Tumor derived UBR5 promotes ovarian cancer growth and metastasis through inducing immunosuppressive macrophages

Abstract

Immunosuppressive tumor microenvironment (TME) and ascites-derived spheroids in ovarian cancer (OC) facilitate tumor growth and progression, and also pose major obstacles for cancer therapy. The molecular pathways involved in the OC-TME interactions, how the crosstalk impinges on OC aggression and chemoresistance are not well-characterized. Here, we demonstrate that tumor-derived UBR5, an E3 ligase overexpressed in human OC associated with poor prognosis, is essential for OC progression principally by promoting tumor-associated macrophage recruitment and activation via key chemokines and cytokines. UBR5 is also required to sustain cell-intrinsic β-catenin-mediated signaling to promote cellular adhesion/colonization and organoid formation by controlling the p53 protein level. OC-specific targeting of UBR5 strongly augments the survival benefit of conventional chemotherapy and immunotherapies. This work provides mechanistic insights into the novel oncogene-like functions of UBR5 in regulating the OC-TME crosstalk and suggests that UBR5 is a potential therapeutic target in OC treatment for modulating the TME and cancer stemness.

Fig. 1. Attenuated OC growth and peritoneal implantation of ID8/Ubr5^−/− in mice.

a Deletion of Ubr5 in ID8 was verified by Western blot. b Representative micrographs of ID8 cell morphology. Scale bars: 200 μm. c, d To evaluate spontaneous metastasis, 5 × 10⁶ ID8 cells were i.v. injected to C57BL/6 female recipient mice (n = 3 mice per group). c Quantification of Muc16⁺ tumor cells. d Representative images (left) and quantified values (right) of metastatic nodules in lung or liver of recipient mice. e Protein expression of β-catenin, Cytokeratin-18, ZEB1, ZEB2, and Vimentin in ID8 cells was evaluated by western blot. f Tumors from orthotopic syngeneic model were resected and measured 8 weeks after tumor inoculation (n = 5 mice per group). g Metastatic nodules in peritoneum, omentum, mesentery and diaphragm were quantified (n = 6 mice per group). h Kaplan–Meier curves showing overall survival of mice (ID8/GFP n = 18, ID8/Ubr5^−/− n = 13 mice per group), P < 0.0001, log-rank test. i Proportion of tumor cells in ascites at indicated times after tumor implantation (n = 5 mice per group). j Ascitic fluid volumes were measured at day 50 (n = 5 mice per group). k Peritoneal metastases were evaluated at day 50 (n = 5 mice per group). l Kaplan–Meier curves showing the survival rates of mice (n = 15 mice per group), P < 0.0001, log-rank test. m Protein expression of UBR5 in ID8/GFP, ID8/Ubr5^−/−, hUBR5 reintroduced ID8/Ubr5^−/− cells was assessed by western blot. n Tumor burdens were evaluated at day 30 (n = 4 mice per group). o Survival rates were quantified (n = 6 mice per group), P < 0.0001, log-rank test. p mRNA expression of Ubr5 in tumor cells was assessed. Data were normalized to puromycin N-acetyl- transferase (PAC) in each sample (n = 5 mice per group). q Proportions of Muc16⁺CD45⁻ tumor cells in ascites were quantified at day 30(n = 5 mice per group). All data are representative of at least two independent experiments with similar results. Data are shown as mean ± SEM, unpaired two-sided Student’s t-test with no correction for multiple comparison, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 2. Analysis of immune cell involvement in regulating ID8/Ubr5^−/− tumor growth.

a–c Peritoneal washes were collected at indicated times from OC bearing mice after peritoneal implantation of ID8 cells (n = 5 mice per group). CD4⁺ (a) /CD8⁺ (b) T cells were analyzed on CD45⁺ infiltrating cells. c Foxp3⁺CD25⁺ regulatory T cells were analyzed on CD45⁺CD3⁺CD4⁺ tumor infiltrating T cells (TILs). d B and T cell deficiency did not mitigate the differences in growth capacity between control and Ubr5^−/− tumors. Proportion of Muc16⁺ CD45⁻ tumor cells in ascites were quantified at day 30 post OC implantation (n = 5 mice per group). e, f Representative FACS images of infiltrated CD11b⁺F4/80⁺ macrophages in peritoneal cavity or lung at day 30 after tumor injection. Proportion of macrophages on CD45⁺ infiltrating cells in peritoneal cavity (e) (n = 5 mice per group), and lung (f) (n = 3 mice per group) were quantified. g Representative images of CD68⁺ macrophages and surrounded Ki67⁺ cells. All panels are the same magnification, scale bars: 50 μm. h–i CD68⁺ cells (h) and Ki67⁺ cells (i) in individual and spheroid populations were quantified (n = 5 mice per group and ten confocal images acquired from each sample). j At day 30 post i.p. injection, spheroids from peritoneal washes were harvested and evaluated by H&E staining (n = 5 mice per group and an average of 10 fields acquired from each sample). Scale bars: 200 μm. In all cases, data are representative of at least two independent experiments with similar results. Data are presented as mean ± SEM, unpaired two-sided Student’s t-test with no correction for multiple comparison, *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 3. Impaired macrophage recruitment and attenuated peritoneal growth of UBR5-deficient OC.

a Representative FACS images and proportion of Muc16⁺CD45⁻ tumor cells (n = 5 mice per group). b Ascitic fluid volumes were measured at day 40 (n = 5 mice per group). c Representative images of CD68⁺ macrophages and surrounded Ki67⁺ cells. All panels are the same magnification, scale bars: 50 μm. d Survival in ID8 bearing mice with or without exogenous TAMs (n = 6 mice per group), log-rank test. e, f Proportion of infiltrated CD11b⁺F4/80⁺ macrophages (e) and Muc16⁺ CD45⁻ tumor cells (f) (n = 5 mice per group). g Representative images of CD68⁺ macrophages and surrounded Ki67⁺ cells (n = 3 mice per group and ten confocal images acquired from each sample). All panels are the same magnification, scale bars: 50 μm. h Spheroids from peritoneal washes (same volume) were harvested and evaluated by H&E staining (n = 3 mice per group and an average of ten fields acquired from each sample). Scale bars: 200 μm. i Survival in ID8 bearing mice with or without LC treatment (n = 5 mice per group), log-rank test. All data are representative of two independent experiments with similar results. Data are presented as mean ± SEM, unpaired two-sided Student’s t-test with no correction for multiple comparison,*P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 4. Functional impairment of macrophages in Ubr5-null tumors.

a Peritoneal macrophages were isolated and starved overnight before loaded into upper chamber with ID8 cells cultured at the bottom. Migrated cells were assessed after 16 h incubation (n = 5 biologically independent samples per group and an average of five fields acquired from each sample). Scale bars: 200 μm. b Heat map representation of differentially expressed M1/M2 genes in naïve macrophages and TAMs. c Representative FACS analysis of intracellular Arginase-1 in CD45⁺ CD11b⁺ F4/80⁺ cells and quantification expressed as mean florescence intensity (MFI) of Arginase-1 (n = 3 mice per group). d Representative FACS analysis and proportion of PD-L1⁺ cells gated on CD45⁺ CD11b⁺F4/80⁺ cells (n = 3 mice per group). e T cell proliferation suppression assay. CFSE-labeled T cells from naïve mice were stimulated with anti-CD3 and CD28 antibodies and co-cultured for 3 days with TAMs isolated from ID8/GFP or ID8/Ubr5^−/− bearing mice at 3:1 ratio (n = 3 mice per group). f Proportion of Muc16⁺CD45⁻ tumor cells with or without exogenous TAMs on day 45 post-tumor inoculation (n = 3 mice per group). g Kaplan–Meier curves showing the survival of ID8 bearing mice with or without exogenous TAMs (n = 5 mice per group), P = 0.0026, log-rank test. Data are representative of two independent experiments with similar results. Data are presented as mean ± SEM, unpaired two-sided Student’s t-test with no correction for multiple comparison,*P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 5. Impaired spheroid formation mediated by p53 in Ubr5-deficient tumor.

a Spheroid cell proliferation assay with quantitative analysis (n = 5 biologically independent samples per group). Scale bars: 25 μm. b, c Representative images of single spheroid, Scale bars: 50 μm, the size of spheroids were statistically evaluated (n = 3 biologically independent samples per group and an average of five fields acquired from each sample). d Western blotting showing the expression of apoptosis-associated protein PARP, cleaved-PARP in ID8. e Apoptosis in lung sections was evaluated by TUNEL staining at indicated time post i.v. injection of ID8 (n = 5 mice per group and an average of five fields acquired from each sample), Scale bar: 400 μm. f Western blot showing p53 protein expression in indicated ID8 cells. g, h Spheroid cell proliferation assay with quantitative analysis (n = 4 biologically independent samples per group). Scale bars: 25 μm. i Western blot showing that knocking down Tp53 in ID8 cells increased β-catenin expression. j, k The relationship between TP53 mutation and UBR5 gene alterations in serous ovarian carcinoma (j) and high-grade serous carcinoma (k) cohorts from the TCGA databases. Data are representative of two independent experiments with similar results. Data are presented as mean ± SEM, unpaired two-sided Student’s t-test with no correction for multiple comparison, *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 6. Effects of tumor-derived paracrine factors on TAMs and spheroids.

a Heat map representation of differentially expressed genes involved in macrophage recruitment, Wnt signaling, and cell adhesion in recovered peritoneal cells from ID8 bearing mice. b ID8 cells were cultured in DMEM overnight and the protein levels of CCL2 and CSF-1 in cell cultures were measured by ELISA (n = 5–6 biologically independent samples per group). c Western blotting showing the protein expression of UBR5/β-catenin/CCL2 in different ID8 cells. d Peritoneal macrophages were harvested and seeded into Transwell inserts with ID8 cell cultured in the bottom. Migrated macrophages were quantified after 16h-incubation (n = 5 biologically independent samples per group). e Representative images of spheroids. Scale bars: 200 μm. f Quantification of spheroid expansion rates (n = 4 biologically independent samples per group). g, h CD68⁺ cells and Ki67⁺ cells in spheroids were quantified (n = 4 mice per group). i Representative images of CD68⁺ macrophages and surrounded Ki67⁺ cells. (n = 4 mice per group and ten confocal images acquired from each sample). All panels are the same magnification, scale bars: 50 μm. j Spheroid retrieved from ID8 bearing mice at day 30 were underwent H&E staining (n = 4 mice per group and an average of five fields acquired from each sample). Scale bars: 200 μm. k Survival in mice bearing ID8 tumors (n = 5 mice per group), log-rank test. All data are representative of at least two independent experiments with similar results. Data are presented as mean ± SEM, unpaired two-sided Student’s t-test with no correction for multiple comparison, *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 7. Effects of human OC-derived UBR5 on TAMs and tumor growth.

a IHC staining of UBR5 and CD68 in primary tumors from OC patients (n = 50 samples). Scale bars: 200 μm. b, c Correlation assessed by Pearson correlation analysis and linear regression analysis between tumor UBR5 expression levels and intratumoral CD68⁺ (b), Ki67⁺ cell density (c) in human EOC patients (n = 50 samples). d Deletion of UBR5 in SKOV3 cells was verified by western blot. Reduced β-catenin and CCL2 expression in SKOV3/UBR5^−/− were determined at protein level, human ovarian surface epithelial cells HOSEpiC were used as control. e Representative micrographs and quantitation of Transwell-migration assay for SKOV3 (n = 4 biologically independent samples per group and an average of five fields acquired from each sample). Scale bars: 50 μm. f On day 21 after tumor implantation, imaging with Luciferase in SKOV3 tumor bearing SCID/Beige mice (n = 3 mice per group). g Representative images and statistical analysis of tumor implantation in mesentery (n = 4 mice per group). h Representative images of CD68⁺ macrophages and surrounded Ki67⁺ cells from peritoneal washes (n = 4 mice per group and ten confocal images acquired from each sample). All panels are the same magnification, scale bars: 50 μm. i Representative FACS images and quantification of infiltrated CD11b⁺F4/80⁺ macrophages in peritoneal cavity (n = 3 mice per group). j Kaplan–Meier curves showing the survival of SKOV3 tumor-bearing mice (n = 10 mice per group). P < 0.0001, log-rank test. k Survival in mice bearing SKOV3/UBR5^−/− with or without TAMs isolated from SKOV3 bearing donor mice were quantified (n = 5 mice per group), log-rank test. In all cases, data are representative of two independent experiments. Data are presented as mean ± SEM, unpaired two-sided Student’s t-test with no correction for multiple comparison, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig. 8. Enhanced therapeutic benefits in OC by targeting tumor-derived Ubr5.

a Dose response curves of ID8 treated with cisplatin for 72 h (n = 5 biologically independent samples per group). The averages of three independent experiments were plotted, data are presented as mean ± SD, P < 0.0001, one-way ANOVA test. b Western blot analysis of cleaved-PARP levels after 24 h of cisplatin (Cis) treatment in ID8 cells. c, d UBR5 overexpression abrogated the therapeutic effect of cisplatin treatment. ID8 bearing mice were treated with cisplatin at 2 weeks after tumor implantation once a week for 3 weeks (n = 5 mice per group). Mouse mortality (c) and body weight (d) were monitored. Data are presented as mean ± SD, unpaired two-sided Student’s t-test. e Anti-PD-1 treatment enhanced the survival of ID8/Ubr5^−/− bearing mice, but not ID8/GFP bearing mice. Mice were treated with anti-PD-1 at indicated times post tumor implantation, and survival rates were quantified (n = 10 mice per group). Data shown is pooled from two independent experiments. f 4H1128z cells administration resulted in long-term survival of mice harboring ID8/Ubr5^−/−. Mice were treated with CAR-T cells 2 weeks after tumor implantation, and survival rates were quantified (n = 10 mice per group). In all cases, data are representative of at least two independent experiments. P values shown in c, e, and f were calculated by log rank test. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.