JQ1 Upregulates FoxO1 Expression To Inhibit The Growth Of Non-small Cell Lung Carcinoma Cells

Background At present,lung cancer is still the main cause of cancer death,and the 5-year survival rate is relatively low.Most lung cancer patients are in advanced stage when they are discovered and diagnosed,so chemotherapy is still the main treatment for lung cancer.JQ1,an inhibitor of bromodomain and extraterminal(BET)domain proteins,has shown anticancer effect on lung cancer;however,its mechanism hasn't been fully elucidated.The Forkhead transcription factor Fox O1 plays an important role in the survival of tumor cells.Previous studies have demonstrated that Fox O1 plays an important inhibitory role in the growth of non-small cell lung cancer.Purpose In this study,we aimed to elucidate the role of Fox O1 in the growth inhibitory effect of JQ1 on non-small cell lung carcinoma cells(NSCLCs).Methods 1.H1299,Calu1,H157 and A549 cells were cultured with various concentrations of JQ1 for 3 days.The cell viability in each condition versus no drug condition was evaluated by SRB assay.H1299 cells were treated with JQ1 for 0-24 h,then We examined the expression of apoptosis-related by western blotting.2.The basal Fox O1 protein expression levels in these four NSCLC cell lines were examined by western blotting.H1299,H157 and Calu-1 cells were cultured with JQ1 for 24 h,then we detected the Fox O1 m RNA and protein expression by q RT-PCR and wsetern blot,respectively.3.H1299 and H157 cells were transfected with BRD2,BRD3 or BRD4 si RNAs for 48h.Next,we detected the expression of Fox O1.H157 cells were transfected with BRD2,BRD3 or BRD4 si RNAs,followed by treatment with or without JQ1 for 24 h,and then we examined BRD2,BRD3,BRD4 and Fox O1 protein levels.4.H157 and H1299 cell lines transfected with Fox O1 si RNAs or its control for 24 h were reseeded to 96-well plates and treated with or without JQ1 on the following day and subjected to a 3-day SRB assay.5.H157 and H1299 cell lines infected with adenovirus encoding an active form of Fox O1(Ad-CA)or its control(Ad-Ctrl)for 24 h were reseeded to 96-well plates and treated with or without JQ1 on the following day and subjected to a 3-day SRB assay.Results 1.The cell number of H1299,Calu-1,H157 and A549 significantly decreased after JQ1 treatment for 3 days in a dose-dependent manner.The protein levels of cleaved PARP and cleaved caspase 9 were significantly increased after JQ1 treatment for 12�24h in H1299 cells.2.The m RNA levels of Fox O1 increased significantly in H1299,H157 and Calu-1 cell lines treated with JQ1.western blot analysis showed that the protein levels of Fox O1 increased significantly in a dose-dependent manner.3.Knockdown of BRD2,BRD3,BRD4 does not increase Fox O1 expression in H1299 and H157 cells.Knockdown of BRD2,3,or 4 by si RNA,JQ1 still upregulated Fox O1 protein levels in H157 cells.4.Knockdown of Fox O1 expression by si RNA promoted the growth of H1299 and H157 cells.And knockdown of Fox O1 expression partially reversed the growth inhibitory effect of JQ1 compared with control in H1299 and H157 cell lines.5.Overexpression active Fox O1 inhibited H1299 and H157 cell growth.Moreover,JQ1 induced growth inhibition was further enhanced by overexpression of active Fox O1.Conclusions JQ1 upregulates Fox O1 expression to inhibit the growth of non-small cell lung carcinoma cells.The increase of Fox O1 expression by JQ1 is not by inhibiting the BET protein.Our results suggest a new strategy for enhancing JQ1's anticancer effect by co-targeting Fox O1.