The Spindle Assembly Checkpoint and Aneuploidy

In: Aneuploidy: Etiology, Disorders and Risk Factors ISBN: 978-1-62100-070-9

Editors: Salvatore de Rossi and Filippo Bianchi ©2012 Nova Science Publishers, Inc.

Chapter III

The Spindle Assembly

Checkpoint and Aneuploidy

Juliana Faria1, Joana Barbosa

1, Inês M. B. Moura

1,

Rui M. Reis2,3

and Hassan Bousbaa*,1,4

1Centro de Investigação em Ciências da Saúde (CICS), Instituto Superior de Ciências da

Saúde – Norte, CESPU, Gandra PRD, Portugal 2Life and Health Sciences Research Institute (ICVS), Health Sciences School,

University of Minho, Braga, Portugal 3Molecular Oncology Research Center, Barretos Cancer Hospital,

Barretos, São Paulo, Brazil 4Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP),

Porto, Portugal

Abstract

Abnormal chromosome number, or aneuploidy, is commonly observed in most solid

tumors, and results from mis-segregation of whole chromosomes in a phenomenon

referred to as chromosome instability (CIN). Dysregulation of the spindle assembly

checkpoint (SAC) is thought as one of the mechanisms underlying CIN. The SAC is a

signaling pathway that prevents precocious chromosome segregation until all

chromosomes of a dividing cell are aligned at the metaphase plate. While complete loss of

the SAC activity is lethal due to massive mis-segregation, partial loss of the SAC is a

common feature of many aneuploid tumor cells allowing them to gain or lose a small

number of chromosomes. We review our current knowledge on the molecular

mechanisms of SAC and discuss its contribution to CIN as well as its potential as a

suitable target in cancer therapy.

* Corresponding author: Prof. Hassan Bousbaa, Centro de Investigação em Ciências da Saúde (CICS), Instituto

Superior de Ciências da Saúde - Norte, CESPU, Rua Central de Gandra, 1317, 4585-116 Gandra PRD,

Portugal. Phone: +351 – 224157186. Fax: +351 – 224157102, [email protected].

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Juliana Faria, Joana Barbosa, Inês M. B. Moura et al. 60

Introduction

The cell cycle is a ubiquitous and complex process that is required for cell growth,

proliferation, genetic material transmission and tissue regeneration (Schafer, 1998). It consists

of temporally coordinated events that ensure proper embryogenesis and subsequent cell

differentiation. Four checkpoint mechanisms are responsible for its tight control: DNA

damage checkpoints at G1/S, S and G2/M, and the spindle assembly checkpoint (SAC)

during mitosis (Tyson and Novak, 2008). These checkpoint mechanisms consist of complex

signaling cascades that only allow cells to progress through the cell cycle if their requirements

are met (Rieder, 2011; Tyson and Novak, 2008).

The spindle assembly checkpoint (SAC) is a surveillance mechanism that is constitutively

expressed during the transition from prometaphase to metaphase in eukaryotic dividing cells

(Kops et al., 2005). It detects improper kinetochore-microtubule attachments, imposing a

mitotic delay by preventing anaphase onset, in order to allow cells to correct them. This ‗wait

anaphase‘ mechanism is sustained until all chromosomes are correctly connected to the

microtubule network, bi-oriented and aligned at the metaphase plate (Logarinho and Bousbaa,

2008; May and Hardwick, 2006; Rieder et al., 1994; Zich and Hardwick, 2010). Therefore,

the SAC activity accounts for equal chromosome segregation to cell progeny and, hence, for

an effective reduction in mitotic error rates. Not surprisingly, weakened SAC activity has

been reported in many aneuploid tumors (Bannon and Mc Gee, 2009; Chi and Jeang, 2007;

Dalton and Yang, 2009; Suijkerbuijk and Kops, 2008). Given its importance in genomic

stability and cancer prevention, we will focus on the molecular mechanism of SAC activity,

its relation to cancer, and its use in current anti-cancer strategies.

The Molecular Mechanism of SAC

In order to be accurately segregated at the onset of anaphase, chromosomes must attach,

through their sister kinetochores, to the microtubules emanating from the opposite poles of

the mitotic spindle. This bi-orientation ensures their alignment at the metaphase equator so

that each chromatid is transported toward the corresponding pole to be delivered to the future

daughter cell. However, attachment of chromosomes to microtubules is a stochastic and

asynchronous event and, upon nuclear envelope breakdown at prometaphase, many

chromosomes experience improper attachments before successful bi-orientation. Such mis-

attachments include monotelic attachment (with one kinetochore of a chromosome attached to

microtubules from one pole and its sister unattached), syntelic attachment (with two sister

kinetochores attached to microtubules from the same pole), and merotelic attachment (with a

sister kinetochore attached to microtubules from both poles). These erroneous attachments, if

left undetected and uncorrected, can lead to chromosome mis-segregation and genomic

instability. Fortunately, they are detected by the SAC, which delays anaphase onset until all

mis-attachments are corrected. Next, we will address the mechanism by which the SAC halts

mitosis to prevent precocious sister chromatid separation. The components involved in the

The Spindle Assembly Checkpoint and Aneuploidy 61

SAC molecular pathway have been reviewed elsewhere (Cheeseman and Desai, 2008;

Musacchio and Salmon, 2007).

Sister chromatids are held together at the centromere by a ring-like structure consisting of

a complex of cohesin proteins synthesized in S phase (Marangos and Carroll, 2008;

Suijkerbuijk and Kops, 2008; Zhou et al., 2002). Their separation at the onset of anaphase

requires degradation of one of cohesin subunits, Scc1, which is promoted by the proteolytic

activity of separase (Bannon and Mc Gee, 2009; Bolanos-Garcia and Blundell, 2010;

Nasmyth, 2005; Przewloka and Glover, 2009). This caspase-like protein is normally kept

inactive by Securin. When all chromosomes are aligned at the metaphase plate with correct

bipolar attachment to spindle microtubules, Securin becomes ubiquitinated by the anaphase

promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase (Logarinho and

Bousbaa, 2008; Morgan, 1999; Reddy et al., 2007; Stegmeier et al., 2007). Ubiquitination

targets securin for degradation by the 26S proteasome (Decordier et al., 2008; Suijkerbuijk

and Kops, 2008). Separase is then activated and cleaves Scc1, no longer holding sister

chromatids together and, thus, anaphase begins (Bannon and Mc Gee, 2009; Bharadwaj and

Yu, 2004; Bolanos-Garcia and Blundell, 2010; Morgan, 1999; Nasmyth, 2005). Degradation

of cyclin B is also accomplished through APC/C-mediated ubiquitination, leading to the

inactivation of cyclin-dependent kinase 1 (Cdk1) and subsequent mitotic exit (Logarinho and

Bousbaa, 2008; May and Hardwick, 2006; Schmidt and Medema, 2006; Suijkerbuijk and

Kops, 2008).

The main downstream target of the spindle assembly checkpoint is Cdc20, a protein

required for APC/C activation. Once the nuclear envelope is broken down, SAC proteins,

namely Mad2, Bub3, and BubR1, are recruited to the outer kinetochore surface of all

unattached chromosomes. These proteins use the kinetochore as a platform to generate, in

near equal stoichiometry, the so called mitotic checkpoint complex (MCC) that diffuses

through the cytosol to prevent Cdc20 from activating the APC/C (Figure 1) (Sudakin et al.,

2001). This diffusible inhibitory signal is generated as long as unattached or mis-attached

kinetochores are present, hence preventing anaphase onset until all chromosomes achieve

correct attachment to bipolar spindle and align at the metaphase equator (Bharadwaj and Yu,

2004; Kops et al., 2005; May and Hardwick, 2006; Zich and Hardwick, 2010). Additionally,

according to the Mad2-template model, a closed conformation of Mad2 (C-Mad2) in complex

with Mad1 resides at unattached kinetochores and serves as receptor to convert cytosolic open

conformation of Mad2 (O-Mad2) into C-Mad2 bound to Cdc20. This latter leaves the

kinetochore and promotes inhibitory signal amplification by converting more O-Mad2 into C-

Mad2 in the cytosol (De Antoni et al., 2005). The C-Mad2 form is a more potent inhibitor of

APC/C in vitro given its higher affinity for Cdc20 (Chan et al., 2005; Musacchio and Salmon,

2007; Suijkerbuijk and Kops, 2008).

The nature of the signal that triggers SAC response is still controversial. It is more likely

to be the result of a redundant combination of both absence of kinetochore-microtubule

attachment and of the lack of physical tension between sister kinetochores (Bharadwaj and

Yu, 2004; May and Hardwick, 2006; Pinsky and Biggins, 2005).

When all chromosomes undergo bipolar attachments to spindle microtubules, tension

between sister kinetochores promotes SAC silencing and anaphase onset (Schmidt and

Medema, 2006; Zhou et al., 2002). Anaphase inhibitory complexes are then disassembled and


SAC proteins withdrawn from the kinetochores, both through free diffusion into the cytosol

and through motor protein-mediated transport along microtubules to the spindle poles (Lu et

al., 2009). Mad1 and Mad2 become undetectable at the kinetochores, while Bub1 and BubR1

levels are diminished three to four-fold (Chan and Yen, 2003; Zhou et al., 2002).

When cells are not capable of satisfying the SAC after a long mitotic arrest, they may

have different fates: some undergo apoptotic death during mitosis, others exit mitosis but die

via apoptosis in G1 phase, and others exit mitosis but are tetraploid and reproductively dead

(Niikura et al., 2007; Suijkerbuijk and Kops, 2008). In this context, Bub1 and BubR1 have

been shown to play an important role in eliminating cells that adapt to prolonged mitosis and

undergo defective mitotic events (Suijkerbuijk and Kops, 2008).

Figure 1. Molecular basis of spindle assembly checkpoint. Unattached kinetochore activates the SAC

(Checkpoint On) by recruiting the Mad2, BubR1, and Bub3. These proteins form the Mitotic

Checkpoint Complex (MCC), the diffusible inhibitory signal that sequesters Cdc20, keeping the APC/C

inactive thereby preventing it from targeting Securin and Cyclin B for degradation. As a consequence,

sister-chromatid cohesion is maintained and the cell cycle is arrested. The MCC disassembles

(Checkpoint Off) once all kinetochores become properly attached and aligned. Cdc20 is the free to

activate the APC/C which results in Securin and Cyclin B ubiquitination (U) and degradation. Securin

degradation leads to the activation of the protease Separase, which cleaves cohesin, leading to sister-

chromatid separation. Cyclin B degradation decreases the cyclin-dependent kinase (Cdk) 1 activity,

which results in mitotic exit.


SAC Relevance to Aneuploidy and Cancer

Aneuploidy is a common feature in human cancers. It has been more than one century

since Hansemann reported aberrant mitotic figures in cancer cells (Ando et al., 2010; Chi and

Jeang, 2007; Foijer, 2010) and Boveri first hypothesized an association between

chromosomal abnormalities and carcinogenesis (Fang and Zhang, 2011; Foijer, 2010;

Holland and Cleveland, 2009; Thompson et al., 2010). Indeed, genomic instability, frequently

manifested in the form of chromosomal instability (CIN) – a term that may refer to a loss or

gain of complete or partial chromosomes –, is a hallmark of many solid tumours (Fang and

Zhang, 2011; Lopez-Saavedra and Herrera, 2010; Thompson et al., 2010). Cells that have

undergone chromosome gain or loss are said to be aneuploid (Foijer, 2010). It is estimated

that 70-80% of cancers display some degree of aneuploidy, most of them showing both

numerical and structural chromosomal abnormalities (Foijer, 2010). Aneuploidy, resulting

from uncontrolled mitotic division, is believed to confer cells evolutionary advantage,

malignant potential and resistance to chemotherapy (Lopez-Saavedra and Herrera, 2010).

Since it facilitates the acquisition of oncogenes and/or the loss of tumour suppressor genes, its

connection with tumorigenesis was early anticipated (Foijer, 2010). Nevertheless, whether it

is a cause or a consequence of tumorigenesis is still a matter of debate (Holland and

Cleveland, 2009). Several pathways drive CIN in human cancers, but mitosis is the most

likely opportunity for chromosome loss and gain as a result of defects in sister chromatid

cohesion, kinetochore-microtubule attachment and dynamics, SAC activity, as well as an

abnormally elevated centrosome number (Foijer, 2010; Foijer et al., 2008; Lopez-Saavedra

and Herrera, 2010; Thompson et al., 2010). Mutations in genes encoding for regulators of

sister chromatid union were reported, possibly accounting for their premature separation or

for abnormal chromosome disjunction during anaphase (Fang and Zhang, 2011; Foijer, 2010;

Holland and Cleveland, 2009). Moreover, Separase depletion or overexpression was found to

induce tetraploidy, further substantiating the role of cohesion-related elements in CIN

(Thompson et al., 2010). Supernumerary centrosomes, arising from the deregulation of their

duplication cycle or as a consequence of tetraploidy, favor CIN by increasing the

establishment of merotelic interactions. In the presence of a multipolar spindle, the frequency

of anaphase lagging chromosomes rises significantly, surpassing the ability of correction

machinery to effectively repair them before anaphase onset (Fang and Zhang, 2011; Foijer,

2010; Ganem et al., 2009; Holland and Cleveland, 2009; Thompson et al., 2010). Since

perturbations affecting regulators of kinetochore-microtubule attachments (e.g., Aurora B,

Kif2b, MCAK and Hec1) stabilize their interactions, they make the correction of attachment

errors more difficult, contributing to CIN phenotype (Bakhoum et al., 2009; Fang and Zhang,

2011; Foijer, 2010; Green and Kaplan, 2003; Silkworth et al., 2009; Thompson et al., 2010).

Given the SAC essential role in controlling mitotic events and, thus, in maintaining

genomic stability, and since many tumor cells are aneuploid, mutations in SAC genes were

initially suggested as a possible molecular explanation for tumorigenesis. Mutated SAC genes

encoding for altered mitotic checkpoint proteins could explain its inefficiency and, therefore,

could allow for precocious chromosome segregation during mitosis, resulting in an

asymmetrical distribution of genetic material to the daughter cells. Many studies were carried


out in order to establish a causal connection between mutations in genes encoding for SAC

proteins and tumor development (Marchetti and Venkatachalam, 2010).

One of the most illustrative results is the one that links biallelic mutations in the SAC

BubR1-encoding gene, Bub1B, with mosaic variegated aneuploidy (MVA) (Chi and Jeang,

2007; Hanks et al., 2004; Lopez-Saavedra and Herrera, 2010; Rio Frio et al., 2010;

Suijkerbuijk and Kops, 2008; Suijkerbuijk et al., 2010; Thompson et al., 2010). MVA is a

rare autosomal recessive disease that is characterized by a high degree of aneuploidy, mild to

severe physical and mental limitations and a strong predisposition to cancer (Hanks et al.,

2004; Suijkerbuijk and Kops, 2008; Suijkerbuijk et al., 2010; Thompson et al., 2010; Yen and

Kao, 2005). Other studies have identified heterozygous mutations in Bub1 and BubR1 in a

panel of 19 aneuploid colorectal cancer cell lines (Cahill et al., 1999). Mutations have also

been found in Mad2-encoding gene, both in breast cancer (Percy et al., 2000) and gastric

cancer (Kim et al., 2005) cell lines.

However, many other attempts surprisingly failed to find SAC mutations, both in human

cancer cell lines and in tissue samples from oncologic patients (Yen and Kao, 2005). For

instance, none or few mutations were detected in Bub3, BubR1 and Bub1 genes in

glioblastoma, breast, lung, bladder and thyroid cancers (Fagin, 2002; Haruki et al., 2001;

Myrie et al., 2000; Olesen et al., 2001; Ouyang et al., 2002; Reis et al., 2001; Sato et al.,

2000). Bub1 gene mutations were shown to be a rare event in a study using 92 acute myeloid

leukemia specimens and 5 hematopoietic cell lines (Lin et al., 2002), in head and neck

squamous cell carcinoma and lung cell lines (Yamaguchi et al., 1999), in a series of

colorectal, hepatocellular and renal tumors (Shichiri et al., 2002) and in breast and gastric

carcinomas (Langerod et al., 2003; Shigeishi et al., 2001). Also, Mad1 gene was found to

carry few or no mutations in lymphomas, bladder, breast, gliomas (Tsukasaki et al., 2001) and

lung carcinomas (Nomoto et al., 1999), as does Mad2 gene in transitional-cell carcinomas of

the bladder, soft-tissue sarcomas, hepatocellular carcinomas (Hernando et al., 2001), breast,

lung (Gemma et al., 2001; Percy et al., 2000; Takahashi et al., 1999) and digestive tract (Imai

et al., 1999) cancer cells. In addition, no mutations were found in the coding sequences of

Mad2 gene in 11 hepatoma cell lines (Sze et al., 2004). In a study of 8 hepatocellular

carcinoma cell lines and 50 hepatocellular carcinoma specimens, although some polymorphic

base changes were noticed in Bub1, BubR1 and Cdc20, no mutations accountable for SAC

impairment were detected neither in these genes nor in Bub3 or Mad2B (Saeki et al., 2002). It

should be noted that these studies did not cover all possible known SAC genes, meaning that

some of their mutations might be yet to unveil. Furthermore, in the vast majority of cases, the

effect of the gene mutation was not studied at the protein level. Further quantitative and

subcellular localization assays are thus needed to clarify the actual impact of these mutations

(Yen and Kao, 2005). Nevertheless, the low frequency of mutations affecting SAC genes

indicates that they are not the main mechanism through which cells may become aneuploid

(Lopez-Saavedra and Herrera, 2010; Schvartzman et al., 2010; Thompson et al., 2010).

Subsequently, studies concerning expression of SAC components, both at the gene and

protein level, have gained attention, suggesting a correlation between altered expression

levels, compromised SAC activities and tumorigenesis (Chi and Jeang, 2007; Fang and

Zhang, 2011; Holland and Cleveland, 2009; Kops et al., 2005; Suijkerbuijk and Kops, 2008).

The SAC efficiency can be easily evaluated as the ability of a given cell population to sustain


a prolonged mitotic arrest upon exposure to chemical compounds that interfere with

microtubule polymerization and dynamics. Such evaluation has been performed in large

panels of tumor cell lines, as well as in histological samples collected from numerous

patients. SAC impairment was implicated in the resistance to anti-microtubule agents-induced

apoptosis in human lung cancers (Masuda et al., 2003), as well as in breast cancer (Yoon et

al., 2002) and in head and neck squamous cell lines, in which it may contribute to

chromosomal instability (Minhas et al., 2003). Most studies have sought for a molecular

explanation for the SAC weakening. Overexpression of SAC components seems to be more

frequent (Foijer, 2010; Holland and Cleveland, 2009; Liu et al., 2009). Even subtle deviations

in SAC mRNA and protein levels were shown to drive tumorigenesis (Bharadwaj and Yu,

2004). Low BubR1 levels, resulting from biallelic mutations in the Bub1B gene, were

associated with chromosome alignment and segregation defects (Suijkerbuijk et al., 2010); a

significantly reduced Bub1B expression was ascertained as the causative event of aneuploidy

in colorectal adenocarcinomas (Burum-Auensen et al., 2008). On the other hand, Bub1B

expression was shown to be high in 25.9% of 27 salivary duct carcinomas, although it had no

prognostic significance (Ko et al., 2010). Additionally, BubR1 overexpression was

documented in oesophageal squamous cell (Tanaka et al., 2008), thyroid (Wada et al., 2008),

hepatocellular (Liu et al., 2009) and squamous cell carcinomas (Hsieh et al., 2010), as well as

in lung cancers (Seike et al., 2002), where it has been associated with worse prognosis,

carcinogenesis progression and suggested as a possible compensatory mechanism that could

represent a potential tumor biomarker (Hsieh et al., 2010). BubR1 overexpression was

reported in 50.3% of 181 gastric cancer samples, correlating significantly with aneuploidy,

tumor invasiveness, metastasis likelihood and poor prognosis (Ando et al., 2010), and in 68%

of 43 gastric carcinomas (Grabsch et al., 2003). BubR1 overexpression was found to be

closely related to chromosomal instability in bladder cancer (Yamamoto et al., 2007) and in

clear cell kidney carcinomas (Pinto et al., 2008). Although some studies point to diminished

expression and aberrant transcription of the hBub1 gene (Lin et al., 2002), its expression was

found to be up-regulated in follicular thyroid adenomas when compared to adjacent normal

tissues (Wada et al., 2008). Along with that of Bub1B, Bub1 overexpression was also found

in a large panel of breast tumor samples and proposed to be implied in the transition of breast

tissues from normal to benign tumors (Bieche et al., 2011). Bub1 overexpression was also

demonstrated, both at mRNA and protein levels, in salivary gland tumors, where it

contributes to abnormal cell proliferation (Shigeishi et al., 2006). Gastric cancers also

overexpress Bub1 in 84% of 43 samples under analysis (Grabsch et al., 2004). Similar

alterations were detected in Mad2 expression. Mad2l1 gene up-regulation was reported in

Familial Adenomatous Polyposis colorectal adenomas, suggesting that this up-regulation is

associated with adenomatous polyposis coli (APC) gene mutation and may constitute an early

event in colorectal carcinogenesis (Abal et al., 2007). Pronounced Mad2 overexpression has

also been documented in advanced differentiated thyroid carcinomas (Wada et al., 2008),

salivary duct carcinomas (Ko et al., 2010) and lung cancers, in which it has been correlated

with enhanced aggressiveness, shorter survival and identified as a prognostic factor (Kato et

al., 2011). Up-regulated Mad2l2 expression, both at gene and protein levels, was found in

21% of 118 colorectal tumor samples, in which it has been suggested to promote mitotic

aberrancies, chromosomal instability and reduced patient survival (Rimkus et al., 2007). Also,


Mad2 protein overexpression, concomitant with that of Aurora A and Aurora B, was

observed in aneuploid colorectal adenocarcinomas (Burum-Auensen et al., 2008), as well as

in a series of 6 oesophageal squamous cell carcinoma (ESCC) cell lines and 21 ESCC

patients, along with that of BubR1 (Tanaka et al., 2008). Inversely, Mad2 protein levels were

found to be lower in colon cancer cell lines that had been exposed to deoxycholate, a

hydrophobic bile acid associated with cancer risk (Payne et al., 2010). Mad2 decreased levels

were suggested to contribute to an escape from cell death, thus leading to tumorigenesis

(Payne et al., 2010). Mad2 protein was also shown to be underexpressed in 75% of 8

testicular germ cell tumour (Fung et al., 2007) and in 54.5% of 11 aneuploid hepatoma tumor

cell lines that failed to arrest in mitosis (Sze et al., 2004).

Mouse Models Link SAC Dysfunction,

Aneuploidy and Cancer

To further investigate the role of the SAC components in checkpoint signaling and in the

prevention of chromosomal imbalance, mouse models lacking SAC genes were created

(Foijer, 2010; Foijer et al., 2008; Li et al., 2009; Schvartzman et al., 2010; Suijkerbuijk and

Kops, 2008; Thompson et al., 2010; Yen and Kao, 2005). Conventional gene knockouts have

been constructed for almost all SAC known genes, including Mad1, Mad2, Bub1, BubR1,

Bub3 and CENP-E, and hypomorphic alleles for Bub1 and BubR1 have been generated

(Holland and Cleveland, 2009). In spite of being compatible with viability and fertility, their

downregulation increased cancer susceptibility and caused aneuploidy in mouse embryonic

fibroblasts and tissues, in a degree that was dependent on the knocked-out gene and on the

extension to which its expression had been decreased (Holland and Cleveland, 2009; Lopez-

Saavedra and Herrera, 2010). Mouse embryonic cells lacking Mad2 displayed a dysfunctional

SAC, leading to chromosome missegregation and apoptosis (Dobles et al., 2000). BubR1 (+/-

) mouse embryonic fibroblasts were proven to defectively activate SAC and to have reduced

amounts of Securin and Cdc20, predisposing mice to rapid development of lung and intestinal

adenocarcinomas and supporting BubR1 role as a tumor suppressor (Dai et al., 2004). In turn,

although there was not a higher susceptibility to tumor formation, perhaps because of the

presence of a partially functional SAC, increased aneuploidy and premature sister chromatid

segregation were reported in Bub3-haploinsufficient mouse embryonic fibroblasts (Kalitsis et

al., 2005). Analysis of mutant mice has shown Bub1 to be essential in preventing malignant

cell transformation and in mediating cell death upon chromosome missegregation (Jeganathan

et al., 2007).

SAC Components and Perspectives

in Anticancer Therapy

The developments in the knowledge in cell cycle regulation and control have allowed the

conception of several pharmacological approaches aiming at stopping tumor cell


proliferation. Abnormal cell proliferation is frequently associated with altered expression

levels of cell cycle-regulating proteins or with alterations in checkpoint mechanisms. These

alterations constitute an advantage not only for tumor cell progression, but also for the

acquisition of increasingly aggressive phenotypes (De Falco and De Luca, 2010).

Given the role of microtubules and SAC proteins on chromosome segregation and cell

division accuracy, these have become the main targets of anti-cancer treatment strategies.

Current chemotherapy approaches use microtubule-targeting agents (MTAs). MTAs affect

the dynamic equilibrium between microtubule polymers and tubulin heterodimers, and have

been used with a considerable degree of success in a wide range of tumors (Bannon and Mc

Gee, 2009; Fojo and Menefee, 2007; Zhou and Giannakakou, 2005). Vinca alkaloids and

taxanes, like paclitaxel and docetaxel, are amongst the most used drugs in cancer treatment.

The first have an inhibitory action in microtubule polymerization, while the latter stabilize

microtubules. Their effects are more pronounced in mitotic cells, which explains their

classification as anti-mitotic drugs (Yamada and Rao, 2010).

Vinca alkaloids were extracted from Vinca rosea (also known as Catharanthus roseus),

and promote microtubule depolymerization, which causes a prolonged mitotic arrest and

subsequently results in cell death. They are used in solid and hematological tumor treatment

(Jordan and Wilson, 2004; Perez, 2009). Taxanes paclitaxel and docetaxel were isolated from

Taxus brevifolia and Taxus baccata, respectively. They stabilize microtubule polymerization

by interfering with their dynamics, which blocks cell cycle at G2/M phase, leading to cell

death. Taxanes are highly efficient, for instance, in lung, breast and head and neck squamous

carcinomas. MTAs are used either alone or combined with other cytotoxic agents (Perez,

2009). They act by interfering with mitotic spindle dynamics, thereby inducing SAC

activation and mitotic arrest (Zhou and Giannakakou, 2005). However, even though mitotic

arrest avoids cell proliferation, it does not necessarily end in cell death (Riffell et al., 2009).

Some cells are capable of escaping mitosis in the absence of chromosomal segregation or cell

division, in a process termed SAC adaptation or mitotic slippage. These cells exhibit multiple

nuclei and polyploidy, both potential tumorigenesis stages. Accordingly, the fates of mitosis-

arrested cells are still unknown (Riffell et al., 2009). In this respect, small-molecule inhibitors

of the APC/C produce a more efficient retention in mitosis than MTAs do, since proteolysis is

APC/C-dependent and required for mitotic slippage. For this reason, APC inhibitors may

constitute a more powerful strategy to induce and sustain mitotic arrest (Zeng et al., 2010).

Since many tumor cells present defective SAC activity, they sometimes do not respond to

mitotic errors, which affects MTAs efficacy (Bolanos-Garcia, 2009). Hence, the development

and optimization of new therapeutic strategies that target SAC proteins may contribute to a

more successful treatment. While a defective SAC may contribute to aneuploidy and CIN

(Figure 2), a more expressive suppression of its activity leads to cell death as a result of

massive chromosome mis-segregation. In this regard, from all protein kinases validated for

this purpose, those that act in the SAC, in particular Bub1, BubR1 and Mps1, constitute main

drug targets (Bolanos-Garcia, 2009). Therapeutic approaches could thus make use of the

specific targeting of SAC-defective tumor cells.

RNAi-mediated Mps1 depletion has allowed for a decrease in tumor cell viability

(Janssen et al., 2009; Janssen et al., 2011). Mps1 is a crucial SAC component that monitors

chromosome alignment; as such, it influences the stability of kinetochore-microtubule


interactions (Colombo and Moll, 2010). Its inactivation compromises the SAC, originating

alignment errors and decreasing cell viability. In vitro assays have unveiled an orally

bioavailable Mps1 small-molecule inhibitor that selectively reduces tumor cell progression

(Colombo et al., 2010). When compared to cancer cells, normal cells were significantly less

sensitive to Mps1 inhibition, whether through siRNA or by treatment with specific small-

molecule-based inhibitors. If proven to occur in vivo, this difference could illustrate the

potential of SAC selective inhibitors as a therapeutic approach (Colombo and Moll, 2010).

These results corroborate that Mps1 may constitute a suitable target to preferentially eliminate

cancer cells (Colombo et al., 2010; Janssen et al., 2009; Janssen et al., 2011).

Inhibitors that specifically interfere with Aurora kinase activity significantly decrease

viability of cells in rapid division, resulting in a 98% reduction in tumor volume in nude mice

injected with human leukemia cells (Bolanos-Garcia, 2009). In fact, several studies have

demonstrated that cells with abnormal Aurora A or Aurora B expression have mitotic spindle

defects and do not undergo cytokinesis. For that reason, interfering with Aurora kinase

activity has been suggested as a possible strategy for cancer treatment (De Falco and De

Luca, 2010; Deep and Agarwal, 2008). Diverse small-molecule Aurora kinase inhibitors are

under development in clinical trials; pan-Aurora kinase inhibitor Danusertib (PHA-739358)

was the first to be tested in humans (Colombo and Moll, 2010).

Apoptotic cell death was observed when BubR1 or Mad2 protein levels were decreased

or when BubR1 kinase activity was blocked in human cancer cells. Unless when cytokinesis

was also inhibited, apoptotic cell death took place within six cell divisions (Kops et al., 2004;

Michel et al., 2004). This can also be explored in order to inhibit tumor cell proliferation

(Kops et al., 2004; Michel et al., 2004).

Figure 2. Contribution of SAC impairment to aneuploidy. A fully functional SAC ensures accurate

chromosome segregation and genomic stability. In cells with weakened SAC, however, occasional mis-

segregations may escape SAC control and lead to aneuploidy.


A possible strategy consists in Mad2 silencing. siRNA nanoparticle-based strategies were

already tested, leading to an unequal division of genetic material and, consequently, to

apoptosis in colon carcinoma cells (Kaestner et al., 2011). Assays using nude mice to which

Mad2 siRNA containing nanoparticles were systemically administered showed a decrease in

tumor growth, suggesting SAC inhibition as a promising concept for anticancer research

(Kaestner et al., 2011). However, therapeutic strategies regarding SAC inhibition through

RNAi have not been well explored to date (Kaestner et al., 2011).

Conclusion

In spite of their satisfactory pre-clinical effectiveness, the clinical efficacy of the

compounds that interfere with cell cycle falls short of expectations. SAC inhibitors are not

completely efficient by themselves, so they must be tested in combination with standard

chemotherapeutic drugs because of their vast clinical application. Therefore, a reasonable

anti-cancer approach would be the combination of cell cycle-based agents with conventional

chemotherapy, so that the resistance to the drug could be minimized (De Falco and De Luca,

2010; Zhou and Giannakakou, 2005).

Acknowledgments

This work was supported by grant 02-GCQF-CICS-2011N, from Cooperativa de Ensino

Superior Politécnico e Universitário (CESPU), and by grant PTDC/SAU-FCF/100930/2008

from Fundação para a Ciência e Tecnologia (FCT).

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The Spindle Assembly Checkpoint and Aneuploidy