Destabilization of macrophage migration inhibitory factor by 4‐IPP reduces NF‐κB/P‐TEFb complex‐mediated c‐Myb transcription to suppress osteosarcoma tumourigenesis

Abstract

BACKGROUND: As an inflammatory factor and oncogenic driver protein, the pleiotropic cytokine macrophage migration inhibitory factor (MIF) plays a crucial role in the osteosarcoma microenvironment. Although 4-iodo-6-phenylpyrimidine (4-IPP) can inactivate MIF biological functions, its anti-osteosarcoma effect and molecular mechanisms have not been investigated. In this study, we identified the MIF inhibitor 4-IPP as a specific double-effector drug for osteosarcoma with both anti-tumour and anti-osteoclastogenic functions. METHODS: The anti-cancer effects of 4-IPP were evaluated by wound healing assay, cell cycle analysis, colony formation assay, CCK-8 assay, apoptosis analysis, and Transwell migration/invasion assays. Through the application of a luciferase reporter, chromatin immunoprecipitation assays, and immunofluorescence and coimmunoprecipitation analyses, the transcriptional regulation of the NF-κB/P-TEFb complex on c-Myb- and STUB1-mediated proteasome-dependent MIF protein degradation was confirmed. The effect of 4-IPP on tumour growth and metastasis was assessed using an HOS-derived tail vein metastasis model and subcutaneous and orthotopic xenograft tumour models. RESULTS: In vitro, 4-IPP significantly reduced the proliferation and metastasis of osteosarcoma cells by suppressing the NF-κB pathway. 4-IPP hindered the binding between MIF and CD74 as well as p65. Moreover, 4-IPP inhibited MIF to interrupt the formation of downstream NF-κB/P-TEFb complexes, leading to the down-regulation of c-Myb transcription. Interestingly, the implementation of 4-IPP can mediate small molecule-induced MIF protein proteasomal degradation via the STUB1 E3 ligand. However, 4-IPP still interrupted MIF-mediated communication between osteosarcoma cells and osteoclasts, thus promoting osteoclastogenesis. Remarkably, 4-IPP strongly reduced HOS-derived xenograft osteosarcoma tumourigenesis and metastasis in an in vivo mouse model. CONCLUSIONS: Our findings demonstrate that the small molecule 4-IPP targeting the MIF protein exerts an anti-osteosarcoma effect by simultaneously inactivating the biological functions of MIF and promoting its proteasomal degradation. Direct destabilization of the MIF protein with 4-IPP may be a promising therapeutic strategy for treating osteosarcoma.