfection. * vs. the NC, by a one-way ANOVA. Relative mean florecence intesity (MFI) of the GFP reporter gene containing wild-type (WT)- or mutant (M)-E-boxes from the IFNA17 regulatory sequence in C4-2 cells following CSS-containing medium or 10 nM DHT (H) or 20 ng/ml IFNA17 protein (I) treatment for 48 h. * vs. -CSS (H) or -IFNA17 (I); # vs. the WT, by a two-way ANOVA. Relative MFI of the GFP reporter gene containing WT- or M-E-boxes from the IFNA17 regulatory sequence in C4-2 cells co-transfected with the EV or MYCN cDNA vector (J), or NC or MYCN siRNA (K) for 48 h. * vs. the EV (J) or the NC (K); # vs. the WT, by a two-way ANOVA. L Schematic of the predicted E-boxes and an introduced single-binding site mutant in regulatory sequence reporter constructs of human PDL1 (GRCh38:9). ChIP assay showing increased binding of MYCN and acetyl-H3 to predicted E-box2 of PDL1 gene regulatory sequence in C4-2 cells with 20 ng/ml IFNA17 protein treatment for 48 h (M) or in LASCPC01 cells with NC or MYCN siRNA transfection (N). * vs. -IFNA17 (M) or the NC (N), by a one-way ANOVA. Relative MFI of the GFP reporter gene containing WT- or M-E-boxes from the PDL1 regulatory sequence in C4-2 cells with 20 ng/ml IFNA17 protein treatment for 48 h (O) or in LASCPC01 cells with NC or MYCN siRNA transfection (P). * vs. -IFNA17 (O) or the NC (P); # vs. the WT, by a two-way ANOVA. Quantification of the ChIP assay and relative MFI values are presented as the mean ± SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: CHRM4 is correlated with IFNA17 responsiveness in prostate cancer.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).

Fig 4: Target CHRM4 reduces the tumor growth and neuroendocrine differentiation of prostate cancer.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 representative 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.