Inhibition of the FEN1-PBX1 axis elicits cellular senescence in breast cancer via the increased intracellular reactive oxygen species levels

Abstract

Abstract Background Cellular senescence is a state of irreversible cell growth arrest. As such, senescence induction is viewed as an efficacious countermeasure in cancer treatment. Flap endonuclease 1 (FEN1) has been reported to participate in tumor growth, metastasis and immunomodulation. However, the role of FEN1 in cellular senescence of breast cancer and its molecular mechanism remains unclear. Methods In vitro assessments of breast cancer cell senescence and apoptosis were conducted using CCK-8 assay, cell cycle assay, senescence-associated β-galactosidase (SA-β-gal) staining, and cleaved caspase-3 staining. Western blot, dihydroethidium (DHE) staining, RNA-sequencing, quantitative real-time polymerase chain reaction (qRT-PCR), rescue experiments, and dual-luciferase reporter assay were performed to explore the potential target of FEN1. Co-Immunoprecipitation (Co-IP), Chromatin immunoprecipitation (ChIP)-qPCR assay, and immunostaining were used to evaluate the interaction between FEN1 and Pre-B-cell leukemia homeobox transcription factor 1 (PBX1). A xenograft mouse model was employed to validate the effect of FEN1 on breast cancer cell senescence and apoptosis. Results Functional analysis demonstrated that FEN1 suppressed both senescence and apoptosis of breast cancer cells in vitro, while in vivo experiments demonstrated moderate therapeutic effects. Further studies indicated that FEN1 deficiency promoted the aforementioned effects by increasing intracellular reactive oxygen species (ROS) levels. RNA-sequencing and qRT-PCR assays revealed that FEN1 knockdown enhanced the expressions of several senescence-associated secretory phenotype (SASP) factors and resulted in decreased PBX1 level. The rescue experiments by PBX1 overexpression verified that PBX1 mediated the senescence and apoptosis of breast cancer cells induced by FEN1 inhibition. In detail, FEN1 downregulation inhibited the transcription activity of PBX1, which was partially restored by itself overexpression. Of note, FEN1 directly interacted with PBX1. Furthermore, immunostaining illustrated the colocalization of FEN1 and PBX1 in breast cancer cells and tissues. In our local breast cancer cohort, a positive correlation was identified between the expression levels of FEN1 and PBX1. Conclusions Knockdown of FEN1 facilitates breast cancer cell senescence through PBX1 down-regulation mediating increase in intracellular ROS levels. This study reveals FEN1 as a negative regulator of cellular senescence and provides support for pro-senescence cancer therapy. Given that FEN1 knockdown exhibited only moderate in vivo effects, these findings underscore the necessity of combining it with senolytic therapy to enhance therapeutic efficacy.

Keywords

• Cellular senescence
• FEN1
• PBX1
• ROS
• Breast cancer therapy