Androgen receptor signalling in macrophages promotes TREM-1-mediated prostate cancer cell line migration and invasion

Abstract

The androgen receptor (AR) is the master regulator of prostate cancer (PCa) development, and inhibition of AR signalling is the most effective PCa treatment. AR is expressed in PCa cells and also in the PCa-associated stroma, including infiltrating macrophages. Macrophages have a decisive function in PCa initiation and progression, but the role of AR in macrophages remains largely unexplored. Here, we show that AR signalling in the macrophage-like THP-1 cell line supports PCa cell line migration and invasion in culture via increased Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signalling and expression of its downstream cytokines. Moreover, AR signalling in THP-1 and monocyte-derived macrophages upregulates IL-10 and markers of tissue residency. In conclusion, our data suggest that AR signalling in macrophages may support PCa invasiveness, and blocking this process may constitute one mechanism of anti-androgen therapy.

Fig. 1. AR expression in PCa-resident macrophages.

a Immunofluorescence staining of a FFPE prostatectomy specimen from a systemically untreated PCa patient showing the presence of AR in CD163+ cells. Nuclei were stained with DAPI (dark blue), whereas AR and CD163 were visualized in light blue and purple, respectively (scale bar = 100 µm). Lower panel are magnifications of inserts (scale bar = 50 µm). Dotted circles identify DAPI+, AR+ and CD163+ cells. These images are representative of immunofluorescence stainings performed in FFPE prostatectomy specimen from three different patients. Pictures were taken in at least five areas to assess marker expression. b Multiplex immunofluorescence analysis. AMACR staining indicating the tumorous area. Representative image of 200–300 scans. Scale bar = 5000 µm (Left panel), 500 µm (Right panel; insert). c Multiplex immunofluorescence analysis. Representative tumorous area in a FFPE prostatectomy specimen stained for CD163, AR, HLA-DRA and CD14 and all combined. Each triangle represents a positive cell included in the quantification. Representative image of 200–300 scans. Scale bar = 5000 µm (Top left panel), scale bar = 80 µm (inserts). d Quantification of multiplex immunofluorescence analysis. Boxplot (median values with interquartile range) showing fraction of HLA-DR+ and/or CD163+ and/or CD14+ cells expressing AR, in the tumour area, in the 200 µm tumour border zone around the tumour area and in the area distant from the tumour in 20 FFPE prostatectomy specimen. Datapoints show individual patients. p-Values were calculated using a Wilcoxon rank-sum test with a cutoff for significance of 0.05. Source data are provided as a source datafile.

Fig. 2. AR expression and nuclear translocation in THP-1 cells.

a Expression of the macrophage marker CD68 (green) in THP-1 cells at steady state (no treatment) and following 2 days of PMA stimulation. Nuclei were stained with DAPI (dark blue). Scale bar = 50 µm. Representative of three images per condition. b RT-PCR showing AR expression at the RNA level in human cancer cell lines of prostate epithelial (CWR-R1) and monocytic (THP-1^PMA;IFNG;LPS) origin. GAPDH was used as a house-keeping control gene. This experiment was performed two times. c Western blot showing AR expression at the protein level in human cell lines originated from prostate cancer (LNCaP), melanoma (M14) and monocytic leukaemia (THP-1^PMA;IFNG;LPS). β-Actin was used as a loading control. This experiment was performed two times. Source data are provided as a source datafile. d Western blot showing AR expression at the protein level in the subcellular chromatin fraction of THP-1^PMA;IFNG;LPS cells and CWR-R1 human PCa cells upon R1881 stimulation. Pol-II was used as a loading control of the chromatin fraction. This experiment was performed two times. Source data are provided as a source datafile.

Fig. 3. AR signalling in THP-1 cells affects PCa cell line migration.

a Workflow showing the procedure of THP-1 cell differentiation into macrophage-like cells and generation of CM. THP-1 cells were stimulated with PMA (Day 0), IFN-γ and LPS (Day 2) and exposed to Vehicle or Drug on Day 3. After 24 h of stimulation, cells were carefully washed and replenished with fresh medium. Medium was collected as ‘CM’ after 48 h. b Scratch assay of CWR-R1 human PCa cells cultured in normal medium (NM) alone or in combination with CM of THP-1 cells stimulated with vehicle or RD162 at base line (0 h) and after 24 h. Charcoal-stripped (DCC) medium was used as a control. Representative of three different images per condition. Scale bar = 200 µm. c Quantification of three independent scratch assays. Datapoints show mean value of three technical replicates in each experiment. CWR-R1 cell migration is assessed through closure of the scratch after 24 h relative to day 0. Error bars represent the s.e.m.. *: p = 0.02 and **: p = 0.002. p-Values were calculated from means of three biological replicates using a One-way Anova test with a cutoff for significance of 0.05. Source data are provided as a source datafile.

Fig. 4. AR signalling in THP-1 cells affects prostate tumour growth in vivo.

a Representative images of both normal chick embryo chorioallantoic membrane (CAM) and PC3 PCa cells derived tumours growing on the CAMs replenished with CM of DMSO or RD162-treated THP-1^PMA;IFNG;LPS cells, CCL2 or NaCl,. The only tumour grown in NaCl-treated CAM conditions is shown. All other NaCl-treated CAMs showed no sign of tumour growth. Scale bar = 500 µm. b Growth curves of PC3 tumours grafted into CAMs showing tumour volume over time in the different treatment conditions. Datapoints represent the average tumour volume as a percentage of the tumour volume at the start of treatment (EDD10). Error bars represent s.e.m. of 6–10 biological replicates per condition in one experiment with one batch of THP-1^PMA;IFNG;LPS cell CM (DMSO CM and RD162 CM). *: p = 0.02. p-value comparing DMSO CM versus RD162 CM was calculated using a Two-way Anova with a cutoff for significance of 0.05. Source data are provided as a source datafile. c The expression of human vimentin in disseminated PC3 cells into CAM tissue distant from the primary tumour site (normal CAM) in the different treatment conditions and normalized to the reference gene human Cyclophylin A (2^−dCt). Error bars represent the s.e.m. of 5–9 biological replicates per condition in one experiment with one batch of THP-1^PMA;IFNG;LPS cells CM (DMSO CM and RD162 CM). p = 1.0. p-value comparing DMSO CM versus RD162 CM was calculated using a One-way Anova test with a cutoff for significance of 0.05. NS: no significant difference. Source data are provided as a source datafile.

Fig. 5. ChIP-seq analysis shows distinct AR-binding profiles in THP-1 cells.

a Venn diagrams showing the level of overlap between AR peaks in vehicle and R1881 conditions (upper panel), the overlap between H3K27ac peaks in vehicle and R1881 conditions (middle panel) and the overlap between AR and H3K27ac peaks in R1881 conditions (lower panel) in THP-1^PMA;IFNG;LPS cells. b Snapshot of AR and H3K27ac peaks in THP-1^PMA;IFNG;LPS cells. Genomic coordinates, gene name and tag counts are indicated. AR peaks in vehicle and R1881 conditions are depicted in blue and red, respectively. H3K27ac peaks in vehicle and R1881 conditions are depicted in green and purple, respectively. Range of normalized read counts is shown on the y axis. c Genomic distribution of AR-binding sites relative to the most proximal gene in THP-1^PMA;IFNG;LPS cells. d Clustered heatmap depicts all the AR-binding sites in THP-1^PMA;IFNG;LPS cells vertically aligned, within a 5 kb window. H3K27ac peaks are shown for the same genomic locations. e Motif analysis of AR-binding sites in THP-1^PMA;IFNG;LPS cells identifies members of the AP-1 complex, including FOS and JUN. Z-score of enrichment is shown on the x axis. f RNA expression of AR transcriptional activators in single CD14+ and/or CD11b+ cells isolated from PCa biopsies. In the tSNE plot, every dot represents a single cell. g Ingenuity pathway analysis of genes most proximal to AR-binding sites in THP-1^PMA;IFNG;LPS cells identifies TREM-1 as the most-enriched signalling pathway (indicated in red).

Fig. 6. AR regulates expression of TREM-1 associated genes in THP-1 cells.

a RT-QPCR analysis to assess fold change expression of TREM-1 (2^-ΔΔCt) in THP-1^PMA;IFNG;LPS cells upon R1881 stimulation compared to vehicle control (set at 1) and normalized to TBP expression. Datapoints show the mean value of three technical replicates in each experiment, while error bars show the s.e.m. of two independent experiments. Source data are provided as a source datafile. b RT-QPCR analysis to assess the fold change expression (2^−ΔΔCt) upon R1881 exposure of 28 TREM-1-associated genes in THP-1^PMA;IFNG;LPS cells, relative to TBP expression and normalized to vehicle conditions (set at 1). Each datapoint represents the mean relative gene expression in three technical replicates. The mean fold change expression (vehicle over R1881) of all genes is indicated, while error bar show the s.e.m. of two independent experiments. *: p = 0.001. p-Value for the comparison of fold change of all genes separately upon vehicle exposure (set at 1) versus R1881 exposure, calculated using an unpaired parametric Studentʼs t test with a cutoff for significance of 0.05. Source data are provided as a source datafile. c RT-QPCR analysis to assess fold change expression (2^−ΔΔCt) upon R1881 exposure of 5 TREM-1 associated chemokines in THP-1^PMA;IFNG;LPS cells, relative to TBP expression. Green bars show R1881 conditions normalized to vehicle (red box and dotted line). Datapoints show mean values and error bars the s.e.m. of two independent experiments with three technical replicates each. Source data are provided as a source datafile. d Fold change expression (2^−ΔΔCt) of TREM-1 associated chemokines in THP-1^PMA;IFNG;LPS cells normalized to TBP expression. Cells were exposed to scramble peptide or to the inhibitory TREM-1 peptide (iTREM1) in combination with R1881 and/or RD162. Values were normalized to scramble peptide alone. Datapoints show mean values and error bars the s.e.m. of two independent experiments with three technical replicates each. Source data are provided as a source datafile. e Kaplan–Meier curves of disease/progression-free survival in PCa patients in relation to levels of TREM-1 expression (TCGA database, No. of patients = 491). Z-score of ± 1. Fifty-seven cases with a high TREM-1 expression (red line; 18 relapses/disease progressions) and 434 cases with a low TREM-1 expression (blue line; 73 relapses/disease progressions). P = 0.0042. Log-rank chi square test was used to calculate the p-value with a cutoff for significance of 0.05. Source data are provided as a source datafile.

Fig. 7. TREM-1 signalling in THP-1 cells promotes PCa migration and invasion.

a Representative images of three independent scratch assays of CWR-R1 cells cultured in CM of THP-1^PMA;IFNG;LPS cells stimulated with vehicle, RD162, scramble peptide or inhibitory TREM-1 peptide (iTREM-1). Three technical replicates were included in each experiment. Scale bar = 200 µm. b Quantification of three independent migration scratch assays. Closure of the scratch by CWR-R1 cells after 24 h relative to day 0. Datapoints show mean values of three technical replicates in each experiment. Error bars represent the s.e.m. *: p = 0.05, one-way Anova test was used to calculate the p-value with a cutoff for significance of 0.05. Source data are provided as a source datafile. c Representative images of two transwell invasion assays (three technical replicates) of CWR-R1 cells cultured in normal medium alone or in a combination with CM of THP-1^PMA;IFNG;LPS cells stimulated with vehicle, RD162, vehicle CM with scramble peptide or vehicle CM with TREM-1 inhibitory peptide. Cells that invade the matrigel and passed through the membrane after 72 h of culture were stained with crystal violet. d Quantification of the transwell invasion assays. Invasion was quantified by optical density (OD) of crystal violet stained cells. Datapoints show mean values and error bars the s.e.m. of two independent experiments with three technical replicates each. Source data are provided as a source datafile. e Representative images of two transwell migration assays (three technical replicates) of CWR-R1 cells cultured in normal medium alone or in combination with CM of THP-1^PMA;IFNG;LPS cells stimulated with vehicle or RD162. Additionally, CWR-R1 cells cultured in normal medium in combination with CM of vehicle stimulated THP-1^PMA;IFNG;LPS cells alone or supplemented with blocking antibodies against CCL13, CCL2, CCL3, CCL7, CXCL8 or the combination of all. Cells that passed through the membrane after 72 h of culture were stained with crystal violet. f Quantification of the migration transwell assays. Invasion was quantified by optical density (OD) of crystal violet stained cells. Datapoints show mean values and error bars the s.e.m. of two independent experiments with three technical replicates each. Source data are provided as a source datafile.

Fig. 8. AR signalling upregulates tissue macrophage-related markers in THP-1 cells.

a Snapshot of AR and H3K27ac sites proximal to genes associated with macrophage differentiation in THP-1^PMA;IFNG;LPS cells (IL-10, CD209, CD206 and CD163). Genomic coordinates, gene name and tag counts are indicated. AR peaks in vehicle and R1881 conditions are depicted in blue and red, respectively. H3K27ac peaks in vehicle and R1881 conditions are depicted in green and purple, respectively. Range of normalized read counts is shown on the y axis. b RT-QPCR analysis to assess fold change expression (2^−ΔΔCt) of CD163, IL-10, CD206, CD209 in THP-1^PMA;IFNG;LPS cells upon R1881 stimulation, relative to GAPDH expression and normalized to vehicle conditions (dotted line). Datapoints show mean values and error bars the s.e.m. of two independent experiments with three technical replicates each. Source data are provided as a source datafile. c Flow-cytometry analysis of surface markers CD206 and CD163 expression in MDM cells. Unstained cells (upper-left) were used as negative control. Twenty-four hrs vehicle stimulated (upper-right), R1881 (10⁻¹⁰ M) stimulated (lower-left) and simultaneous R1881 (10⁻¹⁰ M) and RD162 (10⁻⁷ M) stimulated cells (lower-right). The percentage of double positive CD163+/CD206+THP-1^PMA;IFNG;LPS cells in the various conditions is represented in bold. Plots are representative for three independent experiments.

Fig. 9. Anti-androgen treatment reduces the number of TAMs in the prostate.

a Graphic visualization of the study design: 10 untreated PCa patients and 10 PCa patients treated with the anti-androgen bicalutamide prior to prostatectomy were matched for Gleason pathology score, serum PSA level, age and TNM classification. b PCA visualization of the Kmean unsupervised cluster analysis. Group 1 and Group 2 classify patients in untreated and treated patients, respectively. Numerical data in Supplementary Table 3. P = 0.024, Pearson correlation test was used to calculate the p-value with a cutoff for significance of 0.05. c Quantification of the percentage of CD163+ TAMs in the entire population of HLA-DR+ and/or CD14+ cells in prostatectomy specimen of untreated and bicalutamide treated patients. Datapoints show individual patients and error bars show the s.e.m. p = 0.0968. Paired parametric Studentʼs t test was used to calculate the p-value with a cutoff for significance of 0.05. Source data are provided as a source datafile.