Chromosomal instability sensitizes patient breast tumors to multipolar divisions induced by paclitaxel.

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.

High nuclear TPX2 expression correlates with TP53 mutation and poor clinical behavior in a large breast cancer cohort, but is not an independent predictor of chromosomal instability.

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.

Living in CIN: Mitotic Infidelity and Its Consequences for Tumor Promotion and Suppression.

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.

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

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 Apc(Min/+) mouse intestinal tumor model, in which effects on tumor initiation versus progression can be discriminated. Apc(Min/+) cells exhibit low CIN, and we generated high CIN by reducing expression of the kinesin-like mitotic motor protein CENP-E. CENP-E(+/-);Apc(Min/+) 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 Apc(Min/+) 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.

Centrosome amplification induces high grade features and is prognostic of worse outcomes in breast cancer.

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.

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.

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.

The ARF tumor suppressor prevents chromosomal instability and ensures mitotic checkpoint fidelity through regulation 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.

Cytotoxicity of paclitaxel in breast cancer is due to chromosome missegregation on multipolar spindles.

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.

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.

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.

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.