FOXP1 transcriptionally activates NUSAP1 to induce M2 polarization of tumor-associated macrophages via glycolysis in hepatocellular carcinoma

Abstract

Background In hepatocellular carcinoma (HCC), the polarization of tumor-associated macrophages (TAMs) towards the M2 phenotype is a key factor driving the formation of an immunosuppressive microenvironment and malignant progression, a process frequently associated with glycolytic metabolic reprogramming. Nucleolar and spindle associated protein 1 (NUSAP1) is aberrantly overexpressed in various malignancies. However, whether it promotes HCC progression by driving TAM M2 polarization through regulating tumor cell glycolysis remains unclear. Methods Bioinformatics analysis was used to examine NUSAP1 expression in HCC and its correlation with M2 TAM infiltration. qRT-PCR and Western blot were employed to validate NUSAP1 expression levels in HCC cell lines. Functional experiments, including flow cytometry, ELISA, colony formation, and Transwell assays, were conducted to investigate the regulatory effects of NUSAP1 on TAM M2 polarization and HCC cell malignant behaviors. GSEA and correlation analyses were performed to explore the association between NUSAP1 and the glycolysis pathway. The impact of NUSAP1 on glycolytic metabolic reprogramming in HCC cells was assessed via Seahorse energy metabolism analysis, ATP/lactate production assays, and detection of key glycolytic protein expression. Furthermore, bioinformatics analysis predicted the transcriptional regulation of NUSAP1 by forkhead box P1 (FOXP1), which was subsequently validated by ChIP and dual-luciferase reporter assays. Finally, in vitro rescue experiments and an allograft mouse tumor model were used to confirm that the FOXP1/NUSAP1 axis induces TAM M2 polarization via glycolysis and accelerates HCC progression. Results NUSAP1 was significantly overexpressed in HCC, and its expression level positively correlated with M2 TAM infiltration. Functional experiments demonstrated that NUSAP1 facilitates HCC progression by driving glycolytic metabolic reprogramming in tumor cells and inducing polarization to the M2 phenotype. Mechanistically, FOXP1, as a transcription factor, binds to the NUSAP1 promoter region and activates its transcription. Both in vitro and in vivo experiments further confirmed that the FOXP1/NUSAP1 axis promotes TAM M2 polarization by activating glycolytic metabolism, thereby accelerating HCC progression. Conclusion This study reveals a novel mechanism by which FOXP1 transcriptionally upregulates NUSAP1 expression, resulting in glycolytic metabolic reprogramming that induces TAM M2 polarization and promotes HCC progression. These findings identify a potential therapeutic avenue for reversing the immunosuppressive microenvironment in HCC.