Abnormal Expression And Modifications Of Proteins In Esophageal Squamous Cell Cancer Using Quantitative Proteomics Approach

Esophageal cancer(EC)is one of the most aggressive cancers worldwide,and 300,000 people die of EC every year in the world,the five-year survival rate of patients is less than 10%.Esophageal Squamous Cell Cancer(ESCC)has the highest clinical incidence of esophageal cancer,accounting for 90% of clinical cases of esophageal cancer.The occurrence of esophageal cancer has a strong regional character,and China is one of its main occurrence places,especially in rural areas of China.Today,the treatment of esophageal cancer mainly depends on surgery,but most of the patients with esophageal cancer are in the terminal stage of cancer when they are found to be ill.The main reasons for the high incidence,and low detection rate of esophageal cancer are insufficient understanding of the causes of occurrence and the molecular mechanism of cancer development.The found of tumor markers or therapeutic targets will help to improve the diagnosis and treatment success rate of esophageal cancer.Protein is the major executor of life activities,also plays an important role in the development of cancer.In recent years,mass spectrometry-based proteomics has gradually become an effective method for identifying and quantifying proteins and post-translational modifications of proteins.In this study,we used quantitative proteomics to study the proteome,post-translational modifications and histone post-translational modifications of SHEEC.By using the quantitative proteomics of SHEEC and SHEE cell lines,we found the activated HIF-1 pathway and glycolysis pathway at the protein level.Following this clue,we then enriched and quantitatively analyzed phosphorylation,lysine acetylation and lysine succinylation,which are closely related to these two pathways.In the analysis of these three post-translational modifications,we found a large number of down-regulated lysine succinylation sites in SHEEC cell line.With the phenomenon of low succinylation as a starting point,we explored the effect of low succinylation on cells.Through the comprehensive description of metabolic pathways,we found that metabolic enzymes in metabolic pathways are riched with down-regulated lysine succinylation,and the decrease of succinyl-CoA may be the cause of down-regulated lysine succinylation.At the same time,we found that lysine succinylation can affect the migration ability of ESCC,and the migration ability of ESCC cells will be significantly inhibited after lysine succinylation up-regulated.When we introduce mutations on metabolic enzymes to mimic the up-regulated lysine succinylation sites,cell migration is also affected.Lysine succinylation also affects the level of histone H3K9me2,and the level of H3K9me2 is related to the migration ability of ESCC.More importantly,the migration of ESCC cells is inhibited in vitro or in vivo once the level of lysine succinylation upregulated.Finally,we confirmed the phenomenon of low succinylation in primary ESCC specimens,and the level of low succinylation was proportional to the high lymphatic metastasis rate.As the H3K9me2 is related to ESCC cell migration,we then analyzed histone post-translational modifications.In total,11 histone modification types were mapped to 115 amino acid sites on histones,including 6 non-acetylated short chain acylation modifications.Then we quantified more than 200 different histone-modified peptides by using a quantitative method based on mass spectrometry Data Indenpent Acquisition(DIA)mode,and finally we found 17 significantly changed histone modification sites on H3 and H4.As the dysregulated histone PTMs were found,we then quantified histone modifications in another two malignant ESCC cell lines with different cell migration abilities.Our study showed that the expression of lysine dimethylation,such as H3K9me2,H4K20me2 and H3K36me2,these PTMs increased with the increase of cell migration ability.Next,we performed the transcriptomic analysis on histone modifying enzyme abundances as a proxy for quantifying their activity levels in concurrent to the proteomic analysis.As a result we found that the regulatory mechanism of histone post-translational modification is complex and needs further more study.Finally,we enriched the binding protein of H3K9 hib with a probe enrichment material,and found that PPP1R10 protein may be a binding protein of H3K9 hib.Through the proteomics,the PTMs proteomics and histone modification analysis,and combined with biochemical analysis,clinical samples,and esophageal cancer cell tumor behavior research,we found that the abnormal esophageal protein molecular networks and PTMs sites including lysine acylation and histone modification of ESCC cell metabolism and the impact of migration.These findings suggest that lysine succinylation and histone modification can regulate the metabolism and migration of ESCC cells,and may provide new evidence for the functional significance of post-translational protein modification in cancer biology.