Integrated Proteogenomic Characterization Of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma（ESCC）is one of the most invasive malignant cancers,and there is still a lack of effective targeted therapies.This study used mass spectrometry-based quantitative proteomics technology and whole exome sequencing technology to draw a panoramic map of proteogenomic of esophageal squamous cell carcinoma for the first time.A total of 291 surgical samples（including the follow-up information for the past 15 years）of patients were involved,including 286 tumor tissues（T）,212 distal normal epithelial tissues（N）,30 lymph node metastasis tissues（LNM）and 28 normal lymph node tissues（LN）.The integrated analysis of the multi-dimensional omics data produced by the above samples obtained the characteristics of ESCC mutation and protein expression.At the same time,the ESCC patients were molecularly typed according to the protein molecular phenotype,and the key molecules related to the prognosis of ESCC were preliminarily verified.Quantitative proteomic results show that,compared with normal esophageal epithelium,cell proliferation,cell migration and invasion,and extracellular matrix（ECM）cytokine secretion are the main molecular features of ESCC,while the formation of keratinized envelope and exogenous compounds metabolism is relatively down-regulated in ESCC,which is the pathways enriched in the esophageal epithelial tissues.We further screened specific molecular markers that are ubiquitously expressed in ESCC,but low or not expressed in normal esophageal epithelial tissues,and localized on the cell surface based on protein expression,these markers provided a chance for the further diagnosis and drug target design.Spatially resolved ESCC proteomics analysis reveals the molecular characteristic phenotypes of ESCC in different tumor microenvironments: In the mid-upper esophagus,long-term mechanical damage and more exogenous stimuli caused damage repair and inflammatory response to accumulate a large number of mutations,which in turn caused the disorder of downstream transcription and translation pathways,and finally caused the occurrence of esophageal cancer.In ESCC that occurs in the lower esophagus,PPAR-regulated lipid metabolism,phosphatidylinositol metabolism,and activation of NOTCH,PI3 K and other pathways have become the main characteristics: correspondingly,its adjacent tissues show significant enrichment of long-chain fatty acid metabolism,signal transduction and substance transport related molecules.It indicates that normal tissues with active lipid metabolism and substance transport may induce tumorigenesis through the activation of NOTCH and PI3 K signaling pathways.The comparative analysis of LNM,primary foci,and normal lymph nodes found that vascular e ndothelial growth factor receptor（VEGFR）,phosphatidylinositol（PI）metabolism and hypoxia activation pathways are the key pathways for LNM to respond to changes in the microenvironment in lymph nodes.We further used proteomics data to divide the ESCC cohort into three molecular subtypes with different survival prognosis,which are highly correlated with the change characteristics of cell cycle（G1）,ECM（G2）and lipid metabolism（G3）.Besides,G1 has the highest overall survival rate and disease-free survival rate,while G3 has the worst prognosis in the entire cohort.We found that proteins involved in lipid droplet（LD）formation and phosphatidylinositol metabolism（such as APOE,PLIN2,VDAC3,and SOAT1）are significantly highly expressed in G2 and G3.In vitro and in vivo experiments confirmed that overexpression of APOE and PLIN2 can promote the formation of LDs and enhance the proliferation of ESCC cells.Using the inhibitor DGAT1/2 for triglyceride synthesis process to treat ESCC cells or mouse tumor-bearing models,it was found that the inhibitor can significantly inhibit the formation of LD and the proliferation of ESCC at the in vitro/in vivo level,indicating that it may become a kind o f potential targeted drugs for ESCC.The above results indicate that ESCC may undergo a series of complex microenvironmental changes in the process of development and metastasis,including hypoxia,low glucose,immune stress,exogenous cytokine stimulation and other conditions that are not conducive to cell survival.PI metabolic pathway and lipid droplets generation are possible signal transduction response pathways,which may promote tumor growth,survival and escape when tumor cells face harsh environments.The in-depth study of this microenvironmental feedback mechanism is of great value for deepening the understanding of the mechanism of cancer recurrence and metastasis.At the same time,there are currently a variety of compounds that have specific inhibitory effects on the above pathways.The key molecules related to PI metabolism and lipid droplets synthesis pathways can also be used as potential ESCC drug targets,which have important clinical translational value.