Profiling Global Kinome Signatures of the Radioresistant MCF-7/C6 Breast Cancer Cells Using MRM-based Targeted Proteomics

Ionizing
radiation is widely used in cancer therapy; however, cancer
cells often develop radioresistance, which compromises the efficacy
of cancer radiation therapy. Quantitative assessment of the alteration
of the entire kinome in radioresistant cancer cells relative to their
radiosensitive counterparts may provide important knowledge to define
the mechanism(s) underlying tumor adaptive radioresistance and uncover
novel target(s) for effective prevention and treatment of tumor radioresistance.
By employing a scheduled multiple-reaction monitoring analysis in
conjunction with isotope-coded ATP affinity probes, we assessed the
global kinome of radioresistant MCF-7/C6 cells and their parental
MCF-7 human breast cancer cells. We rigorously quantified 120 kinases,
of which 1/3 exhibited significant differences
in expression levels or ATP binding affinities. Several kinases involved
in cell cycle progression and DNA damage response were found to be
overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1),
cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic
subunit of DNA-dependent protein kinase. The elevated expression of
CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western
blot analysis. Thus, the altered kinome profile of radioresistant
MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression
and DNA repair in tumor adaptive radioresistance. The unique kinome
profiling results also afforded potential effective targets for resensitizing
radioresistant cancer cells and counteracting deleterious effects
of ionizing radiation exposure.

电离辐射（Ionizing radiation）广泛应用于癌症治疗领域，然而癌细胞常可产生辐射抗性，这会削弱癌症放射治疗的疗效。定量评估辐射抗性癌细胞相较于其辐射敏感对应细胞的全激酶组（kinome）变化，或可为阐明肿瘤适应性辐射抗性的潜在机制、发掘用于肿瘤辐射抗性有效防治的新型靶点提供重要依据。本研究采用程序化多反应监测（multiple-reaction monitoring）分析结合同位素编码ATP亲和探针技术，对辐射抗性MCF-7/C6细胞及其亲本MCF-7人乳腺癌细胞的全局激酶组进行了检测。研究团队严格定量了120种激酶，其中三分之一的激酶在表达水平或ATP结合亲和力上存在显著差异。研究发现，多种参与细胞周期进程与DNA损伤应答的激酶出现过表达或过度激活，包括检查点激酶1（CHK1）、细胞周期蛋白依赖性激酶1与2（CDK1和CDK2），以及DNA依赖性蛋白激酶的催化亚基。通过蛋白质免疫印迹（Western blot）分析，本研究进一步验证了MCF-7/C6细胞中CHK1、CDK1和CDK2的表达上调。综上，辐射抗性MCF-7/C6细胞的激酶组谱改变提示，参与细胞周期进程与DNA修复的激酶在肿瘤适应性辐射抗性中发挥了关键作用。本研究获得的独特激酶组分析结果，也为使辐射抗性癌细胞复敏、抵消电离辐射暴露的有害效应提供了潜在的有效干预靶点。