YY1 enhances HIF-1α stability in tumor-associated macrophages to suppress anti-tumor immunity of prostate cancer in mice

Abstract

Immune checkpoint therapy for prostate cancer (PCa), a classic 'immune-cold' tumor characterized by an immunosuppressive tumor microenvironment, failed previously in clinical trials, but the underlying causes remain elusive. Here we find that YY1⁺, immunosuppressive macrophages aggregate in the hypoxic areas of PCa. Mechanistically, hypoxia promotes the phase separation of YY1 in the nucleus, where YY1 binds to NUSAP1 and promotes the SUMOylation, phase separation and stabilization of HIF-1α. Either myeloid-specific conditional knockout of YY1 or a treatment with tenapanor for decreasing the YY1-NUSAP1-HIF-1α interaction impairs subcutaneous PCa tumor formation in mouse prostate tumor models. Lastly, a first-generation tetrahedral DNA nanostructure based on the proteolysis targeting chimera technique, termed YY1-DcTAC, allows targeting and degrading YY1 in tumor-associated macrophages for inducing antitumor effects and CD8⁺ T cell tumor infiltration in mouse tumor models. In summary, our findings underscore the pivotal role of YY1 in the hypoxia/HIF-1α pathway in tumor-associated macrophages and support the targeting of YY1 for treating PCa.

Fig. 1. YY1⁺ macrophages accumulate in hypoxic tumor tissues.

a Schematic diagram showing the process of imaging mass cytometry (IMC) using prostate tissue. IMC of the indicated markers in prostate cancer (PCa) patient tumors (b) and para-cancerous tissues (c). Pathchs were defined as containing at least 10 cells with a maximum distance of 15 μm between cells involved. Adjacent regions were merged together for downstream analysis. The expression of the marker was normalized using z-score, and a threshold of 0.5 was selected. Dot plots showing the ratio of YY1⁺ or YY1⁻ macrophages and the relative YY1 expression of macrophages in HIF-1α⁺ or HIF-1α⁻ patches. The representative plot and analysis are based on 46 tumor samples and 26 para-cancerous tissues. Dot plots present the mean values ± SD, and the p values are calculated using a two-tailed t-test. The p values of left, middle, and right plots of (b) are 0.002, 0.160, and 0.009, respectively; The p values of left, middle, and right plots of (c) are 0.759, 0.798, and 0.820, respectively. *P < 0.05, ns P > 0.05. Source data are provided as a Source Data file. Mø macrophage.

Fig. 2. Hypoxia enhances YY1 phase separation by inducing YY1 tyrosine phosphorylation in macrophages.

a Immunofluorescence staining in THP-1 cells subjected to 1% O₂ and/or 1,6-hexanediol. The p values were calculated using both two-tailed t-test (P = 0.017 and P < 0.001) and one-way ANOVA followed by Tukey test (P < 0.001). b The localization of YY1 was observed by immunofluorescence after hypoxia with 1% O₂ or reoxygenation with 21% O₂. c Western blot of HIF-1α and YY1 in THP-1 cells under 1% O₂ hypoxia. d qRT‒PCR showing the relative RNA expression of YY1 in THP-1 cells under 1% O₂ hypoxia. e Western blot analysis using an anti-phosphorylated YY1 antibody to detect YY1-immunoprecipitated proteins. f Immunofluorescence staining in THP-1 cells treated with Na₃VO₄ or control solution under normal oxygen conditions (P < 0.001). g Western blot analysis using a tyrosine phosphorylation antibody to detect YY1-immunoprecipitated protein. The bacterial alkaline phosphatase was used as a negative control. h Immunofluorescence staining in THP-1 cells treated with SU6656 or control solution under 1% O₂ hypoxia (P < 0.001). i Western blot analysis using a tyrosine phosphorylation antibody to detect EGFP immunoprecipitated from YY1 full-length (YY1-FL), YY1 mutation 1 (YY1-mut1, including amino acid sites 8 and 383), and YY1 mutation 2 (YY1-mut2, including amino acids 145, 185, 251, and 254)-transfected THP-1 cells. j Immunofluorescence of YY1-FL-, YY1-mut1- and YY1-mut2-transfected THP-1 cells. k The left volcano plot illustrates the differentially expressed genes in the indicated RNA-seq (two-tailed t-test and adjusted with Benjamini and Hochberg method). The right panel shows the overlap indicated to identify Lyn. l, m Co-IP and immunofluorescence of YY1 and Lyn from 1% O₂ hypoxia- or normoxia-induced THP-1 cells. n Immunofluorescence of the indicated markers in THP-1 cells under 1% O₂ hypoxia (P < 0.001). All the immunofluorescence data were analyzed, and twenty cells randomly selected from three repeated groups were counted. Scale bar, 5 μm. Bar graphs (a, b, d, f, h, n) present mean values ± SD, and the p values are calculated using a two-tailed t-test. * P < 0.05. Source data are provided as a Source Data file. 1,6-Hex 1,6-hexanediol, BAP bacterial alkaline phosphatase.

Fig. 3. YY1 stabilizes HIF-1α by promoting its SUMOylation and inhibiting its ubiquitination.

a Venn diagram depicting the overlap between two datasets: (1) RNA-seq analysis comparing oe-YY1 and oe-NC THP-1 cells, and (2) a previously reported RNA-seq dataset from hypoxia-induced macrophages (GSE16099). b GSEA analysis of the HIF-1A targets DN set, comparing RNA-seq data from oe-YY1 THP-1 cells to nc-YY1 THP-1 cells. NES and p value are indicated (phenotype permutation test). c, d Gene ontology analysis of enriched signaling pathways in oe-YY1 or hypoxia-induced THP-1 cells when compared to control cells (hypergeometric test and corrected with Benjamini & Hochberg adjustment). e Western blot showing the expression of HIF-1α and YY1 in 1% O₂ hypoxia-induced THP-1 cells transfected with siYY1 or the normal control. Samples calculated are from the same experiment, and the blots were processed in parallel (n = 3 independent experiments, P < 0.001, P < 0.001, respectively). f qRT‒PCR showing the expression of YY1 and HIF-1α in THP-1 cells (n = 3 independent experiments, P < 0.001, P = 0.220, respectively). g Western blot of HIF-1α expression in 1% O₂ hypoxia-induced THP-1 cells treated with cycloheximide (n = 3 independent experiments). The lower panel presents mean values ± SD at each time point (linear regression between the two datasets using two-tailed t-test, P < 0.001). h Western blot of HIF-1α expression in THP-1 cells treated with MG132 and/or 1% O₂ hypoxia. i, j Immunoprecipitation of HIF-1α followed with western blot of ubiquitin or SUMO2/3 in 1% O₂ hypoxia-induced THP-1 cells. Bar graphs (e, f) present mean values ± SD, and the p values are calculated using a two-tailed t-test. * P < 0.05, ns P > 0.05. Source data are provided as a Source Data file. NES normalized enrichment scores, FDR false discovery rate, CHX cycloheximide.

Fig. 4. YY1 promotes the stability of HIF-1α by binding to NUSAP1.

a Mass spectrometry of proteins immunoprecipitated with YY1 and IgG under CoCl₂ treatment conditions. b In the co-IP experiment using 1% O₂ hypoxia-induced THP-1 cells, the expression levels of YY1, NUSAP1, and HIF-1α proteins were detected in the proteins immunoprecipitated by the YY1 or IgG antibody. c Western blot of HIF-1α in THP-1 cells treated with MG132. d, e Western blot of ubiquitin or SUMO2/3 following HIF-1α IP in hypoxia-induced THP-1 cells. f Diagram of the SAP domain and N/C-terminus of NUSAP1. g, h SUMOylation assays in hypoxia-induced THP-1 cells under the indicated conditions. i CO-IP assays in hypoxia-induced THP-1 cells showing the interactions between Flag-tagged NUSAP1 segments and EGFP-tagged YY1. j CO-IP assays in hypoxia-induced THP-1 cells showing the interactions between EGFP-tagged YY1 segments and Flag-tagged NUSAP1. FL, NTD, and CTD represent the full-length, N-terminal domain, and C-terminal domain of YY1, respectively. k, l Co-IP assays showing the interactions of the indicated HA-YY1 or Flag-NUSAP1 mutant. m, n Co-IP assays showing the interactions of truncated HIF-1α and NUSAP1 proteins in hypoxia-induced THP-1 cells. o Immunofluorescence assays showing the colocalization of the indicated markers in THP-1 cells treated with hypoxia or normoxia. The dye intensity alongside the white lines was calculated and plotted in the right panels. Scale bar, 2 μm. p Schematic diagram of the NUSAP1 IDR. q Fluorescence images of m-cherry showing droplet formation in the medium under different concentrations of NUSAP1-m-cherry protein. Scale bar, 5 μm. r Fluorescence images of EGFP and m-cherry showing the physical colocalization of NUSAP1-m-cherry with the YY1-IDR-EGFP or YY1-non-IDR-EGFP in the medium. Scale bar, 5 μm. Source data are provided as a Source Data file. NTD N-terminal domain, CTD C-terminal domain, FL full-length.

Fig. 5. HIF-1α undergoes SUMOylation-related phase separation under hypoxia.

a Diagram showing the intrinsically disordered protein regions (IDRs) of HIF-1α. b Representative images of phase separation condensates based on HIF-1α-IDR1/2-EGFP with different protein concentrations or with 1,6-hexanediol. Scale bar, 5 μm. c The process of fusion and fission in HIF-1α-IDR1-EGFP-mediated condensates. Scale bar, 1 μm. d Fluorescence intensity of the condensate during fluorescence recovery after photobleaching (FRAP) assay. Scale bar, 1 μm. The average relative fluorescence intensities are plotted as white circles. e Localization of HIF-1α-IDRs-EGFP and HIF-1α-non-IDR-EGFP in THP-1 cells after 4 h of 1% O₂ hypoxia and fixation. Scale bar, 5 μm. f Representative fluorescence images of EGFP and DAPI showing the patterns of HIF-1α mutants. Scale bar, 5 μm. g Immunofluorescence staining of HIF-1α in THP-1 cells treated with TAK-981 or control solution under 1% O₂ hypoxia. Twenty cells randomly selected from three repeated groups were analyzed. Scale bar, 5 μm. The bar graph in the lower panel presents mean values ± SD, and the p values are calculated using a two-tailed t-test (P < 0.001). *P < 0.05. h The predicted SUMOylation sites in HIF-1α based on the SUMOplot Analysis Program. i, j SUMO2/3-mediated SUMOylation and immunofluorescence staining in 1% O₂ hypoxia-induced THP-1 cells with the HIF-1α K477F or K391F mutation. Scale bar, 5 μm. Source data are provided as a Source Data file. IDR intrinsically disordered regions, FL full-length.

Fig. 6. A small molecule inhibitor targeting the YY1–NUSAP1–HIF-1α interaction suppressed PCa progression.

a Schematic diagram showing that tenapanor inhibits the binding of the YY1–NUSAP1–HIF-1α nexus. b, c Co-IP of NUSAP1 showed that its interactions with YY1 and HIF-1α were suppressed by tenapanor. The protein intensity of YY1 and HIF-1α pulled down from NUSAP1 was analyzed with ImageJ and standardized to that of the input samples from the same experiment. The blots were processed in parallel (n = 3 independent experiments, P < 0.001 and P < 0.001). *P < 0.05. d, e Schematic diagram and tumor growth curve of the effects of TEN-M2pep and control medium on subcutaneous tumorigenesis in mice. Each experimental group consisted of five male C57BL/6 mice, all aged 6–8 weeks. The right panels present the mean values ± SD at each time point (n = 5 independent experiments, one-way ANOVA followed by Tukey test based on data in the last time point). f, g GO analysis based on RNA-sequencing data of CD45-sorted cells from TEN-M2pep-treated subcutaneous tumors showing enriched immune response-related pathways and representative upregulated genes (two-tailed t-test). h, i Immunohistochemical staining and flow cytometry of subcutaneous tumors showing the infiltration of CD8⁺ T cells in the indicated groups (n = 3 independent experiments, P = 0.019 and P = 0.002). Bar graphs (c, i) present the mean values ± SD, and the p values are calculated using a two-tailed t-test. * P < 0.05. Source data are provided as a Source Data file.

Fig. 7. Construction and application of YY1-DbTACs@jetPEI and transgenic mice.

a Schematic diagram illustrating the mechanism of YY1-DbTACs-mediated degradation of YY1. b Western blot in THP-1 cells treated with YY1-DbTACs. c Western blot of ubiquitin following HIF-1α IP in hypoxia-induced THP-1 cells treated with YY1-DbTACs. d Experimental flow chart of subcutaneous tumorigenesis in mice using M2pep-YY1/NC-DbTACs@jetPEI. Each experimental group consisted of five male C57BL/6 mice (6–8 weeks). e Tumor volume changes over 21 days following subcutaneous tumor implantation. The lower panel presents the mean values ± SD at each time point. The p values were calculated based on the last time point between two datasets using a two-tailed t-test (n = 5 independent experiments, P < 0.001). *P < 0.05. f Schematic diagram illustrating the mechanism of YY1-DcTACs and its tetrahedral structure. g Confocal microscopy images showing the responsiveness of YY1-DcTACs-FAM/BHQ1 in the indicated cell lines. h Changes in tumor volume within 14 days after injection of YY1/NC-DcTACs via the tail vein in a mouse subcutaneous tumor model. The T lymphocytes were sorted by flow cytometry before testing changes in the ratio of CD3⁺CD8⁺ T cells. The lower panel presents the mean values ± SD at each time point. The p values were calculated based on the last time point between the two datasets (two-tailed t-test, P < 0.001). *P < 0.05. i Schematic diagram illustrating the construction of YY1 transgenic mice with myeloid conditional knockout and the establishment of a subcutaneous tumorigenesis model. j Subcutaneous tumor size in mice after 21 days of tumor formation (n = 5 independent experiments, P < 0.001). Five wild-type male C57BL/6J mice and five C57BL/6J-YY1^em1Cflox male mice were used in the experiment (6–8 weeks old). k T lymphocytes were sorted by flow cytometry in the CD45-gated immune cells after grinding the subcutaneous tumors of transgenic mice into a cell suspension, and changes in the ratio of CD3⁺CD8⁺ T cells were observed (n = 5 independent experiments, P < 0.001). Bar graphs (h, j, k) present the mean values ± SD, and the p values are calculated using a two-tailed t-test. * P < 0.05. Source data are provided as a Source Data file.