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FANCD2 limits replication stress and genome instability in cells lacking BRCA2

Abstract

The tumor suppressor BRCA2 plays a key role in genome integrity by promoting replication-fork stability and homologous recombination (HR) DNA repair. Here we report that human cancer cells lacking BRCA2 rely on the Fanconi anemia protein FANCD2 to limit replication-fork progression and genomic instability. Our results identify a new role of FANCD2 in limiting constitutive replication stress in BRCA2-deficient cells, thereby affecting cell survival and treatment responses.

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Figure 1: FANCD2 restricts replication and prevents accumulation of DNA damage in BRCA2-deficient cells.
Figure 2: FANCD2 is required for BRCA2-deficient cell survival and alters olaparib sensitivity.

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Acknowledgements

We thank S. Bertrand for assistance with the senescence assays. We are grateful to G. Brown and M. Woodcock for assistance with microscopy and FACS analyses, respectively. J.Z. is supported by a Cancer Research UK PhD Studentship. The laboratory of M.T. is funded by Cancer Research UK A17201, the Medical Research Council, the University of Oxford and the EMBO Young Investigator Programme.

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Contributions

M.T., J.M. and J.Z. designed the study and the experiments. J.Z. and M.T. wrote the paper. J.M. and J.Z. performed the experiments. U.M. and F.M.B. analyzed FANCD2 expression levels in cell lines and tumors, respectively.

Corresponding author

Correspondence to Madalena Tarsounas.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Depletion of FANCD2 and BRCA2 leads to increased origin firing in HEK-293T cells.

(a) HEK-293T cells were transfected with siRNAs against BRCA2 and/or FANCD2 and cell extracts were processed for immunoblotting 72 h after transfection. SMC1 was used as a loading control. (b) Quantification of newly fired origins of fiber experiments in Figure 1f. n = 3 (independent experiments). error bars, SEM; *, p < 0.05 (unpaired, two-tailed t test).

Supplementary Figure 2 Loss of FANCD2 increases senescence in BRCA2-deficient MRC-5 cells.

(a) MRC-5 cells were transfected with siRNAs against BRCA2 and/or FANCD2 and cell extracts were processed for immunoblotting 48 h after transfection. SMC1 was used as a loading control. (b) Loss of FANCD2 induces senescence in BRCA2-depleted MRC-5 cells as assessed by β-galactosidase staining. n = 3 (independent experiments); error bars, SEM; ***, p < 0.001; ****, p < 0.0001 (unpaired, two-tailed t test). (c) Representative images of quantification shown in (b).

Supplementary Figure 3 FANCD2 and BRCA2 are synthetic lethal.

(a) H1299 cells harbouring Dox-inducible shRNA against BRCA2 were transfected with two different siRNAs against FANCD2 and processed for immunoblotting 72 h after transfection. SMC1 was used as a loading control. (b) Clonogenic survival assays in H1299 cells transfected with FANCD2 esiRNA. n = 3 (independent experiments); error bars, SEM; ***, p < 0.001 (unpaired, two-tailed t test). (c) BRCA2-proficient (+BRCA2) or -deficient (–BRCA2) DLD1 cells were transfected with esiRNA against FANCD2 and processed for immunoblotting 72 h after transfection. SMC1 was used as a loading control. (d) MDA-MB-231 cells harbouring Dox-inducible shRNA against BRCA2 were transfected with siRNA against FANCD2 and processed for immunoblotting 72 h after transfection. SMC1 was used as a loading control. (e) FANCD2-proficient (+FANCD2) or -deficient (–FANCD2) DLD1 cells were transfected with siRNA against BRCA2 and processed for immunoblotting 72 h after transfection. SMC1 was used as a loading control. (f) Clonogenic survival assays in MDA-MB-231 cells transfected with FANCA siRNA. n = 3 (independent experiments); error bars, SEM; *, p < 0.05 (unpaired, two-tailed t test).

Supplementary Figure 4 Schematic illustrating BRCA2 mutations found in breast tumors.

Breast tumors were analyzed in Figure 2f. H, helical domain. OB, oligonucleotide-binding domain. T, tower domain. NLS, nuclear localization sequence.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1–7 (PDF 766 kb)

Supplementary Data Set 1

Uncropped blots shown in Figure 1a. (PDF 125 kb)

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Michl, J., Zimmer, J., Buffa, F. et al. FANCD2 limits replication stress and genome instability in cells lacking BRCA2. Nat Struct Mol Biol 23, 755–757 (2016). https://doi.org/10.1038/nsmb.3252

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