The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

Robert Hromas, Hyun Suk Kim, Gurjit Sidhu, Elizabeth Williamson, Aruna Jaiswal, Taylor A. Totterdale, Jocelyn Nole, Suk-Hee Lee, Jac A. Nickoloff, Kimi Y. Kong

Research output: Contribution to journalArticle

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Abstract

Background: Proper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5' endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52). Depleting RAD52 can cause synthetic lethality in BRCA1/2 mutant cancers by an unknown molecular mechanism. Methods: We hypothesized that cleavage of stressed replication forks by EEPD1 generates a fork repair intermediate that is toxic when HR-deficient cells cannot complete repair with the RAD52 bypass pathway. To test this hypothesis, we applied cell survival assays, immunofluorescence staining, DNA fiber and western blot analyses to look at the correlation between cell survival and genome integrity in control, EEPD1, RAD52 and EEPD1/RAD52 co-depletion BRCA1-deficient breast cancer cells. Results: Our data show that depletion of EEPD1 suppresses synthetic lethality, genome instability, mitotic catastrophe, and hypersensitivity to stress of replication of RAD52-depleted, BRCA1 mutant breast cancer cells. Without HR and the RAD52-dependent backup pathway, the BRCA1 mutant cancer cells depleted of EEPD1 skew to the alternative non-homologous end-joining DNA repair pathway for survival. Conclusion: This study indicates that the mechanism of synthetic lethality in RAD52-depleted BRCA1 mutant cancer cells depends on the endonuclease EEPD1. The data imply that EEPD1 cleavage of stressed replication forks may result in a toxic intermediate when replication fork repair cannot be completed.

Original languageEnglish (US)
Article number122
JournalBreast Cancer Research
Volume19
Issue number1
DOIs
StatePublished - Nov 16 2017

Fingerprint

BRCA1 Protein
Endonucleases
Homologous Recombination
Radiation
Breast Neoplasms
Proteins
Poisons
Neoplasms
Cell Survival
Recombinational DNA Repair
Synthetic Lethal Mutations
Genomic Instability
DNA Repair
Ovarian Neoplasms
Fluorescent Antibody Technique
Hypersensitivity
Western Blotting
Genome
Staining and Labeling
Mutation

Keywords

  • BRCA1
  • Breast cancer
  • Homologous recombination
  • Non-homologous end joining
  • Replication stress
  • Synthetic lethality

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Hromas, R., Kim, H. S., Sidhu, G., Williamson, E., Jaiswal, A., Totterdale, T. A., ... Kong, K. Y. (2017). The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells. Breast Cancer Research, 19(1), [122]. https://doi.org/10.1186/s13058-017-0912-8

The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells. / Hromas, Robert; Kim, Hyun Suk; Sidhu, Gurjit; Williamson, Elizabeth; Jaiswal, Aruna; Totterdale, Taylor A.; Nole, Jocelyn; Lee, Suk-Hee; Nickoloff, Jac A.; Kong, Kimi Y.

In: Breast Cancer Research, Vol. 19, No. 1, 122, 16.11.2017.

Research output: Contribution to journalArticle

Hromas, R, Kim, HS, Sidhu, G, Williamson, E, Jaiswal, A, Totterdale, TA, Nole, J, Lee, S-H, Nickoloff, JA & Kong, KY 2017, 'The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells', Breast Cancer Research, vol. 19, no. 1, 122. https://doi.org/10.1186/s13058-017-0912-8
Hromas, Robert ; Kim, Hyun Suk ; Sidhu, Gurjit ; Williamson, Elizabeth ; Jaiswal, Aruna ; Totterdale, Taylor A. ; Nole, Jocelyn ; Lee, Suk-Hee ; Nickoloff, Jac A. ; Kong, Kimi Y. / The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells. In: Breast Cancer Research. 2017 ; Vol. 19, No. 1.
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AU - Jaiswal, Aruna

AU - Totterdale, Taylor A.

AU - Nole, Jocelyn

AU - Lee, Suk-Hee

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AB - Background: Proper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5' endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52). Depleting RAD52 can cause synthetic lethality in BRCA1/2 mutant cancers by an unknown molecular mechanism. Methods: We hypothesized that cleavage of stressed replication forks by EEPD1 generates a fork repair intermediate that is toxic when HR-deficient cells cannot complete repair with the RAD52 bypass pathway. To test this hypothesis, we applied cell survival assays, immunofluorescence staining, DNA fiber and western blot analyses to look at the correlation between cell survival and genome integrity in control, EEPD1, RAD52 and EEPD1/RAD52 co-depletion BRCA1-deficient breast cancer cells. Results: Our data show that depletion of EEPD1 suppresses synthetic lethality, genome instability, mitotic catastrophe, and hypersensitivity to stress of replication of RAD52-depleted, BRCA1 mutant breast cancer cells. Without HR and the RAD52-dependent backup pathway, the BRCA1 mutant cancer cells depleted of EEPD1 skew to the alternative non-homologous end-joining DNA repair pathway for survival. Conclusion: This study indicates that the mechanism of synthetic lethality in RAD52-depleted BRCA1 mutant cancer cells depends on the endonuclease EEPD1. The data imply that EEPD1 cleavage of stressed replication forks may result in a toxic intermediate when replication fork repair cannot be completed.

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