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

Abstract

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.

Keywords

• 4‐IPP
• CDK9
• c‐Myb
• MIF
• osteosarcoma
• STUB1