Qiu Wei-Gang, Polotskaia Alla, Xiao Gu, Di Lia, Zhao Yuhan, Hu Wenwei, Philip John, Hendrickson Ronald C, Bargonetti Jill
The Department of Biological Sciences Hunter College, City University of New York, Hunter College-Weill Cornell Belfer Research Building, 413 East 69th, New York, NY 10065, USA; The Graduate Center PhD Program in Biology, City University of New York, New York, NY 10016, USA; Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY 10065, USA.
The Department of Biological Sciences Hunter College, City University of New York, Hunter College-Weill Cornell Belfer Research Building, 413 East 69th, New York, NY 10065, USA.
NPJ Breast Cancer. 2017;3. doi: 10.1038/s41523-016-0001-7. Epub 2017 Jan 19.
Over 80% of triple negative breast cancers express mutant p53. Mutant p53 often gains oncogenic function suggesting that triple negative breast cancers may be driven by p53 protein type. To determine the chromatin targets of this gain-of-function mutant p53 we used inducible knockdown of endogenous gain-of-function mtp53 in MDA-MB-468 cells in conjunction with stable isotope labeling with amino acids in cell culture and subcellular fractionation. We sequenced over 70,000 total peptides for each corresponding reciprocal data set and were able to identify 3010 unique cytoplasmic fraction proteins and 3403 unique chromatin fraction proteins. The present proteomics experiment corroborated our previous experiment-based results that poly ADP-ribose polymerase has a positive association with mutant p53 on the chromatin. Here, for the first time we report that the heterohexomeric minichromosome maintenance complex that participates in DNA replication initiation ranked as a high mutant p53-chromatin associated pathway. Enrichment analysis identified the minichromosome maintenance members 2-7. To validate this mutant p53- poly ADP-ribose polymerase-minichromosome maintenance functional axis, we experimentally depleted R273H mutant p53 and found a large reduction of the amount of minichromosome maintenance complex proteins on the chromatin. Furthermore a mutant p53-minichromosome maintenance 2 direct interaction was detected. Overexpressed mutant p53, but not wild type p53, showed a protein-protein interaction with minichromosome maintenance 2 and minichromosome maintenance 4. To target the mutant p53- poly ADP-ribose polymerase-minichromosome maintenance axis we treated cells with the poly ADP-ribose polymerase inhibitor talazoparib and the alkylating agent temozolomide and detected synergistic activation of apoptosis only in the presence of mutant p53. Furthermore when minichromosome maintenance 2-7 activity was inhibited the synergistic activation of apoptosis was blocked. This mutant p53- poly ADP-ribose polymerase -minichromosome maintenance axis may be useful for theranostics.
超过80%的三阴性乳腺癌表达突变型p53。突变型p53常常获得致癌功能，这表明三阴性乳腺癌可能由p53蛋白类型驱动。为了确定这种功能获得性突变型p53的染色质靶点，我们在MDA-MB-468细胞中对内源性功能获得性mtp53进行诱导敲低，并结合细胞培养中的氨基酸稳定同位素标记和亚细胞分级分离。我们对每个相应的互作数据集的70000多个总肽段进行了测序，能够鉴定出3010个独特的细胞质组分蛋白和3403个独特的染色质组分蛋白。目前的蛋白质组学实验证实了我们之前基于实验的结果，即聚ADP-核糖聚合酶与染色质上的突变型p53呈正相关。在此，我们首次报告参与DNA复制起始的异源六聚体微小染色体维持复合体是一条高度相关的突变型p53-染色质途径。富集分析确定了微小染色体维持成员2-7。为了验证这种突变型p53-聚ADP-核糖聚合酶-微小染色体维持功能轴，我们通过实验耗尽R273H突变型p53，发现染色质上微小染色体维持复合体蛋白的量大幅减少。此外，还检测到突变型p53-微小染色体维持2的直接相互作用。过表达的突变型p53，而不是野生型p53，显示出与微小染色体维持2和微小染色体维持4的蛋白质-蛋白质相互作用。为了靶向突变型p53-聚ADP-核糖聚合酶-微小染色体维持轴，我们用聚ADP-核糖聚合酶抑制剂他拉唑帕尼和烷化剂替莫唑胺处理细胞，仅在存在突变型p53的情况下检测到凋亡的协同激活。此外，当微小染色体维持2-7的活性被抑制时，凋亡的协同激活被阻断。这种突变型p53-聚ADP-核糖聚合酶-微小染色体维持轴可能对治疗诊断有用。
