FBXO28 promotes the malignancy of non-small cell lung cancer through ubiquitination-mediated activation of BRD4

Abstract The E3 ubiquitin ligase FBXO28 has been implicated in the progression of various cancers, yet its precise function and substrates remain poorly understood. In this study, we investigated the role of FBXO28 in non-small cell lung cancer (NSCLC) and its underlying mechanism. We observed an upregulation of FBXO28 in NSCLC, and higher FBXO28 levels correlated with poorer patient outcomes. Functional experiments revealed that silencing FBXO28 inhibited NSCLC proliferation, migration, and invasion and induced apoptosis, whereas overexpressing FBXO28 had the opposite effects. Using coimmunoprecipitation (co-IP) assays, we identified an interaction between FBXO28 and BRD4. Further investigations revealed that FBXO28 mediated the ubiquitination of BRD4 at K395 through a K63-conjugated model. The ubiquitination by FBXO28 does not lead to the degradation of BRD4, but promotes its transcriptional coactivation, as the modulation of FBXO28 affects the expression of BRD4-targeting genes and the binding of BRD4 to the promoters of its target genes. Mechanistically, FBXO28-mediated ubiquitination of BRD4 facilitates the activation of P-TEFb and subsequent phosphorylation of the serine 2 (Ser2) C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) to initiate transcription. Furthermore, in a positive feedback loop, BRD4 binds to the FBXO28 promoter and increased FBXO28 transcription. In summary, our findings highlight the pivotal role of the FBXO28/BRD4 positive feedback loop in driving the malignancy of NSCLC.