RBBP6-induced destabilization of FOXP3 promotes glucose metabolism and malignant progression of HBV-related hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection remains a leading cause of cancer-related mortality, yet the molecular mechanisms driving its progression are incompletely understood. Forkhead box P3 (FOXP3), a transcription factor involved in immune regulation, has emerged as a potential regulator in multiple cancers. However, its role in HBV-related HCC remains unexplored.

Methods

Quantitative reverse transcription PCR (qRT-PCR) and western blotting assays were utilized to assess FOXP3, retinoblastoma-binding protein 6 (RBBP6), and angiogenesis/metastasis-related markers including vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), transforming growth factor β (TGF-β). Functional assays included CCK-8, flow cytometry, colony formation, Transwell, and tube formation assays to evaluate proliferation, apoptosis, migration, invasion, and angiogenesis. Glucose metabolism was analyzed via colorimetric assays. Protein interactions among FOXP3, RBBP6 and regulatory factor X5 (RFX5) were examined using glutathione S-transferase pull-down, co-immunoprecipitation, ubiquitination, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. A xenograft mouse model was used to validate in vivo tumorigenicity of HBV-related HCC cells.

Results

FOXP3 expression was downregulated in HBV-positive HCC tissues and cells, contrasting with RBBP6 upregulation. In addition, FOXP3 overexpression suppressed HepG2.2.15 cell proliferation, migration, invasion, glucose uptake, lactate production, and angiogenesis while promoting apoptosis. Moreover, the result showed that RBBP6 destabilized FOXP3 via ubiquitination, and RBBP6 knockdown inhibited tumorigenicity and glucose metabolism by restoring FOXP3 function. RFX5 transcriptionally activated RBBP6 in HBV-positive HCC cells. Further, the study revealed that RBBP6 knockdown inhibited tumor formation in vivo, accompanied by the increased expression of FOXP3 in the transplanted neoplasms resulting from HBV-positive HCC cells.

Conclusion

RBBP6 ubiquitinated and destabilized FOXP3 to enhance glycolytic metabolism and malignant progression of HBV-related HCC. Targeting the RBBP6-FOXP3 axis may offer a novel therapeutic strategy for HBV-driven HCC.