Lauren M. Zasadil

Cytotoxicity of Paclitaxel in Breast Cancer Is due to Chromosome Missegregation on Multipolar Spindles

The chemotherapy drug paclitaxel causes tumor regression and cell death by inducing high rates of chromosome missegregation on multipolar spindles. The Secret Life of Paclitaxel The classic chemotherapy drug paclitaxel is a standard part of treatment for breast cancer and other malignancies. Although it is commonly understood to act as a microtubule poison and lead to mitotic arrest, this knowledge is largely based on studies of cells in culture, with drug concentrations that may not be realistic. Now, Zasadil and coauthors measured the concentration of paclitaxel in real patients undergoing treatment with the drug, and then investigated the response of cancer cells to paclitaxel at these lower and more realistic concentrations. Unexpectedly, the cells treated under these conditions did not undergo mitotic arrest, but instead proceeded through mitosis with abnormal spindles, resulting in chromosome missegregation, which leads to tumor cell death. This intriguing discovery demonstrates that we may not know as much as we thought about the effects of one of our most common chemotherapy drugs. In addition, the findings from this study may lead to clinical applications both in optimizing the selection of chemotherapy drug combinations and in determining which patients are likely to respond to paclitaxel treatment. 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, most 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 because of chromosome missegregation on highly abnormal, multipolar spindles. This mechanistic insight may be used to improve selection of future antimitotic drugs and to identify a biomarker with which to select patients likely to benefit from paclitaxel.

Chromosome missegregation rate predicts whether aneuploidy will promote or suppress tumors

Significance Aneuploidy, an abnormal chromosome content that commonly occurs because of errors in chromosome segregation, can promote or suppress tumor formation. What determines how aneuploidy influences tumorigenesis has remained unclear. Here we show that the rate of chromosome missegregation, rather than the level of accumulated aneuploidy, determines the effect on tumors. Increasing the rate of chromosome missegregation beyond a certain threshold suppresses tumors by causing cell death. Increasing errors of chromosome segregation did not affect tumor formation caused by genetic mutations that do not themselves alter chromosome inheritance. These results suggest that accelerating chromosome missegregation in chromosomally unstable tumors may be a useful strategy therapeutically. 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.

Up-regulation of the mitotic checkpoint component Mad1 causes chromosomal instability and resistance to microtubule poisons

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.

2n or not 2n: Aneuploidy, polyploidy and chromosomal instability in primary and tumor cells.

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.

High rates of chromosome missegregation suppress tumor progression but do not inhibit tumor initiation

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.

The ARF tumor suppressor prevents chromosomal instability and ensures mitotic checkpoint fidelity through regulation of Aurora B

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.

A Golgi-Localized Pool of the Mitotic Checkpoint Component Mad1 Controls Integrin Secretion and Cell Migration

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, where Mad2 is converted into an inhibitor of the anaphase-promoting complex/cyclosome bound to its specificity factor, Cdc20. During interphase, Mad1 remains tightly bound to Mad2, and both proteins localize to the nucleus and nuclear pores, where they interact with Tpr (translocated promoter region). Recently, it has been shown that interaction with Tpr stabilizes both proteins and that Mad1 binding to Tpr permits Mad2 to associate with Cdc20. 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.

Identification of Selective Lead Compounds for Treatment of High-Ploidy Breast Cancer.

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.

Abstract P3-07-53: Chromosomal instability as a predictor of sensitivity to paclitaxel

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.

Abstract 2526: Polyploidy: A new breast cancer subtype and a lead compound that targets it with high selectivity

Polyploidy, the presence of extra chromosome sets, is a feature of many cancers. Polyploid cells have weaknesses that can be exploited for treatment–termed synthetic lethal. We identified polyploid breast cancers and a polypoid-selective lead compound. METHODS: We performed 6-chromosome FISH on 354 human breast cancers and correlated with breast cancer subtype and clinical outcomes of up to 10 years (cohort 1). To validate findings, we analyzed chromosome-17 ploidy on a second cohort of 1095 samples (cohort 2). To identify a polyploid-selective lead compound, a two-stage chemical screen was performed. First, we analyzed data from 45,342 compounds by the NCI Developmental Therapeutics Program to identify compounds selective for high ploidy cell lines. Second, we performed a secondary screen using matched diploid-tetraploid human cells. RESULTS: FISH analysis demonstrates that 10-14% of breast cancers are polyploid (≥3N). Polyploid tumors are commonly estrogen-receptor negative, but are found amongst all subtypes. Outcomes are worse for polyploid cancers than non-polyploid breast cancers in both cohorts (OS; p=0.008 cohort 1, p=0.049 cohort 2; RFS p=0.008, p=0.003). Our primary in silico screen identified potential polyploid-selective compounds. Of these, 35 were procured and tested in the secondary screen using matched diploid-tetraploid human cell lines. One compound, DPBQ, was found to have selective effects on polyploid cells. DPBQ has 3.6-fold selectivity for inhibiting proliferation of tetraploid over diploid cells. A structure-function analysis defined the key chemical motifs required for maintaining polyploid selectivity. Mechanistically, DPBQ selectively induces p53-activation and apoptosis in polyploid cells. However, other chemicals that cause p53 activation and apoptosis lack such selectivity, indicating that DPBQ operates via a unique mechanism. DISCUSSION: Polyploidy indicates an aggressive subtype of breast cancer. Polyploid cells have phenotypes distinct from normal diploid cells, suggesting an opportunity for selective therapeutics. DPBQ is one compound identified by a two-stage screen. This screen enforced the specificity of the effect for polyploid cells and also the external validity across diverse cancer cell types. Mechanistically, DPBQ selectively induces p53 activation and apoptosis in polyploid cells. It will be important do define the direct molecular target of DPBQ. CONCLUSIONS: Polyploid breast cancers represent a unique high-risk subtype of breast cancer. DPBQ is a lead compound that for selective destruction of polyploid cancers. Citation Format: Mark E. Burkard, Alka Choudhary, Robert F. Lera, Ross Fedenia, Craig Kanugh, Jennifer J. Laffin, Lauren M. Zasadil, Beth A. Weaver, Kari B. Wisinski. Polyploidy: A new breast cancer subtype and a lead compound that targets it with high selectivity. [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 2526. doi:10.1158/1538-7445.AM2014-2526