目的:KRAS经常发生突变,Y盒结合蛋白1(YB-1)在结直肠癌(CRC)中过度表达。突变KRAS(KRASmut)通过MAPK/RSK和PI3K/AKT刺激YB-1,独立于表皮生长因子受体(EGFR)。p21活化激酶(PAK)家族是AKT和RSK上游的开关位点。类黄酮化合物非塞素抑制RSK介导的YB-1信号传导。我们寻求干扰DNA双链断裂(DSB)修复并诱导CRC细胞放射敏感性的最有效的分子靶向方法。独立于KRAS突变状态。
方法:通过Ras-GTP测定和NGS分析KRAS活性和KRAS突变。RSK和AKT(DT)的双重靶向作用,在体外和离体照射后,通过Western印迹测试了非塞汀以及FRAX486靶向PAK和厄洛替尼靶向EGFR对YB-1活性的影响.此外,在表达报告构建体的细胞中测试DT和FRAX486对DSB修复途径的影响,用于DSB修复途径流式细胞术分析.通过γH2AX和克隆形成测定法检查了残留的DSB和克隆形成性,分别。
结果:厄洛替尼在体外和离体KRAS突变条件下既不阻断DSB修复也不抑制YB-1磷酸化。DT和FRAX486有效抑制YB-1磷酸化,而与KRAS突变状态无关,并减少了同源重组(HR)和替代性非同源末端连接(NHEJ)修复。DT和FRAX486抑制CaCo2中的DSB修复,但不抑制等基因KRASG12V细胞中的DSB修复。Fisetin抑制YB-1磷酸化,阻断DSB修复和增加放射敏感性,独立于KRAS突变状态。
结论:非塞素联合放疗可改善CRC放疗反应,无论KRASmut状态如何。
OBJECTIVE: KRAS is frequently mutated, and the Y-box binding protein 1 (YB-1) is overexpressed in colorectal cancer (CRC). Mutant KRAS (KRASmut) stimulates YB-1 through MAPK/RSK and PI3K/AKT, independent of epidermal growth factor receptor (EGFR). The p21-activated kinase (PAK) family is a switch-site upstream of AKT and RSK. The flavonoid compound fisetin inhibits RSK-mediated YB-1 signaling. We sought the most effective molecular targeting approach that interferes with DNA double strand break (DSB) repair and induces radiosensitivity of CRC cells, independent of KRAS mutation status.
METHODS: KRAS activity and KRAS mutation were analyzed by Ras-GTP assay and NGS. Effect of dual targeting of RSK and AKT (DT), the effect of fisetin as well as targeting PAK by FRAX486 and EGFR by erlotinib on YB-1 activity was tested by Western blotting after irradiation in vitro and ex vivo. Additionally, the effect of DT and FRAX486 on DSB repair pathways was tested in cells expressing reporter constructs for the DSB repair pathways by flow cytometry analysis. Residual DSBs and clonogenicity were examined by γH2AX- and clonogenic assays, respectively.
RESULTS: Erlotinib neither blocked DSB repair nor inhibited YB-1 phosphorylation under KRAS mutation condition in vitro and ex vivo. DT and FRAX486 effectively inhibited YB-1 phosphorylation independent of KRAS mutation status and diminished homologous recombination (HR) and alternative non-homologous end joining (NHEJ) repair. DT and FRAX486 inhibited DSB repair in CaCo2 but not in isogenic KRASG12V cells. Fisetin inhibited YB-1 phosphorylation, blocked DSB repair and increased radiosensitivity, independent of KRAS mutation status.
CONCLUSIONS: Combination of fisetin with radiotherapy may improve CRC radiation response, regardless of KRASmut status.