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This file contains Supplementary Table 1 and provides hazard ratios for recurrence free survival and overall survival for polyploidy and clinical variables using a Cox proportional hazard model.

This file contains supplementary figures S1-S4. These illustrate correlation of ploidy from chromosome 17 FSIH versus 6-chromosome FISH, chromosome spreads of cell lines, additional dose-response curves, and data demonstrating DPBQ does not operate by binding DNA or by inhibiting topoisomerase II.

Background Targeting Protein for Xenopus Kinesin Like Protein 2 (TPX2) is a microtubule associated protein that functions in mitotic spindle assembly. TPX2 also localizes to the nucleus where it functions in DNA damage repair during S-phase. We and others have previously shown that TPX2 RNA levels are strongly associated with chromosomal instability (CIN) in breast and other cancers, and TPX2 RNA levels have been demonstrated to correlate with aggressive behavior and poor clinical outcome across a range of solid malignancies, including breast cancer. Methods We perform TPX2 IHC on a cohort of 253 primary breast cancers and adopt a clinically amenable scoring system to separate tumors into low, intermediate, or high TPX2 expression. We then correlate TPX2 expression against diverse pathologic parameters and important measures of clinical outcome, including disease-specific and overall survival. We link TPX2 expression to TP53 mutation and evaluate whether TPX2 is an independent predictor of chromosomal instability (CIN). Results We find that TPX2 nuclear expression strongly correlates with high grade morphology, elevated clinical stage, negative ER and PR status, and both disease-specific and overall survival. We also show that increased TPX2 nuclear expression correlates with elevated ploidy, supernumerary centrosomes, and TP53 mutation. TPX2 nuclear expression correlates with CIN via univariate analyses but is not independently predictive when compared to ploidy, Ki67, TP53 mutational status, centrosome number, and patient age. Conclusions Our findings demonstrate a strong correlation between TPX2 nuclear expression and aggressive tumor behavior, and show that TPX2 overexpression frequently occurs in the setting of TP53 mutation and elevated ploidy. However, TPX2 expression is not an independent predictor of CIN where it fails to outperform existing clinical and pathologic metrics.

Paclitaxel (Taxol) is a cornerstone of cancer treatment. However, its mechanism of cytotoxicity is incompletely understood and not all patients benefit from treatment. We show that patients with breast cancer did not accumulate sufficient intratumoral paclitaxel to induce mitotic arrest in tumor cells. Instead, clinically relevant concentrations induced multipolar mitotic spindle formation. However, the extent of early multipolarity did not predict patient response. Whereas multipolar divisions frequently led to cell death, multipolar spindles focused into bipolar spindles before division at variable frequency, and maintaining multipolarity throughout mitosis was critical to induce the high rates of chromosomal instability necessary for paclitaxel to elicit cell death. Increasing multipolar divisions in paclitaxel resulted in improved cytotoxicity. Conversely, decreasing paclitaxel-induced multipolar divisions reduced paclitaxel efficacy. Moreover, we found that preexisting chromosomal instability sensitized breast cancer cells to paclitaxel. Both genetic and pharmacological methods of inducing chromosomal instability were sufficient to increase paclitaxel efficacy. In patients, the amount of pretreatment chromosomal instability directly correlated with taxane response in metastatic breast cancer such that patients with a higher rate of preexisting chromosomal instability showed improved response to taxanes. Together, these results support the use of baseline rates of chromosomal instability as a predictive biomarker for paclitaxel response. Furthermore, they suggest that agents that increase chromosomal instability or maintain multipolar spindles throughout mitosis will improve the clinical utility of paclitaxel.

Expression of a truncated allele of the Apc tumor suppressor causes intestinal tumors with a low rate of chromosomal instability (CIN). Increasing the rate of CIN suppresses tumor growth without inhibiting tumor initiation in both the small intestine and colon, suggesting that increasing CIN is a useful chemotherapeutic strategy., Aneuploidy, an abnormal chromosome number that deviates from a multiple of the haploid, has been recognized as a common feature of cancers for >100 yr. Previously, we showed that the rate of chromosome missegregation/chromosomal instability (CIN) determines the effect of aneuploidy on tumors; whereas low rates of CIN are weakly tumor promoting, higher rates of CIN cause cell death and tumor suppression. However, whether high CIN inhibits tumor initiation or suppresses the growth and progression of already initiated tumors remained unclear. We tested this using the ApcMin/+ mouse intestinal tumor model, in which effects on tumor initiation versus progression can be discriminated. ApcMin/+ cells exhibit low CIN, and we generated high CIN by reducing expression of the kinesin-like mitotic motor protein CENP-E. CENP-E+/−;ApcMin/+ doubly heterozygous cells had higher rates of chromosome missegregation than singly heterozygous cells, resulting in increased cell death and a substantial reduction in tumor progression compared with ApcMin/+ animals. Intestinal organoid studies confirmed that high CIN does not inhibit tumor cell initiation but does inhibit subsequent cell growth. These findings support the conclusion that increasing the rate of chromosome missegregation could serve as a successful chemotherapeutic strategy.

Background: Paclitaxel is one of the most effective therapies for breast cancer, although many patients do not benefit. Our goal is to identify those who will benefit, by understanding how this drug contributes to chromosomal instability (CIN). CIN is the gradual gain/loss of whole chromosomes that can occur with mitotic errors as tumors proliferate. Some breast cancers inherently have CIN whereas others lack CIN. Previous work suggests low rates of CIN can promote tumor growth by creating genetic diversity. By contrast, high rates of CIN are lethal, apparently due to a high incidence of deleterious karyotypes. We hypothesize that paclitaxel operates by increasing CIN, and that this has preferential anticancer effects in tumors with preexisting low CIN. Methods: To assess rates of underlying CIN in human breast cancer, we performed 6-chromosome FISH on 354 human breast cancers and correlated with outcomes on a cohort with median 8.4 year follow-up. We measured the physiologic levels of paclitaxel that occur in human breast tumors. To do this, we treated 5 women with neoadjuvant paclitaxel 175mg/m2, performed tumor biopsy at 20 hours, performed LC to quantify intratumoral levels and analyzed mitotic spindles by IHC. Additionally, we performed timelapse videomicroscopy to analyze mitosis in fluorescently-labeled breast cancer cells in the laboratory after exposure to these levels. To evaluate whether CIN controls paclitaxel sensitivity we artificially introduced low levels of CIN into breast cancer cell lines by doxycycline-inducible expression of GFP-Mad1, a protein involved in the mitotic checkpoint, and tested whether this enhanced sensitivity to physiologic doses of paclitaxel. Results: A total of 77% (270/349) of breast cancer have detectable underlying CIN, (average percentage of non-modal chromosomes averaged for 6 chromosomes) greater than the normals (n=11). CIN is higher in HER2+ and TNBC subtypes compared to HR+. CIN does not correlate with the proliferation marker, Ki67 (r2 = 0.04), which does not support the idea of a growth advantage. CIN greater than median levels correlated with worse breast cancer-specific survival (p=0.022 log rank), but no difference in OS or RFS. Paclitaxel in human breast cancer reaches a level mimicked by 5-50nM exposure in laboratory experiments. In the laboratory, breast cancer cells exposed to these levels exhibit multipolar divisions, and similar abnormal mitoses can be found in patient tumors. In breast cancer cells lacking CIN, chromosome analysis demonstrates that it can be artificially induced by conditionally expressing GFP-Mad1. Inducing GFP-Mad1 expression increases sensitivity to paclitaxel, demonstrating that CIN enhances taxane sensitivity. Conclusions: These data support the idea that excessively high levels of CIN can be lethal to cancer cells and that paclitaxel enhances CIN. We predict that the anticancer effects of paclitaxel are marked in tumors with intrinsic CIN, as the enhanced levels are lethal. Thus CIN may be an effective biomarker to predict which women will benefit from taxane therapy. Ultimately, this could be applied in the clinic to substantially improve patient care by decreasing primary resistance or by reducing side effects associated with paclitaxel use. Citation Format: Cavalcante LL, Denu R, Zasadil L, Weaver BA, Burkard M. Chromosomal instability as a predictor of sensitivity to paclitaxel. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-53.

The ARF tumor suppressor is best known for its role in stabilizing p53. This study identifies p53-independent functions of ARF in chromosome segregation and the mitotic checkpoint. Mitotic defects caused by loss of ARF are recapitulated by Aurora B overexpression and rescued by partial depletion of Aurora B., The ARF tumor suppressor is part of the CDKN2A locus and is mutated or undetectable in numerous cancers. The best-characterized role for ARF is in stabilizing p53 in response to cellular stress. However, ARF has tumor suppressive functions outside this pathway that have not been fully defined. Primary mouse embryonic fibroblasts (MEFs) lacking the ARF tumor suppressor contain abnormal numbers of chromosomes. However, no role for ARF in cell division has previously been proposed. Here we demonstrate a novel, p53-independent role for ARF in the mitotic checkpoint. Consistent with this, loss of ARF results in aneuploidy in vitro and in vivo. ARF−/− MEFs exhibit mitotic defects including misaligned and lagging chromosomes, multipolar spindles, and increased tetraploidy. ARF−/− cells exhibit overexpression of Mad2, BubR1, and Aurora B, but only overexpression of Aurora B phenocopies mitotic defects observed in ARF−/− MEFs. Restoring Aurora B to near-normal levels rescues mitotic phenotypes in cells lacking ARF. Our results define an unexpected role for ARF in chromosome segregation and mitotic checkpoint function. They further establish maintenance of chromosomal stability as one of the additional tumor-suppressive functions of ARF and offer a molecular explanation for the common up-regulation of Aurora B in human cancers.

Errors in chromosome segregation during mitosis have been recognized as a hallmark of tumor cells since the late 1800s, resulting in the long-standing hypothesis that mitotic abnormalities drive tumorigenesis. Recent work has shown that mitotic defects can promote tumors, suppress them, or do neither, depending on the rate of chromosome missegregation. Here we discuss the causes of chromosome missegregation, their effects on tumor initiation and progression, and the evidence that increasing the rate of chromosome missegregation may be an effective chemotherapeutic strategy.

Mitotic defects leading to aneuploidy have been recognized as a hallmark of tumor cells for over 100 years. Current data indicate that ~85% of human cancers have missegregated chromosomes to become aneuploid. Some maintain a stable, aneuploid karyotype while others consistently missegregate chromosomes over multiple divisions due to Chromosomal INstability (CIN). Both aneuploidy and CIN serve as markers of poor prognosis in diverse human cancers. Despite this, aneuploidy is generally incompatible with viability during development, and some aneuploid karyotypes cause a proliferative disadvantage in somatic cells. In vivo, the intentional introduction of aneuploidy can promote tumors, suppress them, or do neither. Here, we summarize current knowledge of the effects of aneuploidy and CIN on proliferation and cell death in nontransformed cells, as well as on tumor promotion, suppression, and prognosis.

Increased ploidy is common in tumors but treatments for tumors with excess chromosome sets are not available. Here, we characterize high-ploidy breast cancers and identify potential anticancer compounds selective for the high-ploidy state. Among 354 human breast cancers, 10% have mean chromosome copy number exceeding 3, and this is most common in triple-negative and HER2-positive types. Women with high-ploidy breast cancers have higher risk of recurrence and death in two patient cohorts, demonstrating that it represents an important group for improved treatment. Because high-ploidy cancers are aneuploid, rather than triploid or tetraploid, we devised a two-step screen to identify selective compounds. The screen was designed to assure both external validity on diverse karyotypic backgrounds and specificity for high-ploidy cell types. This screen identified novel therapies specific to high-ploidy cells. First, we discovered 8-azaguanine, an antimetabolite that is activated by hypoxanthine phosphoribosyltransferase 1 (HPRT1), suggesting an elevated gene-dosage of HPRT1 in high-ploidy tumors can control sensitivity to this drug. Second, we discovered a novel compound, 2,3-diphenylbenzo[g]quinoxaline-5,10-dione (DPBQ). DPBQ activates p53 and triggers apoptosis in a polyploid-specific manner, but does not inhibit topoisomerase or bind DNA. Mechanistic analysis demonstrates that DPBQ elicits a hypoxia gene signature and its effect is replicated, in part, by enhancing oxidative stress. Structure–function analysis defines the core benzo[g]quinoxaline-5,10 dione as being necessary for the polyploid-specific effects of DPBQ. We conclude that polyploid breast cancers represent a high-risk subgroup and that DPBQ provides a functional core to develop polyploid-selective therapy. Mol Cancer Ther; 15(1); 48–59. ©2015 AACR.

The blockbuster chemotherapy drug paclitaxel is widely presumed to cause cell death in tumors as a consequence of mitotic arrest, as it does at concentrations routinely used in cell culture. However, we determine here that paclitaxel levels in primary breast tumors are well below those required to elicit sustained mitotic arrest. Instead, cells in these lower concentrations of drug proceed through mitosis without substantial delay and divide their chromosomes on multipolar spindles, resulting in chromosome missegregation and cell death. Consistent with these cell culture data, the majority of mitotic cells in primary human breast cancers contain multipolar spindles after paclitaxel treatment. Contrary to the previous hypothesis, we find that mitotic arrest is dispensable for tumor regression in patients. These results demonstrate that mitotic arrest is not responsible for the efficacy of paclitaxel, which occurs due to chromosome missegregation on highly abnormal, multipolar spindles. This mechanistic insight may be used to improve selection of future anti-mitotic drugs and to identify a biomarker with which to select patients likely to benefit from paclitaxel.

Aneuploidy, a chromosome content other than a multiple of the haploid number, is a common feature of cancer cells. Whole chromosomal aneuploidy accompanying ongoing chromosomal instability in mice resulting from reduced levels of the centromere-linked motor protein CENP-E has been reported to increase the incidence of spleen and lung tumors, but to suppress tumors in three other contexts. Exacerbating chromosome missegregation in CENP-E(+/-) mice by reducing levels of another mitotic checkpoint component, Mad2, is now shown to result in elevated cell death and decreased tumor formation compared with reduction of either protein alone. Furthermore, we determine that the additional contexts in which increased whole-chromosome missegregation resulting from reduced CENP-E suppresses tumor formation have a preexisting, elevated basal level of chromosome missegregation that is exacerbated by reduction of CENP-E. Tumors arising from primary causes that do not generate chromosomal instability, including loss of the INK4a tumor suppressor and microsatellite instability from reduction of the DNA mismatch repair protein MLH1, are unaffected by CENP-E-dependent chromosome missegregation. These findings support a model in which low rates of chromosome missegregation can promote tumorigenesis, whereas missegregation of high numbers of chromosomes leads to cell death and tumor suppression.

The mitotic checkpoint is the major cell cycle checkpoint acting during mitosis to prevent aneuploidy and chromosomal instability, which are hallmarks of tumor cells. Reduced expression of the mitotic checkpoint component Mad1 causes aneuploidy and promotes tumors in mice [Iwanaga Y, et al. (2007) Cancer Res 67:160–166]. However, the prevalence and consequences of Mad1 overexpression are currently unclear. Here we show that Mad1 is frequently overexpressed in human cancers and that Mad1 up-regulation is a marker of poor prognosis. Overexpression of Mad1 causes aneuploidy and chromosomal instability through weakening mitotic checkpoint signaling caused by mislocalization of the Mad1 binding partner Mad2. Cells overexpressing Mad1 are resistant to microtubule poisons, including currently used chemotherapeutic agents. These results suggest that levels of Mad1 must be tightly regulated to prevent aneuploidy and transformation and that Mad1 up-regulation may promote tumors and cause resistance to current therapies.

Paclitaxel is a chemotherapeutic, microtubule-stabilizing drug used to treat a variety of cancers, including those of the breast. In the neoadjuvant setting, paclitaxel effects tumor shrinkage in up to half of patients. Identification of a biomarker to predict response to paclitaxel would reduce side effects and improve patient outcomes. It has long been known that micromolar concentrations of paclitaxel cause cells in culture to arrest in mitosis, which can lead to cell death. However, the concentration range found within patient tumors remains unclear, both because it has not been directly measured and because paclitaxel accumulates intracellularly up to 1000 fold. In a pilot clinical trial, we are directly measuring the intratumoral drug concentration in breast tumors following neoadjuvant paclitaxel treatment. Our preliminary data support the hypothesis that treatment of cultured cells with a low nanomolar concentration more accurately models the intratumoral drug levels in breast cancer patients. Unlike treatment with micromolar concentrations, treatment with low nanomolar paclitaxel does not cause a significant mitotic arrest. Rather, cells treated with low nanomolar paclitaxel frequently proceed through mitosis with multipolar mitotic spindles, resulting in chromosome missegregation and the production of two or more daughter cells. This alternative mechanism of action may explain why tumors treated with paclitaxel fail to show an increase in mitotic index. Previous studies have shown that low rates of chromosome missegregation are well tolerated, but high rates of chromosome missegregation cause cell death due to loss of both copies of one or more essential chromosomes. Therefore, the fidelity of chromosome segregation may be of use as a predictor of response to paclitaxel. In chromosomally stable breast cancer cells, exposure to paclitaxel for two days is sufficient to induce aneuploidy, and within three days there is an increase in cell death. However, a subset of cells are able to survive in low nM paclitaxel for over one week, and survival is higher than in cells which exhibit chromosomal instability (CIN), and have a preexisting rate of chromosome missegregation during division. In patients, chromosomal stability is of potential use as a predictive biomarker to select patients most likely to respond to paclitaxel treatment. Citation Format: Lauren M. Zasadil, Mark Burkard, Beth A. Weaver. Clinically relevant paclitaxel concentrations cause chromosome missegregation rather than mitotic arrest. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1750. doi:10.1158/1538-7445.AM2013-1750

Paclitaxel is a microtubule-stabilizing drug that binds to the β-tubulin subunit and accumulates in mitotic cells. As a chemotherapeutic agent, paclitaxel is used for the treatment of a variety of cancers including breast. Currently, no biomarker is available to predict which patients will benefit from paclitaxel treatment. Paclitaxel studies in cells focus primarily on relatively high concentrations of the drug, which cause cells to die following long-term mitotic arrest. The clinically relevant concentration of paclitaxel is currently unknown, although it is widely speculated to be in the low nM range. Cells exposed to low concentrations of paclitaxel complete mitosis with minimal delay, but frequently contain multipolar spindles. In chromosomally stable breast cancer cells, low concentrations of paclitaxel are sufficient to increase aneuploidy within two days of exposure. These data suggest that the mechanism of cell death due to low dose paclitaxel is likely to be quite different from the mechanism of cell death caused by high dose paclitaxel. They also predict that chromosomally instable (CIN) cells may be more sensitive to cell death caused by low doses of paclitaxel. CIN cells have an existing level of chromosomal missegregation, which is likely to be exacerbated by treatment with low doses of paclitaxel. Since missegregation of large numbers of chromosomes per division causes rapid cell death, these cells may be more likely to die after low dose paclitaxel treatment as a result of losing both copies of a vital chromosome. Chromosomally stable cells, which do not normally missegregate chromosomes with each division, would be more resistant to death following paclitaxel treatment than CIN cells. These data suggest that CIN may be used as a biomarker to determine which tumors are most likely to be sensitive to treatment with clinically relevant concentrations of paclitaxel. 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 3064. doi:1538-7445.AM2012-3064

Summary Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the major cell-cycle checkpoint that regulates chromosome segregation during mitosis, the mitotic checkpoint (also known as the spindle assembly checkpoint). During mitosis, Mad1 recruits Mad2 to unattached kinetochores [1, 2], where Mad2 is converted into an inhibitor of the anaphase-promoting complex/cyclosome bound to its specificity factor, Cdc20 [1, 3–6]. During interphase, Mad1 remains tightly bound to Mad2 [2, 3, 7, 8], and both proteins localize to the nucleus and nuclear pores [9, 10], where they interact with Tpr (translocated promoter region). Recently, it has been shown that interaction with Tpr stabilizes both proteins [11] and that Mad1 binding to Tpr permits Mad2 to associate with Cdc20 [12]. However, interphase functions of Mad1 that do not directly affect the mitotic checkpoint have remained largely undefined. Here we identify a previously unrecognized interphase distribution of Mad1 at the Golgi apparatus. Mad1 colocalizes with multiple Golgi markers and cosediments with Golgi membranes. Although Mad1 has previously been thought to constitutively bind Mad2, Golgi-associated Mad1 is Mad2 independent. Depletion of Mad1 impairs secretion of α5 integrin and results in defects in cellular attachment, adhesion, and FAK activation. Additionally, reduction of Mad1 impedes cell motility, while its overexpression accelerates directed cell migration. These results reveal an unexpected role for a mitotic checkpoint protein in secretion, adhesion, and motility. More generally, they demonstrate that, in addition to generating aneuploidy, manipulation of mitotic checkpoint genes can have unexpected interphase effects that influence tumor phenotypes.

Background Centrosome amplification (CA) has been reported in nearly all types of human cancer and is associated with deleterious clinical factors such as higher grade and stage. However, previous reports have not shown how CA affects cellular differentiation and clinical outcomes in breast cancer. Methods We analyzed centrosomes by immunofluorescence and compared to ploidy and chromosomal instability (CIN) as assessed by 6-chromosome FISH in a cohort of 362 breast cancers with median clinical follow-up of 8.4 years. Centrosomes were recognized by immunofluorescence using antibodies for pericentriolar material (PCM; pericentrin) and centrioles (polyglutamylated tubulin). CA was experimentally induced in cell culture by overexpression of polo-like kinase 4 (PLK4). Results CA is associated with reduced all-cause and breast cancer-specific overall survival and recurrence-free survival. CA correlates strongly with high-risk subtypes (e.g. triple negative) and higher stage and grade, and the prognostic nature of CA can be explained largely by these factors. A strong correlation between CA and high tumor ploidy demonstrates that chromosome and centrosome doubling often occur in concert. CA is proposed to be a method of inducing CIN via aberrant mitotic cell divisions; consonant with this, we observed a strong correlation between CA and CIN in breast cancers. However, some CA tumors had low levels of CIN, indicating that protective mechanisms are at play, such as centrosome clustering during mitosis. Intriguingly, some high-risk tumors have more acentriolar centrosomes, suggesting PCM fragmentation as another mechanism of CA. In vitro induction of CA in two non-transformed human cell lines (MCF10A and RPE) demonstrated that CA induces a de-differentiated cellular state and features of high-grade malignancy, supporting the idea that CA intrinsically causes high-grade tumors. Conclusions CA is associated with deleterious clinical factors and outcomes in breast cancer. Cell doubling events are the most prevalent causes of CA in cancer, although PCM fragmentation may be a secondary cause. CA promotes high-risk breast cancer in part by inducing high-grade features. These findings highlight the importance of centrosome aberrations in the biology of human breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2083-x) contains supplementary material, which is available to authorized users.