■聚(ADP-核糖)聚合酶(PARP)抑制剂的临床益处仅限于由于原发性和获得性耐药性而导致BRCA缺乏症的三阴性乳腺癌(TNBC)。因此,迫切需要开发针对对PARP抑制具有抗性的BRCA突变的TNBC肿瘤的替代治疗方案.类似于PARP,聚(ADP-核糖)糖水解酶(PARG)在DNA复制和修复中起作用。然而,关于BRCA1缺陷型肿瘤细胞对PARG抑制的脆弱性的报道相互矛盾.这项研究旨在研究PARG抑制剂COH34和泛素特异性蛋白酶(USP)14抑制剂IU1-248的协同致死作用以及BRCA1突变体的潜在机制。PARP抑制剂抗性TNBC细胞。
■在BRCA突变体中单独或与USP14抑制组合的PARG抑制的细胞毒性,PARP抑制剂抗性TNBC细胞系,HCC1937和SUM149PT,使用细胞活力和增殖测定以及流式细胞术进行分析。通过免疫荧光染色评估IU1-248和COH34协同作用的分子机制,DNA修复报告物测定和蛋白质印迹分析。
■发现HCC1937和SUM149PT细胞对单独的PARG抑制表现出中等响应性。据我们所知,这项研究首次证明IU1-248和COH34的组合在相同的环境中对TNBC细胞产生协同作用。机械上,显示IU1-248对USP14的阻断会增加DNA损伤并促进易错的非同源末端连接(NHEJ),正如γH2AX和53BP1在细胞核中的积累和报道分子测定的激活所证明的那样。此外,证明了NHEJ修复活性的抑制减弱了伴随的PARG和USP14抑制的协同作用。IU1-248通过下调c-Myc的表达促进NHEJ修复。
■USP14抑制可能是扩大BRCA突变体中TNBC中PARG抑制剂效用的合理策略,PARP抑制剂抗性设置。
UNASSIGNED: The clinical benefits of poly(ADP-ribose) polymerase (PARP) inhibitors are limited to triple-negative breast cancer (TNBC) with BRCA deficiency due to primary and acquired resistance. Thus, there is a pressing need to develop alternative treatment regimens to target BRCA-mutated TNBC tumors that are resistant to PARP inhibition. Similar to PARP, poly(ADP-ribose) glycohydrolase (PARG) plays a role in DNA replication and repair. However, there are conflicting reports on the vulnerability of BRCA1-deficient tumor cells to PARG inhibition. This study aims to investigate the synergistically lethal effect of the PARG inhibitor COH34 and the ubiquitin-specific protease (USP) 14 inhibitor IU1-248 and the underlying mechanisms in BRCA1-mutant, PARP inhibitor-resistant TNBC cells.
UNASSIGNED: The cytotoxicity of PARG inhibition alone or in combination with USP14 inhibition in the BRCA-mutant, PARP inhibitor-resistant TNBC cell lines, HCC1937 and SUM149PT, was analyzed using cell viability and proliferation assays and flow cytometry. The molecular mechanisms underlying the synergistic effects of IU1-248 and COH34 were evaluated by immunofluorescence staining, DNA repair reporter assays and Western blot analysis.
UNASSIGNED: It was found that HCC1937 and SUM149PT cells exhibited moderate responsiveness to PARG inhibition alone. To the best of our knowledge, this research is the first to demonstrate that the combination of IU1-248 and COH34 produces synergistic effects against TNBC cells in the same setting. Mechanistically, the blockade of USP14 by IU1-248 was shown to increase DNA damage and promote error-prone non-homologous end joining (NHEJ), as evidenced by the accumulation of γH2AX and 53BP1 in the nucleus and the activation of a reporter assay. Additionally, it was demonstrated that the inhibition of NHEJ repair activity attenuates the synergistic effects of concomitant PARG and USP14 inhibition. IU1-248 promotes NHEJ repair through the downregulation of the expression of c-Myc.
UNASSIGNED: USP14 inhibition may be a plausible strategy for expanding the utility of PARG inhibitors in TNBC in BRCA-mutant, PARP inhibitor-resistant settings.