BRD4 Regulates Gemcitabine Efficacy by Modulating Autophagy Activity in Osteosarcoma Cells [Patient samples]

Therapeutic options for metastatic or recurrent osteosarcoma are limited. Gemcitabine and 12 docetaxel combination is used as second-line therapy for this disease. Bromodomain-containing 13 protein 4 (BRD4) plays essential roles in regulating autophagy in several human diseases and is 14 overexpressed in osteosarcoma. Thus, in the current study, we investigated whether BRD4 is 15 involved in the modulation of gemcitabine-induced autophagy in osteosarcoma. We confirmed 16 that gemcitabine induced autophagic flux in LM7 and CCH-OS-D human osteosarcoma cell lines. 17 Furthermore, we found that gemcitabine induced BRD4 protein and mRNA expression in a time-18 dependent manner in LM7 cells and also in CCH-OS-D cells but only at the mRNA level. Moreover, 19 gemcitabine induced CCAAT/enhancer binding protein beta (C/EBPβ), a transcription factor 20 known to regulate autophagy. We further showed that C/EBPβ induction was mediated by BRD4 21 as inhibition of BRD4 blocked its induction. Furthermore, we demonstrated that gemcitabine-22 induced C/EBPβ bound to BRD4. Because inhibition of BRD4 diminished autophagic flux, we 23 conclude that BRD4 may act as an epigenetic regulator of gemcitabine-induced autophagy in 24 osteosarcoma via C/EBPβ. Additionally, because inhibition of BRD4 in gemcitabine treated 25 osteosarcoma cells led to different outcomes, our data suggests the potential for the BRD4- 26 C/EBPβ interaction to define gemcitabine-induced autophagy outcome in osteosarcoma.