The deubiquitinase OTULIN promotes aerobic glycolysis and proliferation of gastric cancer cells / 肿瘤

ABSTRACT

Objective:To examine the expression of OUT domain deubiquitinase with linear linkage specificity(OTULIN)in gastric cancer tissues and explore the impact of OTULIN silencing on the proliferation of gastric cancer MKN45 and AGS cells as well as its underlying mechanisms. Methods:Immunohistochemical staining was performed to detect the expression level of OTULIN in 73 gastric cancer tissues and 24 normal gastric mucosa.The association between OTULIN expression and the prognosis as well as the clinicopathological features of gastric cancer patients was analyzed.The above results were validated using public data from The Cancer Genome Atlas(TCGA)database and Gene Expression Omnibus(GEO)database.CRISPR/Cas9 gene editing technology was used to construct OTULIN-knockout gastric cancer cells MKN45 and AGS.The efficiency of gene knockout was validated by Western blotting.The effects of OTULIN knockout on the proliferation of gastric cancer cells MKN45 and AGS were assessed by CCK-8 assay and soft agar colony formation assay.Immunoprecipitation-mass spectrometry technique was exploited to identify potential protein substrates interacting with OTULIN and linear ubiquitin molecule INT-Ub.7KR.The changes in the activity of rate-limiting enzymes for glycolysis were measured using pyruvate kinase(PK)activity assay kit and lactate production was analyzed by lactate colorimetric/fluorometric assay kit in OTULIN-depleted cells.The effect of OTULIN on substrate linear ubiquitination was evaluated using co-immunoprecipitation and Western blotting. Results:The expression level of OTULIN in gastric cancer tissues was higher than that in normal gastric mucosa(P=0.004 1)as revealed by immunohistochemical analysis.Patients with higher OTULIN expression in the cancer tissues had a lower suvival time(P=0.007 7).Analysis of datasets from TCGA and GEO databases also confirmed that OTULIN was highly expressed in gastric tissues(P＜0.05)and high OTULIN expression was associated with poor prognosis(P=0.011).Statistical analysis also showed that higher expression of OTULIN was correlated with later TNM stages(P=0.027 3)and was an independent indicator for shorter survival time of gastric cancer patients(P=0.04).Knockout of OTULIN significantly inhibited the proliferation(P＜0.000 1),decreased the activity of PK(P＜0.01)and reduced lactate production(P＜0.01)of gastric cancer cells.OTULIN interacted with several key enzymes in the glycolysis pathway and downregulated the linear ubiquitination levels of these enzymes,including pyruvate kinase M1(PKM1),PKM2,lactate dehydrogenase A(LDHA)and LDHB. Conclusion:OTULIN is a novel biomarker for predicting the prognosis of gastric cancer patients.It activates the glycolytic pathway and promote the progression of gastric cancer possibly by downregulating the linear ubiquitination modification of rate-limiting enzymes in glycolysis.