H3K27ac-induced RHOXF2 activates Wnt2/β-catenin pathway by binding to HOXC13 to aggravate the malignant progression of triple negative breast cancer

Abstract

Triple negative breast cancer (TNBC) is insensitive to conventional targeted therapy and endocrine therapy, and is characterized by high invasiveness and high recurrence rate. This study aimed to explore the role and mechanism of RHOXF2 and HOXC13 on the malignant progression of TNBC. RT-qPCR and western blot were used to detect RHOXF2 and HOXC13 expression in TNBC cells. The proliferation, colony formation, invasion, migration, apoptosis and cell cycle of TNBC cells after transfection were analyzed by CCK-8 assay, colony formation assay, transwell assay, wound healing assay and flow cytometry analysis. Co-Immunoprecipitation and GST pull-down assays were used to analyze the combination between RHOXF2 and HOXC13. ChIP-PCR and luciferase reporter gene assay were used to examine the regulation of H3K27ac on RHOXF2. Besides, the expression of Ki67 and cleaved Caspase3 in tumor tissues of nude mice was determined by immunofluorescence. Results revealed that RHOXF2 and HOXC13 expression was increased in TNBC cells. RHOXF2 knockdown suppressed the proliferation, invasion and migration, as well as induced G0/G1 cell cycle arrest and apoptosis of TNBC cells. Besides, RHOXF2 could bind to HOXC13 and RHOXF2 knockdown suppressed HOXC13 expression in TNBC cells. Furthermore, HOXC13 overexpression reversed the impacts of RHOXF2 downregulation on the proliferation, invasion, migration, G0/G1 cell cycle arrest and apoptosis of TNBC cells. In addition, RHOXF2 silencing limited the tumor volume in nude mice, which was reversed by HOXC13 overexpression. Moreover, RHOXF2 knockdown interfered with Wnt2/β-catenin pathway in vitro and in vivo by binding to HOXC13. Importantly, H3K27ac acetylation could activate the expression of RHOXF2 promoter region. In conclusion, RHOXF2 activated by H3K27ac functioned as a tumor promoter in TNBC via mediating Wnt2/β-catenin pathway by binding to HOXC13, which provided promising insight into exploration on TNBC therapy.

Keywords: HOXC13; RHOXF2; Triple negative breast cancer cells; Wnt2/β-catenin signaling pathway.