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. 2017 Oct;9(10):1398-1414.
doi: 10.15252/emmm.201607446.

BRCA1 and BRCA2 tumor suppressors protect against endogenous acetaldehyde toxicity

Eliana Mc Tacconi  1 , Xianning Lai  1 , Cecilia Folio  1 , Manuela Porru  2 , Gijs Zonderland  1 , Sophie Badie  1 , Johanna Michl  1 , Irene Sechi  1 , Mélanie Rogier  3   4   5   6 , Verónica Matía García  7 , Ankita Sati Batra  8 , Oscar M Rueda  8 , Peter Bouwman  7 , Jos Jonkers  7 , Anderson Ryan  9 , Bernardo Reina-San-Martin  3   4   5   6 , Joannie Hui  10 , Nelson Tang  10 , Alejandra Bruna  8 , Annamaria Biroccio  2 , Madalena Tarsounas  11
Affiliations

BRCA1 and BRCA2 tumor suppressors protect against endogenous acetaldehyde toxicity

Eliana Mc Tacconi et al. EMBO Mol Med. 2017 Oct.

Abstract

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.

Keywords: BRCA1; BRCA2; DNA damage; disulfiram, acetaldehyde dehydrogenase; replication stress.

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Figures

Figure EV1
Figure EV1. Acetaldehyde toxicity to human FANCD2‐deleted human cells and FANCD2 ubiquitylation in BRCA2‐deleted cells
  1. A, B

    Human DLD1 cells in which FANCD2 was deleted with CRISPR/Cas9 and control cells were incubated with the indicated concentrations of cisplatin (A) or acetaldehyde (B) for 6 days before processing for dose‐dependent viability assays. Graphs are representative of two independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. Inset, Western blot detection of FANCD2 expression. SMC1 was used as a loading control.

  2. C

    BRCA2‐proficient (+BRCA2) or BRCA2‐deficient (−BRCA2) DLD1 cells were incubated with 4 mM acetaldehyde for 48 h before being processed for immunoblotting as indicated.

  3. D

    H1299 cells expressing a DOX‐inducible BRCA2 shRNA were grown in the presence or absence of DOX and transfected with control or FANCD2 siRNA before being processed for immunoblotting as indicated. DOX, doxycycline.

Figure 1
Figure 1. BRCA1‐ and BRCA2‐deficient human cells are hypersensitive to acetaldehyde
  1. A

    BRCA2‐proficient (+BRCA2) or BRCA2‐deficient (−BRCA2) human DLD1 cells were incubated with the indicated concentrations of olaparib or acetaldehyde for 6 days before processing for dose‐dependent viability assays. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. Cell extracts prepared at the time of acetaldehyde addition were immunoblotted as indicated. SMC1 was used as a loading control.

  2. B, C

    Human H1299 cells expressing DOX‐inducible BRCA2 or BRCA1 shRNAs were grown in the presence or absence of DOX and incubated with the indicated concentrations of olaparib or acetaldehyde for 6 days, before processing for dose‐dependent viability assays. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. Cell extracts prepared at the time of acetaldehyde addition were immunoblotted as indicated. SMC1 was used as a loading control. DOX, doxycycline.

Figure EV2
Figure EV2. Acetaldehyde and disulfiram induce RAD51 foci in BRCA2‐proficient DLD1 cells

  1. Human DLD1 cells, BRCA2‐proficient (+BRCA2) were incubated with acetaldehyde (4 mM) or disulfiram (10 μM) for 96 h prior to processing for immunofluorescence staining with anti‐RAD51 antibody (green). DNA was counter‐stained with DAPI (blue). Scale bar, 10 μm.

  2. Quantification of percentage of cells with 10 or more RAD51 foci in cells treated as in (A). At least 100 nuclei were quantified for each treatment. Ac, acetaldehyde; Di, disulfiram. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test.

Figure 2
Figure 2. BRCA1‐ and BRCA2‐deficient human cells are hypersensitive to disulfiram
  1. A

    Schematic representation of sources of acetaldehyde and its cellular catabolism. ALDH2, a disulfiram target, is also indicated.

  2. B

    BRCA2‐proficient (+BRCA2) or BRCA2‐deficient (−BRCA2) human DLD1 cells were incubated with the indicated concentrations of disulfiram for 6 days, before processing for dose‐dependent viability assays. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment.

  3. C, D

    Human H1299 cells expressing DOX‐inducible BRCA2 or BRCA1 shRNAs were grown in the presence or absence of DOX and incubated with the indicated concentrations of disulfiram for 6 days, before processing for dose‐dependent viability assays. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. DOX, doxycycline.

Figure EV3
Figure EV3. ALDEFLUOR™ assay in human DLD1 cells treated with disulfiram

  1. Quantification of ALDH activity relative to internal DEAB control in BRCA2‐proficient (+BRCA2) and BRCA2‐deficient (−BRCA2) human DLD1 cells treated with DMSO or disulfiram (10 μM) for 4 days. Error bars represent SD of two independent experiments. P‐values were calculated using a one‐sample t‐test.

Figure 3
Figure 3. Elevated replication stress and G2/M accumulation in BRCA2‐deficient human cells treated with disulfiram or acetaldehyde

  1. Representative images of BRCA2‐proficient (+BRCA2) and BRCA2‐deficient (−BRCA2) human DLD1 cells incubated with 10 μM disulfiram or 4 mM acetaldehyde for 4 days prior to processing for immunofluorescence staining with anti‐RPA antibody (green). DNA was counter‐stained with DAPI (blue). Scale bar, 10 μm.

  2. Quantification of the frequency of BRCA2‐proficient (+BRCA2) or BRCA2‐deficient (−BRCA2) human DLD1 cells with 10 or more RPA foci in cells treated as in (A). Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test.

  3. BRCA2‐proficient (+BRCA2) or BRCA2‐deficient (−BRCA2) human DLD1 cells were treated with 2.5 μM disulfiram or 2 mM acetaldehyde overnight and processed for DNA fiber analysis, followed by quantification of the replication track length. Data were obtained from three independent experiments. Middle line represents median, and the box extends from the 25th to 75th percentiles. The whiskers mark the 10th and 90th percentiles. P‐values were calculated using the Mann–Whitney test. ‐, DMSO; Di, disulfiram; Ac, acetaldehyde.

  4. H1299 cells expressing a DOX‐inducible BRCA2 shRNA were grown in the presence or absence of DOX, with or without siRNA‐mediated depletion of FANCD2. Processing for DNA fiber analysis was followed by quantification of CldU track length to measure replication fork stability under 50 mM acetaldehyde treatment, which blocks replication. Data were obtained from two independent experiments. Middle line represents median, and the box extends from the 25th to 75th percentiles. The whiskers mark the 10th and 90th percentiles. P‐values were calculated using the Mann–Whitney test. DOX, doxycycline.

  5. H1299 cells treated as in (D) were grown in the presence of 1 mM acetaldehyde for clonogenic survival assays. Error bars represent SD of three independent experiments, each performed in triplicate. P‐values were calculated using an unpaired two‐tailed t‐test. DOX, doxycycline.

Figure 4
Figure 4. Disulfiram and acetaldehyde induce DSBs, chromosome aberrations, and checkpoint activation in BRCA2‐deficient human cells

  1. Quantification of the frequency of cells with 10 or more γH2AX foci in BRCA2‐proficient (+BRCA2) or BRCA2‐deficient (−BRCA2) human DLD1 cells incubated with 10 μM disulfiram or 4 mM acetaldehyde for 4 days. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test.

  2. Cells treated as in (A) were incubated overnight with colcemid and metaphase spreads were stained with Giemsa. To quantify chromosome aberrations, approximately 50 metaphases were analyzed for each sample. Middle line represents median, and the box extends from the 25th to 75th percentiles. The whiskers mark the 10th and 90th percentiles. P‐values were calculated using an unpaired two‐tailed t‐test. ‐, DMSO; Di, disulfiram; Ac, acetaldehyde.

  3. Cells treated as in (A) were processed for FACS analyses of DNA content. Quantification of the percentage of cells in G2/M is shown. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test.

  4. Whole‐cell extracts prepared from BRCA2‐proficient or BRCA2‐deficient human DLD1 cells treated as in (A) were immunoblotted as indicated. SMC1, GAPDH, and H2AX were used as loading controls. ‐, DMSO; Di, disulfiram; Ac, acetaldehyde.

  5. BRCA2‐proficient (+BRCA2) and BRCA2‐deficient (−BRCA2) human DLD1 cells were incubated with 10 μM disulfiram or 4 mM acetaldehyde. Whole‐cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. KAP1, SMC1, and GAPDH were used as loading controls. Di, disulfiram; Ac, acetaldehyde.

Source data are available online for this figure.
Figure EV4
Figure EV4. Acetaldehyde and disulfiram treatments induce high levels of DNA breaks and chromosome rearrangements in BRCA2‐deficient DLD1 cells

  1. Representative images of Giemsa‐stained metaphase spreads prepared from BRCA2‐proficient or BRCA2‐deficient human DLD1 cells treated with 10 μM disulfiram or 4 mM acetaldehyde for 4 days, followed by overnight incubation with colcemid. Red arrowheads point to aberrations.

Figure 5
Figure 5. Aldh2 gene deletion in MEFs is synthetic lethal with HR abrogation

  1. Aldh2 +/+ and Aldh2 −/− MEFs were infected with lentiviruses expressing luciferase control or BRCA1 shRNAs, followed by selection with puromycin for 72 h. mRNA was isolated and the level of Brca1 transcript was determined using quantitative PCR. Graphs are representative of two independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment.

  2. Proliferation rates of cells treated as in (A). Three days post‐selection cells were plated in 96‐well plates, and proliferation was determined using a resazurin‐based assay at 24‐h intervals for 4 days. Graphs are representative of two independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. PDL, population doubling.

  3. Aldh2 +/+ and Aldh2 −/− MEFs were infected with CRISPR lentiviruses expressing Cas9‐P2A‐Puro and gRNAs targeting mouse Brca2 or control vectors, followed by selection with puromycin for 72 h. Cell extracts representative of the entire cell population were prepared and immunoblotted as indicated. SMC1 was used as a loading control.

  4. Proliferation rates of cells treated as in (C). Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment.

  5. Aldh2 +/+ and Aldh2 −/− MEFs were infected with retroviruses expressing GFP control or RAD51 shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared and immunoblotted as indicated. GAPDH was used as a loading control.

  6. Proliferation rates of cells treated as in (E). Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment.

Source data are available online for this figure.
Figure EV5
Figure EV5. Decreased proliferation rates in ALDH2‐deficient human fibroblasts upon HR abrogation
  1. A–C

    Human fibroblasts carrying either WT ALDH2 or the E487K homozygous ALDH2 mutation were transfected with control (SCR) or siRNAs against BRCA1 (A), BRCA2 (B), or RAD51 (C). Cells were processed for proliferation assays 48 h after transfection. siRNAs were retransfected at an interval of 4 days. Graphs are representative of three independent experiments, each performed with three technical replicas. Error bars represent SD of three technical replica values obtained from a single experiment. Cell extracts were prepared 48 h after transfection and immunoblotted as shown. SMC1 or GAPDH were used as loading controls.

Figure 6
Figure 6. Acetaldehyde toxicity to BRCA2‐ and BRCA1‐deficient mouse mammary tumor cell lines, including those that acquired olaparib resistance

  1. Dose‐dependent viability assays of mouse mammary tumor‐derived Brca2 −/− and control cell lines treated with olaparib or acetaldehyde at the indicated concentrations for 6 days. Graphs are representative of three independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. KB2P3.4R3 is a BRCA2‐proficient tumor‐derived cell line. KB2P3.4 is a BRCA2‐deficient tumor‐derived cell line.

  2. Dose‐dependent viability assays of Brca1 +/+ and Brca1 −/− mouse mammary tumor‐derived cell lines treated with olaparib or acetaldehyde at the indicated concentrations for 6 days. Graphs are representative of two independent experiments, each performed in triplicate. Error bars represent SD of triplicate values obtained from a single experiment. Brca1 +/+, BRCA1‐proficient mouse tumor‐derived cell line; Brca1 −/− (olap‐sens.), BRCA1‐deficient, olaparib‐sensitive mouse tumor‐derived cell line; Brca1 −/− 53BP1‐def. (olap‐res.), BRCA1‐ and 53BP1‐deficient, olaparib‐resistant mouse tumor‐derived cell line.

  3. Brca1 +/+ and Brca1 −/− mouse mammary tumor‐derived cell lines as in (B) were treated with 0.5 μM olaparib or 2 mM acetaldehyde for 48 h before processing for immunofluorescence staining with anti‐RAD51 antibody (green). DNA was counter‐stained with DAPI (blue). Scale bar, 5 μm.

  4. Quantification of the frequency of cells with 5 or more RAD51 foci in cells treated as in (C). More than 200 nuclei were analyzed for each sample. Graphs are representative of two independent experiments. Error bars represent SD.

Figure 7
Figure 7. Acetaldehyde treatment inhibits growth of BRCA1/2‐deficient tumors in mouse models
  1. A–C

    Mice were injected with Brca1 +/+ (A), Brca1 −/− (B), and Brca1 −/−, 53BP1‐deficient (C) mouse mammary tumor cells. Treatment of tumor‐bearing mice with 80 mg/kg acetaldehyde administered intravenously (i.v.) and 0.33 mg/kg PARP inhibitor Biomarine administered orally was initiated when a tumor mass of 250 mg was detected for each tumor. Tumor weight was assessed on the indicated days after initiation of the treatment. Each experimental group included five mice. Tumor weight inhibition (TWI) was calculated at the nadir of the effect (9 and 10 days of treatment for BRCA2‐proficient and ‐deficient tumors, respectively). Error bars represent SD (n = 5). P‐values for acetaldehyde versus untreated were calculated using an unpaired two‐tailed t‐test: (A), P = 0.17; (B), P = 0.0000047; (C), P = 0.0000039. PARPi, PARP inhibitor; ACE, acetaldehyde.

  2. D, E

    Mice were injected intramuscularly with 5 × 106 human DLD1 cells, BRCA2‐proficient (D) or BRCA2‐deficient (E). Error bars represent SD (n = 5). P‐values for acetaldehyde versus untreated were calculated using an unpaired two‐tailed t‐test: (D), P = 0.15; (E), P = 0.0000043.

  3. F

    PDTCs derived from breast cancer samples as previously described (Bruna et al, 2016) were treated with disulfiram at the indicated doses. Cell survival is represented relative to DMSO control. AB521, ER‐negative, no known BRCA1 alteration; STG201, BRCA1 promoter methylation and loss of BRCA1 expression; VHIO179, BRCA1 germ line mutation and MAD2L2 inactivating mutation (olaparib‐resistant); http://caldaslab.cruk.cam.ac.uk/bcape/. Error bars represent SEM of triplicate values obtained from a single experiment.

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