FBXL6 governs c-MYC to promote hepatocellular carcinoma through ubiquitination and stabilization of HSP90AA1.

Heat shot protein 90 (HSP90) AA1 functions as an onco-protein to regulate the assembly, manipulation, folding and degradation of its client proteins, including c-MYC. However, little is known about the mechanism of HSP90AA1 regulation. Transcriptome RNA-sequencing data of hepatocellular carcinoma (HCC) samples were used to detect the mRNA expression of FBXL6. Immunoprecipitation/Mass Spectrum (IP/MS) method was used to identify the interacting proteins of FBXL6. The co-immunoprecipitation assay was used to determine the interaction between FBXL6 and HSP90AA1. The in vivo ubiquitination assay was performed to determine the regulation of HSP90AA1 by FBXL6. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to determine the transcriptional regulation of FBXL6 by c-MYC. Immunohistochemical (IHC) staining was performed to study the correlation of FBXL6 and HSP90AA1 protein expression in 87 HCC samples. Cell counting and colony formation assays were implemented to detect the biological effects of FBXL6 on the growth of HCC cells in vitro. The effect of FBXL6 on HCC tumor growth in vivo was studied in a tumor xenograft model in mice. Here, we identified the orphan F-box protein FBXL6, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the ubiquitin ligase for HSP90AA1. FBXL6 promoted K63-dependent ubiquitination of HSP90AA1 to stabilize it. Through analysis of the TCGA dataset, we found that FBXL6 was significantly increased in HCC tissues and positively correlated with c-MYC pathway. FBXL6 accumulation in HCC causes the stabilization and activation of c-MYC by preventing HSP90AA1 degradation. The activated c-MYC directly binds to the promoter region of FBXL6 to induce its mRNA expression. Collectively, our data revealed an unknown FBXL6-HSP90AA1-c-MYC axis which might contribute to the oncogenesis of HCC, and we propose that inhibition of FBXL6 might represent an effective therapeutic strategy for HCC treatment. Video abstract.