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

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.