Part ? TMTC3,a Novel ER Chaperone,Regulates EMT Progress In ESCC Via Endoplasmic Reticulum Stress Part ? The Function And Molecular Mechanism Of ABC-X,a Member Of ABC Transporters,in ESCC Chemoresistance

Esophageal squamous cell carcinoma(ESCC)is one of the most common malignant tumor in China,and characterized by the onset of concealment and fast progress.Therefore,it is significantly important for diagnosis and therapy to elucidate the molecular mechanism that contribute to ESCC progression.During the tumor progress,tumor cells are exposed to tumor microenvironment,including oncogenic mutation,high metabolic demand,nutrient deprivation and etc.,which induce the endoplasmic reticulum(ER)stress.In order to adapt ER stress,unfolded protein response(UPR)was induced by tumor cells to provoke autophagy,cellular reprograming,and EMT to promote tumor survival,metastasis and drug resistance.In recent years,several studies have shown that signal molecules in ER stress could act as targets for effective therapy.However,whether ER stress plays an important role in ESCC progress and the molecular mechanism is still unclear.In this study,ESCC cell line KYSE450 was induced into ER stress via serum medium and RNA-seq analysis was conducted,of which ectopic expression of TMTC3 was screened and verified in ESCC cells undergoing ER stress.The transcriptome sequencing of 86 pairs ESCC tissues in our lab indicated that the mRNA level of TMTC3 was significantly upregulated in ESCC tissues.The immunohistochemistry of ESCC tissue microarray containing 75 pairs revealed the protein expression of TMTC3 was higher in tumors than adjacent tissues.Clinically,high TMTC3 expression was strongly associated with poor prognosis in ESCC.Combining with the visualized database from TCGA,the expression of TMTC3 were higher in ESCC,lung SCC,head and neck SCC,with certain specificity for SCCs.Further chromatin immunoprecipitation(ChIP)and dual luciferase reporter assay revealed the transcription of TMTC3 was regulated by TP63,a SCCs-specific transcription factor.Cell experiments in vitro showed that knockdown TMTC3 in ESCC cell lines inhibited the ability of invasion and migration via Transwell assay.Additionally,TMTC3 downregulation significantly suppressed the ability of proliferation by RTCA assay.In order to further verify this conclusion in vivo,we constructed a cell line that stably knockdown TMTC3.The growth of subcutaneous tumor in BalB/C nude mice was obviously reduced in stably knockdown TMTC3 cells than control cells.For the tail vein injection,the metastasis ability to lung,also including liver and lymph node,in TMTC3 downregulation cells was lower than control cells.Since TMTC3 is located at ER and involved in ER stress,immunofluorescence was performed to confirm the co-localization between TMTC3 and ER stress sensors or ER chaperone,glucose-regulated protein 78(GRP78).Further immunoprecipitation experiments showed that TMTC3 as a competitor,resulted in the dissociation of ER sensor-PERK from GRP78,to activate the PERK signaling pathway and promote the nucleus location of ATF4.RNA-seq analysis was conducted in stably knockdown TMTC3 cells to detect the alteration of downstream molecules.The results showed that TMTC3 could positively regulate the expression of FAM3C at both mRNA and protein levels.We also performed ChIP and dual luciferase reporter assay to verify that ATF4 activated the transcription activity of FAM3C.Additionally,the positive correlation between ATF4 and FAM3C was verified via the visualized database from TCGA.Further experiments revealed that TMTC3 promoted the expression of EMT markers via FAM3C in ESCC,such as E-cadherin,N-cadherin and Vimentin.In summary,the expression of TMTC3 was upregulated during ER stress,regulated by SCCs-specific transcription factor TP63.TMTC3 disrupted the interaction between GRP78 and PERK,and increased the nucleus location of ATF4,which activated the transcription activity of FAM3C to promote ESCC progression.This study provided a new sight for the pathogenesis and a new target for therapy in ESCC.Chemoresistance in ESCC patients has become a major reason in failure of chemotherapy and tumor relapse.Thus,it is an urgent problem to elucidate the molecular mechanism of chemoresistance and seek for targets to enhance the chemosensitivity.The ATP-binding cassette(ABC)transporters are involved in multiple progress and are closely related to multidrug resistance of tumors.The most studied ABC transporter members are P-glycoprotein,MRP1,and BCRP,for which targeting molecules have been applied in clinic.However,whether the clinical application of ABC transporters was involved in drug resistance and act as biomarkers and targets in ESCC is still unknown.Based on the previous work performed transcriptome sequencing in 86 pairs ESCC tissues and TCGA visualize database,we found ABC-X,a member of ABC transporters was obviously up-regulated and the expression of ABC-X was specific for squamous cell carcinomas(SCCs)among multiple tumors.However,there was no correlation between the over-expression of ABC-X and copy number amplification.The strongly positive relation between ABC-X and TP63 or SOX2,which are specific transcription factors with amplification in SCCs,was found through two databases and confirmed in ESCC tissues.The transcription activity of ABC-X was increased by TP63 and SOX2 binding through chromatin immunoprecipitation(ChIP)and dual-luciferase reporter assay.Additionally,knockdown TP63 or SOX2 by siRNA could inhibited the mRNA expression of ABC-X.Taken together,high expression of ABC-X in SCCs(including ESCC)was transcriptional activated by TP63 and SOX2.For subcellular location of ABC-X,immunofluorescence(IF)assay showed obvious co-localization between ABC-X and lysosome-associated membrane protein 2(LAMP2),the lysosome marker.Cell experiment showed that knockdown ABC-X by siRNA could inhibit the proliferation ability of ESCC cells,but not affect the invasion and migration ability.In addition,we observed a significant inhibition of proliferation and an obvious increase in apoptosis in response to cisplatin in ABC-X knockdown cells.In vivo experiments,the Xenograft tumor growth was significantly inhibited in ABC-X expression decreasing cells compared with control group.In order to investigate the mechanism of ABC-X,proteomics was analyzed in both stably knockdown ABC-X cells and negative control cells by Mass Spectrometry(MS),then the differentially expressed proteins were screened by the NC/sh ratio less than 0.6.Further GO and Pathway enrichment analysis showed that ABC-X was correlated with the membrane trafficking and vesicle-mediated transport.Additionally,VPS34,a key protein in autophagy,was downregulated in stably knockdown ABC-X cells,suggesting the involvement in autophagy,and further study would be conducted.The previous work suggested ABC-X plays a key role in ESCC chemoresistance,and was expected to be a new target for therapy.To seek for the inhibitor of ABC-X,the homology modeling of ABC-X was established according to the amino acid sequence.Next,molecular docking of ABC-X with 1953 compound approved by FDA was conducted,then 41 molecules were screened according to the prediction of binding affinity.The protein level of ABC-X was detected by ELISA assay in ESCC cells treated with 41 compounds,and 5 compounds were selected for further proliferation assay.Finally,the cell growth rate was obviously inhibited by 24E8,which could increase the sensitivity to cisplatin in ESCC cell lines.In summary,ABC-X is a potential target to solve the chemoresistance of ESCC,but the molecular mechanism remains to be further explored.Our finding suggested that the combination therapy with cisplatin-based chemotherapy plus ABC-X inhibitors is expected to overcome the chemoresistance of patients in clinic.