Epigenetic silencing schlafen-11 sensitizes esophageal cancer to ATM inhibitor

Figure 1 The expression and methylation status of Schlafen-11 in esophageal squamous cell carcinoma. A: Correlation analysis between Schlafen-11 (SLFN11) mRNA expression and methylation levels of 8 CpG sites around transcription start site retrieved from TCGA datasets (n = 171). Scatter plots shown inverse relevance of SLFN11 expression and methylation status in representative CpG sites (cg18108623, cg13341380, and cg26573518); B: Semi-quantitative RT-PCR showing the expression of SLFN11 in esophageal squamous cell carcinoma (ESCC) cell lines before and after treatment with 5-aza-2’-deoxycytidine (5-aza); C: Detection of the methylation status of SLFN11 by methylation-specific polymerase chain reaction (MSP) in ESCC cells; D: Representative MSP results of SLFN11 in esophageal tissue samples. TSS: Transcription start site; KYSE30, KYSE140, KYSE150, KYSE180, KYSE450, KYSE510, KYSE520, and colo680n are ESCC cells; 5-aza: 5-aza-2’-deoxycytidine; GAPDH: Internal control of RT-PCR; H₂O: Double distilled water; (-): Absence of 5-aza; (+): Administration of 5-aza; U: Unmethylated alleles; M: Methylated alleles; IVD: In vitro methylated DNA as methylation control; NL: Normal peripheral lymphocytes DNA as unmethylation control; ED: Esophageal dysplasia; EC: Esophageal squamous cell carcinoma.

Figure 2 Schlafen-11 was correlated with chemoresistance to cisplatin in esophageal squamous cell carcinoma cells. A: MTT assay showing the sensitivity to cisplatin in KYSE30 and KYSE450 cells before and after re-expression of Schlafen-11 (SLFN11), and in KYSE510 cells before and after knockout of SLFN11, data are representative of three independent experiments; B: Representative colony formation assay under the treatment of 1 μmol/L and 2 μmol/L cisplatin for 14 d in esophageal squamous cell carcinoma cells. Each experiment was repeated in triplicate. The average normalized colony efficiency was indicated by a bar diagram. Statistical significance was analyzed by t test (^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001). KO: Knockout; SLFN11: Schlafen-11.

Figure 3 The role of Schlafen-11 on DNA damage repair network in esophageal squamous cell carcinoma cells. A: The levels of Schlafen-11 (SLFN11), p-DNAPKcs, DNAPKcs, and XRCC4 under the treatment of 1 μmol/L cisplatin for 12 h and 24 h in KYSE30 and KYSE450 cells before and after SLFN11 re-expression and in KYSE510 before and after SLFN11 knockout; B: The effects on ATR/CHK1 signaling in KYSE30 and KYSE450 cells after SLFN11 re-expression and in KYSE510 cells after SLFN11 knockout under the treatment of 1 μmol/L cisplatin for 12 h and 24 h; C: The effects on ATM/CHK2 signaling in KYSE30 and KYSE450 cells after the restoration of SLFN11 and in KYSE510 cells after SLFN11 knockout under the treatment of 1 μmol/L cisplatin for 12 h and 24 h. β-actin: Internal control.

Figure 4 Schlafen-11 silencing sensitized esophageal squamous cell carcinoma cells to AZD0156. A: MTT assay to evaluate the sensitivity of esophageal squamous cell carcinoma (ESCC) cells to AZD0156 under the treatment of low dose cisplatin, data are representative of three independent experiments; B: Representative colony formation assay under the treatment of low dose cisplatin, AZD0156 and combined mini-dose cisplatin with AZD0156 for 14 d in ESCC cells, each experiment was repeated in triplicate, the average normalized colony efficiency was indicated by a bar diagram, statistical significance was analyzed by t test (^bP < 0.01, ^cP < 0.001, ^dP < 0.0001); C: The protein levels of ATM/CHK2 signaling and γ-H2AX in ESCC cells under the treatment of low dose cisplatin, AZD0156 and combined cisplatin with AZD0156 in ESCC cells. KO: Knockout; β-actin: Internal control.

Figure 5 Invivo efficacy of sensitivity of Schlafen-11 deficient cells to AZD0156. A: Schematic diagram of xenograft mouse models generation and medication; B: Tumors derived from Schlafen-11 (SLFN11) unexpressed and re-expressed KYSE30 cell xenografts in mice treated as indicated; C: Growth curves of xenograft tumors treated with 2 mg/kg cisplatin, 30 mg/kg AZD0156 and combination of 2 mg/kg cisplatin plus 30 mg/kg AZD0156; D and E: Normalized tumor volume and weight in KYSE30 unexpressed and re-expressed xenografts under different modes of treatment, statistical significance was analyzed by t test (^dP < 0.0001); F: Schematic model of synthetic lethality of SLFN11 with ATM inhibitor in esophageal squamous cell carcinoma cells.