Cancers that develop in BRCA1 mutation carriers are usually near tetraploid/polyploid. This led us to hypothesize that BRCA1 controls the mitotic checkpoint complex, as loss of such control could lead to mitotic errors resulting in tetraploidy/polyploidy with subsequent aneuploidy. We used an in vitro system mimicking pre-malignant conditions, consisting of cell strains derived from the benign counterparts of serous ovarian carcinomas (cystadenomas) and expressing SV40 large T antigen, conferring the equivalent of a p53 mutation. We previously showed that such cells undergo one or several doublings of their DNA content as they age in culture and approach the phenomenon of in vitro crisis. Here we show that such increase in DNA content reflects a cell cycle arrest possibly at the anaphase promoting complex, as evidenced by decreased BrdU incorporation and increased expression of the mitotic checkpoint complex. Down-regulation of BRCA1 in cells undergoing crisis leads to activation of the anaphase promoting complex and resumption of growth kinetics similar to those seen in cells before they reach crisis. Cells recovering from crisis after BRCA1 down-regulation become multinucleated, suggesting that reduced BRCA1 expression may lead to initiation of a new cell cycle without completion of cytokinesis. This is the first demonstration that BRCA1 controls a physiological arrest at the M phase apart from its established role in DNA damage response, a role that could represent an important mechanism for acquisition of aneuploidy during tumor development. This may be particularly relevant to cancers that have a near tetraploid/polyploid number of chromosomes.

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.

Ovarian cancer accounts for 5% of all female cancer-related deaths, more than any other reproductive cancer. Of the various histological subtypes, high-grade serous carcinoma is the most common. Mutations in BRCA1/BRCA2, which are associated with familial predisposition, are present in 10% of cases, while TCGA data demonstrates that P53 is mutated in 96% of high-grade serous ovarian carcinomas. In addition to aberrant P53 expression, severe aneuploidy with a near polyploid number of chromosomes is the only other genetic abnormality associated with these tumors. Ploidy changes occur early in tumor development, suggesting that they play a crucial role in oncogenic transformation. Furthermore, severe aneuploidy is associated with poor clinical outcome in patients, presumably due to its role in treatment resistance. We used an in vitro cell culture model derived from ovarian cystadenomas, the benign counterparts of ovarian carcinomas, to investigate the molecular events leading to such ploidy changes and to develop strategies for their prevention. These cells have a non-functional P53 due to SV40 large T-antigen transfection, resulting in a genetic background similar to high-grade serous ovarian carcinomas and their precursors. We showed that these cells spontaneously undergo a mitotic arrest as they age in culture and approach in vitro crisis and that recovery from this arrest can be induced by treatment with siRNA against BRCA1. Cells become multi-nucleated suggesting that reduced BRCA1 leads to initiation of a new cell cycle without cytokinesis, a mechanism leading to polyploidy. Aurora A kinase inhibition stabilizes the metaphase checkpoint and reduces the population of proliferating polyploid/aneuploidy cells. We propose that a similar mechanism occurs in ovarian carcinoma cells, although not readily appreciated because cells undergoing a mitotic arrest within an immortal cell culture, unlike in our mortal cystadenoma cells, are outgrown by a dividing cell population and therefore fail to accumulate. We tested this hypothesis in SKOV3 cells, a high-grade serous ovarian carcinoma line known to be P53 deficient. BRCA1 down-regulation in SKOV3 cells, similarly to ovarian cystadenoma cells, leads to polyploidy. Evidence that this is due to cytokinesis failure comes from the observation that cells with increased DNA ploidy also show multi-nucleation. BRCA1 expression increases upon Aurora A knockdown in our cell culture model. We therefore hypothesize that forced alterations in Aurora A expression may influence this process by acting, directly or indirectly, as an upstream regulator of BRCA1 expression, thereby preventing polyploidy and stabilizing the genome. Our findings raise the possibility of using Aurora A inhibition to reduce the incidence of aneuploidy in ovarian carcinomas with reduced BRCA1 expression. Minimizing the extent of aneuploidy in these tumors may delay development of treatment resistance, ultimately improving survival. Citation Format: Christine M. Marion, Vanessa Yu, Louis Dubeau. Interplay between Aurora A kinase and BRCA1 promotes genetic stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2387. doi:10.1158/1538-7445.AM2014-2387

Aneuploidy, defined by a chromosome number that is not a multiple of the haploid number, is a hallmark of cancer. In order to study the mechanism of aneuploidy development in ovarian cancer, we used cell strains derived from benign ovarian cystadenomas, the benign counterparts of the type of ovarian cancer that develops in BRCA1 mutation carriers. The cells were transfected with SV40 Large T Antigen, conferring the equivalent of a p53 mutation, which is present in nearly all cancers that develop in BRCA1 mutation carriers. These cells are not immortal and invariably reach crisis after up to 50 population doublings in vitro. We previously showed that this crisis event, which is not triggered by telomere attrition, is associated with a cell cycle arrest at the M phase, and that decreased BRCA1 expression allows cells to overcome this crisis event, resulting in tetraploidy and subsequent aneuploidy. The finding that cells recovering from crisis undergo sudden changes in their ploidy status strongly suggests that such recovery is characterized by initiation of a new cell cycle without first completing cytokinesis. We used time-lapse photography to test this hypothesis. Such studies revealed that a significant proportion of cells treated with siRNA against BRCA1 recovered from a cell cycle arrest during the mitotic phase without undergoing cytokinesis, resulting in bi-nucleation. We further hypothesized that Aurora A plays a role in regulating this process, in part, by acting as an upstream regulator of BRCA1 expression. Here we show that Aurora A protein and kinase activity are both up-regulated while levels of BRCA1 expression are down-regulated in cells as they age in culture. Levels of BRCA1 expression increased upon knockdown of Aurora A kinase, further suggesting an inverse relationship between expression levels of these proteins. Inhibition of Aurora A prevented recovery from mitotic arrest as evidenced by an increase in BubR1 and Mad2, two components of the mitotic checkpoint complex and a decrease in Cdc27, a component of the anaphase promoting complex. Genomic DNA profiles of cells treated with siRNA against Aurora A showed decreased tetraploidy and microscopic examination showed decreased multinucleation. Time-lapse photography of cells approaching crisis, treated with siRNA against Aurora A showed that the M phase arrest was prolonged compared to controls and invariably led to apoptosis in contrast to cells treated with siRNA against BRCA1. Down regulation of Aurora A in a younger cells, not approaching crisis, did not induce a mitotic arrest. We conclude that decreased BRCA1 expression in cells approaching crisis leads to recovery from a mitotic arrest without completion of cytokinesis, leading to tetraploidy and subsequent aneuploidy. Although decreased Aurora A expression does not induce a mitotic arrest, it prevents recovery from such arrest, resulting in cell death from apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2038. doi:1538-7445.AM2012-2038

Aneuploidy, as defined by a chromosome number that is not a multiple of the haploid number, is a hallmark of cancer. We used an in vitro cell culture model where explants of benign epithelial ovarian tumors (cystadenomas) were transfected with an expression vector for SV40 Large T Antigen, allowing bypass of senescence due, in part, to p53 and Rb inhibition by this antigen. We previously showed that such cells eventually reach a crisis event that is associated with a cell cycle arrest at the M phase and is independent of telomere attrition. Cells that recover from this event do not complete cytokinesis and become polyploid, which is a precursor to aneuploidy. Decreased BRCA1 expression facilitates abrogation of mitotic arrest, leading to increased rates of aneuploidy. We hypothesized that Aurora A plays a role in regulating this ploidy-associated crisis event by acting as an upstream regulator of BRCA1 expression. Here we show that Aurora A kinase activity and expression is up-regulated in cells approaching crisis. Over-expression of Aurora A resulted in decreased levels of BRCA1, resulting in increased tetraploidy and aneuploidy, secondary to cells overcoming a ploidy-associated crisis. Levels of BRCA1 expression increased upon knockdown of Aurora A kinase using siRNA, further suggesting an inverse relationship between expression levels of these two proteins. Inhibition of Aurora A in cultured ovarian cystadenomas approaching crisis led to decreased cell proliferation and a concomitant decrease in tetraploidy. We tested the hypothesis that this was associated with an increase in crisis-related apoptosis by knocking down Aurora A kinase using siRNA technologies in cells approaching crisis, followed by western blotting using antibodies against PARP and cleaved CASPASE 7 fragment. The results indeed showed increased levels of these two protein fragments upon Aurora A knockdown, indicating activation of a caspase-regulated apoptotic pathway. Our findings suggest a novel role for Aurora A's influence on BRCA1 expression levels in cells approaching ploidy dependent crisis. This may, in part, explain the increased rate of near polyploidy associated with high grade ovarian carcinomas and may provide insights into the mechanism underlying aneuploidy development in epithelial cancers in general. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1068.