GASC1,a H3K9me3 Histone Demethylase,is Involved In The Maintenance Of H ?Man ESCC Cancer Stem Cells And Regulatory Mechanism Of Its Inhibitors

Background:Esophageal cancer?EC?is the eighth most common cancer worldwide and the sixth leading cause of cancer-related deaths.China is the country with the highest incidence of EC.More than 50%of the newly diagnosed esophageal cancer patients are in China every year.The prognosis of esophageal cancer is very poor,and the five year survival rate is only about 10%^[1].The significant regional distribution difference is the prominent feature of the epidemiology of esophageal cancer,and the difference of the incidence of the high and low incidence area can reach 500 times.Taking Henan as an example,Linzhou,Anyang and other places in northern Henan are with both the highest incidence and the highest mortality rate in the world^[2].For half a century,after several generations of oncology clinical research and prevention experts to explorer,the remarkable achievements has made in the epidemiology of esophageal cancer,early diagnosis,comprehensive treatment and prevention.However it is complex that the pathogenesis and prevention of esophageal cancer,more in-depth studys of the molecular mechanism of esophageal cancer development are needed^[3].Histone demethylase GASC1,also known as GASC1,a high amplified gene in poorly differentiated esophageal squamous cell carcinoma?ESCC?,?Gene Amplified in Squamous Cell carcinoma 1,GASC1?^[4].It cloned from KYSE-150 cell line.Studies have shown that GASC1 is essential to maintain the embryonic stem cells for self renewal and differentiation^[5].GASC1 is reported to modify the key regulatory factors?such as NOTCH1,NANOG,OCT4 and SOX2?for the self-renewal of embryonic stem cells^[6].Esophageal squamous epitheliM is the constantly updated tissue.Therefore,normal stem cells or early progenitor cells with the high potential of proliferation and differentiation are needed to maintain their continuous loss and renew.Cancer stem cells?CSCs?theory suggests that there are"stemness"cells in tumor cells,which are responsible for tumor growth,metastasis and recurrence^[7-9].The methylation status of embryonic stem cells?ESCs?H3K9 is maintained by the complex interaction between transcription factor and histone demethylation enzyme^[10,11].As H3K9 demethylation enzyme,GASC1,studies have shown that it is essential to maintain the characteristics of embryonic stem cells^[6].However,it is unclear whether CSC cell subsets exist in esophageal cancer cells,and if GASC1plays a role in maintaining esophageal cancer CSC,and what mechanism of its small molecule inhibitors in anti-tumor effect^[12].Therefore,the purpose of this study is to explore the roles and molecular mechanisms of GASC1 and its inhibitors in the development of esophageal cancer.We hope to provide the theoretical basis for diagnosis and treatment of esophageal cancer by a new target.Objective:To explore the role and mechanisms of both GASC1 and its small molecular inhibitors in the development of esophageal cancer.The First Chapter:The Relationship between GASC1 Expression and the Clinical Pathological Features of ESCCMethods1.To examine GASC1 expression in well-characterized hMan ESCC cell lines,patient-derived ESCC tumors under conditions that permitted expansion in vitro and the normal hMan immortalized epithelial cells using Western blotting assay.2.The relationship between the GASC1 expression and the clinical pathological features and overall survival in patients with ESCC:GASC1 expression in ESCC carcinoma and adjacent tissues was qualitative analyzed by quantitative fluorescence quantitative PCR?qPCR?,and GASC1 was studied in different stages and differentiation of ESCC by IHC.The relationship between the expression of GASC1and the clinical pathological features and overall survival of the patients was analyzed.Results1.Expression of GASC1 proteins were clearly detected in 3 of 5 established ESCC cell lines?EC9706,KYSE-150,KYSE-30?and 3 of 5 primary ESCC cultures?EC-1,2,3?.However,it was docMented at low level in one normal hMan Immortalized epithelial cells?SHEE?.2.The expression of GASC1 in ESCC carcinoma and adjacent tissues were detected by Western Blot,RT-PCR and IHC methods:there was no significant difference between the cancer and the adjacent mucosa;however GASC1 expression in the ESCC cancer tissue with G3 pathology is significantly higher than G1-2.The expression of GASC1 was associated with lymph node metastasis in patients:the rate of lymph node metastasis was increased in the high GASC1 expression group.3.The Kaplan Meier analysis of total OS?Overall Survival,OS?rate showed that the 2-year OS was 62.6%in GASC1 positive group,81.6%in GASC1 negative group.The Second Chapter:CSCs was Detected in ESCC CellsMethods1.To detect the ALDH⁺subsets in the KYSE-150 cell lines and patient derived ESCC tumor cells by flow cytometry.ALDH⁺ESCC cells and ALDH^-cells were isolated and compared;ALDH⁺ESCC cells and ALDH^-cells in serum-free medium suspended in vitro;to observe the ability to cause tumor by separation of ALDH⁺ESCC cells and ALDH^-cells in the SCID mouse.2.To investigate whether ALDH⁺ESC cells have long-term tumor potential and self-renewal capacity,and evaluate their ability to produce tumors in a continuous transplant.The primary transplanted tumors of ALDH⁺and ALDH^-were obtained from mouse subcutaneous xenografts.The single cell suspension was isolated again,and ALDH⁺and ALDH^-cells were separated by FACS.10⁴ cells were transplanted into the recipient mice again.3.Finally,we detected the ability of ALDH⁺cells to reconstruct tumor heterogeneity in vivo used flow cytometry,hematoxylin and eosin?HE?staining and immunohistochemical?IHC?.Results1.The proportion of ALDH⁺population was detected in all the cells of the newly separated ESCC specimens,ranging from 3.92%to 14.7%.The proportion of ALDH⁺in the KYSE-150 cell line is about 10.1%.2.ALDH⁺and ALDH^-progeny cells were cultured under conditions of non adhesion,serum free and growth factors for 7 days.ALDH⁺group could produce more esospheres at 5000 cell/ml.ALDH⁺cells isolated from esospheres can self renew in vitro,showing almost unlimited growth potential from ALDH⁺population percentage and ESL startup ability.3.Compared with ALDH^-cells or parental cells,the tumor-forming capacity of ALDH⁺cells was significantly increased?1:32;1:2704;1:979?.ALDH⁺cells need to be injected with 10² cells while ALDH^-cells need 10⁴ cells at the same time.The tumor was delayed by injection of low dilution ALDH^-cells and tumor was not found during the experiment period.The size,dynamics and incubation period and tumor formation of tumor growth is related to the tumor of ALDH⁺cells injected.4.The proportion of ALDH⁺cells in the EC-2 and EC-3 cell lines was significant high and with high tumorigenicity.100 ALDH⁺cells were purified from EC-2 and EC-3 cell lines,forming tumors in 2/5 and 4/5 mice,respectively.In contrast,1000 of the purified ALDH^-cells from EC-2 and EC-3 are basically non tumorigenic,but only 1/5 and 2/5 mice form tumors.The formation of transplanted tumor was monitored by angiography system.5.By comparing the changes of biolMinescence signal with ALDH^-,it was found that the fluorescence labeled ALDH⁺cells showed the greater frequency of tumor formation,especially when injected into the lower tumor of cells.In the whole process of tumor formation,ALDH⁺cells showed the ability of self-renewal by preserving graft ability,while ALDH^-rapidly lost their tumorigenesis ability after two rounds.6.ALDH⁺derived xenografts contained 10.1%and 14.7%ALDH⁺cell subsets,similar to EC-2 and EC-3 primordial tumors.In addition,the results of HE and IHC showed that the tumor histology and GASC1 antigens formed by ALDH⁺cells were similar to those of primary tumors.The Third Chapter:The Role of GASC1 in CSCs MaintainingMethods1.GASC1 Expression is ESCC CSCsThe expression of GASC1 in ALDH⁺and ALDH^-tumor cells was detected by qPCR and Western blotting.2.The effect of inhibition of GASC1 on the cloning and self renewal of ALDH⁺ESCC-CSCsThrough Aldefluor analysis,to explore the changes in ALDH⁺proportion in ESCC cells before and after GASC1 knockdown by shRNAs lentivirus.to observe the ability of cells to initiate esospheres of ALDH⁺and ALDH^-cells when they grown in non-adherent serum-free conditions in vitro and expand in subsequent in vitro.Different concentrations of Caffeic acid?CA?were used to treat cell line and primary culture cell line.The nMber of ALDH⁺cells was detected by flow cytometry,and the ability of cells to initiate esospheres was observed.3.Targeting of CSCs by CaA in the ALDH⁺-Derived Xenograft Model In VivoALDH⁺cells were subcutaneously injected into the nude mouse breast fat pad.One week after inoculation,the mice were injected with CaA intraperitoneally?5 mg/kg or 10 mg/kg?,and the control group was injected with saline for three weeks,a total of 8 weeks.The volMe of each transplanted tumor was observed in 8 weeks.After 8 weeks,the mice were killed.The residual tumor was resected and separated into single cells.Aldefluor assay was performed.Further,the expression of ALDH1 in each tumor was detected by IHC.Results1.Compared with ALDH^-subgroup,GASC1 was highly expressed in the ALDH⁺subgroup.2.The expression of GASC1 was silenced by shRNAs's lentivirus.Aldefluor analysis showed that GASC1 gene knockout led to a sharp decrease in the proportion of ALDH⁺cells in EC-1 from 9.8%to 2.05%,while EC-2 decreased from 15.6%to3.93%.In addition,the expression inhibition of GASC1,the ability was decreased in vitro propagation subsequently in the culture with non adherent serum-free culture conditions.3.Compared with the control group,the tumor size of the mice treated with CA was reduced,and the tumor latency increased.After 8 weeks,the proportion of ALDH⁺cells in the residual control group was stable compared with the parent tumor.In contrast,the CA treatment group showed a significant reduction in the proportion of ALDH⁺cells:4.1%?5 mg/kg?and 0.29%?10 mg/kg?.The IHC method showed that ALDH1 staining decreased significantly in the treatment group compared with the control group.The Fourth Chapter:Regulatory Mechanism of GASC1-NOTCH1 Passway in ESCC-CSCs MaintainingMethods1.The effect of GASC1 on the unique gene labels and their function in ALDH⁺ESCC-CSCsThe Agilent whole genome microarray was used to analyze ALDH⁺ESCC-CSCs cells after GASC1 knockout by siRNA interference or CaA treatment for 48 hours,and to evaluate the overlap between down regulated genes.The differentially expressed genes were annotated by GO analysis,and qPCR was used for transcriptional verification,and the important transcription factor NOTCH1 was selected for functional study.2.GASC1 participates in the maintenance of H3K9me3 on the locuses of pluripotency-associated genesTo test the hypothesis that down-regulation of pluripotency-associated genes during GASC1 inhibition is linked to histone modifications,we investigated whether the demethylase inhibitor CA affect selected global histone methylation states in ALDH⁺cells,using AlphaLISA Assay.Quantitative chromatin precipitation?qChIP?was used to detect the change of histone in the GASC1 inhibition process of pluripotency-associated genes promoters.The antibody was used to perform chip analysis to identify the amplification of H3K9me2 or H3K9me3 in the NOTCH1promoter region.To examine whether CA blockage of the GASC1 demethylase function affects xenograft tumor growth via epigenetic modifications,we determine whether CA affects H3K9 methylation globally in vivo.H3K9 methylation levels were analyzed in histone extracts prepared from ALDH⁺-derived xenograft tumors after exposure to CaA by western blot analysis.To examine whether the effects reflect the expression of target genes involved in the repression of growth,we analyzed expression of NOTCH1 and H3K9me3 by using IHC analysis from xenograft tumors.It was verified whether the CA treatment promoted the changes in epigenetic markers of the NOTCH1 promoter:the IHC assay was used to detect the status of H3K9me3 markers in xenotransplantation.Results1.ALDH⁺ESCC-CSCs were analyzed by Agilent whole genome microarray which GASC1 was knockdown by shRNA and CA for 48 hours,we identified 694down-regulated overlapped genes.Using GO analyzed the genes functional annotated differentially expression,we found that overlapping down regulated expression genes had strong functions in aldehyde dehydrogenase?NAD?activity,transcription factor binding/dry maintenance,cell cycle regulation and differentiation.The qPCR results were in accordance with the results of the microarray.Among these genes,NOTCH1gene is an important transcription factor to maintain ESCC cell pluripotent and self renewing.It had been significantly down regulated and had been selected as a target for further research.2.ALDH⁺cell was treat by CA with 48h resulted in a rapid growth of H3K9me3level and it was dose-dependent,while the level of H3K4me2 did not change significantly.GASC1 inhibition leaded to a large increase in the H3K9me3 and H3K9me2 levels in the NOTCH1 promoter region.Multiple concentrations?5,10,20M?CA were exposed or did not expose ALDH⁺cells for 48 hours,and chromatin was prepared for analysis.The results showed that after treatment of ALDH⁺cells with CA,the levels of two and trimethylated H3K9 histone in NOTCH1 increased significantly.The decrease in expression of NOTCH1 in these cells was consistent,but it does not affect the anti-GST drugs that are not targeted.Western blot analysis showed that the tri methylation of H3K9 in the xenografts treated by CA was higher than which in the control group significantly.IHC results showed that the expression level of NOTCH1 in the xenograft treated with CA decreased significantly compared with the control group,while the level of H3K9me3expression increased.After CA treatment,the H3K9me3 discovery in the NOTCH1promoter was greatly increased.Conclusion1.GASC1 up-regulated expression was associated with the poor pathological phenotypes and OS in ESCC.2.We confirmed the population of cancer stem cells?CSC?.in hMan ESCC.3.The expression of GASC1 in ALDH⁺subgroup cells is enhanced,and the inhibition of GASC1 affect the development of esophageal cancer by lowering the maintenance of CSCs.4.GASC1 inhibitor,CA,effectively inhibits the expression of GASC1 in ESCC cells and inhibits the growth of the tumor.5.GASC1 regulate the pluripotency-associated genes expression positively by the promoters H3K9me3 and H3K9me2 demethylation.NOTCH1 and its downstream target gene plays a key role in the maintenance of dry ESCC-CSC,GASC1 blocking by CaA may target gene promoter through epigenetic modification to reduce ESCC CSCs.In s?Mmary,our findings provide a theoretical basis for a new treatment strategy development based on the inhibition of the GASC1 pathway to eliminate ESCC-CSC.