Department of Oncology, Changhai Hospital, Second Military Medical University, Shanghai 200433, P.R. China.
School of Life Sciences, Fudan University, Shanghai 200438, P.R. China.
Oncol Rep. 2019 Oct;42(4):1467-1474. doi: 10.3892/or.2019.7242. Epub 2019 Jul 18.
With the increasing use of poly(ADP‑ribose) polymerase (PARP) inhibitors in cancer therapy, understanding their resistance is an urgent research quest. Additionally, CHFR is an E3 ubiquitin ligase, recruited to double‑strand breaks (DSBs) by PAR. Furthermore, ALC1 is a new oncogene involved in the invasion and metastasis of breast cancer. Moreover, PARylated PARP1 activates ALC1 at sites of DNA damage, yet the underlying mechanism remains unclear. Mass spectrometric analysis, western blot analysis and immunoprecipitation were performed to confirm the interaction between CHFR and ALC1 in the physiological condition. Deletion mutants of CHFR and ALC1 were generated to map the interaction domain. PARP1/2 inhibitors were added to identify the ubiquitination of ALC1 by CHFR. ALC1 half‑life was examined to compare the expression of ALC1 protein in the presence and absence of PARP1/2 inhibitors. The results revealed that the transcriptional level of ALC1 was not upregulated in breast cancer tissues. CHFR interacted with ALC1. The PBZ domain of CHFR, the PMD domain and the MACRO domain of ALC1 domain are the necessary regions for the interaction depending on PAR. Ubiquitination of ALC1 by CHFR was dependent on PARylation and resulted in the degradation of PARylated ALC1. PARP1/2 inhibitors decreased the ubiquitination of PAR‑dependent ALC1, and the expression of ALC1 was upregulated by PARP1/2 inhibitors. Ubiquitination mediated by CHFR resulted in the degradation of ALC1. In conclusion, PARP1/2 inhibitors decrease the ubiquitination of ALC1 leading to the accumulation of ALC1, which affects the therapeutic effects of DNA damage response drugs in breast cancer treatment.
随着聚（ADP-核糖）聚合酶（PARP）抑制剂在癌症治疗中的应用日益增多，了解其耐药性是当务之急。此外，CHFR 是一种 E3 泛素连接酶，可被 PAR 募集到双链断裂（DSB）。此外，ALC1 是一种新的癌基因，参与乳腺癌的侵袭和转移。此外，PAR 化的 PARP1 在 DNA 损伤部位激活 ALC1，但潜在机制尚不清楚。通过质谱分析、western blot 分析和免疫沉淀实验，在生理条件下证实了 CHFR 和 ALC1 之间的相互作用。生成 CHFR 和 ALC1 的缺失突变体以定位相互作用结构域。加入 PARP1/2 抑制剂以鉴定 CHFR 对 ALC1 的泛素化。检查 PARP1/2 抑制剂对 ALC1 半衰期的影响，以比较存在和不存在 PARP1/2 抑制剂时 ALC1 蛋白的表达。结果表明，ALC1 的转录水平在乳腺癌组织中没有上调。CHFR 与 ALC1 相互作用。CHFR 的 PBZ 结构域、ALC1 结构域的 PMD 结构域和 MACRO 结构域是 PAR 依赖性相互作用的必需区域。CHFR 对 ALC1 的泛素化依赖于 PAR 化，并导致 PAR 化的 ALC1 降解。PARP1/2 抑制剂降低了 PAR 依赖性 ALC1 的泛素化，PARP1/2 抑制剂上调了 ALC1 的表达。CHFR 介导的泛素化导致 ALC1 的降解。结论：PARP1/2 抑制剂降低了 ALC1 的泛素化，导致 ALC1 积累，从而影响乳腺癌治疗中 DNA 损伤反应药物的治疗效果。
