Functhional analysis of tumor suppressur CHFR and development of novel daiagnotic and therapeutic strategies

有丝分裂检查点基因CHFR在多种人类癌症中因启动子超甲基化而沉默或发生突变，这表明CHFR是一种重要的肿瘤抑制因子。最近的研究报道CHFR作为E3泛素连接酶起作用，导致靶蛋白的降解。为了更好地了解CHFR如何抑制细胞周期进程和肿瘤发生，我们试图使用亲和纯化结合质谱法鉴定CHFR相互作用蛋白。本研究表明，聚（adp -核糖）聚合酶-1（PARP-1）是一种新的CHFR相互作用蛋白。在表达CHFR的细胞中，有丝分裂应激诱导PARP-1的自芳基化，导致CHFR和PARP-1之间的相互作用增强，PARP-1的多泛素化/降解增加。PARP-1蛋白水平的降低促进了细胞周期在前期停滞，支持表达CHFR的细胞对微管抑制剂具有抗性。相比之下，在chfr沉默的细胞中，有丝分裂应激不诱导多泛素化。因此，PARP-1蛋白水平没有降低，细胞在有丝分裂应激下进入有丝分裂，表明chfr沉默的癌细胞对微管抑制剂敏感。此外，我们发现来自Chfr基因敲除小鼠和Chfr基因沉默的原发性胃癌组织的细胞表达更高水平的PARP-1蛋白，这有力地支持了我们的数据，即Chfr和PARP-1之间的相互作用在细胞周期调节和癌症治疗策略中发挥重要作用。根据我们的研究，我们证明了使用PARP抑制剂联合化疗对微管抑制剂耐药的癌细胞具有显着优势。

The mitotic checkpoint gene CHFR is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Herein, we showed poly(ADP-ribose) polymerase-1(PARP-1) to be a novel CHFR interacting protein. In CHFR expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. By contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFRsilenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. Based on our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.