Runnebohm Avery M, Wijeratne H R Sagara, Justice Sarah A Peck, Wijeratne Aruna B, Roy Gitanjali, Singh Naveen, Hergenrother Paul, Boothman David A, Motea Edward A, Mosley Amber L
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN.
Department of Biology, Marian University, Indianapolis, IN.
bioRxiv. 2024 May 18:2024.05.15.594427. doi: 10.1101/2024.05.15.594427.
Triple negative breast cancer (TNBC), characterized by the lack of three canonical receptors, is unresponsive to commonly used hormonal therapies. One potential TNBC-specific therapeutic target is NQO1, as it is highly expressed in many TNBC patients and lowly expressed in non-cancer tissues. DNA damage induced by NQO1 bioactivatable drugs in combination with Rucaparib-mediated inhibition of PARP1-dependent DNA repair synergistically induces cell death.
To gain a better understanding of the mechanisms behind this synergistic effect, we used global proteomics, phosphoproteomics, and thermal proteome profiling to analyze changes in protein abundance, phosphorylation and protein thermal stability.
Very few protein abundance changes resulted from single or dual agent treatment; however, protein phosphorylation and thermal stability were impacted. Histone H2AX was among several proteins identified to have increased phosphorylation when cells were treated with the combination of IB-DNQ and Rucaparib, validating that the drugs induced persistent DNA damage. Thermal proteome profiling revealed destabilization of H2AX following combination treatment, potentially a result of the increase in phosphorylation. Kinase substrate enrichment analysis predicted altered activity for kinases involved in DNA repair and cell cycle following dual agent treatment. Further biophysical analysis of these two processes revealed alterations in SWI/SNF complex association and tubulin / p53 interactions.
Our findings that the drugs target DNA repair and cell cycle regulation, canonical cancer treatment targets, in a way that is dependent on increased expression of a protein selectively found to be upregulated in cancers without impacting protein abundance illustrate that multi-omics methodologies are important to gain a deeper understanding of the mechanisms behind treatment induced cancer cell death.
三阴性乳腺癌（TNBC）的特征是缺乏三种典型受体，对常用的激素疗法无反应。NQO1是一种潜在的TNBC特异性治疗靶点，因为它在许多TNBC患者中高表达，而在非癌组织中低表达。NQO1生物可激活药物诱导的DNA损伤与鲁卡帕尼介导的PARP1依赖性DNA修复抑制相结合，可协同诱导细胞死亡。
为了更好地理解这种协同效应背后的机制，我们使用了全局蛋白质组学、磷酸蛋白质组学和热蛋白质组分析来分析蛋白质丰度、磷酸化和蛋白质热稳定性的变化。
单药或双药治疗导致的蛋白质丰度变化很少；然而，蛋白质磷酸化和热稳定性受到影响。组蛋白H2AX是在用IB-DNQ和鲁卡帕尼联合处理细胞时被鉴定出磷酸化增加的几种蛋白质之一，证实了这些药物诱导了持续性DNA损伤。热蛋白质组分析显示联合处理后H2AX不稳定，这可能是磷酸化增加的结果。激酶底物富集分析预测双药治疗后参与DNA修复和细胞周期的激酶活性发生改变。对这两个过程的进一步生物物理分析揭示了SWI/SNF复合物结合和微管蛋白/p53相互作用的改变。
我们的研究结果表明，这些药物以一种依赖于在癌症中选择性上调的蛋白质表达增加的方式靶向DNA修复和细胞周期调控，这是典型的癌症治疗靶点，且不影响蛋白质丰度，这说明多组学方法对于深入理解治疗诱导癌细胞死亡背后的机制很重要。
