Branched-chain Amino Acid Catabolism Reprogramming Identified By OMICS Exacerbates Endometrial Cancer Progression

Objective:The incidence rate of endometrial cancer(EC)among gynecological malignancies is the first in developed countries and the second in China.EC is one of the few cancers with significantly increased morbidity and mortality.Surgery is the most effective treatment for EC.However,patients with advanced and recurrent EC have low benefit from surgery,their treatment methods are limited and prognosis is poor.With the promotion and application of molecular subtypes,the diagnosis and treatment of endometrial cancer tends to be more individualized and precise.Molecular subtypes can better judge the prognosis of disease and guide clinical treatment.Even so,there are still many problems to be solved,such as the increase of morbidity and mortality,the absence of effective drugs for patients with advanced and recurrent diseases,and the absence of specific biomarkers to monitor the occurrence and progress of EC.The energy characteristic of tumor is high metabolism and high consumption.Metabolic reprogramming can help cancer cells obtain sufficient energy and material synthesis materials.Branched-chain amino acids(BCAAs)as important nutritional signals play an important mediating role in protein synthesis,glucose homeostasis,antiobesity and nutrient-sensitive signal pathways.BCAAs produce a large number of metabolites during the degradation process,which can affect glucose,fatty acid and other metabolic pathways.Therefore,changes in BCAAs metabolism can not only affect the inherent characteristics of cancer,but also reflect changes in metabolic system related to some cancers.Further study the mechanism of BCAAs in cancer can provide new ideas for cancer treatment.The over-activation of Wnt/β-Catenin signal is related to the occurrence,progression,recurrence and drug resistance of gynecological malignant tumors.When the β-Catenin dependent Wnt signal pathway is activated,it will cause β-Catenin accumulates in the cytoplasm and forms a complex with TCF/LEF as a transcription factor.These complexes are then transferred to the nucleus and participate in gene transcription of regulatory factors encoding cell cycle,and their overexpression is related to a variety of human cancers.Mitochondria constantly undergo the confrontation process of division and fusion to maintain the normal operation of mitochondrial function.Dynamin-related protein 1(DRP1)is recruited to the outer membrane of mitochondria to drive mitochondrial division.The balance of mitochondrial dynamics plays an important role in maintaining mitochondrial function and cell homeostasis.Abnormal mitochondrial dynamics can lead to the occurrence of tumors.The purpose of this study is to explore the role of BCAAs in the development of endometrial cancer,the effect of BCAAs metabolism on the biological behavior of endometrial cancer,and the potential mechanism of BCAAs metabolism key inhibitory enzymes branched-chain keto acid dehydrogenase kinase(BCKDK).Methods:1.We selected fecal samples from clinical patients for 16s DNA sequencing to analyze the changes of gut microbiota in EC patients and the functional prediction of different bacterial groups.2.The blood samples of clinical patients were analyzed by metabolite mass spectrometry,and the content of amino acids in the blood of patients was concerned.3.Proteomics was used to detect the differentially expressed proteins and the enrichment analysis of protein pathways in endometrial carcinoma and adjacent tissues.4.The effect of BCAAs on the tumorigenesis rate was detected by subcutaneous tumor-bearing experiment in BALB/c nude mice fed with different levels of BCAAs.5.The expression of key enzymes of BCAAs metabolism in endometrial carcinoma was detected by qPCR and Western blot.6.The effect of leucine on the proliferation of Ishikawa and HEC-1A cells was detected by MTT assay.7.The effect of leucine on the migration and invasion of EC cell line was detected by Wound-healing assay and Transwell assay.8.Immunohistochemical analysis was used to detect the expression level of BCKDK protein in EC patients and cancer tissues of BALB/c nude mice.9.Colony formation assay and EdU assay were used to detect the effect of interference with BCKDK expression on EC cell proliferation.10.The effect of interference with BCKDK expression on the migration and invasion of EC cells was detected by Transwell assay.11.Cell apoptosis was detected by flow cytometry after BT2 inhibited the expression of BCKDK.12.Immunoprecipitation mass spectrometry(IP-MS)was used to detect the proteins interacting with BCKDK.13.Co-Immunoprecipitation(CO-IP)test was used to verify the possible interaction between BCKDK and proteins.14.The interaction site of BCKDK and interaction protein was detected by fluorescence co-localization.15.The changes of mitochondrial function after knockdown BCKDK were observed by JC-1 mitochondrial membrane potential detection and mitochondrial staining.16.The effect of BCKDK on proliferation and apoptosis pathway and the effect of changing the expression of BCKDK on the expression and activity of interacting proteins were detected by Western blot.Results:1.Through 16s DNA sequencing analysis of clinical fecal samples,it was found that there were differences in the composition of intestinal flora between EC patients and control groups,and these differences may affect branched-chain amino acid metabolism.2.Plasma metabolomics and proteomics results showed that the expression of key enzymes of branched-chain amino acid metabolism and branched-chain amino acid metabolism pathways in EC patients were abnormal.3.Subcutaneous tumorigenesis experiments in BALB/c nude mice found that compared with feeding a diet with high content of BCAAs and feeding a diet with normal content of BCAAs,there was no significant difference in the weight of BALB/c nude mice between the two groups,and the tumor growth rate of BALB/c nude mice with high content BCAAs diet was significantly faster than fed with a normal BCAAs diet.4.Leucine was treated to Ishikawa and HEC-1A cells to observe the effect of leucine on EC cell lines.The results show that leucine can enhance the vitality of EC cells,and can also promote the migration and invasion of EC cells.5.Through the study of BCKDK metabolic pathway,it was found that BCKDK was increased in EC,and BCKDK expression could be promoted by BCAAs.6.The proliferation,migration and invasion of EC cells were enhanced after overexpression of BCKDK.7.When the expression of BCKDK was knockdown by lentivirus infection,the malignant biological behavior was inhibited.Similarly,the same trend was obtained when BT2 which is an inhibitor of BCKDK,was used to inhibit the expression of BCKDK leading to EC cell apoptosis.8.BCKDK interaction proteins were found by IP-MS,and CO-IP showed that BCKDK could interact with β-Catenin and DRP1.9.BCKDK regulates mitochondrial function by affecting the activity of DRP1.After inhibiting BCKDK,mitochondrial membrane potential decreased and mitochondrial morphology changed after knocking down BCKDK.10.BCKDK impact β-Catenin Ser675 site and DRP1 Ser616 site phosphorylation level,thus activating β-Catenin and DRP1 signal pathway continue to play a role in accelerating the proliferation,migration and invasion of EC cells.Conclusion:In BCAAs metabolic pathway,disorder of branched-chain amino acid metabolism leads to BCKDK abnormally elevated,which acts on β-Catenin Ser675 site phosphorylation activates Wnt/β-Catenin promotes the proliferation of endometrial carcinoma.Meanwhile,it can also promote the phosphorylation of DRP1 at Ser616 site,which leads to mitochondrion division and enhances the malignant biological behavior of endometrial carcinoma.