Mijung Kwon

Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes

Multiple centrosomes in tumor cells create the potential for multipolar divisions that can lead to aneuploidy and cell death. Nevertheless, many cancer cells successfully divide because of mechanisms that suppress multipolar mitoses. A genome-wide RNAi screen in Drosophila S2 cells and a secondary analysis in cancer cells defined mechanisms that suppress multipolar mitoses. In addition to proteins that organize microtubules at the spindle poles, we identified novel roles for the spindle assembly checkpoint, cortical actin cytoskeleton, and cell adhesion. Using live cell imaging and fibronectin micropatterns, we found that interphase cell shape and adhesion pattern can determine the success of the subsequent mitosis in cells with extra centrosomes. These findings may identify cancer-selective therapeutic targets: HSET, a normally nonessential kinesin motor, was essential for the viability of certain extra centrosome-containing cancer cells. Thus, morphological features of cancer cells can be linked to unique genetic requirements for survival.

Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function.

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, it is expected the development of a potent, selective small molecule inhibitor of Mps1 would greatly facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 co-crystal structures of novel, selective small molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit, and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells possessing extra centrosomes, an abnormality found in some cancers, Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

Centrosomes and cancer: how cancer cells divide with too many centrosomes

Precise control of centrosome number is crucial for bipolar spindle assembly and accurate transmission of genetic material to daughter cells. Failure to properly control centrosome number results in supernumerary centrosomes, which are frequently found in cancer cells. This presents a paradox: during mitosis, cells with more than two centrosomes are prone to multipolar mitoses and cell death, however, cancer cells possessing extra centrosomes usually divide successfully. One mechanism frequently utilized by cancer cells to escape death caused by multipolar mitoses is the clustering of supernumerary centrosomes into bipolar arrays. An understanding of the molecular mechanisms by which cancer cells can suppress multipolar mitoses is beginning to emerge. Here, we review what’s currently known about centrosome clustering mechanisms and discuss potential strategies to target these mechanisms for the selective killing of cancer cells.

HURP Permits MTOC Sorting for Robust Meiotic Spindle Bipolarity, Similar to Extra Centrosome Clustering in Cancer Cells

Similar to clustering of extra centrosomes in cancer cells, HURP promotes microtubule stability and sorts MTOCs into distinct poles during meiosis.

Mechanisms and Consequences of Cancer Genome Instability: Lessons from Genome Sequencing Studies.

During tumor evolution, cancer cells can accumulate numerous genetic alterations, ranging from single nucleotide mutations to whole-chromosomal changes. Although a great deal of progress has been made in the past decades in characterizing genomic alterations, recent cancer genome sequencing studies have provided a wealth of information on the detailed molecular profiles of such alterations in various types of cancers. Here, we review our current understanding of the mechanisms and consequences of cancer genome instability, focusing on the findings uncovered through analysis of exome and whole-genome sequencing data. These analyses have shown that most cancers have evidence of genome instability, and the degree of instability is variable within and between cancer types. Importantly, we describe some recent evidence supporting the idea that chromosomal instability could be a major driving force in tumorigenesis and cancer evolution, actively shaping the genomes of cancer cells to maximize their survival advantage.

A mitotic kinesin-6, Pav-KLP, mediates interdependent cortical reorganization and spindle dynamics in Drosophila embryos.

We investigated the role of Pav-KLP, a kinesin-6, in the coordination of spindle and cortical dynamics during mitosis in Drosophila embryos. In vitro, Pav-KLP behaves as a dimer. In vivo, it localizes to mitotic spindles and furrows. Inhibition of Pav-KLP causes defects in both spindle dynamics and furrow ingression, as well as causing changes in the distribution of actin and vesicles. Thus, Pav-KLP stabilizes the spindle by crosslinking interpolar microtubule bundles and contributes to actin furrow formation possibly by transporting membrane vesicles, actin and/or actin regulatory molecules along astral microtubules. Modeling suggests that furrow ingression during cellularization depends on: (1) a Pav-KLP-dependent force driving an initial slow stage of ingression; and (2) the subsequent Pav-KLP-driven transport of actin- and membrane-containing vesicles to the furrow during a fast stage of ingression. We hypothesize that Pav-KLP is a multifunctional mitotic motor that contributes both to bundling of interpolar microtubules, thus stabilizing the spindle, and to a biphasic mechanism of furrow ingression by pulling down the furrow and transporting vesicles that deliver new material to the descending furrow.

Using Cell Culture Models of Centrosome Amplification to Study Centrosome Clustering in Cancer

The link between centrosome amplification and cancer has been recognized for more than a century, raising many key questions about the biology of both normal and cancer cells. In particular, the presence of extra centrosomes imposes a great challenge to a dividing cell by increasing the likelihood of catastrophic multipolar divisions. Only recently have we begun to understand how cancer cells successfully divide by clustering their extra centrosomes for bipolar division. Several hurdles to dissecting centrosome clustering include limitations in the methodologies used to quantify centrosome amplification, and the lack of appropriate cell culture models. Here, we describe how to accurately assess centrosome number and create isogenic cell lines with or without centrosome amplification. We then describe how imaging of cell division in these cell culture models leads to identification of the molecular machinery uniquely required for cells with extra centrosomes. These approaches have led to the identification of molecular targets for selective cancer therapeutics that can kill cancer cells with extra centrosomes without affecting normal cells with two centrosomes. We further anticipate that the approaches described here will be broadly applicable for studying the causes and consequences of centrosome amplification in a variety of contexts across cancer pathophysiology, such as cell migration and metastasis.

Abstract C65: Mechanisms to maintain extra centrosomes in cancer cells

Multiple centrosomes in tumor cells create the potential for multipolar divisions that can lead to aneuploidy and cell death. Nevertheless, many cancer cells successfully divide because of mechanisms that suppress multipolar mitoses. Using a genome‐wide RNAi screen in Drosophila S2 cells, we defined several mechanisms that suppress multipolar mitoses. We also found that HSET, a normally non‐essential kinesin motor was essential for the viability of cancer cells containing extra centrosomes. Interestingly, using fibronectin micropatterns, we found that interphase cell shape and adhesion pattern can determine the success of the subsequent mitosis in cells with extra centrosomes. Thus, cell adhesion is an important morphological feature that contributes to centrosome clustering. Importantly, during tumor progression, changes in cell architecture and adhesion patterns, such as loss of E‐cadherin and acquisition of an elongated cell shape, are often observed. This is referred to as the epithelial‐to‐mesenchymal transition (EMT). The presence of extra centrosomes is often correlated with more malignant tumors, which lost their epithelial phenotype most likely through EMT. Thus, we hypothesized that the ability of cells to cluster extra centrosomes varies between epithelial cells and non‐epithelial cells. To test this idea we used a panel of non‐transformed mammalian epithelial and non‐epithelial cell lines and quantified their ability to cluster extra centrosomes. We treated cells with DCB to generate tetraploid cells containing extra centrosomes and followed them by live‐cell imaging. We found that non‐epithelial cells cluster their extra centrosomes much more efficiently than epithelial cells. This finding suggests that loss of cell‐cell adhesion might facilitate centrosome clustering. Consistent with this idea, our preliminary data indicate that induction of EMT in the epithelial cell lines MCF10A and MDCK increases the number of tetraploid cells that undergo a bipolar mitosis. We propose that changes that take place during tumor progression, such as loss of cell‐cell adhesion, might facilitate centrosome clustering and therefore increase the ability of cancer cells to maintain extra centrosomes. We are currently investigating the nature of the changes that occur during EMT that facilitate centrosome clustering. Citation Information: Cancer Res 2009;69(23 Suppl):C65.