b Effects of WT and KR mutant of FOXO3a, BRD4 on the activity of promoter luciferase in the presence or absence of MK2206. to the gene promoter, and induces its transcription. Pharmacological inhibition of either BRD4/FOXO3a association or CDK6 significantly overcomes Turanose the resistance of luminal breast malignancy cells to AKT inhibitors in vitro and in vivo. Our study reports the involvement of BRD4/FOXO3a/CDK6 axis in AKTi resistance and provides potential therapeutic strategies for treating resistant breast cancer. Introduction Breast Turanose cancer is usually a heterogeneous disease1,2, characterized into at least four different subtypes: luminal A, luminal B, ERBB2 overexpression, and basal-like3,4. Mutations of gene, which encodes the catalytic subunit (p110) of PI3K, occur in almost 40% of ER+/luminal subtype. In addition, mutations of and contribute to activation of the phosphatidyl inositol 3-kinase (PI3K)/AKT pathway in this subtype5. The PI3K/AKT pathway has key functions in regulating growth, survival, and metabolism in both normal and malignant cells. For example, AKT inhibits Forkhead box O (FOXO)-induced expression of growth inhibition and apoptosis induction genes by phosphorylating FOXOs and blocking their nuclear translocation6,7. These findings show that activation of the PI3K/AKT pathway is likely a causal genetic event in the luminal subtype of breast cancer; thereby, inhibition of this pathway represents a top priority for therapeutic intervention. Indeed, numerous clinical trials have evaluated the efficacy of over 30 drugs targeting different actions of the PI3K/AKT pathway in breast and other cancers, including several AKT inhibitors (AKTis) such as MK2206, AZD5363, and GSK690693. Although Turanose these inhibitors have shown evidence of suppressing growth and inducing apoptosis of luminal breast malignancy cells, responses of solid tumors to monotherapy have been modest and accompanied by quick emergence of drug resistance. For example, AKT inhibition induces the expression and phosphorylation of HER3, IGF-1R, and insulin receptor through FOXO-dependent transcriptional activation, suggesting that targeting different nodes of opinions regulation of PI3K/AKT inhibition may improve the killing effects of these inhibitors. Intriguingly, FOXOs proteins are usually deemed as tumor suppressors because of their growth-inhibitory and cell death-inducing ability; the functional functions and downstream target genes of FOXOs involved in drug resistance remain obscure. The Malignancy Genome Atlas (TCGA) data also indicate frequent amplification of (40%) and low levels of mutations in luminal-type breast malignancy5. The cyclin D1/CDK4/6 complex phosphorylates the retinoblastoma (Rb) protein, which leads to cell cycle activation8. Results from several studies indicate that and have an important role in estrogen-stimulated proliferation of breast malignancy cells in early to mid G1 phase9,10. Thus, CDK4 and CDK6 represent useful therapeutic targets of ER+ advanced breast malignancy. Turanose Consistent with this idea, combination of a Turanose Itga4 CDK4/6 inhibitor with an aromatase inhibitor achieves significant effect on suppressing advanced ER?+?/luminal subtype of breast cancer11. In addition, a combinatorial drug screen on multiple PIK3CA mutant cancers with decreased sensitivity to PI3K inhibitors revealed that combined CDK4/6-PI3K inhibition synergistically reduces cell viability12. Even though combination of PI3K and CDK4/6 inhibitors overcomes intrinsic and adaptive resistance leading to tumor regressions in PIK3CA mutant xenografts, the molecular mechanism underlying the resistance of AKTi and the synergy seen around the PI3K inhibitors and CDK4/6 inhibitors remain elusive. Recently, inhibitors of BRD4, a BET (bromodomain and extra-terminal domain name) family member, have shown significant effects in hindering tumor growth by suppressing the expression of oncogenes13,14. BRD4 has the capacity to assemble diverse transcriptional complexes on gene super-enhancers and activate RNA polymerase II-dependent transcriptional elongation. In the later, BRD4 was found to preferentially occupy on oncogene super-enhancers and maintain their high expression levels in tumor cells15, explaining why BET inhibitors could specifically suppress tumor cell growth and induce apoptosis. Our recent study also demonstrates that BET inhibitors disrupt the Twist/BRD4 conversation and effectively inhibit invasion and malignancy stem cell-like house of basal-like breast malignancy (BLBC) cells16. Although it is usually well accepted that BRD4-guided gene expression mediates diverse processes during tumor development and progression, whether and how BRD4 assembles transcriptional machinery on chromatin to activate opinions survival genes expression is totally unclear. Here our study discovered the novel role of FOXO3a/BRD4/CDK6 axis in AKTi resistance of luminal breast cancer cells. Results Bromodomain inhibitor enhances growth suppressive effects of AKTi As.