David Pellman

A Mechanism Linking Extra Centrosomes to Chromosomal Instability

Chromosomal instability (CIN) is a hallmark of many tumours and correlates with the presence of extra centrosomes. However, a direct mechanistic link between extra centrosomes and CIN has not been established. It has been proposed that extra centrosomes generate CIN by promoting multipolar anaphase, a highly abnormal division that produces three or more aneuploid daughter cells. Here we use long-term live-cell imaging to demonstrate that cells with multiple centrosomes rarely undergo multipolar cell divisions, and the progeny of these divisions are typically inviable. Thus, multipolar divisions cannot explain observed rates of CIN. In contrast, we observe that CIN cells with extra centrosomes routinely undergo bipolar cell divisions, but display a significantly increased frequency of lagging chromosomes during anaphase. To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number. We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division. These segregation errors are a consequence of cells passing through a transient ‘multipolar spindle intermediate’ in which merotelic kinetochore–microtubule attachment errors accumulate before centrosome clustering and anaphase. These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumours. We propose that this mechanism may be a common underlying cause of CIN in human cancer.

Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells.

A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8–30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.

Absolute quantification of somatic DNA alterations in human cancer

We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.

From polyploidy to aneuploidy, genome instability and cancer

Polyploidy is a frequent phenomenon in the eukaryotic world, but the biological properties of polyploid cells are not well understood. During evolution, polyploidy is thought to be an important mechanism that contributes to speciation. Polyploid, usually non-dividing, cells are formed during development in otherwise diploid organisms. A growing amount of evidence indicates that polyploid cells also arise during a variety of pathological conditions. Genetic instability in these cells might provide a route to aneuploidy and thereby contribute to the development of cancer.

DNA breaks and chromosome pulverization from errors in mitosis

The involvement of whole-chromosome aneuploidy in tumorigenesis is the subject of debate, in large part because of the lack of insight into underlying mechanisms. Here we identify a mechanism by which errors in mitotic chromosome segregation generate DNA breaks via the formation of structures called micronuclei. Whole-chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and often extensive fragmentation of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for ‘chromothripsis’ in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.

Causes and consequences of aneuploidy in cancer

Genetic instability, which includes both numerical and structural chromosomal abnormalities, is a hallmark of cancer. Whereas the structural chromosome rearrangements have received substantial attention, the role of whole-chromosome aneuploidy in cancer is much less well-understood. Here we review recent progress in understanding the roles of whole-chromosome aneuploidy in cancer, including the mechanistic causes of aneuploidy, the cellular responses to chromosome gains or losses and how cells might adapt to tolerate these usually detrimental alterations. We also explore the role of aneuploidy in cellular transformation and discuss the possibility of developing aneuploidy-specific therapies.

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.

Yeast formins regulate cell polarity by controlling the assembly of actin cables

Formins are conserved Rho-GTPase effectors that communicate Rho-GTPase signals to the cytoskeleton. We found that formins were required for the assembly of one of the three budding yeast actin structures: polarized arrays of actin cables. A dominant-active formin induced the assembly of actin cables. The activation and localization of the formin Bni1p required components of the polarisome complex. These findings potentially define the cellular function of formins in budding yeast and explain their involvement in the generation of cell polarity. A requirement for formins in constructing specific actin structures might be the basis for the diverse activities of formins in development.

Microtubule "plus-end-tracking proteins" : The end is just the beginning

There are several key unanswered questions about the +TIPs. One is the mechanism of release of +TIPs from the trailing edge of polymerizing MTs. An important barrier to progress is the absence of an in vitro system that faithfully recapitulates both the binding and release that characterize plus end tracking. As +TIP partners/regulators may be important to reconstitute +TIP treadmilling in vitro, future progress may come from systems with additional purified components, or from ones based on crude extracts. Another question is the in vivo relationship between different +TIPs. It appears that CLIP-170 and EB1 can reside on the same growing MT end (Akhmanova et al., 2001xAkhmanova, A., Hoogenraad, C.C., Drabek, K., Stepanova, T., Dortland, B., Verkerk, T., Vermeulen, W., Burgering, B.M., De Zeeuw, C.I., Grosveld, F., and Galjart, N. Cell. 2001; 104: 923–935Abstract | Full Text | Full Text PDF | PubMed | Scopus (290)See all References)(Akhmanova et al., 2001). Is there cooperation or competition between these proteins, and could such interactions be a nodal point for regulating the repertoire of plus end behaviors?In addition, it is not known whether +TIPs can provide stable attachments. It will be important to determine if their association with MTs is stabilized (i.e., if they treadmill less) when MT plus ends interact with target sites. An alternative, suggested for CLIP-170 at the kinetochore, is that +TIPs mediate the initial attachment but then dissociate (Dujardin et al., 1998xDujardin, D., Wacker, U.I., Moreau, A., Schroer, T.A., Rickard, J.E., and De Mey, J.R. J. Cell Biol. 1998; 141: 849–862Crossref | Scopus (116)See all References)(Dujardin et al., 1998). Real-time methods where the turnover of +TIPs at the MT end can be measured may help distinguish between these possibilities. Although budding yeast is not famous for the awesome power of its cytology, the ability to see single MTs interacting with target sites on the membrane provides unique spatial resolution that should facilitate these experiments. Finally, do the +TIPs only function at the MT plus end? Several +TIPs interact with proteins that associate with or regulate the behavior of the MT minus ends (Chen et al. 1998xChen, X.P., Yin, H., and Huffaker, T.C. J. Cell Biol. 1998; 141: 1169–1179Crossref | Scopus (71)See all References, Chen et al. 2000xChen, C.R., Chen, J., and Chang, E.C. Mol. Biol. Cell. 2000; 11: 4067–4077CrossrefSee all References). This raises the possibility that +TIPs might have an additional role in MT nucleation or in anchoring MT minus ends to the centrosome.Like motors and the proteins involved in microtubule nucleation, the CLIP-170 family and EB1 family proteins are highly conserved. We speculate that these proteins evolved because of the need for devices that distinguish the plus end from the body of the MT. Although the plus end has a unique shape, its large size (>25 nm in diameter) makes it an unwieldy object to recognize at the molecular level. +TIPs may solve this problem; if copolymerized with tubulin, +TIPs may “tag” MT plus ends. By interacting with different partners, +TIPs could serve as molecular adaptors, providing links to a large repertoire of signals and target sites. In the cell, this diversity of plus end interactions may be fundamental to the regional control of MT dynamics, MT attachment, and the assembly of complex MT-based structures necessary for cell division and morphogenesis.

Human TUBB3 Mutations Perturb Microtubule Dynamics, Kinesin Interactions, and Axon Guidance

We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals.