Mixed lineage kinase 3 gene mutations in mismatch
repair deficient gastrointestinal tumours
Se ´rgia Velho1, Carla Oliveira1,2, Joana Paredes1, So ´nia Sousa1, Marina Leite1, Paulo Matos3,
Fernanda Milanezi1, Ana Sofia Ribeiro1, Nuno Mendes1, Danilo Licastro4, Auli Karhu5,
Maria Jose ´ Oliveira1,6, Marjolijn Ligtenberg7, Richard Hamelin8, Fa ´tima Carneiro1,2,9,
Annika Lindblom10, Paivi Peltomaki11, Se ´rgio Castedo1,2, Simo ´ Schwartz Jr12, Peter Jordan3,
Lauri A. Aaltonen5, Robert M.W. Hofstra13, Gianpaolo Suriano1,2, Elia Stupka14,15,
Arsenio M. Fialho16and Raquel Seruca1,2,?
1IPATIMUP—Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal,
2Medical Faculty of the University of Porto, 4200-319 Porto, Portugal,3Centre of Human Genetics, National Health
Institute Dr Ricardo Jorge, 1649-016 Lisbon, Portugal,4CBM S.c.r.l., Area Science Park, Basovizza - SS 14, Km.
163,5, 34012 Trieste, Italy,5Department of Medical Genetics, Haartman Institute, University of Helsinki, 00014
Helsinki, Finland,6NewTherapies Group, INEB—Institute for Biomedical Engineering, Porto, Portugal,7Department of
Human Genetics, UMC Nijmegen, 6500 HB Nijmegen, The Netherlands,8INSERM U434 CEPH, 75010 Paris, France,
9Hospital de S. Joa ˜o, 4200-319 Porto, Portugal,10Department of Molecular Medicine and Surgery, Karolinska
Institutet, S 171 76 Stockholm, Sweden,11Department of Medical Genetics, Biomedicum Helsinki, University of
Helsinki, 00014 Helsinki, Finland,12Centre d’Investigacions en Bioquimica i Biologia Molecular (CIBBIM), Hospital
Universitari Vall d’Hebron, Barcelona 08035, Spain,13Department of Medical Genetics, University Medical Center
Groningen, 9700RB Groningen, The Netherlands,14UCL Cancer Institute, Paul O’Gorman Building, University
College London, Gower Street, London WC1E 6BT, UK,15The Blizard Institute, Barts and The London School of
Medicine and Dentistry, 4 Newark Street, London E1 2AT, UK and16Institute for Biotechnology and BioEngineering
(IBB), Center for Biological and Chemical Engineering, Instituto Superior Tecnico, 1049-001 Lisbon, Portugal
Received October 8, 2009; Revised and Accepted November 26, 2009
Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which
cancer-associated mutations have never been reported. In this study, 174 primary gastrointestinal cancers (48
hereditary and 126 sporadic forms) and 7 colorectal cancer cell lines were screened for MLK3 mutations. MLK3
mutations were significantly associated with MSI phenotype in primary tumours (P 5 0.0005), occurring in 21%
of the MSI carcinomas. Most MLK3 somatic mutations identified were of the missense type (62.5%) and more
than 80% of them affected evolutionarily conserved residues. A predictive 3D model points to the functional
relevance of MLK3 missense mutations, which cluster in the kinase domain. Further, the model shows that
most of the altered residues in the kinase domain probably affect MLK3 scaffold properties, instead of its
kinase activity. MLK3 missense mutations showed transforming capacity in vitro and cells expressing the
mutant gene were able to develop locally invasive tumours, when subcutaneously injected in nude mice.
Interestingly, in primary tumours, MLK3 mutations occurred in KRAS and/or BRAF wild-type carcinomas,
although not being mutually exclusive genetic events. In conclusion, we have demonstrated for the first time
strated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.
?To whom correspondence should be addressed. Tel: þ35 1225570700; Fax: þ35 1225570799; Email: firstname.lastname@example.org
# The Author 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is
Human Molecular Genetics, 2010, Vol. 19, No. 4
Advance Access published on December 3, 2009
In gastrointestinal carcinomas, ?15% of sporadic and 90% of
hereditary non-polyposis colon carcinoma (HNPCC or Lynch
tumours) show microsatellite instability (MSI) in multiple
repeat sequences, due to defects in mismatch repair genes
(MMR). Despite showing distinct molecular mechanisms for
MMR silencing (1,2), MSI sporadic gastric (GC) and colorec-
tal carcinomas (CRC), and hereditary MSI carcinomas encom-
pass very similar molecular mutation profiles in non-coding
and coding repetitive tracts. Those genes that accumulate
mutations in their coding sequences are called target genes
(2). Moreover, MMR deficiency is also known to boost the fre-
quency of point mutations, namely in proto-oncogenes. Spora-
dic MSI CRC harbour activating missense mutations in genes
coding for protein members of the RAS-RAF-MAP kinase
pathway,a crucial pathway
BRAFV600E hotspot mutation has been described in ?40%
of sporadic MSI CRC and point mutations in KRAS are
present in ?20% of the cases (1). Mutations in both genes
have been rarely described in MSI sporadic CRC (1). In
MSI GC, only KRAS mutations have been observed in 20%
of the cases (3). Similarly, 40% of HNPCC cases display
KRAS mutations and BRAF mutations were never found
(4,5). Still, many HNPCC and sporadic gastrointestinal carci-
nomas lack KRAS or BRAF activating mutations, and altera-
tions in other genes involved in the KRAS/BRAF pathway
may represent further genetic events, thereby leading to the
deregulation of this pathway. Recently, MLK3 has been
appointed as a pivotal protein involved in the regulation of
the mitogen-protein (MAP) kinase pathway.
MLK3 contains an N-terminal Src-homology 3 (SH3)
domain, a kinase domain, a leucine zipper, a Cdc42/Rac inter-
active binding (CRIB) motif and a COOH-terminal proline–
serine–threonine rich domain. The SH3 domain of MLK3
can bind a proline residue in a region between the leucine
zipper and the CRIB motif resulting in auto-inhibition (6).
MLK3 is a serine/threonine protein kinase that regulates the
MAPKinase pathway activating ERK, p38 and JNK, in
response to extracellular signals (7,8). Further, MLK3 has
been demonstrated to function as a scaffolding protein,
involved in the formation of a multiprotein complex contain-
ing MLK3/BRAF/RAF1 (7,9,10). The formation of this
complex was shown to be important for the activation of wild-
type BRAF and, consequently, to the activation of ERK sig-
nalling (7,9,10). Furthermore, MLK3 was reported to be
important for the proliferation of tumour cells, bearing either
oncogenic KRAS or neurofibromatosis-1 (NF1) or NF2 inacti-
vating mutations (10).
leads to cell transformation (11). Likewise, over-expression of
the wild-type MLK3 was shown to induce transformation of
NIH3T3 fibroblasts (12). Overall, these data suggest that
MLK3 is likely to be involved in cancer and alterations of
this gene could harbour transforming ability; nevertheless,
no MLK3 gene alterations have ever been described so far
In this study, we aimed at determining whether MLK3 gene
is a target of point mutations in gastrointestinal carcinoma and,
if so, what is the distribution and type of such mutations
among different settings of gastrointestinal cancer, and
whether MLK3 missense mutations encompass transforming
and tumorigenic potential in vitro and in vivo. Moreover, we
assessed the co-existence of mutations in MLK3 and in
KRAS and BRAF genes, which are frequently mutated in
this type of neoplasia.
MLK3 heterozygous somatic mutations cluster in MSI
All MLK3 exons and intron–exon boundaries were screened
for mutations in a series of 174 primary gastrointestinal
cancers [114 MSI cases: 48 hereditary carcinomas (38
Lynch tumours with characterized germline mismatch repair
gene mutations and 10 HNPCC patients that fulfilled the clini-
cal criteria), 36 sporadic CRC and 30 sporadic GC; and 60
MSS sporadic CRC cases] and 7 CRC cell lines: 4 MSI and
3 MSS (Table 1). Whenever a sequence variant was found,
matched constitutional DNA was analysed to exclude germ-
line origin and 100 normal chromosomes were analysed to
exclude polymorphic origin.
Overall, we found that 25/174 (14.4%) primary gastrointesti-
nal carcinomas displayed heterozygous somatic and tumour-
mutant cases were MSI and only a single case was MSS. The
frequency calculated only among MSI cases was 21.1%
(24/114) (Table 1). These results demonstrated that MLK3
ing an MSI phenotype (P ¼ 0.0005). In terms of different MSI
gastrointestinal cancer settings, we found MLK3 somatic
mutations in 25% (12/48) of hereditary (Lynch/HNPCC) carci-
nomas; in 19.4% (7/36) of sporadic CRC and in 16.7% (5/30)
of sporadic GC cases. The frequency of MLK3 mutations was
not significantly different among the three settings of MSI carci-
nomas (P ¼ 0.6527) (Table 1). Within the MLK3 mutant cases,
two MSI carcinomas (case 1 and 17) harboured two different
mutations (Table 2).
MLK3 mutations were also found in MSI CRC cell lines: 2/
4 (RKO and CO115), mimicking the results found in primary
tumours. The three MSS colorectal cancer cell lines (HT29,
Caco2 and SW480) analysed did not show MLK3 mutations
From the 24 MLK3 mutated MSI cases, 15 (62.5%) dis-
played somatic missense mutations (Table 1), which are
depicted in Figure 1. Seventy percent of these missense
domains (Fig. 1) (6,13); and more than 80% affected MLK3
residues which have been evolutionarily conserved as far in
evolution as Drosophila melanogaster (Fig. 1).
Frameshift mutations occurred in 33.3% (8/24) of MLK3
mutated MSI cases, preferentially at repetitive sequences
(six out of eight) of about four to six mononucleotides. This
type of mutations was preferentially clustered in the last
2466delG was the only recurrent mutation and was found in
two different cases (Table 2). A single nonsense mutation
was also found among MLK3 mutated MSI cases (Table 2).
698Human Molecular Genetics, 2010, Vol. 19, No. 4
Frameshift mutations affecting coding repetitive sequences
are among the most common mutations occurring in MSI
cancers, as a consequence of DNA polymerase slippage, fre-
quently generating truncated proteins (2). The functional rel-
evance of these frameshift mutations depends both on the
target gene affected and on the tissue where they occur (14).
In order to get insight on the functional role of MLK3
frameshift mutations, we compared their frequency in coding
repetitive sequences with the frequency of frameshift
mutations occurring in 11 non-coding intergenic repeats (15)
of the same type and size (C/G6) in 30 MSI CRCs. The fre-
quency of frameshift mutations in MLK3 repeats was higher
(25%—6/24) when compared with the frequency found in
Table S3). These results suggest that frameshift mutations
occurring in MLK3 coding repeat sequences are selected for
Table 1. Frequency of cases with MLK3 mutations found in MSI and MSS gastrointestinal tumours and cell lines
Mixed lineage kinase 3 (MLK3)
Wild-type, n (%)
Type of mutation
(% total mut)
Mutant, n (%) Frameshift/nonsense,
n (% total mut)
Lynch/HNPCC (n ¼ 48)
MSI sporadic CRC (n ¼ 36)
MSI sporadic GC (n ¼ 30)
Total number of MSI gastrointestinal tumours (n ¼ 114)
MSS sporadic CRC (n ¼ 60)
Total number of MSI and MSS gastrointestinal tumours (n ¼ 174)
MSI CRC cell lines (n ¼ 4)
MSS CRC cell lines (n ¼ 3)
9 (8/1) (37.5)
The frequency of MLK3 mutations found in primary MSI gastrointestinal tumours (24/114) is significantly different from the frequency of MLK3 mutations in
primary MSS tumours (1/60) (P ¼ 0.0005). P-value was calculated using Fisher ´s exact test and P , 0.05 was taken as statistically significant.
MSS, microsatellite stable; MSI, microsatellite unstable; CRC, colorectal cancer; GC, gastric cancer.
Table 2. Type of MLK3 mutations and association with other genetic alterations in hereditary and sporadic MSI gastrointestinal tumours and colorectal cancer
Tumour typeMut Case MLK3 alterations
KRAS BRAF Altered
Lynch/HNPCC1 c.296 A.G
c, coding DNA; p, protein; X, stop codon; ., substitution; del, deletion; ins, insertion; nd, not determined—patients fulfilled the clinical criteria.
Human Molecular Genetics, 2010, Vol. 19, No. 4699
during the process of tumour development of MSI gastrointes-
tinal cancers. In contrast, missense mutations occur less fre-
quently in MSI cancers, but tend to be oncogenic, as is the
case of mutations affecting KRAS, BRAF and PI3KCA in
MSI CRC (16). Moreover, missense mutations in MLK3 are
far more frequent than frameshift mutations in our series of
MSI gastrointestinal cancers. For these reasons, in the
present study, we decided to address the potential impact of
MLK3 missense mutations only.
Missense mutations in the kinase domain of MLK3 are
predicted to impact protein function
Before addressing, in vitro and in vivo, the potential impact of
MLK3 missense mutations in CRC cells, we decided to run a
predictive analysis by modelling the 3D structure of MLK3
kinase domain, using the crystal structure of the same
domain of MLK1 as template (17). There were three reasons
underlying this choice: (i) a crystal structure of MLK3 is not
available; (ii) MLK1 shows great amino acid homology with
MLK3 (Pdb: 3dtc; 70% amino acid identity with MLK3),
allowing an accurate modelling and (iii) even for MLK1,
only the crystal structure of the kinase domain is available.
acid changes (A165S, R240C, P252H, A296T, A352R and
A356V). These mutations were spread along the kinase domain
and did not occur at specific and highly conserved functional
amino acid positions of the kinase catalytic core. Based on our
predictivemodel, all these
surface-exposed residues, causing overall destabilization of the
molecule (Fig. 2A and B). Mutants A165S, P252H and A352R
bonds with neighbouring residues, producing conformational
changes in the protein backbone (Fig. 2a1, a3, a5). R240C
mutation occurs at a coil region that runs parallel to an alpha
helix in the C-terminal lobe of the MLK3 kinase domain
(Fig. 2a2). The replacement of a larger residue (R) by a smaller
of the steric hindrance. The A at position 296 is located at an
exposed loop of the MLK3 kinase domain. The substitution of
a hydrophobic A residue by and hydrophilic T residue (A296T)
is predicted to destabilize this area of the protein, impacting
most probably the scaffold properties of MLK3 (Fig. 2a4). The
A residue at position 356 is part of an Alanine-rich helix of
MLK3. The substitution of A residue by a V (A356V) does not
fore, the helix is likely to assume a different shape/deformation
and/or suffer a destabilizing effect (Fig. 2a6).
Overall, our predictive MLK3 kinase domain 3D model
shows that mutation at the six abovementioned residues, in
the kinase domain of MLK3, are more likely to disturb the
scaffold properties of the protein rather than its kinase activity.
These in silico observations generated enough evidence to
pursue the potential in vitro and in vivo analysis of MLK3
kinase domain missense mutations.
Cells expressing missense mutations of MLK3 have
transforming potential in vitro
missense mutations, we selected five different mutations,
Y99C, A165S, P252H, R799C, P840L occurring in distinct
domains of the protein. A165S and P252H missense mutations
were chosen among the six mutations occurring at the MLK3
kinase domain and the other three mutations occurred in three
different domains of the protein (Table 2, Fig. 1).
After the generation of vectors expressing the five selected
MLK3 mutations, as well as K144R, a kinase dead form of
MLK3 to be used as a negative control (10), all these con-
structs were transiently transfected in NIH3T3 cells, as well
as the wild-type form of MLK3 and the empty vector
(Mock). A classical fibroblast focus formation assay was
used to assess transforming ability of manipulated cell lines.
Figure 1. Summary of the localization of missense mutations in MLK3 gene found in MSI primary gastrointestinal carcinomas and cell lines. The upper diagram
depicts the protein sequence domain architecture, indicating the affected residue and its amino acid change (top), nucleotide start-end coordinates (top) and
amino acid start-end coordinates (bottom) for each domain. Below, the wild-type amino acid residue is shown for 20 eukaryotic species, and the conservation
for each affected residue is summarized in the bottom two rows, one indicating the conservation in mammalian orthologues (n ¼ 4) and the next in all eukaryotic
homologues (n ¼ 16).
700Human Molecular Genetics, 2010, Vol. 19, No. 4
All missense mutants analysed showed higher transforming
capacity in vitro than the wild-type MLK3 and the kinase
dead MLK3 (P , 0.05; Fig. 3).
Cells expressing missense mutations of MLK3 have
tumorigenic potential in vivo
To further evaluate the tumorigenic potential of MLK3 mis-
sense mutations in vivo, NIH3T3 cells expressing P252H
and R799C mutants, which were the most transforming
mutations in vitro and found in two distinct key functional
domains (the kinase and the P/S/T-rich domains, respectively)
were inoculated subcutaneously in nude mice. Cells expres-
sing wild-type MLK3, the empty vector (Mock) and
HRASV12 were used as controls. Six weeks after inoculation,
neither mice inoculated with MLK3 wild-type expressing
cells, nor those inoculated with the empty vector, generated
tumours. As expected, HRASV12-expressing cells generated
subcutaneous tumours within 4 weeks after inoculation.
Nude mice inoculated with both MLK3 mutants developed
subcutaneous tumours within 5 weeks after inoculation. One
of these mice even presented a fast growing tumour 3 weeks
after inoculation (Fig. 4A). The in vivo experiment was com-
pleted 6 weeks after inoculation and tumours were surgically
Figure 2. 3D model to predict how MLK3 mutations could interfere with the MLK3 protein function. (A) Predictive 3D model of the MLK3 kinase domain,
constructed using the crystal structure from MLK1 (Pdb: 3dtc; 70% amino acid identity with MLK3) as a template (17). Kinase active site, nucleotide binding
loop, hinge region, the N- and C-termini lobes and the six missense mutations identified in this study are highlighted. (a1–a6) Magnified views of the different
MLK3 mutations. Prediction of the impact of the six amino acid substitutions is described in the Discussion section. Dotted yellow lines represent potential
H-bonds. (B) Prediction of mutations effects on the MLK3 kinase domain stability, based on the site directed mutator (SDM) program (37). SS element, sec-
ondary structure element (H, helix, C, coil, L, loop); HB, hydrogen bond; OS, overall stability.
Human Molecular Genetics, 2010, Vol. 19, No. 4701
removed and histopathologically analysed. Mutant MLK3
expressing cells grew into malignant tumours, with a high
number of mitotic figures and an infiltrative pattern of
growth. The number of mitotic figures, per observation field,
in MLK3 mutant tumours was similar to those observed in
HRASV12 tumours. Further, and in contrast to the positive
control HRASV12, both MLK3 mutant tumours showed the
ability to locally infiltrate the surrounding adipose tissue and
muscle (Fig. 4B). Xenografted tumours expressing P252H
showed the most aggressive behaviour, with invasion of back-
bone, bone marrow and spinal cord (Fig. 4C).
Our in vivo data clearly demonstrate the tumorigenic poten-
tial of at least two mutant forms of MLK3, suggesting a role
for this gene in cell transformation and acquisition of a malig-
nant behaviour, namely local invasion. To further support a
role for MLK3 in cell invasion, we quantified in vitro the inva-
sion potential of NIH3T3 cells expressing P252H and R799C,
and the same cell line transfected with the empty vector, using
the matrigel invasion assay. Cells expressing MLK3 mutants
P252H and R799C showed a 3-fold increase ability to
invade in vitro when compared with wild-type MLK3 cells
(data not shown).
MLK3 mutations also occur in MSI gastrointestinal
tumours with wild-type KRAS and BRAF
KRAS and BRAF, two key molecules of the MAPKinase
pathway, are frequently mutated in sporadic MSI gastrointes-
tinal cancer, and KRAS mutations occur frequently in
HNPCCs (3,5). Nevertheless, oncogenic mutations at these
genes only account for 30–50% of the cases (1,3–5,18,19).
Overall, it is described that ?50% of all MSI sporadic CRC,
40% of HNPCC and 30% of sporadic GC harbour oncogenic
mutations in these genes (1,3–5,18,19). As our analysis
revealed that mutations at MLK3 are most probably function-
ally relevant, occurring almost exclusively in MSI carcinomas,
and that MLK3 is described as a component of the multipro-
tein BRAF/RAF1 complex (7,9,10,20), we decided to
analyse how mutations in MLK3 (all types) correlated with
mutations in KRAS and/or BRAF in our series (Table 2 and
In hereditary (HNPCC and Lynch) MSI tumours, 12.5% (6/
48) harboured only MLK3 mutations; 25% (12/48) harboured
only KRAS mutations and 12.5% (6/48) harboured mutations
in both genes. In sporadic MSI CRCs, 5.6% (2/36) harboured
only MLK3 mutations, 16.6% (6/36) harboured only KRAS
mutations, 16.6% (6/36) harboured only BRAF mutations,
5.5% (2/36) harboured mutations in MLK3 and KRAS and
8.3% (3/36) had both MLK3 and BRAF mutations. In MSI
sporadic GC, 13.3% (4/30) harboured only MLK3 mutations,
3.3% (1/30) harboured mutations in both MLK3 and KRAS
genes, and none of the GC tumours harboured only mutations
Overall, 12 out of 24 (50%) of the MLK3 mutations occur
in MSI gastrointestinal carcinomas bearing wild-type KRAS
and BRAF (Table 2 and Fig. 5). Seven of these are of the mis-
sense type (Tables 1 and 2) and three of them showed trans-
forming potential in vitro and/or tumorigenic potential in vivo.
In the last years, our group and others contributed to define the
frequency and type of activating oncogenic mutations in MSI
and MSS sporadic gastrointestinal tumours, namely occurring
in KRAS and BRAF genes, which are members of the
fairly frequent, mutations at MAPK-related genes, like
KRAS and BRAF, are not found in all gastrointestinal
Figure 3. In vitro transforming capacity of mutant MLK3. NIH3T3 cells were transfected with the indicated constructs and then submitted to a Focus Formation
Assay, in order to ascertain the transforming potential of mutant MLK3. Experiments were performed in triplicate. (A) Values in the graph represent the average
fold increase of the number of foci observed when compared with the number of foci formed by NIH3T3 Mock cells. (B) Photographs of representative dishes;
mutants were compared with wild-type for statistical analyses, using Student’s t-test, and P , 0.05 was taken as statistically significant. Wt, wild-type MLK3.
702 Human Molecular Genetics, 2010, Vol. 19, No. 4
tumours (1,3–5,18,19), although MAPK activation is reported
to occur in over 70% of overall CRC cases (22).
MLK3 is known to be required for mitogen activation of
BRAF and ERK (7,9,10,25). So, taken all these observations
together, it is plausible to believe that activation of other
MAPK upstream targets may occur in CRC and, eventually,
in other gastrointestinal cancers. Therefore, we tested whether
MLK3 was a target for mutations in this type of cancers.
Evidence pointing to the transforming potential of MLK3
has been described in the literature. However, MLK3 gene
structural alterations have never been reported in cancer. In
this study, we found, for the first time, MLK3 gene mutations
in hereditary and sporadic primary gastrointestinal tumours.
Moreover, we verified that these mutations did not occur ran-
domly, but were rather clustered in the group of MSI cases.
Further, MLK3 mutations were only found in CRC derived
cell lines with MSI phenotype. This finding is interesting
since, in CRC, it mimics what happens with BRAF gene, in
which mutations also occur only in MSI cases (1,3,18,19)
and, in GC, it mimics what happens with KRAS and
PI3KCA genes, also mutated only in cancer cases with MSI
As BRAF mutations have been described in 60% of mela-
noma (26), we tested whether MLK3 mutations would also
occur in a small series of six melanoma cell lines (M14,
UACC62, SKMEL2, SKMEL5, SKMEL28 and MEWO).
Noteworthy, two MLK3 missense mutations (E121D and
T680M) were found in two of these cell lines (SKMEL2 and
UACC62) (data not shown), raising the hypothesis that
MLK3 mutations might also occur in other types of malignant
cancer associated to the activation of the MAPKinase pathway.
Further supporting the potential relevance of MLK3
mutations is the fact that most of them were missense
(64%), spread throughout the MLK3 gene, and affecting
amino acid residues that are evolutionary conserved, as it
has been previously described for MLK4 in CRC, another
MLK family member (27). Changes in less conserved amino
acids were mainly found in the P/S/T-rich domain. This
result may be related to the fact that this is the less conserved
domain among MLK family and considered to confer function
specificity to each MLK member (8,28).
A 3D predictive modelling of some of the MLK3 missense
mutations revealed that, although located at the kinase domain
of MLK3, the protein phosphorylation was not envisaged to be
modified. Supporting this prediction, HEK293 cell lines tran-
siently transfected with A165S and P252H mutations did not
show increased levels of phosphorylated MLK3, using
western blotting and an antibody for phospho-MLK3 (data
not shown). Rather, the 3D model suggests that MLK3
mutants may interfere with its scaffold properties, and thus
are likely to impact MAPkinase signalling. Further studies
are needed to prove this hypothesis.
The transforming potential of several MLK3 mutations
showed that the introduction of a mutant MLK3, in cells
with an MLK3 wild-type background, was sufficient to
confer significant higher in vitro transforming potential to
these cells in comparison to cells over-expressing wild-type
MLK3. These results demonstrate that mutant MLK3 proteins
are likely to be functionally relevant, at least in vitro.
We additionally demonstrated the relevance of MLK3
mutations in vivo, since nude mice injected with cells expres-
sing mutant MLK3 developed
tumours. Most importantly, we verified that these tumours
had an infiltrative pattern of growth, a neoplastic behaviour
that was not even observed in tumours induced by RASV12
oncogene. The local invasive ability of tumour cells expressing
mutant MLK3, in vivo, was further supported by increased
number of invading cells in matrigel invasion assay. The mech-
anism for invasion mediated by mutant MLK3 remains to be
determined; nevertheless, a role for MLK3 in cell migration
has been previously demonstrated (29). Interestingly, it was
verified that MLK3-depleted cells showed defects in cell
migration, with an increase in the thickness and number of
stress fibres, as well as enlarged focal adhesions and absence
of lamellipodia protrusions. These results showed that MLK3
is pivotal for cells to move. Further, it was proved that these
non-migrating cells presented high levels of activated Rho,
and the mechanism of MLK3-mediated Rho inhibition was
3D model for the MLK3 mutants found in this gastrointestinal
Figure 4. In vivo tumorigenic capacities of MLK3 mutations. (A) Tumour volume was monitored over time and plotted for each mouse. NIH3T3 cells over-
expressing the P252H and R799C MLK3 mutations were able to generate tumours when subcutaneously inoculated in nude mice, as happens with HRASV12.
Cells over-expressing wild-type MLK3 or the empty vector (Mock) were not able to generate tumours at 7 weeks after inoculation. At the end of the seventh
week, one of the two mice inoculated with wild-type MLK3 expressing cells, presented a small tumour, according to the previously shown wild-type MLK3 in
vitro transforming potential. (B and C) Histopathological analysis of tumours over-expressing MLK3 P252H and R799C mutations. (B) Effect of RASV12 (I),
MLK3 P252H (II) and MLK3 R799C (III) transfection on the growth of NIH3T3 cells in nude mice. Tumour cells harbouring MLK3 mutations (V, VI) showed
an infiltrative pattern of growth, with invasion of the surrounding adipose tissue and muscle, in contrast to RASV12 (IV). All the mutations induced high-grade
fibrosarcomas, with a high number of mitotic figures (VII; VIII, IX). (C) MLK3 P252H-overexpressing tumours showed the most aggressive behaviour, with
invasion of backbone, bone marrow and spinal cord.
Human Molecular Genetics, 2010, Vol. 19, No. 4703
Finally, we found that, in MSI primary gastrointestinal
tumours, half of the MLK3 mutations (12/24) were found in
cases harbouring wild-type KRAS or BRAF genes. Nowadays,
and imperative in clinical terms, the presence of mutations in
genes belonging to KRAS/BRAF/MAPkinase pathway raises
important therapeutic implications, namely in the selection
of patients that can benefit from the treatment with mono-
clonal antibodies against EGFR (23,30). Most importantly,
CRCs frequently harbour concomitant alterations in several
genes, that lie up and downstream of KRAS or BRAF signal-
ling cascade, which may represent an important factor for the
acquisition of resistance to current therapies (16,31). This
scenario is particularly relevant in the case of CRC patients,
since the presence of KRAS mutations constitute exclusion
criteria to treat metastatic colorectal patients with anti-EGFR
therapies (30). Since it is known that MLK3 plays a role in
controlling the signal transduction of KRAS and BRAF
(7,9,10), it is tempting to speculate that MLK3 mutations are
likely to constitute, in the near future, a biomarker for thera-
peutic selection of CRC patients for anti-EGFR therapies.
In conclusion, we describe for the first time MLK3 as a new
cancer-related mutated kinase, associated to MSI gastrointesti-
nal tumours and, eventually, to melanoma. Moreover, we show
that missense MLK3 mutations, localized in the different
domains of the protein, harbour transforming and tumorigenic
potential, in vitro and in vivo. Although further studies are
our data put forward a new candidate biomarker and/or an
attractive potential target for future therapy in MSI gastrointes-
tinal tumours, namely in the hereditary setting.
MATERIALS AND METHODS
A total of 174 primary gastrointestinal tumours and seven col-
orectal cancer cell lines—HCT15, HCT116, RKO, CO115,
HT29, SW480 and Caco2, were analysed for MLK3
mutations. Tumour DNA samples were obtained from: Uni-
versity Hospital of Groningen and University Medical
Centre of Nijmegen (The Netherlands), Hospital of S. Joa ˜o
and GDPN from Porto (Portugal), Saint-Antoine Hospital
from Paris (France), University of Helsinki (Finland), Karo-
linska University Hospital from Stockholm (Sweden) and Hos-
pital Universitari Vall d’Hebron from Barcelona (Spain).
Genomic DNA was isolated from macro-dissected frozen or
paraffin-embedded tumour tissues using standard methods.
DNA from normal controls was used for the analyses of 100
chromosomes to exclude polymorphisms. The study protocol
was reviewed and approved by the appropriate Ethics Com-
mittees, and control, constitutional and tumour samples were
obtained with informed consent and in compliance with Hel-
sinki declaration (http://www.wma.net/e/policy/b3.htm). All
cases analysed were characterized for microsatellite status.
Thirty-eight of 48 HNPCC families were characterized for
MSH2, MLH1 or MSH6 germline mutations. KRAS and
BRAF mutations were screened using current protocols.
Additionally, DNA from six melanoma cell lines (M14,
UACC62, SKMEL2, SKMEL5, SKMEL28 and MEWO) was
also analysed for MLK3 mutations.
MLK3 mutation screening
(ENSG00000173327) were screened for mutations. Exon 1
and exon 9 were subdivided in order to allow amplification
by PCR. Primer sequences are described in Supplementary
Material, Table S1. Except for exon 9, a multiplex PCR
approach was used to amplify MLK3 sequence, using the
QuantiTect Multiplex PCR Kit (Qiagen) and following the
manufacturer’s instructions. Exon 9 was amplified using a
PCR Enhancer Solution (Invitrogen) and MgSO4. The purified
PCR product was directly sequenced. Sequence alterations
were validated with a second independent PCR.
exons andintron–exonboundaries ofMLK3
The three-dimensional modelling was performed using the
Swiss-PdbViewer v8.05 program package (32). The 3D
model of the MLK3 kinase domain was constructed using
the crystal structure of the MLK1 as a template (33). The
initial model was subsequently subjected to energy minimiz-
ation by using the GROMOS force field (34), as implemented
in Swiss-PdbViewer. The quality of the structure model was
validated using the PROCHECK suite of programs (35). Con-
sequently, this structure served as the model structure for
mapping MLK3 somatic mutations. Structures were manipu-
lated using the Swiss-PDB viewer and were rendered using
Yasara View program (36).
cDNA constructs and mutagenesis
Wild-type MLK3 and mutant sequences were cloned into
MLK3 sequences (Y99C, K144R, A165S, P252H, R799C
and P840L) were generated by site-directed mutagenesis,
using the MLK3 wild-type sequence, also cloned in
pLENTID6/V5, as a template. Primer sets used to produce
MLK3 mutant sequences are described in Supplementary
Material, Table S2. pLENTID6/V5 empty vector (Mock)
Figure 5. Graphic representation of the frequency of MLK3, KRAS and
BRAF mutations in MSI primary carcinomas. From the left to the right,
columns represent the frequency of cases with KRAS alone or in combination
with MLK3, BRAF alone or in combination with MLK3, and MLK3 alone in
the overall series of primary carcinomas, in the hereditary setting and in spora-
dic forms of colorectal and gastric carcinoma.
704 Human Molecular Genetics, 2010, Vol. 19, No. 4
was obtained by the insertion of a small fragment of cDNA, in
order to circularize the plasmid. The pRK5 vector expressing
Myc-HRASV12 was used as a control in in vivo assays.
Mouse NIH3T3 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) (Gibco, Invitrogen), supplemented
with 10% NCS and 1% penicillin–streptomycin (Gibco, Invi-
trogen). Human HEK293 were maintained in DMEM (Gibco,
Invitrogen) supplemented with 10% FBS and 1% penicillin–
streptomycin (Gibco, Invitrogen). Both cell lines were grown
in a humidified incubator with 5% CO2at 378C.
Transient transfections of NIH3T3 and HEK293 cells were
carried out using Lipofectamine 2000 (Invitrogen), according
with technical information provided by the manufacturer. For
Expression kit (Invitrogen) was used for the transduction
of the MLK3 wild-type and mutant P252H sequences, as
well as the empty vector. Lentiviral transduction was per-
formed following the manufacturer’s instructions. Trans-
duced cells were selected by antibiotic resistance to
blasticidin (12 mg/ml) (Gibco, Invitrogen). The expression
levels of MLK3 in the different clones selected was
measured by western blot.
Focus formation assay
Low passageNIH3T3cells,seededon35 mmdishesat60–80%
expressing GFP protein (pCMV-GFP). Equivalent amounts of
ectopic protein expression between different transfections were
achieved, adjusting the concentration of each individual
plasmidthat was used. When required, the total amountoftrans-
fected DNA was adjusted with an empty vector. Twenty four
hours later, cells were trypsinized, split into two 100 mm
dishes and maintained in DMEM supplemented with 5% (v/v)
new born calf serum (CS; Invitrogen). The medium was
changed every 3 days thereafter. Twenty-nine days after, cells
were fixed with methanol, and the GFP fluorescence of the foci
was confirmed under an inverted fluorescence microscope
(Leica DM 2000). Cells were then stained with 0.4% crystal
violet in methanol, in order to count the foci and photograph
the dishes. The experiments were done in triplicate and the
levels of ectopic protein expression were monitored by western
blot after each transfection.
In vivo assays
Female N:NIH(s)II:nu/nu nude mice are reproduced, main-
tained and housed at IPATIMUP Animal House, sited at the
Medical Faculty of the University of Porto, in a pathogen-free
environment, under controlled conditions of light and humid-
ity. Animal experiments were carried out in accordance with
the Guidelines for the Care and Use of Laboratory Animals,
directive 86/609/EEC. Nude mice, aged 6–7 weeks, were
used for in vivo experiments. Mice were subcutaneously
injected in the dorsal flanks, using a 25-gauge needle, with
2 ? 106of NIH3T3 cells transfected with MLK3 wild-type
sequence, or with the mutants MLK3-P252H or MLK3-
R799C. Cells transfected with the empty vector or with the
mutant HRASV12 were also injected, to be used as negative
and positive controls, respectively. Mice were weighted, and
tumour width and length were measured with calipers every
week. Each mouse was euthanized 2–3 weeks after tumour
development in order to minimize suffering of the mice. The
in vivo experiment was stopped at the seventh week after
inoculation, when the last mice that were inoculated with
mutant cells (MLK3-P252H and MLK3-R799C) had already
developed large and infiltrative tumours. At this time, mice
that were subcutaneously inoculated with cells transfected
with wild-type MLK3 or with the empty vector (Mock) were
also euthanized, in order to allow comparison with mutants,
and tumour development was evaluated. Histopathology of
the tumours was evaluated using 5 mm sections and conven-
tional Hematoxilin and Eosin (H&E) staining.
Matrigel invasion assay
Prior to each experiment, 24-well matrigel-coated invasion
inserts of 8 mm pore size filters (Becton and Dickinson)
were introduced into 24-well plates. For re-hydration, the
inner and outer compartments of the system were filled with
DMEM medium, and incubated for 2 h at 378C. After rehydra-
tion, 7 ? 104cells were added to the upper part of the insert,
re-suspended in DMEM supplemented with 1% FBS, whereas
the lower part was filled with DMEM supplemented with 10%
FBS, and incubated for 24 h at 378C. Non-invasive cells were
removed and filters were washed in PBS, fixed in methanol
and mounted inVectashield
2-phenylindole (DAPI, Vector Laboratories, Burlingame,
CA). Invasive cells were counted (?20 objective), corre-
sponding to the DAPI-counterstained nuclei, which represent
cells that passed through the pores of the filter.
The statistical analysis was performed using Student’s t-test or
Fisher’s exact test, when appropriated. Differences were taken
to be statistically significant when P , 0.05.
Supplementary Material is available at HMG online.
We wish to thank Filipa Sousa and Marta Novais for technical
Conflict of Interest statement. None declared.
This work was supported by Grants from The Portuguese
Foundation for Science and Technology (FCT) (Project
Human Molecular Genetics, 2010, Vol. 19, No. 4705
PTDC/SAU-OBD/68310/2006), The Swedish Cancer Society Download full-text
and the Sixth Framework Programme from EU-FP6 (Project
Program Cie ˆncia 2007 (FCT) for C.O., P.M., J.P. and M.J.O.
Funding to pay the Open Access publication charges for this
article was provided by The Portuguese Foundation for
Science and Technology (FCT) through project PTDC/SAU-
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