ACSS2/AMPK/PCNA Pathway Driven Proliferation And Chemoresistance Of Esophageal Squamous Carcinoma Cells Under Nutrient Stress

【 Objective 】 To explore the molecular mechanism of ACSS2/AMPK/PCNA signaling pathway involved in proliferative maintenance and regulation of chemotherapy resistance in esophageal squamous cell carcinoma in nutrient stress.【Methods】(1)Immunohistochemical method was used to compare and analyze the expression of ACSS2 in tumor tissues and adjacent normal tissues of 28 patients with esophageal squamous cell carcinoma.Combined with nutrient stress treatment(low serum concentration),the proliferation of esophageal squamous cell carcinoma cell line TE-1,ECA-109 and normal esophageal squamous cell Het-1A were explored by CCK8.The expression of ACSS2 in Het-1A,TE-1 and ECA-109 cells were detected by RT-PCR and Western blotting.(2)Under the conditions of normal culture and nutrient deficiency,the CCK8 method was used to detect the proliferation of esophageal squamous carcinoma cells before and after the down-regulation of ACSS2(siRNA-ACSS2 treatment),and the effect of siRNA-ACSS2 treatment on cell cycle distribution was investigated by flow cytometry.Western blotting was used to detect the potential relationship between ACSS2 expression and PCNA under nutrient deficiency conditions.TUNEL and Annexin V-FITC/PI were used to detect the effect of nutrient deficiency treatment and ACSS2 expression on the apoptosis of esophageal squamous carcinoma cells in cisplatin.Combined with cisplatin and siRNA-ACSS2 treatment,immunofluorescence and Western blotting were used to detect the relationship between ACSS2 and PCNA,p-ATM,γH2AX and other proteins related to DNA repair in esophageal squamous cell carcinoma.The correlation of PCNA with p-ATM and γH2AX in esophageal squamous carcinoma cells was detected by Western blotting after siRNA-PCNA and DDP treatment.(3)Western blotting detected the activation of AMPK signaling pathway in TE-1,ECA-109 cells after inhibition of ACSS2 treatment or not;Using AMPK V signaling inhibitor Dorsomorphin to verify the effect of AMPK signaling pathway on PCNA expression in ESCCs.(4)The expression levels of ACSS2,PCNA and Ki-67 in cancer tissues of 28 cases of ESCC patients were graded after immunohistochemistry.The chi-square test was used to analyze the relationship between ACSS2 with age,sex,tumor differentiation,tumor size,lymph node metastasis,TNM,PCNA,and Ki-67 of these patients.Kaplan-Meier analysis of the relationship between ACSS2,PCNA,Ki-67 with prognosis.【Results】(1)ACSS2 was highly expressed in esophageal cancer tissues and cells,while the lack of nutrition has no significant change in the proliferation ability of esophageal cancer cell lines and the expression of ACSS2 was increased.ACSS2 expressed in cancer tissues and adjacent tissues of esophageal cancer patients,and the expression intensity was higher in cancer tissues.The expression of ACSS2 in esophageal cancer cell lines TE-1 and ECA-109 was higher than that in normal esophageal squamous cells Het-1A.The proliferative capacity of TE-1 and ECA-109 cells decreased by 10.06 ± 0.59% and 7.68 ± 0.64%,respectively,and the proliferation ability of Het-1A cells decreased 41.64 ± 0.72%.The expression levels of ACSS2 mRNA in TE-1 and ECA-109 cells were significantly increased in the short term,while the protein expression levels were multiplied in both short-term and long-term.There was no significant change in mRNA levels and protein levels of normal esophageal squamous cells under NS.(2)ACSS2 promotes proliferation and DNA repair of esophageal cancer cells through PCNA.Interfering with the expression of ACSS2 in TE-1 and ECA-109 cells,the cell proliferation ability was decreased in the normal and nutrient deficiencies,and the proliferative capacity was the lowest in the case of nutrient deficiency.Compared with the NS+NC group,it was further decreased by 23.87 ± 0.14% and 16.95 ± 0.53% respectively.Flow cytometry showed G2/M block in the cell cycle after interfering with ACSS2.The expression of PCNA was down-regulated with the decrease of ACSS2 expression.The results of TUNEL and Annexin V-FITC/PI showed that the NS and the expression of ACSS2 had no significant effect on the apoptosis of ECA-109 cells.Under the condition of adding DDP,the apoptosis rate of ACSS2 interference group was significantly increased,and it was most obvious in the case of nutrient deficiency.Under normal and NS,the expression of ACSS2 has no effect on DNA damage-related proteins p-ATM,γH2AX and anti-apoptotic protein Bcl-xL.After adding DDP and down-regulating the expression of ACSS2,the expression of p-ATM,γH2AX increased significantly,the expression of Bcl-xL showed a downward trend.After the addition of DDP,when the expression of PCNA was interfered,and the expression of p-ATM and γH2AX was increased.(3)ACSS2 and AMPK synergistically regulate the expression of PCNA.NS can cause accumulation of p-AMPK,and the expression of p-AMPK was decreased after interfering with ACSS2.After the addition of AMPK signaling pathway inhibitor Dorsomorphin in esophageal cancer cells under normal and nutrient deficiencies,the expression of PCNA was decreased,as well as the expression of ACSS2.(4)The expression of ACSS2 was related to the prognosis of patients.In 28 patients with ESCC,the expression intensity of ACSS2 was positively correlated with the expression of PCNA and Ki-67,but not with age,gender,tumor differentiation,tumor size,lymph node metastasis,and TNM staging.Patients with low expression of ACSS2 have longer disease-free survival time.【Conclusions】In the absence of nutrients,ACSS2 and AMPK signaling pathways in esophageal squamous cell carcinoma synergistically maintain PCNA expression,thereby promoting cell proliferation and cisplatin resistance.