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RASSF1A–LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2

Nature Cell Biology volume 16, pages 962–971 (2014)Cite this article

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An Erratum to this article was published on 27 March 2015

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Abstract

Genomic instability is a key hallmark of cancer leading to tumour heterogeneity and therapeutic resistance. BRCA2 has a fundamental role in error-free DNA repair but also sustains genome integrity by promoting RAD51 nucleofilament formation at stalled replication forks. CDK2 phosphorylates BRCA2 (pS3291-BRCA2) to limit stabilizing contacts with polymerized RAD51; however, how replication stress modulates CDK2 activity and whether loss of pS3291-BRCA2 regulation results in genomic instability of tumours are not known. Here we demonstrate that the Hippo pathway kinase LATS1 interacts with CDK2 in response to genotoxic stress to constrain pS3291-BRCA2 and support RAD51 nucleofilaments, thereby maintaining genomic fidelity during replication stalling. We also show that LATS1 forms part of an ATR-mediated response to replication stress that requires the tumour suppressor RASSF1A. Importantly, perturbation of the ATR–RASSF1A–LATS1 signalling axis leads to genomic defects associated with loss of BRCA2 function and contributes to genomic instability and ‘BRCA-ness’ in lung cancers.

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Figure 1: LATS1 regulates RAD51 nucleofilament formation in response to replication stress in an homologous-recombination-independent manner.
Figure 2: LATS1 interacts with CDK2 in response to genotoxic stress modulating its kinase activity towards BRCA2.
Figure 3: Tumour suppressor RASSF1A stimulates LATS1–CDK2 interaction in response to ATR activation.
Figure 4: Deletion of the RASSF1A–LATS1 axis compromises the stability of nascent DNA at stalled forks.
Figure 5: Deletion of the RASSF1A–LATS1 axis induces chromosomal aberrations.
Figure 6: RASSF1A methylation correlates with increased CNV in lung cancer patients.
Figure 7: Model of RASSF1A–LATS1–CDK2 signalling and the protection of stalled replication forks.

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  • 11 March 2015

    In the original version of this Article, the bar chart in Fig. 6 was incorrect. This has now been corrected in all online versions of this Article.

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Acknowledgements

The authors would like to acknowledge T. Helleday for helpful discussions, M. Woodcock for fluorescence-activated cell sorting analysis and A. G. Abraham for useful comments on the manuscript. Lats1−/− MEFs were provided by T. Xu and myc–LATS1 constructs by H. Silljé. This work was funded by Cancer Research UK A19277, Vertex and the Medical Research Council (MRC).

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Authors and Affiliations

  1. Department of Oncology, CRUK/MRC Oxford Institute, University of Oxford, Oxford OX3 7DQ, UK

    Dafni-Eleftheria Pefani, Robert Latusek, Isabel Pires, Anna M. Grawenda, Karen S. Yee, Garth Hamilton, Ester M. Hammond & Eric O’Neill

  2. The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK

    Louise van der Weyden

  3. Dunn School of Pathology, South Parks Road, University of Oxford, Oxford OX1 3RE, UK

    Fumiko Esashi

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Contributions

D-E.P. designed and performed most of the experiments, analysed data and contributed to writing the paper; R.L. performed initial experiments for LATS1–CDK2 interaction, γH2AX analysis, pBRCA2 western blots and I-SceI assay; I.P. transferred the DNA fibre technology; K.S.Y. performed initial immunoprecipitation experiments for RASSF1A effect on LATS1–CDK2 interaction. G.H. performed the in vitro kinase assays; A.M.G. performed the TCGA analysis; L.v.d.W. isolated Rassf1A−/− mouse embryonic fibroblasts; F.E. provided BRCA2 reagents and advice; E.M.H. helped with reagents and advice; E.O’N. conceived, coordinated and supervised the project, designed experiments, analysed data and wrote the paper.

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Supplementary Figure 1 LATS1 ablation results in defective repair kinetics and induces a G2/M arrest.

(a) DNA content analysis at the indicated time points of U2OS cells treated with siRNA against LATS1 or control siRNA after exposure to 4 Gy Ionizing Radiation (γIR). The percentage of cells at the different phases of the cell cycle was quantified and presented. (b) Quantification of γH2AX foci in Lats1+/+, Lats1−/−, Lats1−/−mycLATS1, Lats1−/−mycLats1KD MEFs after exposure to 10 Gy γIR at the indicated time points. Bar-graphs indicate the percentage (%) of cells stained positive for γH2AX in 15 fields of view from a representative experiment out of n = 3 experiments. Error bars indicate Standard Error of the Mean (s.e.m.). Statistical significance was determined by a two-tailed t-test. ∗P < 0.05 (c) Lats1+/+, Lats1−/− and Lats1−/− cells expressing wt hLATS1 (Lats1−/−mycLATS1) or a hLATS1 kinase dead derivative LATS1-D846A (Lats1−/−LATS1KD) were treated with 10 Gy γIR. Total cell extracts were isolated at the indicated time points after irradiation and probed with the indicated antibodies (full panel of antibodies for Fig. 1b) (d,e) U2OS cells were transfected with siRNA against LATS1 or Non Targeting siRNAi (siNT) and exposed to 4 Gy γIR. Cells were either harvested at the indicated time points after irradiation and analysed with Western Blotting for the expression of the indicated antibodies (d) or fixed and processed for immunofluorescence for γH2AX foci formation (e). Bargrahs derived from 15 fields of view from a representative experiment and error bars indicate s.e.m. Statistical significance was determined by a two-tailed t-test, ∗P < 0.05. (f) Lats1+/+, Lats1−/−, Lats1−/−mycLATS1 and Lats1−/−hmycLATS1KD MEFs were treated with 10 Gy γIR and after the indicated time points single-stranded DNA fragments were separated from matured DNA by alkaline single cell electrophoresis. Quantification of the percentage of comet tail DNA after alkaline comet assay for each condition is shown next to representative images. 50 cells were counted per condition in n = 3 independent experiments. Error bars derive from n = 3 independent experiments and represent standard deviation. Statistical significance was determined by a two-tailed t-test. ∗P < 0.05.

Supplementary Figure 2 LATS1 is necessary for the establishment of RAD51 foci in response to genotoxic stress, independently of YAP.

(a) Representative plots of two-colour fluorescence analysis for U2OS cells stably transfected with DR-GFP (related to Fig. 1c). The percentage of green fluorescent cells falling above the diagonal for each transfection is indicated. (FL1) Green fluorescence; (FL2) orange fluorescence. (b) Lats1+/+, Lats1−/−, Lats1−/−mycLATS1 and Lats1−/−mycLATS1KD MEFs were exposed to 10 Gy γIR. After the indicated time points cells were fixed and assessed for RAD51 foci by immunofluorescence. The percentage of RAD51 positive cells (> than 9 RAD51 foci/cell) is presented. (c) Quantification graphs and representative pictures of RAD51 foci in Lats1−/− MEFs or WT MEFs treated with siNT, siMST2 or siYAP after 4 h exposure to 2 mM HU are shown. Error bars represent standard deviation from n = 3 independent experiments. Statistical significance was determined by a two-tailed t-test ∗P < 0.05,∗∗P < 0.01. Scale bars, 10 μm.

Supplementary Figure 3 LATS1 compromises CDK2 kinase activity to S3291-BRCA2 in response to stress.

(a) Detection of pS3291-BRCA2 in lysates of HT1080 cells treated with the indicated siRNAs and expressing Myc-LATS1 or control plasmid. Line graphs represent time course measurement of pS3291-BRCA2 levels at indicated times post 4 Gy γIR in cycling cells (bottom) or nocodozol treated cells (top). In pS3291-BRCA2 quantification plots, error bars represent the variation in the densitometry of the representative image. Quantification of cycling cells also presented in Fig. 2e in a different scale. (b) In vitro kinase assay of immunoprecipitated CDK2 from Lats1+/+ and Lats1−/− MEFs incubated with C-terminal BRCA2 purified peptide (His-TR2) in the presence of 32[P]ATP. Reactions were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was visualized by phosphoimage detection (PMI imager, Bio-Rad) and probed with the indicated antibodies.

Supplementary Figure 4 RASSF1A/LATS1 signalling is necessary for the stability of nascent DNA during replication stress.

(a,b) CIdU tract length distributions and representative pictures from DNA fibres from Lats1+/+ and Lats1−/− MEFs (a) and Rassf1A+/+, Rassf1A−/− MEFs (b). Sketch above delineates experimental design. Bar-graphs derived from a representative experiment. 100 tracks were analysed per condition. Mean track lengths and standard deviation given in parenthesis derived from n = 3 independent experiments. Statistical significance was determined by a two-tailed t-test. (c) Examples of DNA fibre images from the indicated cells and conditions. The length of CIdU and IdU tracts are presented in Fig. 4a and Supplementary Fig. 5. (d) Representative images from Rassf1A+/+, Rassf1A−/−, Rassf1A−/−FLAGR1A and Rassf1A−/−FLAG−R1A−A133S MEFs. Length of CIdU and IdU tracts were analysed in Fig. 4c and Supplementary Fig. 5. Scale bar, 10 μm.

Supplementary Figure 5 Deletion of RASSF1A/LATS1 axis compromises fork recovery after stress.

(a) IdU tract length distributions from DNA fibres of Lats1+/+, Lats1−/− and Lats1−/−mycLATS1 after release from 4 h treatment with 2 mM HU, to determine the ability of the cells to recover after replication stress. (b) IdU tract length distributions of Rassf1A+/+, Rassf1A−/−, Rassf1A−/−FLAG−R1A and Rassf1A−/−FLAG−R1A−A133S MEFs after release from 2 mM HU. 100 tracks were analysed in each condition in n = 3 independent experiments. Statistical significance was determined by a two-tailed t-test. Bar-graphs derived from a representative experiment. 100 tracks were analysed per condition. Mean track lengths and standard deviation given in parenthesis derived from n = 3 independent experiments. Statistical significance was determined by a two-tailed t-test. Sketch delineates experimental design.

Supplementary Figure 6 Chromosomal instability accumulates after exposure to replication stress in the absence of RASSF1A/LATS1 signalling.

(a) Representative pictures from metaphase spreads from Lats1+/+, Lats1−/−, Rassf1A+/+ and Rassf1A−/− untreated MEFs quantified for chromosomal abnormalities on Fig. 5a, b. (b) Western Blot analysis for LATS1 and RASSF1A levels in U2OS cells that were examined for chromosomal aberrations in Fig. 5c. (c) Western Blot analysis to assess LATS1 depletion and FLAG-R1A overexpression in H1299 cells used for metaphase spreads in Fig. 5d. (d) U2OS cells treated with Non Targeting siRNA, siRNA against LATS1 or siRNA against RASSF1A were treated or not with HU and released for 16 h prior fixation. DNA was stained with DAPI and cells positive for micronuclei (MN) were scored. 300 cells were scored per condition in n = 3 independent experiments. Error bars represent standard deviation. Statistical significance was determined by a two-tailed t-test. Scale bar, 10 μm.∗∗P < 0.01,∗∗∗P < 0.001.

Supplementary Figure 7 Loss of RASSF1A does not correlate with increased mutation frequency.

Correlation of RASSF1 promoter methylation (assessed with illumina HM450) with genomic mutation count in lung adenocarcinoma datasets from the cancer genome atlas database (TCGA, Provisional). A total of 115 patients with Lung Adenocarcinoma were analysed using the Fisher’s exact test. Absolute numbers and P values are presented in the table.

Supplementary Figure 8 Original uncropped images of western blots.

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Pefani, DE., Latusek, R., Pires, I. et al. RASSF1A–LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2. Nat Cell Biol 16, 962–971 (2014). https://doi.org/10.1038/ncb3035

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