Abstract
Abstract Objective: All-trans retinoic acid (ATRA) is a widely-used differentiation drug that can effectively induce osteogenic differentiation of osteosarcoma cells, but the underlying mechanism remains elusive, which limits the clinical application for ATRA in osteosarcoma patients. In this study, we identified E2F1 as a novel regulator involved in ATRA induced osteogenic differentiation of osteosarcoma cells. Methods: Osteosarcoma cell lines were used as our research models. (1) Protein levels were detected by Western blotting; (2) The interactions between E2F1 and RARalpha were determined by immunofluorescence, immunoprecipitation and GST pull-down assay; (3) The mRNA levels were determined by RT-PCR; (4) Related proteins and genes were investigated via transfection; (5) RARE activity were detected by luciferase reporter assay. (6) Alkaline phosphatase activity was assessed by colorimetric assay using BCIP/NBT Alkaline Phosphatase Color Development kit. Results: We observed that osteosarcoma cells are coupled with individual differences in the expression levels of E2F1 in patients, and E2F1 impairs ATRA-induced differentiation of osteosarcoma cells. Moreover, remarkable anti-proliferative and differentiation-inducing effects of ATRA treatment are only observed in E2F1 low to negative expressed primary osteosarcoma cultures. These results strongly suggested that E2F1 may serve as a potent indicator for the effectiveness of ATRA treatment in osteosarcoma. Interestingly, E2F1 is found to down-regulate retinoic acid receptor (RAR), a key factor determines the effectiveness of ATRA. E2F1 specifically binds to RAR and promotes its ubiquitination-mediated degradation; as a consequence, RAR-mediated differentiation is inhibited in osteosarcoma. Conclusion: E2F1 may serve as a potent biomarker as well as a therapeutic target for ATRA-based differentiation therapeutics, and raise the hope of using differentiation-based approaches for osteosarcoma patients. Citation Format: Qian Zhou, Meidan Ying, Bo Yang, Qiaojun He. E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma by promoting ubiquitination-mediated degradation of RARa. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1142.
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