Fanconi anemia signaling network regulates the spindle assembly checkpoint

Abstract

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.

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.

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.

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.

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.

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.