Tian Wenjuan, Ji Zhaodong, Wang Jingshu, Meng Jiao, Bi Rui, Ren Yulan, Shan Boer, Yang Gong, Wang Huaying
Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, China.
Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China.
Front Oncol. 2022 Oct 12;12:1018034. doi: 10.3389/fonc.2022.1018034. eCollection 2022.
This study was aimed to profile hotspot exonuclease domain mutations (EDMs) of the DNA polymerase ϵ gene () in endometrial cancer (EC) and to investigate the effects of EDMs on tumor cell behavior and catalytic activities of Polϵ.
sequencing was performed in tumor tissue samples from patients with EC to identify hotspot EDMs. Bioinformatics tools were used to select the potential pathogenic EDMs. The association of EDMs with the clinical outcomes of patients was assessed. EC cells were transfected with wildtype or variants to examine the effects of the EDMs on EC cell behavior, including cell cycle, migration, and invasion. Co-immunoprecipitation was employed to obtain FLAG-tagged wildtype and mutant catalytic subunits of Polϵ, followed by the assessment of polymerase and exonuclease activities.
In addition to previously reported P286R and V411L, R375Q and P452L were identified as novel, and deleterious hotspot EDMs of EC. Patients in EDM group had significantly better clinical outcomes than the rest of the cohort. Compared with wildtype POLE, overexpression of POLE variants promoted cisplatin resistance, G0/G1 cell cycle arrest, and cell migration and invasion in EC cells. Overexpression of POLE variants significantly increased the abundance of 3'-OH and upregulated the expression of DNA mismatch repair genes in HEK293T cells. Compared with wildtype Polϵ, Pol ϵ mutants exhibited undermined polymerase and exonuclease abilities in the presence of mismatched nucleotides in HEK293 cells.
We characterized the of hotspot exonuclease domain mutations in the DNA polymerase ϵ gene and identified P286R, V411L, R375Q, and P452L as pathogenic hotspot EDMs in endometrial cancer. These hotspot EDMs are associated with the malignant behavior of endometrial cancer cells and favorable prognosis in patients, suggesting that affects a wide range of cellular processes beyond DNA replication and proofreading.
本研究旨在分析子宫内膜癌（EC）中DNA聚合酶ϵ基因（）的热点外切核酸酶结构域突变（EDMs），并研究EDMs对肿瘤细胞行为及Polϵ催化活性的影响。
对EC患者的肿瘤组织样本进行测序以鉴定热点EDMs。使用生物信息学工具筛选潜在的致病性EDMs。评估EDMs与患者临床结局的相关性。用野生型或变体转染EC细胞，以检测EDMs对EC细胞行为的影响，包括细胞周期、迁移和侵袭。采用免疫共沉淀法获得带FLAG标签的Polϵ野生型和突变型催化亚基，随后评估聚合酶和外切核酸酶活性。
除先前报道的P286R和V411L外，R375Q和P452L被鉴定为EC新的有害热点EDMs。EDM组患者的临床结局明显优于队列中的其他患者。与野生型POLE相比，POLE变体的过表达促进了EC细胞对顺铂的耐药性、G0/G1细胞周期阻滞以及细胞迁移和侵袭。POLE变体的过表达显著增加了3'-OH的丰度，并上调了HEK293T细胞中DNA错配修复基因的表达。与野生型Polϵ相比，在HEK293细胞中存在错配核苷酸的情况下，Pol ϵ突变体表现出受损的聚合酶和外切核酸酶能力。
我们对DNA聚合酶ϵ基因的热点外切核酸酶结构域突变进行了特征分析，并确定P286R、V411L、R375Q和P452L为子宫内膜癌致病性热点EDMs。这些热点EDMs与子宫内膜癌细胞的恶性行为及患者的良好预后相关，表明其影响范围超出了DNA复制和校对的广泛细胞过程。
