Department of Cancer Biology and Genetics, The Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
Department of Outcomes and Translational Sciences, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
Int J Mol Sci. 2022 Mar 28;23(7):3729. doi: 10.3390/ijms23073729.
Wwox-deficient human cells show elevated homologous recombination, leading to resistance to killing by double-strand break-inducing agents. Human Wwox binds to the Brca1 981-PPLF-984 Wwox-binding motif, likely blocking the pChk2 phosphorylation site at Brca1-S988. This phosphorylation site is conserved across mammalian species; the PPLF motif is conserved in primates but not in rodents. We now show that murine Wwox does not bind Brca1 near the conserved mouse Brca1 phospho-S971 site, leaving it open for Chk2 phosphorylation and Brca1 activation. Instead, murine Wwox binds to Brca1 through its BRCT domain, where pAbraxas, pBrip1, and pCtIP, of the A, B, and C binding complexes, interact to regulate double-strand break repair pathway response. In Wwox-deficient mouse cells, the Brca1-BRCT domain is thus accessible for immediate binding of these phospho-proteins. We confirm elevated homologous recombination in Wwox-silenced murine cells, as in human cells. Wwox-deficient murine cells showed increased ionizing radiation-induced Abraxas, Brca1, and CtIP foci and long resected single-strand DNA, early after ionizing radiation. Wwox deletion increased the basal level of Brca1-CtIP interaction and the expression level of the MRN-CtIP protein complex, key players in end-resection, and facilitated Brca1 release from foci. Inhibition of phospho-Chk2 phosphorylation of Brca1-S971 delays the end-resection; the delay of premature end-resection by combining Chk2 inhibition with ionizing radiation or carboplatin treatment restored ionizing radiation and platinum sensitivity in Wwox-deficient murine cells, as in human cells, supporting the use of murine in vitro and in vivo models in preclinical cancer treatment research.
Wwox 缺陷的人类细胞显示出同源重组的增加，导致对双链断裂诱导剂的杀伤产生抗性。人类 Wwox 与 Brca1 981-PPLF-984 Wwox 结合基序结合，可能阻止 Brca1-S988 上的 pChk2 磷酸化位点。该磷酸化位点在哺乳动物物种中是保守的；PPLF 基序在灵长类动物中保守，但在啮齿动物中不保守。我们现在表明，鼠 Wwox 不会结合保守的鼠 Brca1 磷酸化 S971 位点附近的 Brca1，从而为 Chk2 磷酸化和 Brca1 激活留出空间。相反，鼠 Wwox 通过其 BRCT 结构域与 Brca1 结合，A、B 和 C 结合复合物中的 pAbraxas、pBrip1 和 pCtIP 在此处相互作用以调节双链断裂修复途径反应。在 Wwox 缺陷的小鼠细胞中，Brca1-BRCT 结构域因此可立即与这些磷酸化蛋白结合。我们证实 Wwox 沉默的小鼠细胞中同源重组增加，就像在人类细胞中一样。Wwox 缺陷的小鼠细胞在电离辐射后早期显示出增加的 Abraxas、Brca1 和 CtIP 焦点和长切除的单链 DNA，导致同源重组增加。Wwox 缺失增加了基础水平的 Brca1-CtIP 相互作用和 MRN-CtIP 蛋白复合物的表达水平，该复合物是末端切除的关键参与者，并促进了 Brca1 从焦点中的释放。抑制 Brca1-S971 的磷酸化 Chk2 延迟末端切除；通过将 Chk2 抑制与电离辐射或卡铂联合治疗相结合来延迟过早的末端切除，恢复了 Wwox 缺陷的小鼠细胞中的电离辐射和铂敏感性，就像在人类细胞中一样，支持在临床前癌症治疗研究中使用小鼠体外和体内模型。
