The Role Of Metabolic Enzymes ASS1 And PYCR1 In Oesophageal Squamous Cell Carcinoma

Cancer cells develop under limited nutrient and oxygen microenvironment caused by relative insufficient vascularization."Metabolic adaptation" helps tumor cells continue to survive and develop under these “harsh” conditions.Similar to most solid tumors,oesophageal squamous cell carcinoma(OSCC)survives and develops in the environment of low oxygen and glucose,however,the mechanism of metabolic reprogramming of OSCC in response to adverse environment is not clear.By integrating metabolomic and genomic profiling,we found metabolic enzymes argininosuccinate synthetase 1(ASS1)and pyrroline-5-carboxylate reductase 1(PYCR1)were enhanced under environment of low glucose or oxygen.The influence of these two key metabolic enzymes on metabolic reprogramming of OSCC was further analyzed,and the IGF1R-c MYC axis regulating their expression in OSCC was identified.On the basis above,the role of IGF1R-c MYC-ASS1/PYCR1 on the development of OSCC was studied.The aim of our study is to explain the molecular mechanism under biological behavior of OSCC from the perspective of metabolic reprogramming,and to provide theory foundation for early diagnosis,treatment and molecular intervention to OSCC.Part Ⅰ: The expression of ASS1/PYCR1 in OSCC and their clinical significance in OSCC.Objective: To verify the expression of ASS1 and PYCR1 in tissues and cells of OSCC and analyze their clinical significance.Methods: Upregulated metabolic enzymes in OSCC was detected by RNA-seq.The expression of ASS1 and PYCR1 was detected by immunohistochemical staining and western blotting in OSCC tissues and cells.Meanwhile,q PCR was performed to examine the expression of ASS1 and PYCR1 in OSCC cells,and the correlation between ASS1/PYCR1 expression and OSCC development was analyzed.Results: Upregulated metabolic enzymes ASS1 and PYCR1 were detected by RNA-seq in OSCC tissues,and in OSCC cell lines cultured under low oxygen and glucose environments.IHC and WB confirmed the expression of ASS1/PYCR1 in both OSCC and normal esophageal epithelial tissues and cells.Furthermore,the expression of ASS1/PYCR1 in OSCC tissues and cells was higher than that in normal esophageal squamous epithelium,and the expression level was related to the cancer stage.Meanwhile,q PCR analysis showed that ASS1 and PYCR1 were highly expressed in OSCC cells,and the expression of two enzymes was further enhanced with the extension of glucose and oxygen deprivation time.Conclusions: The expression of ASS1 and PYCR1 in OSCC tissues is higher than that in normal adjacent esophageal epithelial tissues,and the high expression level is related to cancer stage.ASS1 and PYCR1 expression were increased in OSCC cells under deprived oxygen and glucose conditions.Part Ⅱ: Effects of ASS1 and PYCR1 on altered metabolism of OSCC cells.Objective: To analyze the metabolic abnormalities of OSCC under low oxygen and glucose conditions,and to study roles of ASS1 and PYCR1 in OSCC metabolism abnormalities.Methods: Metabolomics technology was used to detect the abnormal accumulation of metabolites under the conditions of oxygen and glucose poor.The possible metabolic pathways involved in these metabolites were analyzed.And the influence of ASS1 and PYCR1 on those metabolic pathways were also analyzed.Results: Under limited glucose and oxygen conditions,abnormal metabolites of OSCC mainly involved arginine/proline and aspartic acid metabolism,which showed abnormal accumulation of aspartic acid,arginine or proline intermediate metabolites.Further knockdown of ASS1 and PYCR1 showed a decrease in arginine or proline levels in OSCC.However,the changed metabolism of OSCC under limited glucose and oxygen environments were not completely the same,showing that the level of arginine did not increase under low glucose microenvironments,but the metabolism of proline and aspartic acid was activated.Conclusions: Under hypoxia,the increased expression of ASS1 and PYCR1 changes the arginine and proline metabolism of OSCC cells,respectively,and there is a close relationship between arginine and proline metabolism.In low glucose condition,the high expression of PYCR1 changed the proline metabolism of OSCC cells,while ASS1 increased slightly and did not significantly change the arginine metabolism.Part Ⅲ: Molecular mechanisms under ASS1 and PYCR1 expression.Objective: To study the molecular regulation mechanism of ASS1 and PYCR1 expression.Methods: A public database was used to predict the possible binding sites of transcription factors to ASS1 and PYCR1,the authenticity of the sites was confirmed by Ch IP assay,and the direct transcriptional regulatory relationship between transcription factors and ASS1/PYCR1 was confirmed by luciferase reporter gene assay.The upstream regulatory relationship between transcription factors and ASS1/PYCR1 was further verified by immunoblotting.Results: IGF1 R locates on the membrane surface of OSCC cells,senses external pressure signals,and affects the overall phosphorylation levels of Ras/MAPK and JAK/STAT signaling pathways,thereby activating downstream molecules of the pathways.The activated signaling pathway increases the phosphorylation level of c MYC,which promotes the transcriptional activity of c MYC and ultimately promotes the transcription of metabolic enzymes ASS1 and PYCR1.Conclusions: IGF1 R affects the phosphorylation level of c MYC through the classical signaling pathways Ras/MAPK and JAK/STAT,and ultimately promotes the transcription of ASS1 or PYCR1 under low glucose and oxygen conditions.Part Ⅳ: The effect of IGF1R-ASS1/PYCR1 axis on the cell functions to OSCCs.Objective: To study the effect of IGF1R-ASS1 /PYCR1 axis on the function of OSCC in vitro.Methods: By interfering or enhancing the expression of IGF1R/ASS1/PYCR1 in OSCC cell lines ECA109 and KYSE150 under low glucose and oxygen mircoenvironments,respectively,the effects of IGF1R-ASS1 /PYCR1 regulation on the proliferation and migration of tumor cells were detected.Results: CCK8 and transwell assays showed that down-regulation of ASS1/PYCR1 expression could inhibit the proliferation and migration of ECA109 and KYSE150 cells,and overexpression of IGF1 R restored the function of the inhibited cells.Conclusions: In vitro,cell experiments have confirmed that the regulation of IGF1R-ASS1 /PYCR1 can affect the function of OSCC.Part Ⅴ: The effect of IGF1 R,ASS1 and PYCR1 on OSCCs in vivo.Objective: To study the effects of IGF1 R,ASS1 and PYCR1 on subcutaneous tumor formation and chemotherapy resistance of OSCC cells in vivo.Methods: OSCC cells with stable down-regulation of IGF1 R,ASS1 and PYCR1 were obtained by transfection of sh RNAs-lentivirus,and then subcutaneous tumor-forming model was established by subcutaneous inoculation of stable cell lines and control cells in nude mice,and tumor growth was observed.The expression of IGF1 R,ASS1 and PYCR1 proteins were examined by IHC and WB assays to verify the regulatory relationship of IGF1R-ASS1/PYCR1 in subcutaneous transplanted tumors.Further combined with chemotherapy drug cisplatin,the effect of IGF1R-ASS1 /PYCR1 regulation on drug resistance of OSCC was explored.Results: In vivo nude mice subcutaneously transplanted tumor model,the tumor volume in the sh RNA-IGF1R/ASS1/PYCR1 group was significantly reduced compared with that in the NC-sh RNA group.IHC and WB results showed that the expression of IGF1 R,ASS1 and PYCR1 proteins in sh RNA-IGF1 R group was lower than that in the control group.After treated with chemotherapy drug cisplatin,the tumor volume of IGF1R/ASS1/PYCR1-KD group was further reduced.Conclusions: High expression of IGF1 R,PYCR1 or ASS1 contributes to cisplatin resistance of OSCC cells in vitro under low glucose and oxygen conditions.In vivo,the high expression of IGF1 R,PYCR1 and ASS1 promotes the growth of OSCC and enhances the resistance to chemotherapy.