Fanconi anemia signaling network regulates the spindle assembly checkpoint

Fanconi anemia (FA) is a heterogenous genetic disease with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair; however, it is incompletely understood why FA-deficient cells also develop gross aneuploidy, leading to cancer. Here, we systematically evaluated the role of the FA proteins in chromosome segregation through functional RNAi screens and analysis of primary cells from patients with FA. We found that FA signaling is essential for the spindle assembly checkpoint and is therefore required for high-fidelity chromosome segregation and prevention of aneuploidy. Furthermore, we discovered that FA proteins differentially localize to key structures of the mitotic apparatus in a cell cycle–dependent manner. The essential role of the FA pathway in mitosis offers a mechanistic explanation for the aneuploidy and malignant transformation known to occur after disruption of FA signaling. Collectively, our findings provide insight into the genetically unstable cancers resulting from inactivation of the FA/BRCA pathway.

Fanconi anemia (FA) is a genetic disorder resulting from germline mutations in 1 out of 15 known FA or FA-like genes (FANCA–FANCP) (1–3). In addition to bone marrow failure and congenital malformations, patients with FA develop various malignancies, including acute leukemias and squamous cell carcinomas (4, 5). Mutations in the FA network are etiologically implicated in a significant proportion of inherited breast, pancreatic, and ovarian cancers (6–12). The high risk of malignant transformation in FA-deficient cells is due to genomic instability characterized by impaired DNA repair, chromosome breakage, and gross aneuploidy (13–15). Although the role of FA signaling in interphase DNA cross-link repair is well established (5, 16), the origins of gross aneuploidy in FA-deficient cells are incompletely understood.

The spindle assembly checkpoint (SAC) is the tumor suppressor signaling network that ensures high-fidelity chromosome segregation during mitosis to prevent aneuploidy (17, 18). It is established that FA-deficient cells have a high frequency of aneuploidy and micronucleation, which are symptoms of chromosome missegregation (13, 14, 19–23). However, the potential role of the FA pathway in the SAC has not been systematically analyzed. Our recent work in a genetically engineered murine model of FA that recapitulates the malignant hematopoietic manifestations of human FA (24) highlighted the correlation among gross aneuploidy, myelodysplasia, and leukemia in FA (14). Interestingly, we observed dysregulation of SAC proteins MAD2 and BUBR1 in this mouse model (D.W. Clapp, unpublished observations). Therefore, we hypothesized that the FA pathway may be essential for the SAC. Here we describe the results of comprehensive studies that address this hypothesis.

Functional RNAi screen reveals role for FA signaling in the SAC. We generated an FA siRNA library against the 15 known human FA gene products and successfully validated these siRNAs via immunoblotting (Supplemental Figure 1, A–N; supplemental material available online with this article; doi: 10.1172/JCI67364DS1). To examine the status of the SAC upon FA knockdown, we performed a functional RNAi screen as described previously (25, 26). HeLa cells were transfected with arrayed library siRNAs, challenged with taxol to activate the SAC through disruption of spindle microtubules, fixed, and imaged (Figure 1A). Negative control cells arrested in prometaphase due to SAC activation by taxol (Figure 1B). RNAi knockdown of the SAC regulator MAD2 resulted in multinucleation of taxol-treated cells due to SAC inactivation (Figure 1B and refs. 25, 26). Remarkably, we found that RNAi silencing of each FA gene except FANCM inactivated the SAC, as evidenced by multinucleation in response to taxol, similar to MAD2 (Figure 1, C and D, and Supplemental Figure 2, A and B). Off-target siRNA effects are a risk in mitotic RNAi screens, and MAD2 has been shown to be particularly susceptible (27). Thus, for each of the FA proteins identified as SAC regulators in the screen, we performed immunoblotting to show that MAD2 is not nonspecifically knocked down by FA siRNAs (Supplemental Figure 3, A–J) or tested multiple unique siRNA sequences targeting each FA gene product (Supplemental Figure 1, A–D). When multiple siRNAs were tested, the severity of SAC failure correlated with the degree of target protein knockdown (Supplemental Figure 1, A–D). Since patients with FA frequently develop aneuploidy-associated hematopoietic malignancies (4, 28), we tested whether the FA pathway controls the S AC in primary hematopoietic cells. Indeed, shRNA knockdown of FANCA disrupted the SAC in primary human CD34⁺ cells, as shown by microscopy-coupled flow cytometry analysis (Figure 2). When treated with taxol, cycling FANCA-knockdown CD34⁺ cells became multinucleated (Figure 2 and Supplemental Figure 4). These data support the hypothesis that FA signaling is essential for the human SAC.

Figure 1

FA signaling is essential for the SAC. (A) RNAi screen schematic. (B) Microscopy-based screen readout. HeLa^{GFP-H2B/mCherry-αtubulin} cells arrest in prometaphase upon taxol exposure. CDC25A knockdown results in premitotic arrest (25). MAD2 siRNA induces multinucleation due to SAC failure. Original magnification, ×200 (BD Pathway 855). (C) Representative images of cells transfected with indicated siRNAs and treated with taxol. Note multinucleation induced by FANCA and FANCC siRNAs and MAD2 siRNA (positive control). Original magnification, ×200 (BD Pathway 855). (D) Quantification of screen results. *P < 0.0001 (1-way ANOVA); n = 9 counts per siRNA. All bars represent mean values ± SEM.

Figure 2

FANCA is essential for the SAC in primary human CD34⁺ cells. (A) CD34⁺ cell experiment schematic. (B) Microscopy-coupled flow cytometry allows quantification of SAC failure in cycling CD34⁺ cells. (C) Quantification of SAC failure in cycling FANCA shRNA–transduced CD34⁺ cells. *P = 0.029 (2-tailed t test); n = 6. All bars represent mean values ± SEM.

Spindle checkpoint failure in cells from patients with FA. To validate the RNAi screen, we tested SAC activity in primary fibroblasts isolated from patients with FA of all available (12 out of 15) FA complementation groups (Figure 3A and see Supplemental Table 1 for list of FA mutations). Consistent with the RNAi screen results, primary fibroblasts of all tested FA complementation groups became multinucleated in response to taxol, while fibroblasts obtained from healthy individuals arrested in prometaphase (Figure 3, B and C; Supplemental Figure 5; and Supplemental Figure 6A). We confirmed SAC failure in primary FA cells using quantitative flow cytometry (Supplemental Figure 6D). Importantly, stable expression of the missing FANCA protein in primary FA-A fibroblasts not only rescued hypersensitivity to mitomycin known to result from loss of FA signaling (Supplemental Figure 7), but also the SAC failure following exposure to taxol (Figure 3D and Supplemental Figure 6B). We also found that FANCA is required for mitotic arrest in response to nocodazole, another spindle poison that is mechanistically different from taxol (Figure 3E and Supplemental Figure 6C). Collectively, these findings confirm an essential role for FA signaling in the human SAC using primary cells from patients with FA.

Figure 3

Spindle checkpoint failure in primary human FA cells. (A) Experiment schematic. (B) FA fibroblasts fail to arrest in mitosis upon taxol exposure and generate multinucleated cells (arrows). Original magnification, ×200 (Applied Precision personalDX). (C) Quantification of SAC defects in cells from patients with FA. *P < 0.01 (1-way ANOVA with post-hoc Bonferroni’s correction); n = 10–15 counts per genotype. (D) Ectopic expression of FANCA rescues the SAC defect in FA-A fibroblasts exposed to taxol. *P = 0.0038 (2-tailed t test); n = 15 counts. (E) FANCA-deficient patient fibroblasts fail to arrest at the SAC in response to nocodazole. *P = 0.021 (2-tailed t test); n = 15 counts. All bars represent mean values ± SEM.

FA proteins localize to mitotic spindle and centrosomes during cell division. We then asked whether FA proteins localize to the mitotic apparatus in a cell cycle–dependent manner, which would be predicted based on their newly found role in the SAC (Figures 1–3). We found that 8 FA proteins (FANCA, FANCB, FANCE, FANCG, FANCL, FANCD1, FANCD2, and FANCN, also known as PALB2) localize to centrosomes during mitosis. We validated this finding by high-resolution immunofluorescence of 7 endogenous FA proteins (FANCA, FANCB, FANCE, FANCG, FANCN, FANCD2, and FANCD1) and 2 GFP-fused FANCG and FANCL proteins (Figure 4, A, C, and D; Supplemental Figures 8 and 9; and G. Nalepa, R. Enzor, and D.W. Clapp, unpublished observations). Additionally, FANCC and, to a lesser degree, FANCA localized to the mitotic spindle in a cell cycle–dependent manner (Figure 4, A and B, and Supplemental Figure 10). This experiment revealed that FA proteins are found on the mitotic apparatus during cell division (Figure 4D), consistent with a role for the FA pathway in the SAC.

Figure 4

FA pathway proteins associate with centrosomes and mitotic spindle during cell division. (A) Endogenous FANCA and (C) GFP-FANCG localize to centrosomes during mitosis. Original magnification, ×1,000 (Applied Precision personalDX). (A) A fraction of FANCA also localizes to the spindle, which emanates from the centrosomes during prophase and metaphase. (B) Endogenous FANCC colocalizes with α-tubulin at the spindle in early mitosis; midzone spindle during late anaphase; and midbody during telophase. Original magnification, ×1,000 (Applied Precision personalDX). (D) Subcellular localization of FA proteins during mitosis.

Failure of centrosome maintenance in FA cells. Given the newly identified role of the FA pathway in mitosis (Figures 1–3) and centrosomal localization of multiple FA proteins (Figure 4 and Supplemental Figures 8 and 9), we questioned whether the FA network is functionally important for maintenance of normal centrosome number (1 or 2 centrosomes per cell, depending on cell cycle stage). This question is clinically significant because extra centrosomes cause aneuploidy and cancer through promotion of merotelic kinetochore-spindle association (29). When we immunostained with endogenous pericentrin (a known centrosome marker) in FA cells, we found that primary fibroblasts from patients with FA contained abnormally high numbers of centrosomes compared with control fibroblasts (Figure 5, A, B, and D). We also noted structural nuclear abnormalities (multinucleated cells and micronuclei) in patient fibroblasts from all tested FA complementation groups cultured ex vivo without spindle poisons (Figure 5, C and E), consistent with previous reports (20–22, 30). We found that HeLa cells transfected with siRNAs against FA genes acquire extra centrosomes as well (Supplemental Figure 11, A and B). We concluded that FA-deficient cells accumulate supernumerary centrosomes. While a previous study suggested that one FA protein (FANCD1, also known as BRCA2) is critical for centrosome maintenance and localizes to centrosomes (15), our findings suggest that other FA proteins are also essential for maintaining normal centrosome numbers. Our preliminary experiments suggest that passage through mitosis is essential for centrosome accumulation in FA-deficient cells (G. Nalepa, R. Enzor, and D.W. Clapp, unpublished observations). Further work will be needed to validate this observation.

Figure 5

Abnormal centrosome counts and spontaneous micronucleation in cells of patients with FA. (A) Fibroblasts obtained from healthy controls contain 1–2 centrosomes per cell, as shown by endogenous pericentrin immunofluorescence (red). (B) Fibroblasts isolated from patients with FA contain supernumerary centrosomes (white arrows). (C) Fibroblasts from patients with FA have abnormal nuclear structures and undergo spontaneous micronucleation (yellow arrows). Original magnification, ×200 (Applied Precision personalDX). (D and E) Increased fraction of cells with abnormal centrosome counts and abnormal nuclei in primary fibroblasts from patients with FA. *P < 0.05 compared with WT (1-way ANOVA with post-hoc Bonferroni’s correction). All bars represent mean values ± SEM.

The FA pathway, genomic instability, and cancer. The aneuploidy and multinucleation consistently observed in FA-deficient cells (20–23, 30) are suggestive of abnormal cell division (31), but the role of the FA pathway in mitosis has previously not been well defined. Several publications revealed a biochemical interaction between FA proteins and the key mitotic cyclin-dependent kinase CDK1 (32, 33), while other work established an essential role for CDK1 in the SAC (34). A previous study in a murine model highlighted the functional cross-talk between the SAC pathway and FANCD1, a breast cancer tumor suppressor subsequently discovered to be an FA protein (35). Recently, it was proposed that some FA proteins resolve replication stress-induced chromatin bridges during anaphase (19, 20) and regulate cytokinesis (30). The data presented here, combined with these previous investigations, clearly demonstrate that abnormal cell division occurs in the absence of FA signaling. The SAC is a key tumor suppressor signaling network that protects cells from aneuploidy by ensuring accurate chromosome segregation, and SAC regulators have been implicated in a number of cancers (17, 18). Thus, we suggest that the newly discovered role for the FA signaling network in regulating the SAC (Figures 1–3) may contribute to the increased risk of aneuploidy and malignant transformation in FA-deficient cells.

Additionally, we hypothesize that error-prone mitosis may exacerbate mutagenesis resulting from disrupted DNA damage repair in FA-deficient cells. Recent elegant work showed that micronuclei generated through abnormal mitoses are sites of intense DNA breakage and mutagenesis, culminating in chromosome pulverization (36) and cancer (37). FA-deficient cells are prone to spontaneous multinucleation (Figure 5A; Supplemental Figure 11, C and E; and ref. 22), presumably due to a combination of weakened SAC (Figures 1–3), acquisition of supernumerary centrosomes (Figure 5 and Supplemental Figure 11), and failed cytokinesis (30). We propose that DNA damage in the micronuclei of FA-deficient cells cannot be effectively resolved due to the crucial role of the FA pathway in interphase DNA damage repair (reviewed in ref. 2). Ultimately, this vicious cycle of abnormal cell division followed by multinucleation and mutagenesis may lead to cell death or genomic instability and transformation in FA-deficient cells (ref. 31 and Figure 6).

Figure 6

Multiple cell cycle defects may lead to aneuploidy in FA-deficient cells. Loss of the FA signaling pathway results in abnormal mitosis due to (A) SAC failure (Figures 1–2); (B) presence of supernumerary centrosomes (Figure 5 and Supplemental Figure 11), leading to merotelic kinetochore-spindle attachment, as proposed by Ganem et al. (29); and (C) premature cytokinesis, as reported by Vinciguerra et al. (30). The failed mitosis produces multinucleated cells with ongoing mutagenesis in the micronuclei (36). Since FA-deficient cells cannot efficiently recognize and repair DNA damage (reviewed in refs. 1, 2, 43), this process leads to accumulation of mutations and either cell death or malignant transformation. Centrosomes are shown in yellow, kinetochores are shown in red, DNA is shown in gray, and micronuclei undergoing mutagenesis are shown in purple.

In summary, our study identifies a novel role for the FA signaling network in mitosis. The discovery of SAC failure in FA-deficient cells advances our understanding of chromosomal instability in patients with FA. As somatic inactivation of the FA pathway occurs in a spectrum of spontaneous malignancies, our studies are relevant to the pathogenesis and treatment of cancers in the general population.

View Supplemental data

This work was supported by the NIH/NCATS KL2 TR000163 Clinical and Translational Sciences Award, Indiana Pediatric Scientist Award (IPSA) K12, and Morris Green Research Fellowship to G. Nalepa; by the NIH/NCATS TL1 TR000162 Clinical and Translational Sciences Award and NIH/NIGMS GM077229-02 MSTP Award to R. Enzor; by the NIH R01s CA138237-01 and CA155294-01 to D.W. Clapp and H. Hanenberg; and by the BMBF grant “Inherited bone marrow failure syndromes” and DFG SPP1230 to H. Hanenberg. We are grateful to Shaochun Bai (Indiana University, Indianapolis, Indiana, USA) for sequencing the FANCA and FANCC genes in the patient-derived cell lines used in this study, confirming our retroviral complementation studies. We thank Claire Walczak (Indiana University, Bloomington, Indiana, USA) for HeLa cell lines stably expressing GFP-γtubulin/GFP-CENPA and cells stably expressing GFP-H2B/mCherry-αtubulin. We are grateful to H.E. Broxmeyer, E.F. Srour, L.S. Haneline, and D.A. Ingram (all at Indiana University School of Medicine, Indianapolis, Indiana, USA) for critical reading of the manuscript. We thank D. Schindler (University of Wuerzburg, Wuerzburg, Germany) for sharing unpublished mutations that confirmed our retroviral complementation results. We thank the patients with FA and families, Fanconi Anemia Research Fund Inc., and German family organizations “Aktionskreis FA e.V.” and “Deutsche FA Hilfe e.V.” for their support of our research. We acknowledge the In Vivo Therapeutics Core of Indiana University Simon Cancer Center as well as Arthur Baluyut (St. Vincent Hospital, Indianapolis, Indiana, USA) for umbilical cord blood samples. Indiana University is a Center for Excellence in Molecular Hematology (P30).

Address correspondence to: D. Wade Clapp, Indiana University School of Medicine, Herman B Wells Center for Pediatric Research, 1044 W. Walnut Street, R4-402, Indianapolis, Indiana 46202, USA. Phone: 317.278.9290; Fax: 317.274.0138; E-mail: dclapp@iu.edu. Or to: Grzegorz Nalepa, Indiana University School of Medicine, Department of Pediatrics, Division of Pediatric Hematology-Oncology, Herman B Wells Center for Pediatric Research, 1044 W. Walnut Street, R4-421, Indianapolis, Indiana 46202, USA. Phone: 317.278.9846; Fax: 317.274.0138; E-mail: gnalepa@iu.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2013;123(9):3839–3847. doi:10.1172/JCI67364.

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