Part 1:APC/C-CDH1-regulated IDH3? Coordinates With The Cell Cycle To Promote Cell Proliferation Part 2:The Function And Molecular Mechanism Of Tumor Antigen In Esophageal Squamous Cell Carcinoma

Cell division is composed of a series of precisely regulated events and intracellular metabolism is closely related to the living state of cells,thus,metabolic activities are often accompanied with cell cycle progression.With two essential co-activators CDC20 and CDH1,the E3 ubiquitin ligase APC/C targets crucial cell cycle proteins for proteasomal degradation to ensure accurate timing of mitotic events temporally and spatially.Besides canonical functions of APC/C in controlling cell cycle transition,APC/C-CDH1 takes PFKFB3 as a substrate to impact glycolysis and targets GLS1 to affect glutaminolysis,describing an additional function in linking cell cycle and metabolic network in human cancers.Here,we screened proteins containing both KEN box and D box in 2,752 metabolic transporters and enzymes,and those whom had more than 20%copy number variation frequency in our previous sequencing data in ESCC(Esophageal squamous cell carcinoma)were selected.Finally,17 genes were put forward as candidates.As the regulation of APC/C on TCA cycle remains unknown,we observed that the changing trend of ?-KG,fumarate and malate were significantly correlated with S phase in ESCC cells,suggesting the potential linkage between cell cycle and TCA cycle.Thus,we focused on IDH3? which encoding one of the rate-limiting enzymes in TCA cycle to explore the regulation between TCA cycle and cell cycle.To ensure IDH3? is a bona fide APC/C substrate as expected,we conducted immunoprecipitation assay and found endogenous IDH3? was interacted with APC/C.We synchronized cells to check the oscillation of IDH3? protein level during cell cycle progression and found that IDH3P appeared to be fluctuated in a cell cycle-dependent way,peaking in late G1/S and decreasing in mitosis.Besides,our results uncover that IDH3? degraded through APC/C-CDH1-mediated ubiquitin-proteasomal manner.To explore the biological function of IDH3? in tumor progression,we analyzed S phase distribution by flow cytometry and EDU assay,we demonstrated that overexpression of IDH3P accelerated G1/S transition,contributing to the promotion of cell proliferation in vitro and in vivo.As IDH3 was one of the important rate-limited enzyme in TCA cycle,we examined the intracellular a-KG fumarate and malate level after upregulating IDH3?,and found they were mildly increased.Furthermore,IDH3a and IDH3y overexpression increased the amount of S phase cells,and a-KG,fumarate and malate levels were significantly increased in IDH3a and IDH3y overexpression cells compared to the control cells.These results demonstrated that IDH3?,as a component of IDH3,would contribute to oxidative metabolism in TCA cycle.Since a-KG was a crucial metabolite for cell proliferation and the a-KG level was upregulated by IDH3?,we confirmed that the promotion on cell growth of IDH3? was partially depended on a-KG production.Interestingly,IDH3? could increase PFKFB3 protein level and enhance glucose uptake.Next,we performed immunohistochemistry assay to evaluate the expression level of IDH3? in 340 paired samples of ESCC tissues,matched adjacent normal tissues and lymph node metastasis tissues.Consistently,IDH3? was expressed at higher levels in ESCC tumor tissues and metastatic lymph nodes than in adjacent normal tissue.Importantly.IDH3? overexpression was strongly associated with poor overall survival and disease free survival of ESCC patients.Multivariate Cox regression survival analysis reported strong correlation between IDH3? positive expression and shorter overall survival,showing that IDH3? expression was an independent prognostic factor for outcome in overall survival in ESCC.Taken together,our findings confirmed that IDH3? was a new APC/C-CDH1 substrate and expressed in a cell cycle-dependent manner,which demonstrated a new cross-talk regulation between cell cycle and TCA cycle.Given that the relationship between cellular metabolism and cell cycle machinery was bidirectional,here we not only displayed the regulation of APC/C on IDH3?,but also revealed the ability of IDH3? to promote G1/S transition,resulting in increased cell proliferation in vitro and in vivo.These results presented a new two-way regulation between cell cycle and TCA cycle.In addition?IDH3? overexpression resulted in elevation of PFKFB3 protein level.These observations raised a novel concept that there existed regulation between key enzymes in TCA cycle and glycolysis.Importantly,high expression level of IDH3? was significantly correlated with poor survival among patients with ESCC,indicating that IDH3?-dependent metabolic alteration contributes to ESCC progression and IDH3?can serve as a promising prognostic molecular marker.Esophageal cancer is a common gastrointestinal cancer with a sixth leading cause of cancer mortality.Half of global esophageal cancer cases happen in China and esophageal squamous cell carcinoma(ESCC)is the major subtype histologically.Accounting for the high frequency of metastasis and relapse,ESCC has poor prognosis even after combined therapies.To have a better understanding of ESCC progression,a number of whole-genome analysis of ESCC have been undertaken in China,which would provide effective approaches to improve diagnosis and therapy.Taking advantage of the whole genome sequencing(WGS),whole-exome sequencing(WES)and comparative genomic hybridization(CGH),our previous work conducted whole-genome analysis in ESCC patients and found the tumor antigen MAGE-C3(Melanoma-associated Antigen C3),which occurred 8.0%mutation and 8.6%copy number amplification in the ESCC sequencing cohort,suggesting an important role of MAGE-C3 during ESCC progression.We also found that the mutation and copy number variation of MAGE-C3 were happened in different cancers from some public databases,indicating that MAGE-C3 may play important role in multiple cancers.Thus,we focused on MAGE-C3 and detected its protein level in ESCC tissues and adjacent tissues by immunohistochemistry.It turned out that MAGE-C3 was overexpressed in ESCC tissue(P<0.001)and high expression of MAGE-C3 was significantly associated with poor survival in ESCC patients(P=0.042),implying a promoting role of MAGE-C3 in prognosis.In the function study,we found that MAGE-C3 overexpression enhanced ESCC cell invasion and migration,while it had no influence in cell proliferation.Furthermore,MAGE-C3 overexpressed cell promoted lung metastasis of ESCC after tail vein injection.To further clarify the function of MAGE-C3 in cells,we performed RNA-seq in MAGE-C3 stably overexpressed cells and control cells,then the differentially expressed genes were analyzed.In GO pathway enrichment analysis,we observed that the differentially expressed genes caused by MAGE-C3 overexpression were enriched in IFN(Interferon)pathway.Since the type ? IFN participated in immune regulation in cells,we set about to displore the influence of MAGE-C3 in IFN-? pathway.Firstly,we adopted KYSE30,which was sensitive with IFN-? treatment,for following study.And we foud that the IFN-? treatment on KYSE30 was dose and time dependent.Next,we investigated the effect of MAGE-C3 on the IFN-? pathway in KYSE30 using optimal IFN-? concentration and time(20 ng/ml,24 h).MAGE-C3 overexpression profoundly elevated the phosphorylation of STAT1 at 701 and the luciferase units of GAS(Gamma Activation Site)in presence with IFN-?.In conclusion,overexpressed MAGE-C3 heightened the activation level of IFN-? pathway under IFN-? induction.Additionally,the mRNA and membrane level of PD-L1,which was the downstream of IFN-?,were also increased by MAGE-C3.Given that IFN-? pathway was involved in immunoregulation,we co-cultured MAGE-C3 stably knockdown ESCC cells with activated PBMC(Peripheral Blood Mononuclear Cell)from healthy donators and found that MAGE-C3 knockdown cells presented higher apoptosis.These results suggested an immune suppressive function of MAGE-C3.In addition,we co-cultured MAGE-C3 stably knockdown ESCC cells with activated Jurkat T cell,and the supernatant lymphocyte factors were detected by ELISA.The rsults showed that the levels of TNF-? and IFN-?,which promote immune function,were increased,while the levels of IL-10 and IL-1?,which inhibit immune function,were decreased.These results hinted that MAGE-C3 could regulate T cell cytokine secretion.To confirm the role of MAGE-C3 in normal immune environment,we used murine melanoma cell line B16F10 for study.The stable MAGE-C3-overexpressed B16F10 cells generated more lung metastasis than control cells in tail vein injection.Next,we evaluated the tumor-infiltrating T lymphocytes in lung metastasis loci by immunohistochemistry and flow cytometer.We observed that CD8+T cell/T cell ratio was decreased and PD-1+CD8+T cell/CD8+T cell ratio was increased in lung metastasis loci compared with control group.Besides,we noted that VIM which related with EMT(Epithelial-Mesenchymal Transition)was appear among the differentially expressed genes in RNA-seq assay.Then,we checked the mRNA level and protein level of Vimentin after overexpressing or knocking down MAGE-C3.We found that the phosphorylation of STAT3 at 705 and the protein level of Vimentin were upregulated after overexpressing MAGE-C3,while the protein level of E-cadherin was downregulated,suggesting the impact of MAGE-C3 in improving cell invasion and migration.Taken together,we uncovered that MAGE-C3 regulated metastasis by modulating EMT-related proteins and suppressing the immune function in tumor microenvironment by activating IFN-y pathway,which complementally promoted cancer progression.