Andrew J. Holland

Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis

The mitotic checkpoint is a major cell cycle control mechanism that guards against chromosome missegregation and the subsequent production of aneuploid daughter cells. Most cancer cells are aneuploid and frequently missegregate chromosomes during mitosis. Indeed, aneuploidy is a common characteristic of tumours, and, for over 100 years, it has been proposed to drive tumour progression. However, recent evidence has revealed that although aneuploidy can increase the potential for cellular transformation, it also acts to antagonize tumorigenesis in certain genetic contexts. A clearer understanding of the tumour suppressive function of aneuploidy might reveal new avenues for anticancer therapy.

Cep152 interacts with Plk4 and is required for centriole duplication

Cep152, the orthologue of Drosophila Asterless, is a Plk4 target that functions with Plk4 in centriole assembly.

Losing balance: the origin and impact of aneuploidy in cancer

Most solid human tumours are aneuploid, that is, they contain an abnormal number of chromosomes. Paradoxically, however, aneuploidy has been reported to induce a stress response that suppresses cellular proliferation in vitro. Here, we review the progress in our understanding of the causes and effects of aneuploidy in cancer and discuss how, in specific contexts, aneuploidy can provide a growth advantage and facilitate cellular transformation. We also explore the emerging possibilities for targeting the cause or consequences of aneuploidy therapeutically.

Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E

Opposing roles of Aurora kinases and protein phosphatase 1 (PP1) during mitosis have long been suggested. Here, we demonstrate that Aurora kinases A and B phosphorylate a conserved residue on the kinetochore motor CENP-E. PP1 binds CENP-E via a motif overlapping this phosphorylation site and binding is disrupted by Aurora phosphorylation. Phosphorylation of CENP-E by the Auroras is enriched at spindle poles, disrupting binding of PP1 and reducing CENP-Es affinity for individual microtubules. This phosphorylation is required for CENP-E-mediated towing of initially polar chromosomes toward the cell center. Kinetochores on such chromosomes cannot make subsequent stable attachment to spindle microtubules when dephosphorylation of CENP-E or rebinding of PP1 to CENP-E is blocked. Thus, an Aurora/PP1 phosphorylation switch modulates CENP-E motor activity as an essential feature of chromosome congression from poles and localized PP1 delivery by CENP-E to the outer kinetochore is necessary for stable microtubule capture by those chromosomes.

Polo-like kinase 4 kinase activity limits centrosome overduplication by autoregulating its own stability

Plk4 phosphorylates itself in trans to prevent accumulation and self-limit kinase activity, which may be important for regulating centriole duplication.

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

Chromoanagenesis and cancer: mechanisms and consequences of localized, complex chromosomal rearrangements

Next-generation sequencing of DNA from human tumors or individuals with developmental abnormalities has led to the discovery of a process we term chromoanagenesis, in which large numbers of complex rearrangements occur at one or a few chromosomal loci in a single catastrophic event. Two mechanisms underlie these rearrangements, both of which can be facilitated by a mitotic chromosome segregation error to produce a micronucleus containing the chromosome to undergo rearrangement. In the first, chromosome shattering (chromothripsis) is produced by mitotic entry before completion of DNA replication within the micronucleus, with a failure to disassemble the micronuclear envelope encapsulating the chromosomal fragments for random reassembly in the subsequent interphase. Alternatively, locally defective DNA replication initiates serial, microhomology-mediated template switching (chromoanasynthesis) that produces local rearrangements with altered gene copy numbers. Complex rearrangements are present in a broad spectrum of tumors and in individuals with congenital or developmental defects, highlighting the impact of chromoanagenesis on human disease.

Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells

Inducible degradation is a powerful approach for identifying the function of a specific protein or protein complex. Recently, a plant auxin-inducible degron (AID) system has been shown to degrade AID-tagged target proteins in nonplant cells. Here, we demonstrate that an AID-tagged protein can functionally replace an endogenous protein depleted by RNAi, leading to an inducible null phenotype rapidly after auxin addition. The AID system is shown to be capable of controlling the stability of AID-tagged proteins that are in either nuclear or cytoplasmic compartments and even when incorporated into protein complexes. Induced degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 min and proceeding to completion with first-order kinetics. AID-mediated instability is demonstrated to be rapidly reversible. Induced degradation is shown to initiate and continue in all cell cycle phases, including mitosis, making this system especially useful for identifying the function(s) of proteins of interest during specific points in the mammalian cell cycle.

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.

Requirements for NuMA in maintenance and establishment of mammalian spindle poles

Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.