A cancer-associated polymorphism in ESCRT-III disrupts the abscission checkpoint and promotes genome instability

Abstract

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the endosomal-sorting complexes required for transport (ESCRT) machinery and is tightly regulated by charged multivesicular body protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4C (rs35094336, CHMP4C^T232) increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: The CHMP4C^T232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4C^T232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome missegregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint and suggest that dysregulation of abscission by CHMP4C^T232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.

Keywords: CHMP4C; ESCRT pathway; abscission checkpoint; cancer; genome instability.

Fig. 1.

The CHMP4C A232T substitution reduces ALIX binding. (A) β-Galactosidase activity assays of yeast cotransformed with the indicated full-length CHMP4C constructs fused to VP16 and full-length ALIX or CHMP4C fused to GAL4 (mean ± SD, n = 3). (B) Competitive fluorescence polarization binding assay with an ALIX construct spanning the Bro1 and V domains (residues 1–698) binding to fluorescently labeled CHMP4C^A232 peptide (residues 216–233) competing with the indicated unlabeled CHMP4C peptides. Curves are from a representative experiment. K_i values are expressed as mean ± SD. n ≥ 7. (C, Left) Superposition of ALIX Bro1 domain (gray) complexes with CHMP4C^A232 (green) and CHMP4C^T232 (orange) peptides. (Right) Enlarged view of the CHMP4C peptide superposition highlights the reduction in CHMP4C^T232 helicity. (D) Binding interfaces between ALIX (gray) and CHMP4C^A232 (green) (Left) and CHMP4C^T232 (orange) (Right). Figures show equivalent intermolecular distances (in angstroms) for different atoms of CHMP4C residue W231. See also SI Appendix, Fig. S1 and Table S1.

Fig. 2.

CHMP4C T232 does not support the abscission checkpoint. (A) HeLa cells stably expressing siRNA-resistant HA-CHMP4C constructs were treated with the indicated siRNA and stained for α-tubulin. (Upper) Percentage of midbody-arrested cells. Data are mean ± SD from more than three separate experiments with n > 900 cells. (Lower) Representative immunoblots from A. (B) Asynchronous HeLa mCherry-tubulin cells stably expressing siRNA-resistant HA-CHMP4C constructs were transfected with the indicated siRNA, and midbody abscission times were scored from three or more separate experiments. Here and throughout, box edges mark the 25th and 75th quartiles, and whiskers mark fifth and 95th percentiles. Horizontal bars denote the median. Mean times ± SD were nontargeting (NT): 110 ± 47 min; siCHMP4C: 75 ± 26 min; HA-CHMP4C^A232 + siCHMP4C: 108 ± 47 min; HA-CHMP4C^L228A,W231A + siCHMP4C: 86 ± 33 min; HA-CHMP4C^T232 + siCHMP4C: 81 ± 27 min; and HA-CHMP4C^−INS + siCHMP4C: 87 ± 44 min. See also Movies S1–S6. (C) HeLa cells stably expressing siRNA-resistant HA-CHMP4C constructs and YFP-Lap2β were transfected with the indicated siRNA. Resolution time of Lap2β bridges were quantified from three or more separate experiments. Mean times ± SD were nontargeting (NT): 721 ± 333 min; siCHMP4C: 296 ± 187 min; HA-CHMP4C^A232 + siCHMP4C: 853 ± 474 min; HA-CHMP4C^L228A,W231A + siCHMP4C: 415 ± 317 min; HA-CHMP4C^T232 + siCHMP4C: 396 ± 394 min; and HA-CHMP4C^−INS + siCHMP4C: 421 ± 341 min. See also Movies S7–S12. (D) HA-CHMP4C recruitment to midbodies was determined by staining for DNA (DAPI, blue), α-tubulin (red), and HA (green) from three or more separate experiments. (Scale bars, 5 µm.) (E) Data represent the staining intensity of HA normalized to background measurements. The mean value is marked. P values were calculated using two-way ANOVA and Sidak’s multiple comparisons test (A and E) or one-way ANOVA vs. control (HeLa siNT) (B and C); ***P < 0.001; ns, not significant. Immunoblots for B and C are shown in SI Appendix, Fig. S2.

Fig. 3.

Cells lacking the abscission checkpoint exhibit elevated genome damage and are sensitized to replication stress. (A) HCT116 cells with (WT) or without (δCHMP4C) endogenous CHMP4C expressing the indicated HA-CHMP4C construct were stained for 53BP1 (green), α-tubulin (red), and DNA (DAPI, blue). (Scale bars, 20 μm.) Insets show gray-scale images of boxed cells. (B, Upper) Numbers of 53BP1 foci per cell were determined from three independent experiments and binned into the designated categories. Shown are the mean ± SD from >900 cells. P values were calculated using two-way ANOVA and Sidak’s multiple comparisons test comparing each category to control (WT); *: 0–2 foci; #: more than six foci; *^/#P < 0.05; **^/## = P < 0.005; ***^/### = P < 0.001. (Lower) Representative immunoblots from B, Upper. (C) Cells stably expressing the indicated HA-CHMP4C construct were treated with the indicated siRNA for 48 h and then with DMSO or 30 nM aphidicolin for 24 h. The number of cells connected by midbodies was scored. (D) HCT116^WT or HCT116^δCHMP4C cells expressing the indicated HA-CHMP4C constructs were cultured in the continuous presence of DMSO (blue trace) or 30 nM aphidicolin (red trace), and cell numbers were determined at the indicated time points. Plotted are the mean ± SD from three independent experiments. See also SI Appendix, Fig. S7A.

Fig. 4.

Defects in chromosome segregation and the abscission checkpoint synergize to impair cell growth. (A) HCT116 cells expressing histone H2B-mCherry were grown in the continuous presence of DMSO (blue bar) or 0.1 μM reversine (red bar), and anaphase segregation defects were scored. Data shown are the mean ± SD from n > 130 cells from three separate experiments. See also SI Appendix, Fig. S7 A and B and Movies S16–S19. (B and C) HCT116^WT or HCT116^δCHMP4C cells were cultured for 48 h in the continuous presence of DMSO (blue) or 0.1 μM reversine (red). Metaphases were enriched by overnight treatment with nocodazole, and chromosome number was determined. Plots show all data with medians marked. Extreme aneuploidy is defined as chromosome numbers above 48 or below 37 (solid lines). Representative metaphase spreads can be seen in C. (Scale bars, 10 μm.) Data were collected from more than four independent experiments. (D, Upper) HCT116 cells expressing histone H2B-mCherry were depleted of p53 by shRNA treatment (shp53), and anaphase segregation defects were scored. Data are expressed as the mean ± SD, n > 200, from four separate experiments across two shRNA transductions. See also SI Appendix, Fig. S7C and Movies S20–S23. (Lower) Representative immunoblots from D, Upper. (E) HCT116^WT or HCT116^δCHMP4C cells expressing the indicated CHMP4C construct were grown in the continuous presence of either DMSO (blue traces) or 0.1 μM reversine (red traces) (Upper Row) or were depleted of p53 by shRNA treatment (shCtrl, blue; shp53, red) (Lower Row), and cell numbers were determined at the indicated time points. Data are the mean ± SD from more than three independent experiments. P values were calculated using two-tailed unpaired Student’s t test; **P < 0.005, ***P < 0.001. See also SI Appendix, Fig. S9A.