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. 2018 Dec 1;143(11):2932-2942.
doi: 10.1002/ijc.31659. Epub 2018 Oct 3.

Mechanism of cytokinesis failure in ovarian cystadenomas with defective BRCA1 and P53 pathways

Theresa Austria  1 , Christine Marion  1 , Vanessa Yu  1 , Martin Widschwendter  2 , David R Hinton  3 , Louis Dubeau  1
Affiliations

Mechanism of cytokinesis failure in ovarian cystadenomas with defective BRCA1 and P53 pathways

Theresa Austria et al. Int J Cancer. .

Abstract

We previously described an in vitro model in which serous ovarian cystadenomas were transfected with SV40 large T antigen, resulting in loss of RB and P53 functions and thus mimicking genetic defects present in early high-grade serous extra-uterine Müllerian (traditionally called high-grade serous ovarian) carcinomas including those associated with the BRCA1 mutation carrier state. We showed that replicative aging in this cell culture model leads to a mitotic arrest at the spindle assembly checkpoint. Here we show that this arrest is due to a reduction in microtubule anchoring that coincides with decreased expression of the BUB1 kinase and of the phosphorylated form of its substrate, BUB3. The ensuing prolonged mitotic arrest leads to cohesion fatigue resulting in cell death or, in cells that recover from this arrest, in cytokinesis failure and polyploidy. Down-regulation of BRCA1 to levels similar to those present in BRCA1 mutation carriers leads to increased and uncontrolled microtubule anchoring to the kinetochore resulting in overcoming the spindle assembly checkpoint. Progression to anaphase under those conditions is associated with formation of chromatin bridges between chromosomal plates due to abnormal attachments to the kinetochore, significantly increasing the risk of cytokinesis failure. The dependence of this scenario on accelerated replicative aging can, at least in part, account for the site specificity of the cancers associated with the BRCA1 mutation carrier state, as epithelia of the mammary gland and of the reproductive tract are targets of cell-nonautonomous consequences of this carrier state on cellular proliferation associated with menstrual cycle progressions.

Keywords: BRCA1; P53; aneuploidy; cytokinesis failure; ovarian carcinoma.

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Figures

Figure 1:
Figure 1:. Reduced microtubule anchoring in aging ovarian cystadenomas.
A: Confocal images of ovarian cystadenomas in prometaphase/metaphase stained with a fluorescent antibody against BUB3 (red) and counterstained with DAPI (blue) showing representative examples of predominantly nuclear (left) and cytoplasmic (right) distribution of BUB3. B: Cystadenomas of low (<35 population doublings) versus high (>50 population doublings) replicative ages were examined by confocal microscopy as in (A); mitotic cells were scored based on presence of predominantly nuclear versus cytoplasmic distribution of BUB3; the numbers on the stacked bars represent the total number of cells for each parameter. C: Western blots of either whole cellular protein extracts or of proteins extracted from the cytoplasmic fraction only of cultured cystadenomas with less than 35 versus more than 50 population doublings were probed with antibodies against the indicated proteins; GAPDH was used as loading control. D: Western blots of nuclear and whole cell protein extracts of cystadenomas of low and high replicative ages probed with an antibody against EB1; Ku70 and GAPDH are used as loading controls for nuclear and cytoplasmic proteins, respectively. E: Confocal microscopy images showing examples of ovarian cystadenomas undergoing mitosis with and without EB1 localization to the kinetochore; immunopositivity for BUB3 is used as a surrogate marker for the kinetochore. F: Cystadenomas of low versus high replicative ages were examined by confocal microscopy as in (E) and the mitotic cells were scored based on presence versus absence of colocalization of EB1 and BUB3 to the kinetochore; the numbers on the stacked bars represent the total number of cells for each parameter. Magnification bars: 6 microns.
Figure 2:
Figure 2:. Mechanism of escape from the spindle assembly checkpoint in cells with reduced BRCA1 expression.
A: Confocal images of cells stained with DAPI showing a normal metaphase (left) and a metaphase undergoing cohesion fatigue (right). B: Cystadenomas of low (<35 population doublings) versus high (>50 population doublings) replicative ages were examined by confocal microscopy and the mitotic cells were scored based on presence of either normal metaphases versus metaphases showing evidence of cohesion fatigue; the numbers on the stacked bars represent the total number of cells for each parameter. C: Western blot of nuclear or total protein extracts obtained from cystadenomas treated with siRNA directed against either BRCA1 or GFP (control) and probed with an antibody against EB1; a probe for Ku70 was used to evaluate loading of nuclear proteins while a probe for GAPDH was used to evaluate loading of cytoplasmic protein extracts. D: Cells stained with an antibody against EB1 (green) and counterstained with DAPI (blue) were examined by confocal microscopy and photographed under constant exposure; these representative images illustrate the increase in the size of EB1 signals in cells treated with siRNA directed against BRCA1 compared to cells treated with siRNA directed against GFP. E: Cystadenomas of low versus high replicative ages were treated with siRNA directed against either BRCA1 or GFP and examined by confocal microscopy; the mitotic cells were scored based on presence of either normal metaphases versus metaphases showing evidence of cohesion fatigue; the numbers on the stacked bars represent the total number of cells for each parameter. Magnification bars: 6 microns in (A) and 8 microns in (D).
Figure 3:
Figure 3:. Consequences of BRCA1 down-regulation on formation of bridges between chromosome plates.
A: Examples of mitotic cells in anaphase showing varying degrees of chromosome bridging seen by confocal microscopy after staining with DAPI; magnification bars: 8 microns. B: Cystadenomas of low (<35 population doublings) versus high (>50 population doublings) replicative ages were examined by confocal microscopy as in (A); cells undergoing anaphase were scored based on presence of either absent/minor or severe chromosome bridging; the numbers on the stacked bars represent the total number of cells for each parameter.
Figure 4:
Figure 4:. Consequences of BRCA1 down-regulation on cytokinesis failure.
A: Cystadenomas of high replicative ages were treated with siRNA directed against either BRCA1 or GFP and examined by time-lapse photography over a 20-hour period; all cells undergoing mitosis were followed and scored as showing either completion of cytokinesis or cytokinesis failure; the numbers on the stacked bars represent averages of the total number of cells for each parameter; the error bars represent variation in independent observations of the entire 20-hour video by 3 observers. Selected frames from the time-lapse microscopy video used are shown in panel (B), illustrating a mononuclear cell subsequently undergoing a mitotic arrest followed by cytokinesis failure resulting in a multi-nucleated interphase cell. The entire video is submitted as supplemental data.
Figure 5:
Figure 5:. Working model for interplay between cell-nonautonomous and cell-autonomous mechanisms of cancer predisposition in BRCA1 mutation carriers.
The menstrual cycle, under the influence of ovarian granulosa cells and cells from the anterior pituitary, control the proliferation of tissues that have an elevated cancer risk in BRCA1 mutation carriers . These cell-nonautonomous signals are amplified in such carriers resulting in accelerated replicative aging. P53 alterations, which are frequent in these tissues in BRCA1 mutation carriers, eventually lead to mitotic arrest in aging cells due to defective microtubule anchoring. Cohesion fatigue is the most likely outcome in tissues with premature replicative aging due to increased menstrual cycle activity in the absence of a germline BRCA1 mutation, but some cells abruptly exit the arrest due to mitotic slippage resulting in cytokinesis failure and binucleation due to interference of lagging chromosomes or microtubules at the site of cell cleavage. The presence of a germline BRCA1 mutation not only intensifies accelerated replicative aging, but also leads to recovery from the mitotic arrest due to uncontrolled kinetochore attachments resulting in the formation of chromosome bridges; this significantly increases the rate of cytokinesis failure, leading to polyploidy and, subsequently, aneuploidy and malignant transformation.
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