Characterization of hotspot exonuclease domain mutations in the DNA polymerase ϵ gene in endometrial cancer

Wenjuan Tian; Zhaodong Ji; Jingshu Wang; Jiao Meng; Rui Bi; Yulan Ren; Boer Shan; Gong Yang; Huaying Wang

doi:10.3389/fonc.2022.1018034

Characterization of hotspot exonuclease domain mutations in the DNA polymerase ϵ gene in endometrial cancer

Front Oncol. 2022 Oct 12:12:1018034. doi: 10.3389/fonc.2022.1018034. eCollection 2022.

Authors

Wenjuan Tian^{1

2}, Zhaodong Ji³, Jingshu Wang⁴, Jiao Meng^{2

5}, Rui Bi⁶, Yulan Ren^{1

2}, Boer Shan^{1

2}, Gong Yang^{2

4

5}, Huaying Wang^{1

2}

Affiliations

¹ Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, China.
² Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China.
³ Department of Clinical Laboratory, Huashan Hospital, Fudan University, Shanghai, China.
⁴ Central Laboratory, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai, China.
⁵ Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, China.
⁶ Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China.

Abstract

Objective: This study was aimed to profile hotspot exonuclease domain mutations (EDMs) of the DNA polymerase ϵ gene (POLE) in endometrial cancer (EC) and to investigate the effects of EDMs on tumor cell behavior and catalytic activities of Polϵ.

Methods: POLE 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 POLE or POLE 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.

Results: In addition to previously reported P286R and V411L, R375Q and P452L were identified as novel, and deleterious POLE 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.

Conclusion: We characterized the of hotspot exonuclease domain mutations in the DNA polymerase ϵ gene and identified P286R, V411L, R375Q, and P452L as pathogenic POLE hotspot EDMs in endometrial cancer. These hotspot EDMs are associated with the malignant behavior of endometrial cancer cells in vitro and favorable prognosis in patients, suggesting that POLE affects a wide range of cellular processes beyond DNA replication and proofreading.

Keywords: 3’-5’-Exonuclease; DNA polymerase epsilon; DNA replication; endometrial cancer; hotspot exonuclease domain mutations.