CRL4-DCAF13 Ubiquitin Ligase Regulates Metastasis Of Non-small Cell Lung Cancer

Background: Tumor metastasis is the main cause of death in cancer patients.Controlling metastasis is the key to cure cancer,but there is still no effective anti-metastasis treatment.This study intends to explore new mechanisms of metastasis and regulation of NSCLC from the perspective of post-translational modification of proteins,which will have important guiding significance and clinical value for future development of effective treatment strategies.Protein ubiquitination is an important post-translational modification of proteins.Ubiquitin ligases are key enzymes that regulate the ubiquitination of proteins.Among them,cullin-RING ubiquitin ligases(CRLs)are the largest number of ubiquitin ligase families in the human body,and their members pass different cullin scaffold proteins(such as cullin-1,cullin-2,cullin-3 and cullin-4A)./4B,etc.)and RING protein and substrate recognition subunits combine to form ligase complexes;dysfunctional CRLs have been shown to be closely related to tumors.Our team has long been involved in the study of cullin 4-RING ubiquitin ligase(CRL4)and tumor pathogenesis.Given the pivotal role of CRL4 substrate recognition subunit DCAF protein(at least 50 members)in regulating the biological function of CRL4,this study focused on DCAF protein and carried out related work.Objective: To explore a new mechanism of metastasis regulation and potential intervention targets for non-small cell lung cancer based on DCAF protein.Methods: Firstly,the TCGA public cancer database was used to identify members of the DCAF protein family that were highly expressed in lung cancer and potentially related to prognosis,and then further validated by fresh clinical tumor tissue samples; human lung cancer cell line A549 and highly metastatic Mouse disseminated lung adenocarcinoma cell line 889 DTC was used as a cell model.RNA interference was used to knock down the intracellular DCAF protein.DCAF protein was identified in lung adenocarcinoma by cell proliferation assay,cell clone formation assay,cell scratch assay,and Transwell cell migration assay.The effects of in vitro migration,proliferation,survival,and migration of cancer cells;identification of tumor metastasis-associated DCAF protein substrates in combination with various biochemical techniques;establishment of conditional DCAF knockout mouse models by CRISPR gene editing techniques.Results: Through the above work,the study obtained the following results: 1.It was found that DCAF13 protein is highly expressed in non-small cell lung cancer tissues and suggests poor prognosis.2.Confirmed that down-regulation of intracellular DCAF13 protein can significantly inhibit the migration of non-small cell lung cancer cells in vitro,but has little effect on tumor cell proliferation and survival.These results suggest that DCAF13 protein has the potential to promote lung cancer metastasis in vivo.3.Identification of DCAF13 can interact with DDX47 helicase(a tumor "dry" related protein)and promote its intracellular polyubiquitination,but this ubiquitination modification does not promote the DDX47 solution.Helicase degradation,thus suggesting that the ubiquitination of DDX47 helicase,is involved in the regulation of downstream signaling pathways in a non-degradative form.4.It was confirmed that DCAF13 mediated the ubiquitination of DDX47 helicase at position 250 is lysine(K250),and the type of linkage between ubiquitin molecules(Linkage)is mainly K63.5.The successful establishment of a conditional DCAF13 knockout mouse model laid a foundation for the subsequent identification of the impact of DCAF13 protein on the occurrence,development and metastasis of non-small cell lung cancer.Conclusions: This study found that DCAF13 was abnormally high in non-small cell lung cancer tissues;it was confirmed that DCAF13 regulates the in vitro migration of non-small cell lung cancer cells and promotes the ubiquitination of DDX47 helicase.