Effects And Mechanism Of Acetylation On AKR1C1 In Promoting Lung Cancer Metastasis

IntrodutionMetastasis accounts for more than 90%of Non-Small Cell Lung Cancer(NSCLC)related death.Once distant metastasis occurs,the five-year survival rate of NSCLC patients will drop from 50%to less than 10%.Although a large number of NSCLC patients have benefited from targeted therapies that constrain the abnorml proliferation of tumor cells,there is still a lack of effective therapeutic drugs for patients with metastases.Studying key pathways and proteins that regulate NSCLC metastasis will hopefully find potential targets for interventional metastasis.In our previous study,AKR1C1 promotes NSCLC metastasis by activating the STAT3 signaling pathway and the downstream target genes of STAT3.AKR1C1 is a member of the human aldo-keto reductase protein family that catalyzes NADP+-dependent reduction,and thus plays essential roles in the metabolisms of steroid hormones,prostaglandins and polycyclic aromatic hydrocarbons.However,our recent study uncovered that depriving AKR1C1 of its reductase activities(by utilizing 5-BPSA or reductase activity-loss mutants)could minimally affect cell motility and STAT3 activation,indicating that the canonical enzymatic activities are dispensable for AKR1C1 to promote NSCLC metastasis.We therefore concluded that there must be other regulatory mechanisms underpinning the pro-metastatic effects of AKR1C1.Acetylation is a highly conserved post-translational modification,which plays a key role in regulating a myriad of biological functions,such as enzymatic activity,protein stability and regulating tumor malignant proliferation and metastasis.During our investigation,a high abundance of lysine acetylation was observed in AKR1C1 through mass spectrometry.Therefore,we speculate that whether the acetylation of AKR1C1 is involved the metastasis of NSCLC;if it is involved,and is there any new therapeutic interventions for regulating the metastasis of NSCLC based on AKR1C1 acetylation?ObjectiveThe research of this thesis aims to explore the acetylation modification of AKR1C1 and its role in metastasis of NSCLC;Explore the mechanism of regulating the metastasis of NSCLC based on the acetylation of AKR1C1 protein.Contents and MethodsIn this study,we investigate the acetylation modification of AKR1C1 protein and its specific sites.We study the effects of acetylation modification of AKR1C1 protein on NSCLC cell migration,enzymatic activity and protein stability.We identify the deacetylase of AKR1C1 protein and explore the effects of acetylation of AKR1C1 protein on STAT3 signaling pathway.Immunoprecipitations and Mass spectrometry were used to investigate the acetylation of AKR1C1 protein and its specific acetylated sites;Transwell assays were used to study whether AKR1C1 deacetylation could influence the metastasis of NSCLC cells;Immunoprecipitation,Western Blot and quantitative Real-Time PCR(qRT-PCR)were used to determine the effects of deacetylation of AKR1C1 on STAT3 pathway;Purified protein technology was used to detect whether AKR1C1 acetylation is dependent on its reductase activity;Western Blot analysis was utilized to investigate the effects of acetylation of AKR1C1 on its protein stability;Co-Immunoprecipitation and Transwell assays were introduced to identify the potential deacetylase and its effects and mechanism in inhibiting the metastasis of NSCLC.ResultsAccording to our research,we find AKR1C1 is an acelyated protein and it is acetylated at lysines 185 and 201.Acetylation of AKR1C1 is fundamental for its pro-metastatic ability in NSCLC cells.Acetylation of AKR1C1 plays a signaling role in activating STAT3 pathway in NSCLC while deacetylation of AKR1C1 losts its function.Acetylation has no effects on the reductase activity of AKR1C1,the Kcat/Km of AKR1C1-2KR is 0.7 ± 0.1 min-1μM-1 and it is no significant difference with AKR1C1-WT.Acetylation has no effects on the protein stability of AKR1C1.SIRT2 binds to and deacetylates AKR1C1 protein,it suppresses the metastatic ability of AKR1C1 in NSCLC through STAT3 pathway.ConclusionsThe pro-metastasis factor AKR1C1 undergoes acetylation on two lysine sites,lysine 185 and lysine 201.Acetylation of AKR1C1 has little effects on its enzymatic activity and its protein stability,but is fundamental for its pro-metastatic ability in NSCLC.SIRT2 binds to and deacetylates AKR1C1.Deacetylation of AKR1C1 could abolish the interaction between AKR1C1 and STAT3,inhibit the activation of STAT3 pathway and suppress the pro-metastatic ability of AKR1C1.