Accurate progression of mitosis is pivotal to ensure a correct euploid karyotype. However, many human diseases including cancer and neurodegenerative diseases are characterized by aneuploidy. A common cause for aneuploidy in human cancer cells is chromosomal instability (CIN) (Lengauer et al. 1997). One can distinguish between structural chromosomal instability (S-CIN) that describes the susceptibility to structural rearrangements including translocations, deletions, inversions and duplications of chromosomal parts (Ricke et al.
2008; Thompson et al. 2010) and whole chromosomal instability (W-CIN), which is defined as the perpetual gain or loss of whole chromosomes during mitosis. In a typical aneuploid cancer cell, one chromosome in every one to five cell divisions becomes missegregated (Lengauer et al. 1997; Thompson & Compton 2008). It is thought that these low missegregation rates allow the acquirement of new cancer phenotypes and the adaptation to the environment (Thompson et al. 2010). In contrast, high rates of chromosome missegregation induced by a highly deregulated mitosis are lethal (Kops et al. 2004).
Furthermore, CIN and aneuploidy is of disadvantage for tumor growth, probably caused by metabolic changes and proteotoxic stress (Torres et al. 2007; Sheltzer & Amon 2011; Ertych et al. 2014).
Introduction
23 The molecular mechanisms causing CIN in human cancer cells are not well understood, but could involve abnormalities during interphase as well as various alterations in mitotic progression (Orr & Compton 2013).
A controversially discussed mechanism is an impaired spindle assembly checkpoint (SAC).
The SAC senses improper kinetochore-microtubule attachments and maintains genomic stability by delaying the metaphase-to-anaphase-transition until all chromosomes are amphitelically attached (Fig. 1.5). A defective SAC leads to a premature anaphase onset in the presence of faulty kinetochore-microtubule attachments, resulting in chromosome missegregation. However, in human cancer cells exhibiting CIN, a weakened SAC due to mutations in SAC related genes is rarely found (Tighe et al. 2001; Barber et al. 2008) and the complete loss of SAC function was even shown to be lethal (Kops et al. 2004).
Chromosome missegregation can also result from the presence of supernumerary centrosomes, which occur from an aberrant cytokinesis or from defects in centrosome biogenesis or centrosome amplification during interphase. In principal, cells containing more than two centrosomes can build up a multi-polar spindle resulting in massive chromosome missegregation. However, this was shown to be unviable for progenies arising from multi-polar cell divisions (Ganem et al. 2009). Instead, supernumerary centrosomes often cluster to form a pseudo bi-polar spindle (Brinkley 2001). But still, the transient occurrence of a multi-polar spindle promotes transient spindle geometry defects, erroneous kinetochore-microtubule attachments and lagging chromosomes (Ganem et al. 2009), leading to chromosome missegregation.
Lagging chromosomes are widely recognized as a cause for chromosomal instability and arise from merotelic kinetochore attachments (Fig. 1.5a). In this case, one kinetochore is concomitantly attached to spindle microtubules emanating from the two opposing spindle poles (Cimini et al. 2001; Cimini et al. 2002; Thompson & Compton 2008). Normally, sister chromatids are attached to opposing poles of the spindle, known as amphitelic attachments.
During chromosome alignment, one kinetochore becomes attached first and orients towards the spindle pole (Rieder & Salmon 1998). This monotelically attached chromosome moves poleward until microtubules bind to the unattached kinetochore, resulting in an amphitelic attachment and chromosome bi-orientation (Rieder & Salmon 1998; Cimini et al. 2002). But errors in kinetochore attachment can occur, including syntelic attachments, where both sister chromatids are attached to spindle microtubules emanating from the same spindle pole. In addition, merotelic attachments are often detectable in cancer cells (Fig. 1.5a). Merotelic attachments support chromosome alignment and the establishment of the metaphase plate, but these errors are not detected by the SAC and lead to a chromosome remaining near the spindle equator (Cimini et al. 2001) (Fig. 1.5b). During the following cytokinesis, the lagging chromosome is distributed onto one of the daughter cells by chance.
24
Figure 1.5: Classification of kinetochore-microtubule attachments. (a) Amphitelic attachments describe the state in which both sister kinetochores are attached to spindle microtubules emanating from the opposing spindle poles. In case of monotelic attachments, only one kinetochore is attached to microtubules emanating from one spindle pole, while syntelic attachments refer to the attachment of both sister kinetochores to spindle microtubules emanating from the same spindle pole. Lagging chromosomes arise from merotelic attachments, which describes the attachment of sister kinetochores to spindle microtubules emanating from the opposing spindle poles, whereby one kinetochore is also attached to microtubules from both spindle poles. (b) Merotelic attachments lead to the generation of lagging chromosomes during anaphase. The chromatid is randomly segregated onto the daughter cells.
During progression through mitosis, erroneous attachments can be corrected. Monotelic attachments will be sensed by the SAC (Rieder et al. 1995), whereas syntelic attachments generate low tension between sister kinetochores (Pinsky & Biggins 2005; Nezi & Musacchio 2009). Like syntelic attachments, merotelic attachments can be resolved by an error correction machinery involving the Aurora B kinase (Cimini et al. 2003; Knowlton et al. 2006;
Holland et al. 2009). Aurora B is localized to the inner centromere and phosphorylates outer kinetochore components like Ndc80 (Cheeseman et al. 2006; DeLuca et al. 2006), Dam1 (Cheeseman et al. 2002), Ska1 (Chan et al. 2012; Schmidt et al. 2012) and MCAK (Gorbsky 2004), thereby destabilizing kinetochore-microtubule attachments. However, increased rates of the generation of erroneous microtubule-kinetochore attachments might overload the error correction machinery leading to the persistence of lagging chromosomes.
Increased microtubule plus-end assembly rates constitute a novel route to chromosomal instability, recently described by our lab (Ertych et al. 2014). MIN/MSI and CIN cell lines were analyzed regarding their microtubule plus-end assembly rates during mitosis. These analyses revealed elevated rates in all analyzed CIN cell lines. It is assumed, that increased
Introduction
25 microtubule plus-end assembly rates lead to transient spindle geometry defects, which result in hyper-stable kinetochore-microtubule attachments, the occurrence of lagging chromosomes and CIN. In turn, restoration of proper microtubule assembly rates by genetic means or chemicals suppresses the CIN phenotype. Vice versa, an increase of microtubule plus-end assembly rates by genetic means also induced CIN and aneuploidy (Ertych et al.
2014).
Furthermore also abnormalities in interphase might contribute to whole CIN. In fact, replication stress during S-phase was shown to affect chromosome segregation but this observation is still debated (Bakhoum et al. 2014).