Sarah L. Thompson

Examining the link between chromosomal instability and aneuploidy in human cells

Solid tumors can be highly aneuploid and many display high rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). In principle, aneuploidy is the consequence of CIN, but the relationship between CIN and aneuploidy has not been clearly defined. In this study, we use live cell imaging and clonal cell analyses to evaluate the fidelity of chromosome segregation in chromosomally stable and unstable human cells. We show that improper microtubule–chromosome attachment (merotely) is a cause of chromosome missegregation in unstable cells and that increasing chromosome missegregation rates by elevating merotely during consecutive mitoses generates CIN in otherwise stable, near-diploid cells. However, chromosome missegregation compromises the proliferation of diploid cells, indicating that phenotypic changes that permit the propagation of nondiploid cells must combine with elevated chromosome missegregation rates to generate aneuploid cells with CIN.

Mechanisms of Chromosomal Instability

Most solid tumors are aneuploid, having a chromosome number that is not a multiple of the haploid number, and many frequently mis-segregate whole chromosomes in a phenomenon called chromosomal instability (CIN). CIN positively correlates with poor patient prognosis, indicating that reduced mitotic fidelity contributes to cancer progression by increasing genetic diversity among tumor cells. Here, we review the mechanisms underlying CIN, which include defects in chromosome cohesion, mitotic checkpoint function, centrosome copy number, kinetochore-microtubule attachment dynamics, and cell-cycle regulation. Understanding these mechanisms provides insight into the cellular consequences of CIN and reveals the possibility of exploiting CIN in cancer therapy.

Proliferation of aneuploid human cells is limited by a p53-dependent mechanism

After chromosome missegregation, the growth of nondiploid cells is inhibited thanks to a p53-dependent mechanism.

Genome stability is ensured by temporal control of kinetochore-microtubule dynamics

Most solid tumours are aneuploid and many frequently mis-segregate chromosomes. This chromosomal instability is commonly caused by persistent mal-oriented attachment of chromosomes to spindle microtubules. Chromosome segregation requires stable microtubule attachment at kinetochores, yet those attachments must be sufficiently dynamic to permit correction of mal-orientations. How this balance is achieved is unknown, and the permissible boundaries of attachment stability versus dynamics essential for genome stability remain poorly understood. Here we show that two microtubule-depolymerizing kinesins, Kif2b and MCAK, stimulate kinetochore–microtubule dynamics during distinct phases of mitosis to correct mal-orientations. Few-fold reductions in kinetochore–microtubule turnover, particularly in early mitosis, induce severe chromosome segregation defects. In addition, we show that stimulation of microtubule dynamics at kinetochores restores stability to chromosomally unstable tumour cell lines, establishing a causal relationship between deregulation of kinetochore–microtubule dynamics and chromosomal instability. Thus, temporal control of microtubule attachment to chromosomes during mitosis is central to genome stability in human cells.

Chromosome missegregation in human cells arises through specific types of kinetochore–microtubule attachment errors

Most solid tumors are aneuploid, and many missegregate chromosomes at high rates in a phenomenon called chromosomal instability (CIN). CIN reflects the erosion of mitotic fidelity, and it correlates with poor patient prognosis and drug resistance. The most common mechanism causing CIN is the persistence of improper kinetochore–microtubule attachments called merotely. Chromosomes with merotelic kinetochores often manifest as lagging chromosomes in anaphase, suggesting that lagging chromosomes fail to segregate properly. However, it remains unknown whether the lagging chromosomes observed in anaphase segregate to the correct or incorrect daughter cell. To address this question, we tracked the segregation of a single human chromosome during cell division by using LacI-GFP to target an integrated LacO array. By scoring the distribution of each sister chromatid during mitosis, we show that a majority of lagging chromosomes in anaphase segregate to the correct daughter cell. Instead, sister chromatids that segregate erroneously frequently do so without obvious evidence of lagging during anaphase. This outcome is expected if sister kinetochores on a chromosome bind microtubules oriented toward the same spindle pole, and we find evidence for syntelic kinetochore attachments in cells after treatments that increase missegregation rates. Thus, lagging chromosomes in anaphase are symptomatic of defects in kinetochore–microtubule attachment dynamics that cause chromosome missegregation associated with CIN, but the laggards rarely missegregate.

Chromosomes and cancer cells

Two prominent features of cancer cells are abnormal numbers of chromosomes (aneuploidy) and large-scale structural rearrangements of chromosomes. These chromosome aberrations are caused by genomic instabilities inherent to most cancers. Aneuploidy arises through chromosomal instability (CIN) by the persistent loss and gain of whole chromosomes. Chromosomal rearrangements occur through chromosome structure instability (CSI) as a consequence of improper repair of DNA damage. The mechanisms that cause CIN and CSI differ, but the phenotypic consequences of aneuploidy and chromosomal rearrangements may overlap considerably. Both CIN and CSI are associated with advanced stage tumors with increased invasiveness and resistance to chemotherapy, indicating that targeted inhibition of these instabilities might slow tumor growth. Here, we review recent efforts that define the mechanisms and consequences of CIN and CSI.

Targeting the Cyclin E-Cdk-2 Complex Represses Lung Cancer Growth by Triggering Anaphase Catastrophe

Purpose: Cyclin-dependent kinases (Cdk) and their associated cyclins are targets for lung cancer therapy and chemoprevention given their frequent deregulation in lung carcinogenesis. This study uncovered previously unrecognized consequences of targeting the cyclin E–Cdk-2 complex in lung cancer. Experimental Design: Cyclin E, Cdk-1, and Cdk-2 were individually targeted for repression with siRNAs in lung cancer cell lines. Cdk-2 was also pharmacologically inhibited with the reversible kinase inhibitor seliciclib. Potential reversibility of seliciclib effects was assessed in washout experiments. Findings were extended to a large panel of cancer cell lines using a robotic-based platform. Consequences of cyclin E–Cdk-2 inhibition on chromosome stability and on in vivo tumorigenicity were explored as were effects of combining seliciclib with different taxanes in lung cancer cell lines. Results: Targeting the cyclin E–Cdk-2 complex, but not Cdk-1, resulted in marked growth inhibition through the induction of multipolar anaphases triggering apoptosis. Treatment with the Cdk-2 kinase inhibitor seliciclib reduced lung cancer formation in a murine syngeneic lung cancer model and decreased immunohistochemical detection of the proliferation markers Ki-67 and cyclin D1 in lung dysplasia spontaneously arising in a transgenic cyclin E–driven mouse model. Combining seliciclib with a taxane resulted in augmented growth inhibition and apoptosis in lung cancer cells. Pharmacogenomic analysis revealed that lung cancer cell lines with mutant ras were especially sensitive to seliciclib. Conclusions: Induction of multipolar anaphases leading to anaphase catastrophe is a previously unrecognized mechanism engaged by targeting the cyclin E–Cdk-2 complex. This exerts substantial antineoplastic effects in the lung. Clin Cancer Res; 16(1); 109–20

Anaphase Catastrophe Is a Target for Cancer Therapy

Neoplastic cells are genetically unstable. Strategies that target pathways affecting genome instability can be exploited to disrupt tumor cell growth, potentially with limited consequences to normal cells. Chromosomal instability (CIN) is one type of genome instability characterized by mitotic defects that increase the rate of chromosome mis-segregation. CIN is frequently caused by extra centrosomes that transiently disrupt normal bipolar spindle geometry needed for accurate chromosome segregation. Tumor cells survive with extra centrosomes because of biochemical pathways that cluster centrosomes and promote chromosome segregation on bipolar spindles. Recent work shows that targeted inhibition of these pathways prevents centrosome clustering and forces chromosomes to segregate to multiple daughter cells, an event triggering apoptosis that we refer to as anaphase catastrophe. Anaphase catastrophe specifically kills tumor cells with more than 2 centrosomes. This death program can occur after genetic or pharmacologic inhibition of cyclin dependent kinase 2 (Cdk2) and is augmented by combined treatment with a microtubule inhibitor. This proapoptotic effect occurs despite the presence of ras mutations in cancer cells. Anaphase catastrophe is a previously unrecognized mechanism that can be pharmacologically induced for apoptotic death of cancer cells and is, therefore, appealing to engage for cancer therapy and prevention. Clin Cancer Res; 17(6); 1218–22. ©2011 AACR.

Yet1p–Yet3p interacts with Scs2p–Opi1p to regulate ER localization of the Opi1p repressor

A major phospholipid regulatory circuit in yeast is controlled by Scs2p, an ER membrane protein that binds the transcriptional repressor protein Opi1p. Here we show that the Yet1p–Yet3p complex acts in derepression of INO1 through physical association with Scs2p–Opi1p.

Abstract 4600: Examining the selective contribution of chromosomal instability to tumor evolution and prognosis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The specific role of Chromosomal Instability (CIN) in tumorigenesis has been a matter of conjecture. In part, this is due to the challenge of directly observing dynamic whole-chromosome mis-segregation events as well as the lack of firm understanding of the mechanisms that lead to CIN in cancer. These shortcomings have long prevented the ability to distinguish the role of CIN, which consists of increased rates of chromosome mis-segregation, from that of aneuploidy, which is a state of non-diploid chromosome number. To overcome these limitations, we use human-derived cancer cell lines to understand the biological basis of chromosome mis-segregation. We then extend these findings to tumor samples from patients diagnosed with Diffuse Large B-Cell Lymphoma to examine the selective contribution of CIN to tumor prognosis and evolution. We show that, in human cancer cell lines, chromosome mis-segregation occurs primarily as a result from defective microtubule dynamics, which prevent the correction of erroneous attachments of microtubules to chromosomes during mitosis. These attachment errors lead to the formation of lagging chromosomes and chromatin bridges during anaphase. Strikingly, restoring normal microtubule dynamics significantly decreases chromosome mis-segregation frequencies and suppresses CIN. We then use lagging chromosomes and chromatin bridges as morphological features to examine the selective contribution of CIN to tumor prognosis. Hematoxylin and Eosin-stained samples from a cohort of 54 patients diagnosed with Diffuse Large B-Cell Lymphoma (DLBCL) are used to examine the relationship between frequencies of chromosome mis-segregation and patient prognosis, overall survival, and response to treatment. We show that a two-fold increase in the frequency of chromosome mis-segregation leads to a 24% decrease in overall survival and 48% decrease in relapse-free survival after treatment. The hazard ratio (HR) of death in patients with increased chromosome mis-segregation is 2.31 and these patients are more likely to present with higher tumor stage, exhibit tumor bone marrow involvement, and receive a higher International Prognostic Index (IPI) score. In summary, this work demonstrates that CIN primarily arises due to defects in the attachments of microtubule to chromosomes. Furthermore, increased rates of chromosome mis-segregation in DLBCL substantiate inferior outcome and poor prognosis. This is likely due to increased heterogeneity of tumor cells leading to a larger predilection for adaptation in response to external pressures such as metastasis and drug treatments. We propose that targeting CIN would yield improved prognosis and enhanced response to chemotherapeutic drugs. 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 4600. doi:1538-7445.AM2012-4600