HORIZON DISCOVERY
Using Rac1 isogenics to model tumor metastasis
Ceri Wiggins, Holly Astley, Claire Mahoney, David Sorrell, Chris Torrance and Rebecca Foster
Introduction
DIC
(B)
Immunofluoresce
nce
Rac1 (+/+)
The Rho family of GTPases regulate a wide range of normal cellular functions including cell
growth and cytoskeleton dynamics, but it is becoming increasingly apparent that when
deregulated, proteins such as Rac1 contribute to tumorigenesis, invasion and metastasis1.
Over-expression of Rac1 has been reported in human breast cancer and coincides with cancer
progression, metastasis and resistance to chemotherapy1.
The aim of this study was to generate a pair of X-MAN® (gene-X, Mutant And Normal) cell lines
in order to provide a genetically clean model system to investigate how Rac1 activation affects
the migratory properties of human breast epithelial cells.
(A)
(A)
(B)
Q61
MCF10A Rac1 (+/+)
P<0.001
exon 3
Q61L
exon 3
LoxP
Promoter Trapping
Selection Marker
LoxP
Homologous
Recombination
MCF10A Rac1 (-/+)
Right
homology ITR
arm
Rac1 (Q61L/+)
Figure 2. Constitutive Rac1 activation results in an altered cell morphology, the appearance of large
vacuoles and disruption of the cytoskeleton. Cells grown on coverslips were either imaged directly by
differential interference contrast microscopy (DIC) (A) or were fixed, stained to reveal the microtubules (red),
actin filaments (green) and DNA (blue), and imaged by confocal microscopy (B). White arrows indicate
examples of membrane ruffling.
Rac1 (+/+)
Rac1 (Q61L/+)
(4) Rac1 (Q61L/+) cells show altered MEK/ERK signalling
To identify signalling molecules upregulated by Rac1, pathway analysis was performed on key
components of the MEK/ERK cascade, which have been linked to cell migration in various cell
types (Figure 5). Under normal culture conditions Rac1 (Q61L/+) cells showed higher basal
MEK/ERK signalling; and in contrast to parental cells, withdrawal of serum led to heightened
pathway activation. However, growth under low anchorage conditions increased flux down the
MEK/ERK pathway in both parental and Rac1 (Q61L/+) cells. These results suggest that
constitutive activation of Rac1 markedly alters the response of cells to distinct external stimuli.
(3) Rac1 (Q61L/+) cells show increased motility and adhesion
Rac1 activation has previously been associated with increased cell motility, and as such the
ability of Rac1 (Q61L/+) cells to migrate was assessed in two assay formats - wound healing and
Boyden chamber. In both assays, cells with constitutively active Rac1 showed a higher degree of
migration, when compared to the parental cell line (Figure 3).
(A)
Q61L
Rac1 (+/+)
Rac1 (Q61L/+)
(B)
P<0.05
exon
MCF10A Rac1 (Q61L/+)
Promoter Trapping
Selection Marker
LoxP
Cre-mediated
excision
Q61L
T=0
LoxP
exon
Figure 1. Generation and validation of the MCF10A Rac1 (Q61L/+) line. (A) Generation of the MCF10A Rac1
(Q61L/+) cell line using Horizon’s gene-engineering technology: An AAV vector was used to introduce the
mutation into MCF10A cells by homologous recombination. Expression of the selection resistance gene was
driven by the Rac1 endogenous promoter, leading initially to a knock-out of the targeted allele. Subsequent
Cre-mediated excision of the selection marker via flanking LoxP sites resulted in expression of the mutated
allele. (B) Quantification of active GTP-bound Rac1 using the G-LISA™ Rac1 Activation Assay Kit
(Cytoskeleton).
(2) Constitutively active Rac1 leads to morphological and cytoskeletal changes
Introduction of the Q61L mutation resulted in dramatic phenotypic changes (Figure 2). Rac1
(Q61L/+) cells exhibited a change in shape, from elongated to spherical/oval, and intracellular
organisation was severely disrupted in a high proportion of cells by the presence of
large/multiple vacuoles. Immunofluorescence analysis of actin filaments and microtubules also
revealed microtubule disorganisation and evidence of pronounced membrane ruffling,
properties which have been previously associated with Rac1 activation2, 3.
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e [email protected]
w www.horizondiscovery.com
Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
+ 20 hours
ITR
Left
homolog
y arm
(B)
Figure 4. Rac1 (Q61L/+) cells are more adhesive. (A) Cells were plated in triplicate at 7.5x104 cells/well into
24-well plates. At the indicated time intervals, non-adherent cells were removed and the wells washed with
PBS. Cells remaining in the well were quantitated by sulforhodamine (SRB) assay. (B) Cells were plated in low
anchorage 6-well plates, incubated for 24 hours and imaged.
Results
(1) Generation of an MCF10A cell line with an activating Rac1 mutation
Using the rAAV gene-engineering technology, a predicted activating mutation (Q61L) was
knocked-in to one allele of the Rac1 locus of non-tumorigenic human breast epithelial cells
(MCF10A) to generate a heterozygous mutant cell line (Q61L/+) with an identical background to
the parental cells they were derived from (Figure 1). A Rac1 G-LISA assay was used to measure
intracellular GTP-bound Rac1 to assess what effect introducing this mutation has on Rac1
activity. This revealed that the modified Rac1 (Q61L/+) line has approximately three times the
amount of activated Rac1, compared to parental Rac1 (+/+) cells.
(A)
Figure 3. Rac1 (Q61L/+) cells show increased motility in wound healing and Boyden chamber migration
assays. (A) A single scratch was made through a confluent cell layer and images taken immediately (t=0) and
then after a further 20 hours. (B) Cells were seeded into the upper part of a Boyden chamber (Trevigen;
3455-096-K), and allowed to migrate towards serum free media (negative control) or 10% FBS (chemoattractant) for 24 hours. Cells that migrated into the bottom chamber were quantitated with calcein AM.
Data is expressed as a fold increase in migration to chemoattractant, relative to the negative control.
Since the ability to migrate is dependent on traction gained by adhesion, the adhesive
properties of Rac1 (Q61L/+) cells was examined using an adhesion assay. As expected, these
cells were able to adhere to a 2D surface more rapidly than parental cells, as well as showing
increased cell-cell adhesion when cultured in suspension conditions (Figure 4).
Figure 5. Rac1 (Q61L/+) cells have higher basal MEK/ERK signalling and respond differently to serum
withdrawal and anchorage-independent growth. Cells were cultured in 6-well plates with/without serum
(A) or under standard compared to low-anchorage conditions (B) for 24 hours. Pathway markers were then
analysed by Western blot.
Conclusions
• X-MAN® Rac1 isogenic cell lines are a valuable tool to study the effects of constitutively
activating the Rac1 pathway, without the caveats associated with transfection-based overexpression approaches
• Constitutively active Rac1 has significant effects on cell morphology/architecture, motility
and intracellular signalling networks
• This platform can easily be exploited to profile molecularly targeted agents aimed at reducing
tumor invasion and metastasis
1. Parri M, Chiarugi P: Cell Communication and Signaling (2010) 8:23
2. Ridley AJ, Hall A: Cell (1992) 70:389-399
3. Ridley AJ, Patterson AF, Johnston SL, Diekmann D, Hall A: Cell (1992) 70:401-410