SPOP downregulation promotes bladder cancer progression based on cancer cell-macrophage crosstalk via STAT3/CCL2/IL-6 axis and is regulated by VEZF1

Abstract

Background: Cancer cells are intimately intertwined with tumor microenvironment (TME), fostering a symbiotic relationship propelling cancer progression. However, the interaction between cancer cells and tumor-associated macrophages (TAMs) in urothelial bladder cancer (UBC) remains poorly understood. Methods: UBC cell lines (5637, T24 and SW780), along with a monocytic cell line (U937) capable of differentiating into macrophage, were used in a co-culture system for cell proliferation and stemness by MTT, sphere formation assays. VEZF1/SPOP/STAT3/CCL2/ IL-6 axis was determined by luciferase reporter, ChIP, RNA-seq, co-IP, in vitro ubiquitination, RT-qPCR array and ELISA analyses. Results: We observed the frequent downregulation of SPOP, an E3 ubiquitin ligase, was positively associated with tumor progression and TAM infiltration in UBC patients and T24 xenografts. Cancer cell-TAM crosstalk promoting tumor aggressiveness was demonstrated dependent on SPOP deficiency: 1) In UBC cells, STAT3 was identified as a novel substrate of SPOP, and SPOP deficiency increased STAT3 protein stability, elevated chemokine CCL2 secretion, which induced chemotaxis and M2 polarization of macrophage; 2) In co-cultured macrophages, IL-6 secretion enhanced UBC cell proliferation and stemness. Additionally, transcription factor VEZF1 could directly activate SPOP transcription, and its overexpression suppressed the above effects in UBC cells. Conclusions: A pivotal role of SPOP in maintaining UBC stemness and remodeling immunosuppressive TME was revealed. Both the intrinsic signaling (dysregulated VEZF1/SPOP/STAT3 axis) and the extrinsic cues from TME (CCL2-IL-6 axis based on macrophages) promoted UBC progression. Targeting this crosstalk may offer a promising therapeutic strategy for UBC patients with SPOP deficiency.

Keywords: SPOP; STAT3/CCL2/IL-6 axis; VEZF1; bladder cancer; tumor-associated macrophage.

Figure 1

SPOP level was negatively correlated with tumor grade, stage and clinical outcome in UBC patients. A-B, Analysis of SPOP mRNA levels in public UBC datasets: TCGA-BLCA (A) and GSE13507 (B). C, Quantitative assessment of SPOP mRNA levels in 24 paired UBC (T) and adjacent normal (N) tissues from our cohort. D-E, The quantification (D) of Western blotting analysis (E) of SPOP protein levels in 24 paired UBC and adjacent normal tissues. F, Representative IHC staining for SPOP in low- and high-grade UBC tissues. Scale bar, 50 μm; the inset, 250 μm. G-H, SPOP protein levels by IHC in 94 UBC specimens, stratified by tumor grade (G) and tumor stage (H) from our cohort. I, Kaplan-Meier survival plot depicting cumulative overall survival of 94 UBC patients, stratified by SPOP protein levels using IHC staining scores. Data were shown as mean ± SD and analyzed by unpaired t test in (A-B, and G-H) or by paired t test in (C-D). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2

SPOP overexpression suppressed UBC growth, stemness and TAM infiltration. A-C, SPOP overexpression inhibited T24 xenograft growth. The growth curves (A), photograph (B) and weights (C) of T24 xenografts with SPOP overexpression (SPOP) and control (CV) were shown. n = 6 mice per group. The ectopic expression of SPOP in T24 cells was confirmed by Western blotting analysis (A, inset). D-F, Comparisons of the proliferative (Ki67⁺, D) and cancer stemness (CK5⁺, E) indices between SPOP and CV groups, with representative IHC and IF staining data (F). FLAG-tagged SPOP (red), CK5 (green) and F4/80 (white) with DAPI counterstaining (blue) were shown in F. Scale bar, 20 μm. G, Western blotting analysis of stemness markers in T24-CV and -SPOP xenografts. H-J, Comparative assessment of macrophage (F4/80⁺, H) and M2-like macrophage (CD206⁺, I and J) number in SPOP and CV groups, with representative IHC and IF staining data shown in F and I. Scale bar, 20 μm. K, Representative IHC staining for SPOP, CD68 and CD206 in human low-grade (upper panels) and high-grade (lower panels) UBC tissues. Scale bar, 50 μm; the inset, 250 μm. L-M, Kaplan-Meier survival plots for 94 UBC patients, stratified by CD68⁺ (L) and CD206⁺ (M) macrophage numbers, according to the IHC staining data, showing cumulative overall survival. N-O, Correlations between SPOP levels and CD68⁺ (N) and CD206⁺ (O) cell number/field in 94 UBC specimens, as determined by IHC staining. Data were shown as mean ± SD and analyzed by unpaired t test in (A, C-E, H and J). r, Pearson correlation coefficient. **, p < 0.01; ***, p < 0.001.

Figure 3

Interactional regulation between UBC cells and macrophages in cancer proliferation and stemness. A, MTT assay on T24 cells (CV and SPOP) after co-cultured with or without U937 cells in a non-contact system for 4 days. B-C, Quantification (B) and representative images (C) of mCherry-labeled T24 spheres (CV and SPOP) co-cultured with or without U937 cells for 6 days. Scale bar, 200 μm. D, Western blotting analysis of stemness markers in T24 cells (CV and SPOP) co-cultured with or without U937 cells in a non-contact system for 4 days. E, Stable knockdown of SPOP in 5637 cells using two different shRNAs (sh1 and sh2) by Western blotting analysis. shNC represented the negative control. F, MTT assay on control and two SPOP knockdown 5637 cells for 3 days, after co-cultured with or without U937 cells in a non-contact system for 4 days. G-H, Quantification (G) and representative images (H) of mCherry-labelled control and SPOP knockdown 5637 spheres co-cultured with and without U937 cells for 10 days. Scale bar, 200 μm. I, Western blotting analysis of stemness markers in 5637 cells (shNC, sh1 and sh2) co-cultured with or without U937 cells in a non-contact system for 4 days. J-K, RT-qPCR analysis of U937 cells co-cultured in a non-contact way with T24 cells (CV and SPOP; J) and 5637 cells (shNC and sh1; K) to assess expression of pan-marker (CD68), M1-like marker (CD86 and iNOS), and M2-like markers (CD163, CD206 and CCL22) in macrophages. Cell culture medium alone (RPMI1640) served as the negative control. Data were presented as mean ± SD and analyzed by unpaired t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p ≥ 0.05. #, p < 0.05; ##, p < 0.01; ###, p < 0.001; with vs without co-cultured U937 cells.

Figure 4

SPOP mediated the degradation of STAT3 protein. A, Heatmap displaying DEGs between SPOP knockdown (sh1) and control (shNC) 5637 cells as determined by RNA-seq (|Fold change| ≥ 1.50; p < 0.05). B, KEGG enrichment analysis identified the pathways affected by SPOP knockdown in 5637 cells. C, GSEA analysis on the RNA-seq data. D, The structure of STAT3 protein, indicating the location of a putative SBC motif. E, Sequence alignment of STAT3 protein across different species, highlighting the presence of a putative SBC motif with conserved nonpolar (Φ) and polar (π) residues, as well as serine (S) and threonine (T) residues. F-G, Western blotting analysis of STAT3 protein levels in 5637 cells following transient transfection with siRNAs targeting CUL3 (siCLU3) (F) or RBX1 (siRBX1) (G), compared to siRNA control (siNC). H, Western blotting analysis of HA-tagged wild-type STAT3 and its SBC motif mutants, along with FLAG-tagged SPOP in 293T cells co-transfected with the indicated plasmids. I, Co-IP assay in 293T cells transiently transfected with HA-tagged STAT3 (WT, ΔSBC, and S514A) and FLAG-tagged SPOP, using a FLAG-tag antibody for IP and an HA-tag antibody to detect ectopic STAT3. WCL, whole cell lysate. J, Co-IP assay in T24 cells transiently transfected with FLAG-tagged SPOP, using a FLAG-tag antibody for IP and a STAT3 antibody to detect endogenous STAT3. K, Co-IP assay in T24 cells using a STAT3 antibody for IP and a SPOP antibody to detect endogenous SPOP. L, Western blotting analysis on WCL and anti-HA tag IP from 293T cells co-transfected with indicated plasmids, including HA-tagged STAT3 (WT, ΔSBC and S514A), FLAG-tagged SPOP, and Myc-tagged ubiquitin. M-N, Western blotting analysis (M) and quantification (N) for HA-STAT3 WT and its mutants (ΔSBC and S514A) protein levels in 293T cells, after treatment with 100 μg/ml cycloheximide (CHX) for the indicated timepoints. The 0-h timepoint was used as a normalization control. O-P, Western blotting analysis (O) and quantification (P) of endogenous STAT3 protein levels in SPOP-knockdown (sh1) and control (shNC) 5637 cells after CHX treatment for the indicated timepoints.

Figure 5

STAT3 inhibition by SPOP diminished macrophage-mediated UBC cell proliferation and stemness. A, MTT assay on 5637 cells (shNC, sh1, and sh1+shSTAT3), after 4-day co-culture with or without U937 cells in a non-contact system. B-C, Representative images (B) and quantification (C) of mCherry-labelled 5637 spheres (shNC, sh1, and sh1+shSTAT3) after 10-day co-culture with or without U937 cells. Scale bar, 200 μm. D, Western blotting analysis of stemness markers in 5637 cells (shNC, sh1, and sh1+shSTAT3) co-cultured with or without U937 cells. E, Quantification of U937 cell migration in a transwell system in response to the CM from T24 cells (CV and SPOP) and 5637 cells (shNC, sh1, and sh2). F-H, A Venn diagram (F) illustrated the RT-qPCR array for 48 cytokines/chemokines in T24 cells (CV and SPOP) (G) and 5637 cells (shNC and sh1) (H). The undetected genes, such as CXCL9 and IL-33 in T24 cells and CXCL13, IL-15, IL-22, IL-33, PDGFβ and TNF in 5637 cells, were not shown. Blue, downregulated genes; red, upregulated genes. I, ELISA for CCL2 secretion in the CM of T24 cells (CV and SPOP) and 5637 cells (shNC, sh1, and sh2). J, Quantification of U937 cell migration in a transwell system in response to the CM from 5637 cells (shNC, sh1, and sh1 treated with RS 504393 (2 μM), a CCL2 receptor inhibitor). K, CCL2 mRNA levels in 5637 cells (shNC, sh1, and sh1+shSTAT3) by RT-qPCR assay. sh1 or sh2, SPOP-knockdown; shNC, shRNA control. Data were presented as mean ± SD and analyzed by unpaired t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p ≥ 0.05. #, p < 0.05; ##, p < 0.01; ###, p < 0.001; with vs without co-cultured U937 cells.

Figure 6

Macrophage-secreted IL-6 enhanced UBC cell proliferation and cancer stemness, which was negatively regulated by SPOP. A, The heatmaps of RT-qPCR arrays for 48 cytokines/chemokines in U937 cells co-cultured with T24 cells (CV and SPOP; left panel) or 5637 cells (shNC and sh1; right panel). The undetectable genes (CXCL9, IL-15, IL-33, PDGFβ, and TNF in T24 cells and CSF3, IL-15, IL-22, PDGFβ, and TNF in 5637 cells) were not shown. B, A Venn diagram of macrophage-derived chemokine/cytokine genes, mediated by SPOP in UBC cells. Blue, downregulated genes; red, upregulated genes. C, ELISA for IL-6 secretion from U937 cells co-cultured with T24 cells (CV and SPOP), as well as 5637 cells (shNC, sh1 and sh2). D-F, Assessment of UBC cell proliferation and stemness by MTT assay (D), Western blotting analysis for stemness marker protein levels (E), and quantification of mCherry-labelled 5637 spheres (F) between 5637 shNC and sh1 cells, co-cultured with or without U937 cells or treated with IL-6 (50 ng/ml) for 4 days. ##, p < 0.01; ###, p < 0.001; or $$$, p < 0.001; co-culture with U937 or treatment of IL-6 vs vehicle control. G-I, Assessment of UBC cell proliferation and stemness by MTT assay (G), Western blotting analysis for stemness marker protein levels (H), and quantification of mCherry-labelled 5637 spheres (I) between 5637 shNC and sh1, co-cultured with or without U937 cells for 4 days. The addition of α-IL-6 neutralizing antibody (100 ng/ml) or isotype control IgG in the presence of U937 cells. sh1, SPOP-knockdown; shNC, shRNA control. Data were presented as mean ± SD and analyzed by unpaired t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p ≥ 0.05.

Figure 7

SPOP was a direct downstream target of VEZF1 in UBC cells. A, A Venn diagram illustrating a significantly positive association between transcriptional factors from Human TFome library and SPOP mRNA level in four UBC datasets. B, Heatmap depicting the correlations between four transcriptional factors and SPOP mRNA level in eight UBC public datasets, determined by Pearson correlation analysis. C, Pearson correlation of SPOP and VEZF1 mRNA levels in TCGA-BLCA dataset. D-E, VEZF1 mRNA levels in normal bladder and UBC samples from TCGA-BLCA (D) and GSE13507 (E) datasets. F-G, Western blotting analysis (F) and quantification (G) of SPOP protein levels in 24 paired UBC (T) and adjacent normal (N) tissues. H-I, The depletion of VEZF1 reduced SPOP at mRNA (H) and protein level (I) in T24 cells. J, VEZF1 binding consensus sequence from JASPAR database. K, Schematic representation of four putative VEZF1 binding sites within intron 1 of SPOP gene, predicted by JASPAR. Three amplicons for ChIP assay were indicated by blue arrow, with mutation sequences for each binding site marked in blue. Ex, exon. L, Luciferase activity assay on different SPOP reporters, containing wild-type (WT) or four mutant VEZF1 binding sites (MUT) following VEZF1 overexpression in 293T cells. CV, control vector. M, ChIP assay by VEZF1 antibody on SPOP gene promoter in T24 cells, with IgG as a negative control. Data were shown as mean ± SD and analyzed by unpaired t test in (D-E, H, L-M) or by paired t test in (G). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p ≥ 0.05. #, p < 0.05; ##, p < 0.01; MUT vs WT-reporter luciferase activity with VEZF1 overexpression.

Figure 8

VEZF1 suppressed macrophage-mediated UBC proliferation and stemness. A, Western blotting analysis confirming the ectopic expression of VEZF1 in T24 cells. B, MTT assay on T24 cells (CV and VEZF1) after 96 h co-cultured with or without U937 cells in a non-contact system. C, Sphere assay performed on mCherry-labelled T24 cells (CV and VEZF1) co-cultured with or without U937 cells at day 6. D, Western blotting analysis for stemness markers in T24 cells with VEZF1 overexpression co-cultured with or without U937 cells. E, SPOP depletion in T24 cells with VEZF1 overexpression using siRNA (siSPOP) by RT-qPCR analysis. siNC, negative control. F, RT-qPCR analysis on U937 cells co-cultured in a non-contact way with T24 cells with VEZF1 overexpression (VEZF1; CV, negative control)/SPOP depletion (siSPOP; siNC, negative control) to assess expression of pan-marker (CD68), M1-like marker (CD86 and iNOS), and M2-like markers (CD163, CD206 and CCL22) in macrophages. Cell culture medium alone (RPMI1640) served as the negative control. G, Expression of SPOP, VEZF1, CD68 and CD206 in 94 human UBC tissues by IHC staining. Representative images from two UBC patients were displayed. Scale bar, 50 μm; the inset, 250 μm. H-J, The association of VEZF1 and SPOP protein levels (H), CD68⁺ macrophage number (I) and CD206⁺ macrophage number (J) in 94 human UBC patients. K, Kaplan-Meier survival plot of cumulative overall survival of 94 UBC patients, divided by SPOP^LowVEZF1^Low vs Others. Data were presented as mean ± SD and analyzed by unpaired t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p ≥ 0.05.