Fig. 5: ZFP82 inhibits HDAC3 and promotes mutation p53 degradation.

Reduction of ZFP82 and p53 induces esophageal tumorigenesis. A In String database (https://string-db.org), the nucleotide exchange factor of HSP70, HSPA4 is the downstream target of HDAC3. B Co-IP assay verifies the binding of HSP70 and HDAC3. Over-expression ZFP82 disrupted the HSP70-HDAC3 complex. C Western-blot verified in resistant TE6/R cell lines, ZFP82 induced CHIP expression and down regulated HSP70 and p53^R248Q. D TE6/R cells were treated with CHX 10 μg/mL, and time course western blot suggests ZFP82 re-expression increased CHIP expression and induced p53^R248Q and HDAC3 degradation. E Survival analysis using TCGA database. The patients are sub-stratified into four groups, as the data shown, patients with high expression of both ZFP82 and p53 have the highest survival rate, p < 0.05. F, G The pathological correlation between ZFP82 expression and p53 in esophageal cancer was investigated. Patients were stratified into the following groups: stage Ⅰ, stage Ⅱa, stage Ⅱb (ZFP82 plus wtp53 (high)) and stage Ⅱb (ZFP82 plus wtp53 (low)). Tumor tissues from esophageal cancer patients were used to generate gene expression by qPCR. Data showed that patients with stage Ⅰ and stage Ⅱa were all survived within 5 years, stage Ⅱ patients with increased expressions of ZFP82 and p53 showed relatively good prognostic outcomes, with a 5-year survival rate exceeding 80%. The 5-year survival rate was < 50% for stage Ⅱ patients with low expressions of ZFP82 and p53 (log-rank test P¼ 0.00387). H Overexpress of ZFP82 and HDAC3 knockdown potentiates 5-FU-induced apoptosis. Stable ZFP82-expressing KYSE960/R cells were transfected with shHDAC3, and then treated with 5-FU. DNA damage of cells was determined by the TUNEL assay. * p < 0.01. I Overexpress of ZFP82 with HDAC3 knockdown potentiates 5-FU-induced cell apoptosis in TE6/R cells. Stable ZFP82-expressing TE6/R cells were transfected with shHDAC3, and then treated with 5-FU. DNA damage of cells was determined by the TUNEL assay. * p < 0.01. J In vivo tumorigenicity assay, KYSE960/R and TE/6 cells were subcutaneous injected into nude mice, representative bioluminescent images of subcutaneous tumor outgrowth are shown. In vivo tumor growth indicates that ectopic expression of ZFP82 or knockdown of HDAC3 significantly inhibits the tumor growth compared to vector, significantly increased chemo-sensitivity of KYSE960 cells to 5-FU. K The overexpression of ZFP82 substantially enhances the chemosensitivity of KYSE960/R and TE6/R cell lines. Cells were stably transfected and subsequently injected subcutaneously into the left flank of athymic nude mice. Following a four-week period post-inoculation, animals presenting with tumors of similar dimensions (ranging from 100 to 200 mm³) were selected for chemotherapy with 5-FU at a dosage of 25 mg/kg, administered with a 2-day interval over a period of 8 weeks. Tumor volume measurements were conducted over a 12-week duration. The error bars depicted in the corresponding figures represent the standard deviation of the mean.