TRIM65-BTG3-HIF-1α axis drives osteosarcoma progression via M2 macrophage polarization and angiogenesis

Background and Objectives Osteosarcoma (OS) is an aggressive bone cancer with a poor prognosis, especially in metastatic cases. The tumor microenvironment (TME), particularly tumor-associated macrophages (TAMs), plays a crucial role in its progression. This study aimed to investigate the role and mechanism of the tripartite motif-containing 65 (TRIM65), B-cell translocation gene 3 (BTG3), hypoxia-inducible factor 1α (HIF-1α; TRIM65-BTG3-HIF-1α) signaling axis in regulating TAMs and angiogenesis in OS. Methods Bioinformatic analysis of The Cancer Genome Atlas (TCGA) and TARGET-OS datasets identified M2 macrophage-related genes. Clinical OS samples were analyzed via immunohistochemistry (IHC), immunofluorescence (IF), messenger ribonucleic acid (mRNA) sequencing, and data-independent acquisition (DIA) quantitative proteomics. In vitro functional assays (cell counting kit-8 [CCK-8], colony formation, transwell migration/invasion, tube formation) and in vivo subcutaneous xenograft models in nude mice were performed using OS cell lines (U-2 OS [U2OS], sarcoma osteogenics [SAOS], human OS (HOS), 143B) and the human fetal osteoblast (hFOB) normal osteoblast line. Macrophage polarization was induced from Tohoku Hospital pediatrics-1 (THP-1) cells. Molecular interactions were assessed by co-immunoprecipitation (Co-IP), ubiquitination assays, and luciferase reporter assays. Results High infiltration of M2 macrophages correlated with poor patient prognosis. BTG3 was identified as a key M2-related gene exhibiting a "transcriptional upregulation with translational downregulation" pattern in OS. TRIM65, an E3 ubiquitin ligase upregulated in OS, directly interacted with and promoted the ubiquitin-mediated degradation of the BTG3 protein. Knockdown of TRIM65 inhibited OS cell proliferation, migration, invasion in vitro , and tumor growth in vivo . BTG3 acted as a negative regulator of HIF-1α transcriptional activity. Loss of BTG3 led to HIF-1α activation, which in turn promoted M2 polarization and enhanced migration of macrophages. Furthermore, conditioned medium from BTG3-deficient cells significantly promoted endothelial cell migration and tube formation, indicating enhanced angiogenesis. This pro-angiogenic effect was dependent on HIF-1α activation. Conclusions We delineate a novel TRIM65-BTG3-HIF-1α signaling axis in OS. TRIM65 degrades BTG3, leading to HIF-1α activation, which drives M2 macrophage polarization and angiogenesis, ultimately fostering tumor progression. Targeting this axis may offer a promising therapeutic strategy for OS.