OTUD1 Antagonizes BH3 Mimetic Inhibitor Induced Cell Death Through Regulating MCL1 Protein Stability

Objective Drug resistance is a key problem in tumor therapy.Many studies indicated that the BCL-2 family plays an important role in tumor cell survival and drug resistance.The BCL-2 family can be divided into anti-apoptotic subfamily and pro-apoptotic subfamily according to their functions.MCL1 belongs to the BCL-2 anti-apoptotic subfamily.MCL1 was first discovered from human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway.MCL1 plays key roles in embryonic development,lymphatic system development,cell growth,cell survival,and so on.Some tumor cells up-regulated MCL1 gene expression level to inhibit apoptosis induced by chemotherapy drug.Intervention of MCL1 gene in cancer cells greatly sensitized to chemotherapy drugs.Therefore,researchers have paid greatly attention in functional research and targeted drug design of MCL1.The functions of MCL1 are regulated at transcription level and post-translational level.Ubiquitination has been shown to play an important role in regulating the functions of MCL1 protein.Studies indicated that MCL1 protein was ubiquitinated by several ubiquitin ligases,which in turn leading it to be degraded in a proteasome dependent manner.However,whether and how deubiquitinating enzyme regulates MCL1 still need to investigated.Our work will not only uncover the mechanism of how deubiquitinating enzyme regulates MCL1,but also investigate the role of deubiquitinating enzyme in tumor drug resistance.Methods1.Transfected the deubiquitinases gene expression plasmids and the control plasmid into He La cells respectively,36 hours later,cells were harvested.The protein level of MCL1 were analyzed using Western Blot;2.Constructed sh RNA clones of deubiquitinase.Packing indicated sh RNA lentivirus,and infected cells to knock down the deubiquitinase.Then the protein level of MCL1 were analyzed using Western Blot;3.The deubiquitinase and MCL1 plasmid were co-transfected into cells.Then the cells were treated with protein synthesis inhibitor Cycloheximide for indicated time.Then the cells were harvested and the protein level of MCL1 were analyzed using Western Blot;4.Constructed deubiquitinase enzymatic activity mutant and related domain-deletion expression clones,and transfected these expression clones into cells.Then the cells were harvested and the protein level of MCL1 were analyzed using Western Blot;5.The deubiquitinase and MCL1 plasmid were co-transfected into 293 T cells.Then the cells were harvested to performed immunoprecipitation to detect whether the deubiquitinase interacts with MCL1;6.The control vector,the deubiquitinase expression vector and its related enzyme activity mutation expression vector were co-transfected into 293 T cells with MCL1 and ubiquitin expression vector into cells respectively.Then the cells were harvested and the ubiquitination level of MCL1 were analyzed using Western Blot;7.Deubiquitinase overexpression or knockdown cells lines were treated with the BH3 mimetic inhibitor ABT-263 or ABT-263 and Sorafenib.Then the cell viability were determinted using Cell Titer-Glo kit.8.Analyzing the relationship between the expression level of deubiquitinase gene and the survival rate of cancer patients using bioinformatics method.Results1.After screening,we found that deubiquitinating enzyme OTUD1 significantly increased the protein level of MCL1 in different cell lines;2.Knockdown OTUD1 reduced the protein level of MCL1;3.Overexpression of OTUD1 increased the protein stability of MCL1;4.Overexpression of OTUD1 and its the enzymatic activity domain significantly increased the protein level of MCL1,while the enzymatic activity mutant of OTUD1 and its N-terminal domain did not upregulated the protein level of MCL1;5.The results of co-immunoprecipitation experiments indicated that both OTUD1 and its enzymatic activity domain interacted with MCL1;6.The results of co-immunoprecipitation experiments indicated that OTUD1 deubiquitinated MCL1,while the enzymatic active mutant of OTUD1 lost this function;7.Overexpression of OTUD1 inhibited the toxicity of ABT-263 towards tumor cells.Consistently,knockdown OTUD1 enhanced the toxicity of ABT-263 towards tumor cells.Moreover,overexpression of OTUD1 reduced the toxicity of ABT-263 and Sorafenib towards tumor cells.8.High expression of OTUD1 is an indicator of poor prognosis of patients of liver cancer,ovarian cancer,and certain subtypes of breast cancer and cervical cancer.ConclusionOTUD1 interacted with MCL1 and promoted the deubiquitination of MCL1,thereby increasing the stability of MCL1 protein.Functional studies have shown that OTUD1 affected the toxicity of ABT-263 and its combination with Sorafenib towards tumor cells.Bioinformatics analysis indicated that the high expression of OTUD1 is negatively correlated with the prognosis of various tumor patients.