3. Molecular mechanisms triggering CIN
3.6. Deregulation of the cell cycle arrest pathways
A malfunction of cell cycle regulators (e.g. transcription factors or cyclins) can also contribute to CIN and tumorigenesis. One of the central players in the maintenance of the genome stability and tumor suppression is p53. Inactivation of p53 due to
Introduction
2011). p53 haploinsufficiency results in Li-Fraumeni syndrome associated with a very high predisposition to tumorigenesis (Varley, 2003). Moreover, p53 mutants frequently not only lose tumor suppression function but even obtain an oncogenic potential (Brosh and Rotter, 2009). However, targeted inactivation of p53 alone is not sufficient to promote CIN (Bunz et al., 2002). Instead, CIN was shown to develop upon inactivation of both the Mad2- and p53-dependent checkpoints (Burds et al., 2005). Therefore, p53 loss is not sufficient to promote CIN and requires additional changes (for example, on mitotic level) in human cells.
In turn, p53 proficiency is important for abrogating the proliferation of cells with abnormal karyotypes (Andreassen et al., 2001; Donehower et al., 1995; Ganem and Pellman, 2007; Livingstone et al., 1992), when missegregation even of a few chromosomes triggers p53 accumulation in the nucleus (Thompson and Compton, 2010). This p53 activation prevents highly missegregating tetraploid cells from further proliferation already after the first tetraploid mitosis (Kuffer et al., 2013). Thus, it becomes clear why tetraploid progeny has been analyzed mostly in p53-negative cells so far (Fujiwara et al., 2005; Lv et al., 2012; Vitale et al., 2010). Interestingly, p53-proficient tetraploid cells that escape the arrest fate maintained chromosomal stability, suggesting absence of other defects that can contribute to CIN (Ho et al., 2010).
The activity of p53 can be attenuated or completely abolished upon overexpression of its inhibitors Mdm2 and MdmX that mediate p53 export from the nucleus and monoubiquitination for proteasome degradation (Badciong and Haas, 2002; Moll and Petrenko, 2003). Overexpression of Mdm2 was reported to facilitate tumorigenesis (Wade and Wahl, 2009). Moreover, Mdm2 overexpression in mouse leads to multiple centrosomes and multipolarity in mitosis, and, subsequently, CIN exactly as p53 absence (Carroll et al., 1999). Similarly, Mdm2-overexpressing mice have a higher incidence of aberrant karyotypes and develop cancers (Wang et al., 2008b). Accordingly, Mdm2 heterozygous murine cells are chromosomally stable (Wang et al., 2006). However, lack of MdmX complemented with loss of p53, instead, manifests in CIN and even faster tumor development than due to loss of p53 alone (Matijasevic et al., 2008). Although it remains enigmatic which effects of Mdm2 and
Introduction
A downstream target of p53, p21, serves as a direct inhibitor of Cdk1 and an executor of p53-mediated arrest: activation of p21 even in absence of p53 is sufficient to suppress aneuploidy (Barboza et al., 2006). Decrease in p21 levels strongly correlates with CIN in high and low grade premalignant liver lesions as well as hepatocarcinomas (Lee et al., 2009b), suggesting potential role of p21 deregulation in the development of liver cancers.
Other tumor suppressor Rb protein, which is mutated in retinal cancer (retinoblastoma) and some other cancers, act as a regulator of E2F family of transcription factors. Deregulation of Rb pathway and abnormal activation of E2F transcription factors lead to E2F-dependent Mad2 overexpression, causing CIN in p53-deficient cells (Hernando et al., 2004; Schvartzman et al., 2011). In addition, proper function of Rb is important to limit the proliferation of tetraploid cells (Andreassen et al., 2001; Borel et al., 2002).
Deregulation of cyclins was also reported to promote CIN: for example, steady expression of cyclin E, a regulator of Cdk2 (cyclin-dependent kinase 2) leads to abnormalities in S-phase, CIN and tumorigenesis (Spruck et al., 1999; Willmarth et al., 2004). Notably, in this case the S-phase defect does not manifest in abnormally high centrosome numbers that could explain CIN. However, another study reports centrosome overamplification upon cyclin E overexpression (Nakayama et al., 2000). Continuous expression of another regulator of G1 to S transition cyclin D1 was linked to enrichment of the genes of the CIN signature (Casimiro et al., 2012; Casimiro and Pestell, 2012).
Apart from the defects triggered by defects of the above-mentioned cell cycle regulators, some other mutations and deregulations associated with aneuploidy and CIN were described. The examples include: Notch pathway in meningiomas (Baia et al., 2008), tumor suppressors BRCA1 and BRCA2 in breast cancer (Joukov et al., 2006; Miyoshi et al., 2002; Popova et al., 2012), transcription factor c-Myc (Menssen et al., 2007), GTPase Ran-binding protein RanBP1 (Tedeschi et al., 2007), FoxM1 (Laoukili et al., 2005; Teh et al., 2010), DNA damage response kinase ATM (Shen et al., 2005) and many others. For many of them the direct mechanistic link between genetic and expression changes and CIN remains unclear.
Introduction
In conclusion, much insight has been gained into the mechanisms driving faithful chromosome segregation and the maintenance of the numerical chromosomal stability. First, the CIN-associated defects can arise on the mitotic level through malfunctions of the mitotic spindle, sister chromatid cohesion, KT-MT attachment error correction or the SAC, manifesting as chromosome missegregation. Second, attenuated response to chromosome missegregation can allow proliferation of abnormal karyotypes. Currently, a large body of clinical evidence suggests that CIN is the dominant cause of tumor unresponsiveness to therapy. Thus, targeted manipulation of the chromosome segregation machinery and other CIN signature genes and pathways can be used for the therapeutic purposes in cancer treatment.
Aim of This Study
Aim of This Study
Whole chromosome instability (CIN) is a common hallmark of many cancers that is associated with a poor clinical prognosis. In recent years, significant advance has been made in deciphering the mechanisms leading to persistent chromosome missegregation. A route to CIN through a tetraploid intermediate formation has been proposed previously. Mounting evidence supports the view that tetraploidy is a transient state that results in aneuploidy, CIN, and, eventually, in tumorigenesis, at least in p53-deficient cells (Fujiwara et al., 2005; Lv et al., 2012). However, the route from tetraploidy to aneuploidy and CIN remains largely elusive. Initially, multiple centrosomes were suspected to be the major cause of CIN in tetraploid cells. Yet, the fact that multiple rounds of cytokinesis failures do not establish centrosome amplification (Krzywicka-Racka and Sluder, 2011) argues against the role of multiple centrosomes as the sole source of CIN in tetraploid progeny.
The aim of my study was to determine which adaptations allow the cell proliferation after tetraploidization and what mechanisms contribute to chromosomal instability in posttetraploid progeny. Furthermore, I aimed to investigate whether single tetraploidization alone is sufficient to trigger CIN and whether it depends on p53 presence and function in posttetraploid cells. In more detail, my objective was to: 1. Generate posttetraploid progenies (PTs) after induced cytokinesis failure in stable
diploid cell lines.
2. Investigate the chromosome segregation fidelity in the PTs in comparison to progenitor diploid and tetraploid (immediately after cytokinesis failure) cell lines. 3. Assess the contribution of extra centrosomes to CIN in PTs.
4. Explore further alterations that can be involved in CIN development such as: 4.1. Changes in the microtubule dynamics and spindle geometry.
4.2. Alterations in the spindle assembly checkpoint.
Results