Chromosome segregation errors: a double-edged sword - TI Pharma

tumor cells, leaving diploid cells relatively unaffected. In line with this hypothesis, it has been shown
that introduction of extra chromosomes in diploid cells directly leads to prolonged mitotic timing,
since these cells need more time to congress their chromosomes to the spindle equator 539 . Indeed,
time lapse imaging of our panel of cell lines revealed an enhanced timing from nuclear envelope
breakdown (NEB) until anaphase in the tumor cell lines when compared to diploid RPE-1 cells (Fig.5A).
MCF-7, SW480, U2OS and Hela cells took on average 55, 81, 34 and 55 minutes respectively to
complete mitosis, whereas RPE-1 cells only needed ~29 minutes (Fig.5A). As expected 119 , treatment
with 30 nM compound-5 reduced mitotic timing for all tumor cell lines with more than 15 minutes,
whereas RPE-1 cells only reduced their time in mitosis with ~2 minutes on average (Fig.5A).
On top of the relatively big effect on mitotic timing, chromosome congression in tumor cells might
also be more affected by partial Mps1 inhibition when compared to diploid cells due to Mps1’s
role in chromosome alignment 116 . Already without the addition of compound-5, tumor cells
displayed many more chromosome misalignments (31%-44%) when compared to diploid RPE-1
cells (13%), when we allowed cells to align their chromosomes in the presence of the proteasome
inhibitor MG132 (Fig.5B). Addition of 30 nM compound-5 severely enhanced the number of tumor
cells with chromosome misalignments (49%-95%), whereas RPE-1 cells were only mildly affected
(23%) (Fig.5B).As an alternative of using tumor cells, which could have accumulated non-related
defects that render them sensitive to Mps1 inhibition, we also induced transformation in BJ-Tert
cells in three consecutive steps 583 . First, we inactivated p53 by introducing stable p53 knockdown.
Next, we expressed a mutant form of HRAS (HRASV12) known to induce transformation and
finally we combined this with inhibition of the Rb tumor suppressor pathway by transducing
A
B
% of cells with misalignments
Time from NEB-anaphase (minutes)
n=93
100n=39 80
60
40
20
0
- +
RPE
350
325
300
275
250
225
200
175
150
125
100
75
50
25
0
n=86
n=92
- +
MCF-7
- + - + - + - + - + 30nM Comp.5
RPE
n=78
n=56
- +
SW480
n=87
- +
U2OS
MCF-7
+ 30’ MG132
n=38
n=131
n=75
- +
Hela
SW480
n=92
n=101
n=100
n=136
- +
HCT-116
- +
LS174T
U2OS
30nM
Comp.5
Hela
WT
p53KD
p53KD
HRASV12
p53KD
HRASV12
E1A
nM compound-5
0 10 20 30
BJ-Tert
Figure 5. Low dose Mps1 inhibition specifically affects mitotic timing and chromosome alignment in tumor cells
A) Live cell analysis (tagged H2B) of the mitotic duration of indicated cell lines in the presence or absence of compound-5. Mitotic
duration was determined as the time in minutes from chromosome condensation (nuclear envelope breakdown (NEB)) to the
start of anaphase. Dot plot is shown with each dot representing one cell. Average is indicated +/- SEM. B) Quantification of
chromosome alignment of indicated cell lines after 30 minutes of MG132 treatment in the presence or absence of compound-5.
N indicates number of cells. C) Colony formations of indicated BJ-Tert cell lines in the presence of increasing concentrations
compound-5.
C
123
Mps1 inhibition kills aneuploid cells
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