The Role Of CUL4A In Cisplatin Resistance In Gastric Cancer

Objective: Gastric cancer(GC)is the second most common cancer in China.As a highly heterogeneous disease,GC progresses rapidly and is prone to develop chemo-resistance,resulting in a disappointing therapeutic outcome as well as a poor prognosis.Thus,it is of great clinical significance to identify the key factors in chemo-resistance to improve the therapeutics for GC.Dys-regulation of protein ubiquitination has been found to be involved in many malignancies,but its role in chemo-resistance remains largely elusive.As a key component of the Cullin-RING ubiquitin ligase 4A complex,Cullin 4A(CUL4A)acts as a scaffold protein.Its N-terminus binds to the adaptor protein DDB1,through which to recruit different substrate receptors(DCAFs)for the recognition of their corresponding substrates,while its C-terminal interacting with the ubiquitin conjugating enzyme(E2)upon binding to ROC1/RBX1.Thus,CUL4A brings the substrate and E2 close to each other,so as to transfer the activated ubiquitin molecule attached on E2 to the substrate.The substrates of CUL4A ubiquitin ligase are involved in a wide range of biological processes,such as signal transduction,transcriptional regulation,cell cycle regulation,maintenance of genomic stability and embryonic development.Our recent immunohistochemistry results showed that the level of CUL4A was associated with the chemotherapeutic outcomes of GC patients.Patients with low expression level of CUL4A were more resistance to chemotherapy,suggesting a potential role of CUL4A in modulating chemo-resistance.In this study,we further explored the molecular mechanism underpinning the cisplatin resistance involving CUL4A in GC,providing a novel insight into the development of chemo-sensitizing drugs for GC.Methods: 1.The expression levels of CUL4A in gastric cancer cell lines and normal gastric epithelial cells were determined by Western Blotting.2.The previously constructed CUL4A-knockdown plasmid in the laboratory,along with the lentivirus packaging plasmids,were transfected into the 293 T cells using the transfection reagent Lipofectamine TM3000,to generate the lentiviral particles(p LKO-Tet-On-sh CUL4A)with the ability to silence the expression of CUL4A.Subsequently,gastric cancer cell lines were infected with sh CUL4A lentiviral particles to establish stable gastric cancer cell lines with CUL4A knocked-down.Western Blotting was used to determine the levels of CUL4A in control and CUL4A-knockdown cells.3.After the lentiviral mediated knockdown of CUL4A,the proliferation rate of gastric cancer cells was measured using CCK8 method,and the cell cycle and apoptosis assays were carried out by flow cytometry.4.The sensitivity of gastric cancer cells to cisplatin was determined by CCK8 assay after knocking down of CUL4A.5.Using the method mentioned above in “2”,SNU-1 cells were infected with lentiviral particles to establish stable SNU-1 cell line containing tetracycline inducible CUL4A knockdown construct.Upon knocking down CUL4A in these SNU-1 cells by tetracycline adduction,the total proteins(control group and CUL4A-knockdown group)were collected and subsequently undergone the i TRAQ protein mass spectrometry to identify the potential substrate proteins of CUL4A ubiquitin ligase.6.After knocking down CUL4A in SNU-1 and MKN45 cells,Western Blotting and q RT-PCR were employed to determine the m RNA and protein levels of the above potential substrate proteins,among which CLIC4 was validated to be a potential substrate protein due to its elevated protein level but no change at the m RNA level.7.The validation of CLIC4 as a substrate for CUL4A ubiquitin ligase was further performed by Co-IP,Ubiquitin and half-life assays.8.The cisplatin sensitivity was determined after further knocking down CLIC4 in CUL4A-knockdown SNU-1 and MKN45 cells by CCK8 assay.Result: 1.CUL4A was highly expressed in gastric cancer cell lines SNU-1,SGC-7901,MGC-803,MKN45,AGS and HGC-27.2.Knockdown of CUL4A had little effect on cell proliferation,cell cycle and apoptosis in SNU-1 and MKN45 cells,but significantly enhanced the cisplatin resistance of these cells.3.CLIC4 was identified as a novel substrate of CUL4A ubiquitin ligase in gastric cancer cells.4.The phenotype of cisplatin resistance by knocking down of CUL4A in gastric cancer cells was rescued after further knockdown of CLIC4 in these cells.Conclusions: 1.Knocking down the expression level of CUL4A in gastric cancer cells can significantly enhance the resistance of the cells to cisplatin,indicating that CUL4A plays an important role in modulating chemotherapy resistance in gastric cancer.2.CUL4A ubiquitin ligase controls the steady-state level of CLIC4 by mediating its ubiquitination,thereby modulating the cisplatin resistance in gastric cancer cells..