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

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 impact on patient prognosis. 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 cell proliferation, migration, invasion, and induced apoptosis, while overexpression of FBXO28 had the opposite effects. Using tandem-affinity-purification mass-spectrum (TAP-MS) and co-immunoprecipitation (co-IP) assays, we identified an interaction between FBXO28 and BRD4. Further investigations showed that FBXO28 mediated the ubiquitination of BRD4 through a K63-conjugated model. This K63-conjugated ubiquitination of BRD4 appeared to influence the expression of genes targeted by BRD4, revealing FBXO28's role in mediating the transcriptional co-activation of BRD4. 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), initiating transcription. Besides, FBXO28 was found to promote the formation of liquid-liquid phase separation within the BRD4 complex, potentially enhancing transcription. Furthermore, in a positive feedback loop, we discovered that BRD4 bound to the FBXO28 promoter and increased the transcription of FBXO28. In summary, our findings highlight the pivotal role of the FBXO28/BRD4 positive feedback loop in driving the malignancy of NSCLC.