YY1 complex in M2 macrophage promotes prostate cancer progression by upregulating IL-6

Abstract

Background: Tumor-associated macrophages are mainly polarized into the M2 phenotype, remodeling the tumor microenvironment and promoting tumor progression by secreting various cytokines.

Methods: Tissue microarray consisting of prostate cancer (PCa), normal prostate, and lymph node metastatic samples from patients with PCa were stained with Yin Yang 1 (YY1) and CD163. Transgenic mice overexpressing YY1 were constructed to observe PCa tumorigenesis. Furthermore, in vivo and in vitro experiments, including CRISPR-Cas9 knock-out, RNA sequencing, chromatin immunoprecipitation (ChIP) sequencing, and liquid-liquid phase separation (LLPS) assays, were performed to investigate the role and mechanism of YY1 in M2 macrophages and PCa tumor microenvironment.

Results: YY1 was highly expressed in M2 macrophages in PCa and was associated with poorer clinical outcomes. The proportion of tumor-infiltrated M2 macrophages increased in transgenic mice overexpressing YY1. In contrast, the proliferation and activity of anti-tumoral T lymphocytes were suppressed. Treatment targeting YY1 on M2 macrophages using an M2-targeting peptide-modified liposome carrier suppressed PCa cell lung metastasis and generated synergistic anti-tumoral effects with PD-1 blockade. IL-4/STAT6 pathway regulated YY1, and YY1 increased the macrophage-induced PCa progression by upregulating IL-6. Furthermore, by conducting H3K27ac-ChIP-seq in M2 macrophages and THP-1, we found that thousands of enhancers were gained during M2 macrophage polarization, and these M2-specific enhancers were enriched in YY1 ChIP-seq signals. In addition, an M2-specific IL-6 enhancer upregulated IL-6 expression through long-range chromatin interaction with IL-6 promoter in M2 macrophages. During M2 macrophage polarization, YY1 formed an LLPS, in which p300, p65, and CEBPB acted as transcriptional cofactors.

Conclusions: Phase separation of the YY1 complex in M2 macrophages upregulated IL-6 by promoting IL-6 enhancer-promoter interactions, thereby increasing PCa progression.

Keywords: cytokines; macrophages; prostatic neoplasms; tumor microenvironment.

Figure 1

In vivo YY1 overexpression increased M2 macrophage infiltration and prostate cancer progression. (A–C) Flow cytometry assay of CD163 in peritoneal cavity–derived and bone marrow–derived macrophages (PCDM and BMDM) derived from transgenic mice overexpressing YY1 or wild-type mice. (D) RM-1 cells were subcutaneously injected into transgenic mice overexpressing YY1 or wild type, and tumor volume was examined during the next 4 weeks. (E, F) Multiplex fluorescence immunohistochemistry staining of the indicated subcutaneous tumor tissues showed the expression of YY1 and the infiltration of F4/80⁺CD163⁺ and F4/80⁺CD86⁺ macrophages, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. (G) Flow cytometry of harvested subcutaneous tumors showing the CD163, CD4, and CD8 proportion in tumor-infiltrated immune cells. (H) ELISA showed the IFNγ expression in tumor tissue suspension and the indicated mice serum. (I) RM-1 cells were subcutaneously injected into oe-YY1 mice, and clodronate liposome/PBS liposome was intraperitoneally injected 7 days before subcutaneous tumorigenesis. (J) RM-1 subcutaneous tumorigenesis in oe-YY1 chimeric and wild-type mice. (K) RM-1 cells were injected into the tail vein of oe-YY1 transgenic mice aged 6–8 weeks followed by the indicated treatments to construct pulmonary metastasis model (6 mice every group), and Kaplan-Meier survival plot visualized the survival proportion of indicated groups. Premixed M2pep-siYY1 was injected by tail vein every 4 days at a dose of 15 mL/kg 2 weeks after the tumor cells were injected. Anti-PD-1 was given intraperitoneally every 4 days at a dose of 8 mg/kg 2 weeks after the tumor cells were injected. (L) CT scan of the lung was conducted 30 days after the tumor cells were injected. The black arrow points to the visible pulmonary nodules under CT scan. (M) Representative images show the H&E staining and immunohistochemistry staining of CD4 and CD8 in the indicated groups. Scale bar, 50 µm. *p<0.05.

Figure 2

YY1 was positively correlated with CD163⁺ M2 macrophages in human prostate cancer. (A) Immunohistochemistry staining of YY1 and CD163 were conducted in large slices of prostate cancer tissue. Black arrow points to representative cells expressing YY1 in tumor stroma area. Scale bar, 50 µm. (B) Representative images of three patients with multiplex fluorescence immunohistochemistry (IHC) showed a high expression of YY1 on CD163⁺ M2 macrophages in tumor stroma. (C) The left panel is the representative image of prostate cancer, in which blue is 4′,6-diamidino-2-phenylindole, orange is CD163, and green is YY1. The right panel displays a corresponding diagram derived from the R script based on the coordinate of positive cells in multiplex fluorescence IHC, in which red refers to CD163 and YY1 double-positive cells and green refers to YY1 single-positive cells. Scale bar, 20 µm. (D) Representative image of CD163 and YY1 IHC staining from tissue microarray containing a prostatic tumor, metastatic, and normal tissue. Red dashed line marks the tumor stroma area. Scale bar, 50 µm. (E, F) YY1 score in tumor stroma and CD163 density (cells/high-power field (HPF)) in the tissue microarray of prostate cancer were evaluated by pathologists. (G, H) Higher expression of YY1 shows a worse biochemical recurrence (BCR)–free survival (p=0.020), while the patients with high expression of both YY1 and CD163 have significantly worse BCR-free survival (p=0.004). *p<0.05.

Figure 3

YY1 was upregulated by IL-4/STAT6 pathway and participated in M2 macrophage polarization. (A) Morphological changes (from suspended cells to antennal, spindle-shaped adherent cells) on the induction of M0 cells to M2 macrophages. (B) Higher proportion of CD206 in M2 macrophages (91.0% vs 9.0%) compared with M0 cells (46.9% vs 53.1%) was observed by flow cytometry. (C) Quantitative reverse transcription (qRT)-PCR analysis showed the different mRNA expressions of CD163 and YY1 in M2 or M0 cells. (D) Western blotting assay of CD163 and YY1 in M2 or M0 cells. (E) Representative multiplex fluorescence immunohistochemistry images of YY1 and CD206 colocalization in oe/nc-YY1 M2 macrophages. Scale bar, 20 µm. (F) M2-like morphological change was displayed on YY1 overexpression in M0 macrophages. (G) mRNA expression of M1 (IL-12 and TNF-α) and M2 markers (IL-10 and ARG1) was tested by qRT-PCR in oe/nc-YY1 M0 macrophages. (H) Higher proportion of CD206 in oe-YY1 M0 cells (82.1% vs 17.9%) than in nc-YY1 (47.2% vs 52.8%) was observed by flow cytometry. (I) Luciferase assay showed that IL-4 increased the activity of YY1 promoter in M0 macrophages. (J) ChIP-qPCR showed significantly increased interaction between YY1 and STAT6 in IL-4 stimulated M0 macrophages. (K) High p-STAT6 and YY1 expression in IL-4 stimulated M0 macrophages as showed by western blotting. *p<0.05.

Figure 4

YY1 increased M2 macrophage-induced PCa malignancy. (A) The migration of prostate cancer cells DU145 was increased when treated with conditioned medium (CM) from M2 macrophages compared with treatment with M0 CM. (B–F) The migration (B) and proliferation (C) of DU145 cells was increased on treatment with the CM of YY1 overexpressed M2 macrophages, while there is a decrease in the migration (D), proliferation (E), and colony formation (F) of DU145 cells on treatment with the CM of YY1 knocked down M2 macrophages. (G) Primary cancer cells from patients with prostate tumor were co-cultured with CM from peripheral blood mononuclear cell (PBMC)–derived macrophages, and representative images displayed the increasing sphere formation ability in those cultured with oe-YY1 PBMC-derived macrophage CM. (H) RM-1 cells mixed with oe-YY1 M2 macrophages (M2 RAW264 cells) or control M2 RAW264 cells were subcutaneously injected into the backs of C57BL/6 mice, and tumor volume was examined during the next 4 weeks. Scale bar, 50 µm. (I) RM-1 cells mixed with oe-YY1 M2 RAW264 cells or control M2 RAW264 cells were injected into the caudal vein of C57BL/6 mice, and pulmonary metastasis nodules were counted after 6 weeks. Arrow directed to the representative nodules. (J, K) Gene Ontology and gene set enrichment analyses displayed that the upregulated genes in LNCaP cells treated with oe-YY1 M2 macrophage CM were enriched in the NF-κB and JAK-STAT pathway components. (L) The expression of p-STAT3 and p65 in LNCaP cells treated with oe-YY1 M2 macrophage CM was increased as shown by western blot. *p<0.05.

Figure 5

IL-6 upregulation by YY1 in M2 macrophages increased prostate cancer malignancy. (A, B) Cytokine microarray assay shows the highest increase of IL-6 in oe-YY1 M2 macrophage CM (A), and reduction of IL-6 in si-YY1 M2 macrophage CM (B). (C–E) qRT-PCR followed by western blotting assay showed the changes in IL-6 in oe-YY1 (C), sg-YY1 (D), and si-YY1 (E) in M2 macrophages. (F–J) Colony formation (F), proliferation (G), migration (H), and wound healing (I) of DU145 cells after adding an exogenous recombinant IL-6 (rIL-6) to si-/nc-YY1 M2 macrophage CM. (J–M) Colony formation (J), proliferation (K), migration (L), and wound healing (M) of DU145 cells after adding the IL-6 antibody to oe-/nc-YY1 M2 CM. *p<0.05.

Figure 6

YY1 regulates IL-6 expression by modulating p300, p65, and CEBPB. (A) YY1, CEBPB, and p65 were predicted on the upstream of IL-6 promoter according to the JASPAR database. (B) The table and schematic show the predicted interaction between YY1 with p300, p65, and CEBPB. (C–E) Chromatin immunoprecipitation (ChIP)-qPCR showing promoter 1–promoter 4 (P1–P4), four sites in IL-6 promoter, as the binding sites of YY1 (C), p65 (D), and CEBPB (E, F). ChIP-on-ChIP analysis showed the interaction between IL-6 P1 with CEBPB, p300, and p65, respectively, in the YY1 binding protein. (G–J) Binding of YY1 (G), CEBPB (H), p300 (I), and p65 (J) to the IL-6 promoter are shown by the Luciferase assay. (K–M) Co-immunoprecipitation, followed by western blotting assay, of YY1, p65, CEBPB, and p300 shows the interaction between them. (N, O) Decrease in the immunoprecipitation between YY1, CEBPB, and p65 on the knockdown of p300 (N) and use of a YY1 complex inhibitor hypericin (O). (P) IL-6 mRNA expression was tested by qRT-PCR in M2 macrophages treated with hypericin at different doses (0, 20, 40 mol/mL). (Q, R) Migration (Q) and proliferation (R) of cultured prostate cancer cells on the treatment of the CM of M2 macrophages with different doses of hypericin (0, 20, 40 mol/mL). *p<0.05.

Figure 7

YY1 promotes IL-6 transcription by M2-specific enhancer. (A) Based on the single-cell RNA sequencing data set, regulons were ranked by difference of the regulon specificity score between tumor and normal prostate tissue from high to low, and the top 20 different regulons were used to plot the heatmap. (B) An M2-specific IL-6 enhancer was located by comparing H3K27ac ChIP-seq data for THP-1 cells and M2 macrophages. (C) Distribution of H3K27ac and YY1 ChIP-seq signals around M2-specific enhancers in M2 macrophage and THP-1 cells. Top panel: normalized ChIP-seq signals around M2-specific enhancers. Bottom panel: heatmap of ChIP-seq signals; each row represents an M2-specific enhancer. All rows are sorted according to the signal values. (D) IL-6 enhancer location and its interaction with IL-6 promoter as displayed by the Integrative Genomics Viewer. (E) Plasmids containing four segments (regions E1–4) of the identified enhancer. (F, G) Binding of region E1 with YY1 as shown by the luciferase assay (F) and chromosome conformation capture experiment (G) after the co-transfection of vector oe-YY1 or nc-YY1 into 293 T cells. (H, I) qRT-PCR assay (H) and ELISA analysis (I) showed a significant decrease in the expression of IL-6 on CRISPR/Cas9 knocking out region E1 (sg-E1) compared with the negative control group. (J, K) The enhancer signaling inhibitor JQ1 decreases IL-6 expression (J) and M2 markers (K) tested by the qRT-PCR assay. (L, M) The migration of DU145 cells were tested on treatment with JQ1/nc (L) and sg-E1/sg-nc M2 macrophage CM (M, N). JQ1, CPI-637, and blank control medium were intraperitoneally injected for 18 days into mice with subcutaneous tumors, and tumor volume was measured after 3 weeks. *p<0.05.

Figure 8

YY1 formed liquid–liquid phase separation during M2 macrophage polarization. (A) Immunofluorescence assay using confocal fluorescence microscopy showed patterns of YY1 droplets distribution in M0 (PMA-THP-1 cells) and M2 macrophages (IL-4 stimulated PMA-THP-1 cells). Scale bar, 5 µm. (B) PONDR VSL2 score of YY1 displayed according to the database of pondr.com, with the purple bar showing the sites of intrinsically disordered regions (IDRs) and the green bar showing segments of IDR-EGFP and non-IDR-EGFP. (C) Representative images of phase separation droplets based on IDR-EGFP with different protein concentrations or with 1,6-hexanediol (1,6-Hex). Two panels on the left show the images when either YY1 or non-IDR-EGFP alone was added. (D) Live M2 macrophages transfected with YY1-IDR-EGFP, YY1-EGFP, and YY1-non-IDR-EGFP were observed under confocal fluorescence microscope. Scale bar, 2 µm. (E) The process of fusion and fission in the YY1-IDR-EGFP mediated droplets. (F) Fluorescence intensity of the condensate during fluorescence recovery after photobleaching assay. The white circle in the top left panel shows the photobleaching targeted focus range where it is bleached for 1 s. Scale bar, 2 µm. (G) YY1-IDR-EGFP was displayed to localize on the M2 macrophage nuclear when the cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 µm. (H) The numbers of droplets per cell were detected in the M2 macrophages knocking out YY1-IDR (sg-YY1-IDR) or negative control (sg-NC). Scale bar, 5 µm. (I) Immunoprecipitation with the EGFP antibody followed by western blot in YY1-IDR-EGFP transfected M2 macrophages. (J) Colocalization of YY1 droplets with p300, p65, and CEBPB in M2 macrophages. Scale bar, 5 µm. (K) Liquid–liquid phase separation droplets of YY1 were inhibited by using siRNA to reduce p300, p65, and CEBPB expression or inhibitor of YY1 complex in M2 macrophages, respectively. Scale bar, 5 µm. (L–N) The mRNA expression of M2 markers (IL-10 and ARG1) and IL-6 was tested by qRT-PCR in the indicated groups. *p<0.05.