Chapter 8

Kinesin-13 Microtubule Depolymerizing

Proteins as Targets for Cancer Therapy

Anutosh Ganguly and Fernando Cabral

8.1 Introduction

The kinesin-13 family of molecular motors contains three closely related members:
Kif2a, Kif2b, and Kif2c (also commonly called mitotic centromere associated
kinesin or MCAK). Unlike most kinesin motors that use ATP to power the transport
of cargo along microtubules, kinesin-13 proteins use ATP to catalyze the loss of
tubulin subunits from microtubule ends [1]. These motors differ structurally from
conventional kinesins by having an internal, rather than N- or C-terminal, motor
domain leading to their alternate designation as Kin I (for internal) or M-type (for
middle) kinesins [2, 3]. All three kinesin-13 motors are present during interphase,
but their main activity is seen during mitosis when they are believed to play overlapping
yet distinct roles in cell division [4].
Elevated levels of kinesin-13 family members have been found in an increasing
number of patients with a variety of cancers, and the levels of these proteins appear
to predict tumor aggressiveness and poor patient survival. Altered levels of Kif2c
have also been shown to affect the sensitivity of cells to microtubule inhibitors, a
class of chemotherapeutic drugs that has been successfully used against various
forms of cancer for the last 50 years. Because a number of excellent reviews on
kinesin-13 motors have recently been published [5–7], this article will focus on the

A. Ganguly
Department of Microbiology and Infectious Diseases, Snyder Institute, University of Calgary,
Calgary, AB T2N4Z6, Canada
F. Cabral (*)
Department of Integrative Biology and Pharmacology, University of Texas Medical School,
Houston, TX 77030, USA
e-mail: fernando.r.cabral@uth.tmc.edu

© Springer Science+Business Media Dordrecht 2015 117

F. Kozielski (ed.), Kinesins and Cancer, DOI 10.1007/978-94-017-9732-0_8
118 A. Ganguly and F. Cabral

potential for using these proteins as markers for tumorigenesis and metastasis, as
well as on the possibility of targeting them to treat cancer and overcome some forms
of drug resistance.

8.2 Cellular Localization

During interphase Kif2a is primarily found in the centrosome. In mitosis it appears on

spindle microtubules but remains concentrated in the spindle poles (centrosomes).
Kif2a localization was also reported in the spindle midzone and midbody during
anaphase and telophase. Trace amounts of Kif2a were detected in kinetochores, but
its presence was only apparent after microtubules were depolymerized with drugs
like nocodazole [8].
Kif2b also localizes most strongly to centrosomes during interphase and to spindle
poles during mitosis, but can additionally be found associated with microtubules [4].
There is some staining of kinetochores that is most clearly seen during prophase
and diminishes as cells progress to metaphase. In the later stages of mitosis, it is
associated with the midbody region as well as separating spindle poles. Because
Kif2b is a low abundance protein in most cells, studies were carried out using
GFP-Kif2b and thus, the localization should be regarded as tentative because other
studies have shown that GFP fusions with proteins such as EB1 or MCAK can lead
to alterations in localization and/or degradation [9, 10]. Although kinesins generally
bind to microtubules via a direct interaction with their motor domains, it was
recently shown that Kif2b binding to spindle fibers is substantially reduced when
Cep170 is depleted [11]. This finding opens up the possibility that the localization
and activity of the kinesin-13 family of motor proteins may be regulated in part by
binding partners.
During interphase, MCAK (Kif2c) is transported into the nucleus by an import
signal-mediated process [12]; however, a substantial fraction remains in the cyto-
plasm where it is attached to the centrosome and is weakly bound to microtubules
[13]. In prophase, it is found predominantly in kinetochores and spindle poles but is
again weakly associated with microtubules. It remains bound to these structures
during prometaphase, but once chromosomes are aligned at metaphase, kinetochore
staining becomes almost undetectable and staining of spindle poles and fibers is
greatly reduced. Analysis of synchronized CHO cells stably transfected with
flag-tagged human MCAK indicates that the protein is synthesized throughout the
cell cycle, reaches its highest accumulation in mitosis, and is then rapidly degraded
at the metaphase to anaphase transition [13]. Immunofluorescence microscopy
demonstrated that FLAG-tagged MCAK parallels the behavior of the endogenous
protein and is only weakly associated with the mitotic spindle and midbody following
metaphase. In contrast, GFP-tagged MCAK has been shown to be strongly associated
with the spindle and chromosomal structures throughout mitosis [14]. These differences
in localization can potentially be explained by the finding that fusion with GFP
prevents the degradation of MCAK [10].
8 Kinesin-13 Microtubule Depolymerizing Proteins as Targets for Cancer Therapy 119

8.3 Function

Kif2a has been shown to catalyze microtubule depolymerization [1], and its
depletion arrests cells in mitosis [8]. Cells depleted of Kif2a are defective in spindle
pole separation and accumulate monopolar spindles. In addition, very long microtu-
bules are formed that include those that are found in the central spindle [8, 15].
Interestingly, it was discovered that nocodazole treatment of cells lacking Kif2a
partially restores spindle bipolarity, suggesting that Kif2a depleted cells have some
form of hyperstabilized microtubules [4, 8]. Depletion of MCAK partially restores
bipolar spindle assembly in Kif2a-depleted cells [8]. This observation suggests that
Kif2a acts together with MCAK to control bipolar spindle assembly, but each
performs a distinct and potentially antagonistic function that necessitates an optimal
balance between the two proteins.
Kif2b is a very low abundance protein that is primarily located in the outer kinet-
ochore during prometaphase where it has been shown to correct kinetochore micro-
tubule attachment errors during the early stages of mitosis [4, 16]. The recruitment
of Kif2b to kinetochores is mediated by CLASP1 in a process that appears to be
regulated by Aurora B kinase. A high concentration of Aurora B on the kinetochore
during prometaphase promotes the binding of Kif2b to CLASP1; but as kineto-
chores on sister chromatids stretch apart when the chromosomes congress at the
metaphase plate, they are exposed to a lower Aurora B activity that allows astrin to
displace Kif2b from CLASP1. Under these high tension conditions, kinetochore
microtubules become more stable and MCAK may then provide the activity needed
to correct any further improper attachments as cells approach metaphase [17].
In support of this idea, it was recently shown that MCAK is required in the final step
of a mechanism needed to convert a kinetochore that is bound to the lateral wall
of a microtubule to an end-on microtubule attachment [18]. It should be noted
that another study did not find a stable association of Kif2b with CLASP1 but did
find an association with Cep170, a microtubule binding protein that could provide
an additional binding target for Kif2b [11]. It thus appears that Kif2b localization
may involve multiple proteins and the regulation of its activity may be controlled by
phosphorylation and perhaps other posttranslational modifications.
In addition to its role in correcting aberrant chromosome attachments, Kif2b is
needed for bipolar spindle assembly. Depletion of Kif2b leads to a high incidence of
monopolar spindles that arise transiently but then progress to bipolarity. Nonetheless,
Kif2b depletion produces cells with an increased frequency of lagging chromo-
somes, presumably due to persistent errors in kinetochore microtubule attachment
[16]. Similar to the ability of MCAK depletion to restore spindle bipolarity in kif2a
depleted cells [8], depletion of MCAK was again able to partially restore bipolarity
in Kif2b depleted cells, but it was not able to prevent the increased frequency of
lagging chromosomes in those cells. Although the mechanism for this effect is
unknown, the observations indicate that Kif2b and MCAK may have overlapping
functions in correcting kinetochore microtubule attachment errors but could
potentially also have antagonistic functions needed for bipolar spindle assembly.
120 A. Ganguly and F. Cabral

The possibility of overlapping functions in correcting chromosome misalignment is

supported by the observation that overexpression of Kif2b or MCAK (but not Kif2a)
has been shown to suppress the incidence of lagging chromosomes in tumor cell
lines [16].
Kif2c/MCAK is the most well studied member of the kinesin-13 family and is
named for its prominent location in the centromeres of mitotic chromosomes.
Like other members of this family, it is a homodimeric protein with microtubule
depolymerizing activity. Unlike conventional kinesins that have N- or C-terminal
motor domains, MCAK lacks the ability to transport cargo. Instead, it binds to
microtubules and diffuses bidirectionally to the microtubule ends where it can
catalyze microtubule depolymerization by promoting a curved conformation of the
protofilaments [19–21]. The presence of lagging chromosomes in MCAK-inhibited
cells led to an early suggestion that, during anaphase, the protein might be responsible
for poleward movement of sister chromatids by catalyzing microtubule depolymer-
ization at the kinetochores using a Pacman-like mechanism [14]. However, later
studies indicated that lagging chromosomes result from improper kinetochore
microtubule attachments formed earlier in mitosis [22], and the role of MCAK is to
sever the improper microtubule attachments and thus allow chromosomes to correctly
align at the metaphase plate [23]. This view is consistent with the observation that
the protein is largely degraded at the metaphase-to-anaphase transition [13].
Given its prominent role in ensuring faithful chromosome segregation, it is not
surprising that MCAK is essential for normal mitotic progression. Depletion of
MCAK increases the mitotic index and produces cells with longer and more abundant
astral fibers, resulting in a “hairy” spindle morphology [24, 25]. These abnormal
spindles may potentially be explained by the observations that MCAK stimulates
kinetochore microtubule turnover [26] and stimulates microtubule detachment from
centrosomes and spindle poles [27]. Although depletion of MCAK hinders mitotic
progression, many cells nevertheless escape the mitotic checkpoint with missegre-
gated chromosomes resulting in an increase in aneuploidy [23, 24].
For normal mitosis to occur, MCAK must be tightly regulated and this is
accomplished by posttranslational modifications such as phosphorylation and by
regulating the degradation of the protein. Although moderate levels of overexpression
(e.g. two times endogenous) can be tolerated, higher levels (6–8 times endogenous or
more) inhibit cell proliferation [27]. These high levels of expression delay CHO
cells in prometaphase, deplete microtubules, disrupt spindle assembly, and lead to
the formation of unstable multinucleated cells. MCAK was found to continuously
accumulate starting in G1 phase and reach its maximum level during the early stages
of mitosis. It is then degraded at the metaphase to anaphase transition by the protea-
some [13]. The signal for degradation appears to involve phosphorylation of residue
S628 by an unknown kinase, and this phosphorylation may additionally trigger
release of MCAK from the kinetochore once the chromosome has aligned on the
metaphase plate [10]. It was speculated that degradation of MCAK serves not only
to maintain a proper protein levels, but also to prevent the carryover of multiphos-
phorylated species of the protein created during mitosis into the next cell cycle.
8 Kinesin-13 Microtubule Depolymerizing Proteins as Targets for Cancer Therapy 121

MCAK activity is also regulated by phosphorylation through the action of

multiple kinases including Aurora A, Aurora B, cdk1/cyclin B1, and Plk1 [28].
For example, it has been found that Aurora B phosphorylates human MCAK at S95
and S192, sites that were originally identified as T95 and S196 in Xenopus MCAK [29].
The mutation S95A changes kinetochore binding affinity while the mutation S192A
reduces the preference of MCAK for the kinetochore, increases its localization
along chromosome arms, and inhibits its microtubule depolymerase activity [29–31].
Recently, it was shown that Aurora A kinase also phosphorylates MCAK in its
C-terminal region to control ran-dependent spindle bipolarity [32]. A more detailed
discussion of the effects of MCAK phosphorylation can be found in several excellent
reviews [5, 33, 34].

8.4 Clinical Studies of Kinesin-13 Proteins

There is growing evidence that kinesin-13 family proteins could play a role in
tumorigenesis. Kinesin-13 s are needed to assemble a normal spindle apparatus and
to ensure the fidelity of chromosome separation during mitosis. Abnormalities in
mitotic progression can cause aneuploidy that can further lead to transformation of
normal somatic cells into cancer cells. It is therefore not surprising that overexpres-
sion of kinesin-13 s has been reported in a number of metastatic tumors. For exam-
ple, Kif2a was found to be overexpressed in 70 % of 44 patients with oral carcinoma,
and high expression was associated with lymph node metastasis [35]. A possible
role for the protein in cancer cell invasion was strengthened by an ex-vivo experiment
showing that a tongue squamous cell carcinoma cell line (Tca8113) transfected with
si-RNA to deplete Kif2a exhibited decreased motility [35]. Similarly, transfection
with miR-183, a micro RNA that decreased Kif2a levels and inversely correlated
with the metastatic potential of lung cancer cells, inhibited the migration and
invasion capacity of HeLa cells [36].
Overexpression of MCAK has also been reported in tumors, most notably those
of the breast and gastrointestinal track. MCAK was among the proteins found to be
overexpressed in breast tumor cells [37] and a later microarray analysis of samples
derived from 81 patients showed high levels of MCAK in breast tumors but unde-
tectable levels in normal tissues except the testis [38]. The latter authors further
demonstrated that siRNA-mediated depletion of MCAK inhibited the proliferation
of T47D and HBC5 breast cancer cell lines and that MCAK expression could be
suppressed by p53.
Experiments using RT-PCR revealed elevated MCAK in 66 % of 65 patients with
gastric cancer and the high levels were linked to lymphatic invasion, lymph node
metastasis, and poor prognosis [39]. Moreover, a gastric cancer cell line stably
transfected with MCAK exhibited increased proliferation and greater motility in
trans-well assays. Similarly, MCAK was overexpressed in 91 of 120 patients with
colon cancer where it was linked to node metastasis, venous invasion, peritoneal
dissemination, and poor survival [40]. A second study involving 176 patients also
122 A. Ganguly and F. Cabral

saw increased MCAK expression in colorectal cancer as well as in pancreatic,

gastric, breast, and head and neck cancer [41]. The authors examined expression of
NY-CO-58, a tumor antigen derived from MCAK [42], and found it to be elevated
in all tumor types, but most strongly in breast, colorectal, and gastric cancer.
Moreover, nearly 50 % of colorectal patient peripheral blood mononuclear cells
(PBMCs) exposed to peptide antigens exhibited CD4+ T cell responses, suggesting
that MCAK could be used for immunological approaches to treatment. This possi-
bility was further strengthened by direct experiments showing that a cytotoxic T-cell
response against cells with high MCAK expression could be elicited by ex-vivo
exposure of PBMCs to peptide antigens derived from MCAK [43].
More recent studies point to the possibility that elevated expression of MCAK
and perhaps other kinesin-13 proteins will be associated with many other types of
cancer. High MCAK levels have been reported in castration-resistant and
chemotherapy-resistant prostate cancer, and the knockdown of MCAK led to the
growth arrest of prostate cancer cells in culture [44]. MCAK was also shown to be
upregulated an average of fourfold in glioma samples taken from 40 patients [45].
High levels were associated with a poor survival rate and it was suggested that
MCAK could be used as a marker for prognosis.
Despite the increasing number of reports showing elevated kinesin-13 proteins in
tumor samples, a causal link between these changes and tumor progression has not
been firmly established. It was shown that MCAK levels rise during the cell cycle
and become maximal at mitosis [13]. Other kinesin-13 family members may exhibit
similar patterns of expression. Thus, the elevated levels of these proteins in tumors may
simply reflect the higher mitotic and proliferative activity of tumor versus normal
tissue. Nonetheless, high expression of MCAK in tumors and the appearance of
peptides on the cell surface could open the possibility of targeting MCAK using
immunotherapy. In addition, the strong correlation between elevated MCAK and
poor survival in cancer patients could make this protein a useful prognostic marker.

8.5 Role of Kinesin-13 Proteins in Cancer Progression

From the preceding discussion it is evident that kinesin-13 proteins play important
roles in cell division by maintaining spindle integrity and normal spindle function.
Overproduction as well as depletion of kinesin-13 proteins can affect spindle assem-
bly demonstrating that the levels and activity of these proteins must be tightly
regulated to ensure normal cell division. Kif2b and MCAK appear to act primarily
at kinetochores to correct aberrant microtubule attachments. Thus, changes in the
activities of these proteins would be expected to delay the congression of chromo-
somes to the metaphase plate, engage the mitotic checkpoint and ultimately lead to
apoptosis. However, depending on the severity of the defect, some cells might slip
through the checkpoint and continue to progress through the cell cycle as aneuploid
cells with unstable genomes that ultimately lead to cancer. The potential link
8 Kinesin-13 Microtubule Depolymerizing Proteins as Targets for Cancer Therapy 123

between kinesin-13 proteins and cancer progression was recently strengthened by

the identification of a nonsynonymous single-nucleotide polymorphism in the
coding region of MCAK that is strongly associated with colorectal cancer [46] and
by the finding that overexpression of Kif2a is associated with tumor progression and
metastasis [35]. Kif2a is primarily located at spindle poles and it appears to mainly
act to promote spindle bipolarity. Thus, alterations in Kif2a activity can also cause
defective mitotic progression and the inaccurate segregation of chromosomes that
ultimately promote tumorigenesis.

8.6 A New Role for Kinesin-13 s in Microtubule

Detachment from Spindle Poles

Kinesin-13 family proteins are thought to act by altering microtubule dynamics that
may be necessary for bipolar spindle assembly and proper microtubule attachment
to the kinetochores of segregating chromosomes. This view appears to be consistent
with experiments showing that kinesin-13 proteins increase the frequency of catas-
trophe and reduce the frequency of rescue at microtubule plus-ends [25] and that
MCAK or Kif2b inhibition reduces kinetochore fiber microtubule turnover [16, 26,
47]. However, some recent observations have begun to question the role of microtu-
bule dynamics in mitosis. While it remains likely that some level of microtubule
dynamicity is needed for successful cell division, a scan of the literature reveals that
cells can have widely different levels of dynamicity yet all the cells divide normally
[48–51]. More recent studies show that cells treated with low concentrations of
microtubule drugs exhibit suppressed microtubule dynamics but no change in their
ability to proliferate [52]. Conversely, paclitaxel-dependent cell lines with near
normal microtubule dynamics cannot proliferate when the drug is absent, but are
able to proliferate with suppressed dynamics when the drug is present [53]. These
observations led the authors to conclude that microtubule dynamics are not as
important for mitosis as is currently thought, and to look for other actions of the
drugs that might explain their ability to block cell division. It was found that low drug
concentrations that suppressed microtubule dynamics did not inhibit cell division
but strongly inhibited cell migration. The higher drug concentrations needed to
inhibit cell division in normal cells and promote cell division in paclitaxel-dependent
mutants, on the other hand, affected the detachment of microtubules from centro-
somes and spindle poles.
Microtubule detachment is a poorly studied but important phenomenon that is
cell cycle regulated. The frequency of detachment is low during interphase but
greatly increases when cells enter mitosis [52, 54]. The frequency of detachment is
also increased by treatment with drugs such as vinblastine, colchicine, nocodazole,
and others that inhibit microtubule assembly; but it is suppressed by drugs such as
paclitaxel and epothilones that promote microtubule assembly [52, 53]. Detached
microtubules are relatively short-lived as they can depolymerize from both
124 A. Ganguly and F. Cabral

plus- and minus-ends, and they are able to translocate away from the centrosome
by an as yet unknown mechanism [52, 55]. These actions generate microtubule
fragments of variable length that may play a critical role in microtubule turnover
and spindle formation.
Although the mechanism of microtubule detachment is not yet well understood,
recent experiments have implicated kinesin-13 family members that are located at
the centrosome. It is noteworthy that MCAK function at kinetochores is well
studied, but its function at the spindle poles is still largely unexplored. There is
now strong evidence that one of its roles is to catalyze microtubule detachment.
This finding is consistent with the ability of MCAK to catalyze catastrophe from
both plus- and minus-ends of the microtubule, and with its elevated activity during
mitosis due to cell cycle dependent accumulation. Direct evidence that MCAK is
involved in microtubule detachment came from experiments showing that the
frequency of detachment was elevated in cells overexpressing MCAK [27] and
suppressed in cells depleted of MCAK [24]. Moreover, depletion of MCAK was
able to reverse the paclitaxel-dependence phenotype and allow the proliferation of
CHO cell lines with elevated frequencies of detachment resulting from mutations in
tubulin or overexpression of a neuronal and testis specific β-tubulin isotype. It is
noteworthy that growth of the paclitaxel dependent cell line with the stronger
phenotype was only partially restored by MCAK depletion, suggesting that MCAK
may be only one of multiple proteins at spindle poles that are involved in microtubule
detachment. Given its strong localization to spindle poles, Kif2a may be another
kinesin involved in detachment, but this prediction is yet to be tested.

8.7 Kinesin-13 Affects Sensitivity to Microtubule Inhibitors

The involvement of kinesin-13 proteins such as MCAK in microtubule detachment

has important therapeutic implications. While most current models of the mitotic
spindle envision continuous microtubules connecting spindle poles with chromo-
somes, some older work on mitotic spindles along with more recent studies of
meiotic spindles have detected the presence of numerous microtubule fragments and
this has led to the idea that spindle fibers consist of both continuous and overlapping
fragmented microtubules connected by motor proteins [56–59]. Some of these frag-
ments appear to be nucleated from the sides of existing microtubules catalyzed by a
protein called augmin [60], but it is likely that microtubule detachment also plays an
important role in their formation. The premise for therapy directed at the mitotic
spindle is that a proper mixture of fragmented and continuous microtubules is
needed for successful cell division. Too many, very short fragments may produce a
weak unstable spindle; but too few fragments may produce an overly stable spindle
that is unable to remodel during the various stages of mitosis.
Given the ability of proteins like MCAK to catalyze microtubule detachment,
one would predict that inhibition of its protein level or activity would lead to
8 Kinesin-13 Microtubule Depolymerizing Proteins as Targets for Cancer Therapy 125

hyperstable spindles whereas overexpression or activation of MCAK would lead

to unstable spindles. A number of studies support this prediction. For example,
MCAK depletion leads to the formation of “hairy” spindles that have abnormally
long astral fibers [4, 24, 25, 61, 62], Kif2a regulates microtubule length in the central
spindle as well as the overall spindle size [15, 63], and Kif2b depletion leads to
disorganized spindles with a three to fourfold increase in microtubule polymer [4].
These observations suggest that targeting kinesin-13 activity to disrupt spindle
function should be an effective strategy to kill tumor cells. Unfortunately, such a
direct approach currently suffers from a lack of drugs with the needed specificity.
On the other hand, drugs that target microtubules are in widespread use and they
also affect microtubule detachment suggesting that kinesin-13 manipulation may
affect the sensitivity of cells to microtubule inhibitors. Thus, tumors that overex-
press kinesin-13 proteins would be predicted to have a high frequency of microtu-
bule detachments making the cells more sensitive to drugs like vinblastine that
increase detachment, but more resistant to drugs like paclitaxel that suppress detach-
ment. In fact, support for these predictions has already appeared. It was reported
that the effects of Kif2a depletion (expected to produce fewer detachments) can be
counteracted by the presence of nocodazole (an inducer of detachment) [4, 8],
and that depletion of a kinesin-13-like protein in Drosophila S2 cells increased
resistance to colchicine whereas overexpression of the protein increased sensitivity
to the drug [64]. Similarly, overexpression of Kif2b has been reported to increase
the sensitivity of spindle microtubules to nocodazole-induced depolymerization
whereas Kif2b deficient cells experienced delayed kinetochore microtubule depoly-
merization [16]. It was further reported that paclitaxel increases the density of
microtubules at spindle poles at the expense of kinetochore fibers and that MCAK
depletion increases astral but reduces K-fiber fluorescence [47]. Consistent with
these observations, it was directly shown that overexpression of MCAK increased
the frequency of microtubule detachment, decreased microtubule polymer levels,
and made cells resistant to paclitaxel and epothilone A, but more sensitive to
colcemid [27]. Depletion of MCAK lowered the frequency of detachment and made
cells more sensitive to paclitaxel [24].
The results argue that all three known kinesin-13 proteins can affect the sensitivity
of cells to microtubule inhibitors, likely by a mechanism that affects the detachment
of microtubules from spindle poles. Thus, efforts to therapeutically target kinesin-13
proteins may potentially be used in combination with existing microtubule inhibitors
to enhance tumor eradication. Treatments to enhance kinesin-13 levels or activity
would be expected to increase microtubule detachment and thereby make cells
more sensitive to drugs that also increase detachment (vinca alkaloids, colchicine
analogs, etc.); but treatments to inhibit kinesin-13 would decrease detachment and
increase sensitivity to drugs such as the taxanes and epothilones that also decrease
detachment. Levels or activity of kinesin-13 proteins could also potentially be used
as a predictive marker for tumor sensitivity to microtubule inhibitors. Tumors with
elevated kinesin-13 proteins are predicted to be relatively resistant to taxanes and
epothilones, but tumors with low kinesin-13 proteins are expected to be relatively
126 A. Ganguly and F. Cabral

resistant to vinca alkaloids and colchicine analogs. Many of these predictions are
still in need of experimental and clinical verification, but a clearer picture on the
role of kinesin-13 proteins in cancer therapy is now emerging.

8.8 Drugs That Bind Kinesin-13 Proteins

Unfortunately, drugs that specifically bind to Kif2a or Kif2b and interfere with their
function have not yet been identified, but a group of compounds known as sulfoqui-
novosylacylglycerols (SQAGs, Fig. 8.1) have been shown to interact with MCAK. In
vitro analysis suggested that the binding site is a 14 amino acid sequence present in
the neck linker domain that together with the motor domain is involved in MCAK
activity and binding to microtubules [65]. However, cells treated with SQAGs are
blocked in both mitotic and S phases of the cell cycle indicating that the drugs are
likely to have additional cellular targets [66, 67]. Although none of these drugs are
currently in clinical trials, SQAGs may yet prove to be useful drugs for cancer
chemotherapy and may lead to the development of compounds that more specifically
inhibit MCAK.
Another potential group of inhibitors is represented by thiazole compounds that
were originally introduced as Eg5 inhibitors but have been shown to strongly inhibit
the MT-simulated ATPase activity of MCAK. The structures of some of these agents
are shown in Fig. 8.2. Compound A is about tenfold more selective for MCAK
compared to Eg5, and does not appear to inhibit other kinesins like Kif5A, Kif5B,
Kif1B, CENP-E, Kif3A, and MKLP-1 [68]. Compound B, with an IC50 value of
160–170 nM, is the most potent inhibitor of MCAK [68] (reviewed by Good and
Kozielski [67]). The activities of these compounds against other members of the
kinesin-13 family have not yet been evaluated and thus the specificity for MCAK is
not established.

SO3Na

HO
O

HO O OR2
OH OR1
Fig. 8.1 Structures of the
Sulfoquinovosylacylglycerol
group of kinesin-13 R1=H, R2=CO(CH2)16CH3
inhibitors. Members of this R1=H, R2=CO(CH2)7CH=CH(CH2)7CH3
group of compounds R1=H, R2=CO(CH2)16CH3
differ primarily at positions
R1 and R2 R1,R2=CO(CH2)16CH3
8 Kinesin-13 Microtubule Depolymerizing Proteins as Targets for Cancer Therapy 127

a b

S S
Cl
N N
N
N
N

Fig. 8.2 Structures of thiazole inhibitors of kinesin-13 s

8.9 A Model for Predicting the Effects of Kinesin-13

Intervention to Treat Cancer

The spindle can be viewed as a metastable structure that requires rapid remodeling
in order to carry out its mission of segregating chromosomes prior to cell division.
The model in Fig. 8.3 assumes that spindle function relies on dynamic events that
occur not only at the plus ends (growth and shrinkage), but also, and perhaps more
importantly, on sliding of microtubule fragments present in the kinetochore fibers
connecting spindle poles to kinetochores. A proper mixture of fragmented and
continuous microtubules ensures a level of plasticity that promotes efficient
chromosome capture and segregation as well as successful cell division. However,
tubulin mutations that stabilize microtubules, treatment with drugs like paclitaxel
that promote microtubule assembly, or depletion of kinesin-13 proteins such as
MCAK can inhibit the production of microtubule fragments leading to stabilized
spindles that are functionally less efficient. Conversely, destabilized spindles can
arise from destabilizing tubulin mutations, treatment with drugs like colcemid or
vinca alkaloids that inhibit microtubule assembly, or by overexpression of kine-
sin-13 proteins like MCAK. If the defect is relatively small (weak mutations, lower
drug concentrations, small changes in kinesin-13 production) cell division may still
occur, but there may be a higher incidence of aneuploidy. Stabilized spindles would
make the cells relatively resistant to drugs like colcemid and vinca alkaloids;
whereas the destabilized spindles would confer resistance to drugs like paclitaxel
and epothilones.
More severe defects (stronger mutations, higher drug concentrations, larger
changes in kinesin-13 production) can produce very stable or unstable spindles
unable to efficiently segregate chromosomes. This would lead to prolonged mitotic
checkpoint activation and cell death or to cells that exit mitosis but fail to divide.
The highly aneuploid cells could themselves die in subsequent cell cycles or
contribute to the formation or aggressiveness of malignant cells as has been inferred
by clinical observations linking overexpression of kinesin-13 proteins to tumor cell
metastasis and poor patient prognosis. Cells that form very unstable spindles
frequently have a paclitaxel dependence phenotype in which spindle structure and
successful cell division can be restored by the presence of paclitaxel (or other drugs
128 A. Ganguly and F. Cabral

Too Many Fragments Normal Cell Too Few Fragments

Increasing Severity of Defect

Cmd Treatment Ptx Treatment

Tubulin Mutation Tubulin Mutation
MCAK MCAK
PtxR CmdR

Abnormal spindle Normal spindle Abnormal spindle

Ptx Treatment Cmd Treatment

MCAK MCAK
PtxD CmdD
No cell division Successful cell division No cell division

Fig. 8.3 Model for the effects of various mitotic spindle modifiers on cell proliferation.
The top row shows the effects of various treatments on the production of microtubule fragments at
the spindle poles. Minor effects on cell division but significant effects on drug sensitivity (e.g.
paclitaxel resistance, PtxR; or colcemid resistance, CmdR) can occur when the perturbations are not
too severe. The bottom row shows the expected effects of more severe perturbations on spindle
structure and cell division that can lead to drug dependent phenotypes (e.g. paclitaxel dependence,
PtxD; or colcemid dependence, CmdD). Treatments potentially able to correct those defects and
restore cell division are indicated

that inhibit the production of microtubule fragments), or by depleting MCAK, and

perhaps other kinesin-13 proteins. Cells that form very stable spindles, on the other
hand, can be rescued by treating with drugs like colcemid that destabilize microtu-
bules, or by overexpressing MCAK (and perhaps other kinesin-13 proteins) that
increase the production of microtubule fragments.
Although the model emphasizes treatments that have already been shown to
affect fragment production through detachment of microtubules from spindle poles,
it provides a framework for predicting the effects of modulating other proteins or
drugs that may also interfere with microtubule detachment. For example, overex-
pression of β3-tubulin has been shown to destabilize microtubules and produce
paclitaxel resistant/dependent cells that can be rescued by depleting MCAK [24, 69].
Similarly, overexpression of β5-tubulin can increase microtubule detachment, produce
microtubule fragments, and confer a paclitaxel dependence phenotype [70, 71].
On the other hand, overexpression of β6-tubulin produces microtubule fragments by
a different mechanism and does not confer paclitaxel resistance or dependence [72].
It will be of interest to test whether other proteins such as augmin, katanin, and
spastin that have been implicated in microtubule fragment formation can affect cell
sensitivity to microtubule inhibitors.
8 Kinesin-13 Microtubule Depolymerizing Proteins as Targets for Cancer Therapy 129

8.10 Summary and Perspectives

The previous discussion focused on the possibility of targeting kinesin-13 proteins

to kill tumor cells, alter their sensitivity to microtubule inhibitors, or reverse
resistance to drugs that target microtubules. Although there are a few drugs that can
inhibit these kinesins, they lack specificity and there is a pressing need to discover
better agents. Indirect methods of targeting kinesin-13 s could potentially include
inhibiting kinases that are known to activate, inhibit, alter the localization, or affect
the degradation of the proteins, but these approaches are likely to produce unwanted
side effects due to the fact that kinases typically have multiple substrates. In fact,
mitotic kinase inhibitors have thus far had very limited success in treating cancer [73].
Molecular genetic approaches to directly alter kinesin-13 levels and interfere with
tumor growth is theoretically possible, but current methods for achieving this in
patients are also limited.
On the other hand, kinesin-13 status has very significant implications for the
sensitivity of cells to currently used and future mitotic inhibitors that target micro-
tubules and spindle assembly. Thus, assaying the levels of kinesin-13 proteins in
tumors may aid in predicting the dosage of microtubule inhibitors that will be effec-
tive in patients. It is also possible that weak kinesin-13 inhibitors that by themselves
are unable to prevent tumor growth, may work synergistically to enhance the effec-
tiveness of microtubule inhibitors. As mentioned earlier, kinesin-13 s may provide
effective targets for developing immunotherapeutic approaches to treat tumors that
overexpress those proteins; and they may potentially be used as markers for patient
prognosis.
In addition to targeting kinesin-13 s to kill tumor cells, inhibiting their activity
may reduce tumor malignancy. Several studies cited earlier demonstrated that tumor
cells that overexpress kinesin-13 proteins tend to be more aggressive and that reduc-
ing the production of Kif2a or MCAK can inhibit cell motility [35, 36, 39, 74].
These actions are likely to be mediated by changes in the microtubule cytoskeleton.
Low concentrations of microtubule drugs have been shown to suppress microtubule
dynamics and inhibit cell migration without affecting cell division [52, 75].
The need for dynamic microtubules appears to be most acute at the trailing edge of
migrating cells where the cytoskeleton needs to be remodeled in order to allow tail
retraction and forward movement [76]. Given that kinesin-13 proteins have been
shown to catalyze catastrophe and inhibit rescue at microtubule ends, they are likely
to have important effects on the ability of microtubules to remodel. Thus, treatments
aimed at inhibiting kinesin-13 proteins would be expected to interfere with tumor
cell metastasis and invasion of neighboring tissues. In addition, it has been shown
that suppressing microtubule dynamics in vascular endothelial cells inhibits their
migration, suggesting that therapies aimed at kinesin-13 proteins may also limit
tumor growth by inhibiting angiogenesis [77].
130 A. Ganguly and F. Cabral

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