Dittmann Klaus, Mayer Claus, Paasch Angela, Huber Stephan, Fehrenbacher Birgit, Schaller Martin, Rodemann H Peter
Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Germany.
Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Germany.
Radiother Oncol. 2015 Sep;116(3):431-7. doi: 10.1016/j.radonc.2015.08.016. Epub 2015 Aug 27.
EGFR is translocated into the cell nucleus in response to irradiation, where it is involved in regulation of radio-sensitivity. The aim of this study is to elucidate the functional role of nuclear EGFR.
To identify EGFR-bound nuclear proteins and mRNAs, Maldi-TOF analysis and mRNA gene arrays were used. Complex formation of proteins was shown by confocal microscopy, immunoprecipitation and Western blotting. The effect of EGFR binding to mRNAs was exhibited by quantitative RT-PCR. Cellular endpoints were shown by Western blotting, mitochondrial mass quantification, lactate quantification and clonogenic survival assays.
Maldi-TOF analysis of proteins bound to nuclear EGFR in response to irradiation showed colocalization with Lamin A and heterogeneous nuclear ribonucleoproteins. Confocal microscopy and Western blotting confirmed this colocalization. Both Lamin A and heterogeneous nuclear ribonucleoproteins are involved in mRNA processing. To support a role of nEGFR in this context after irradiation, we isolated EGFR-bound mRNA and observed an EGFR kinase-dependent mRNA stabilizing effect. With the help of DNA microarrays, we identified mRNAs associated with the Warburg effect that were bound to nuclear EGFR. In this context, we observed radiation-induced HIF1α expression, which triggers inhibition of pyruvate dehydrogenase and blocks the tricarboxylic acid cycle. Consequently, we detected mitophagy and increased lactate production, which is associated with increased treatment resistance. Reduction of nEGFR decreased radiation-induced expression of Hif1α and lactate production.
We showed that nuclear EGFR selectively binds and stabilizes mRNA involved in the Warburg effect in response to irradiation. As a consequence, cells switch from aerobic to anaerobic glucose metabolism, which can be prevented by HIF1α inhibitor BAY87-2243, Dasatinib, Erlotinib or EGFR siRNA.
表皮生长因子受体（EGFR）在受到辐射后会转位至细胞核内，参与放射敏感性的调节。本研究旨在阐明细胞核内EGFR的功能作用。
采用基质辅助激光解吸电离飞行时间质谱分析（Maldi-TOF）和mRNA基因芯片来鉴定与EGFR结合的核蛋白和mRNA。通过共聚焦显微镜、免疫沉淀和蛋白质印迹法显示蛋白质复合物的形成。通过定量逆转录聚合酶链反应（RT-PCR）展示EGFR与mRNA结合的作用。通过蛋白质印迹法、线粒体质量定量、乳酸定量和克隆形成存活试验显示细胞终点。
对受辐射后与细胞核内EGFR结合的蛋白质进行Maldi-TOF分析，结果显示其与核纤层蛋白A和不均一核核糖核蛋白共定位。共聚焦显微镜和蛋白质印迹法证实了这种共定位。核纤层蛋白A和不均一核核糖核蛋白均参与mRNA加工。为支持辐射后细胞核内EGFR在此过程中的作用，我们分离了与EGFR结合的mRNA，并观察到一种EGFR激酶依赖性的mRNA稳定作用。借助DNA微阵列，我们鉴定出与瓦伯格效应相关且与细胞核内EGFR结合的mRNA。在此背景下，我们观察到辐射诱导的缺氧诱导因子1α（HIF1α）表达，其触发丙酮酸脱氢酶的抑制并阻断三羧酸循环。因此，我们检测到线粒体自噬和乳酸生成增加，这与治疗抗性增加相关。细胞核内EGFR的减少降低了辐射诱导的Hif1α表达和乳酸生成。
我们表明，细胞核内EGFR在受到辐射后选择性地结合并稳定参与瓦伯格效应的mRNA。结果，细胞从有氧葡萄糖代谢转变为无氧葡萄糖代谢，而这可通过HIF1α抑制剂BAY87-2243、达沙替尼、厄洛替尼或EGFR小干扰RNA（siRNA）来预防。
