A functional genetic screen reveals new regulators ofb1-integrin activity
Teijo Pellinen1,2,3,*, Juha K. Rantala1,*, Antti Arjonen1,2, John-Patrick Mpindi1,3, Olli Kallioniemi1,3,* andJohanna Ivaska1,2,4,*,`
1Medical Biotechnology, VTT Technical Research Centre of Finland, 20521, Turku, Finland2Centre for Biotechnology, University of Turku, 20520, Turku, Finland3Institute for Molecular Medicine Finland (FIMM), Biomedicum 2U, 00014 University of Helsinki, Helsinki, Finland4Department of Biochemistry and Food Chemistry, University of Turku, 20520, Turku, Finland
*These authors contributed equally to this work`Author for correspondence ([email protected])
Accepted 29 September 2011Journal of Cell Science 125, 649–661� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.090704
Summaryb1 integrins constitute a large group of widely distributed adhesion receptors, which regulate the ability of cells to interact with theirsurroundings. This regulation of the expression and activity of integrins is crucial for tissue homeostasis and development andcontributes to inflammation and cancer. We report an RNA interference screen to uncover genes involved in the regulation of b1-
integrin activity using cell spot microarray technology in cancer cell lines. Altogether, ten cancer and two normal cell lines were used toidentify regulators of b1 integrin activity. Cell biological analysis of the identified b1-integrin regulatory genes revealed that modulationof integrin activity can influence cell invasion in a three-dimensional matrix. We demonstrate with loss-of-function and rescue
experiments that CD9 activates and MMP8 inactivates b1 integrins and that both proteins associate with b1 integrins in cells.Furthermore, CD9 and MMP8 regulate cancer cell extravasation in vivo. Our discovery of new regulators of b1-integrin activityhighlight the complexity of integrin activity regulation and provide a set of new genes involved in regulation of integrin function.
Key words: Integrin activity, RNAi, Invasion
IntroductionIntegrins are a family of cell surface adhesion receptors that
regulate the ability of cells to interact with their surroundings
(Gahmberg et al., 2009; Legate et al., 2009). In humans, there are
24 different integrins and 12 of these are a and b1 heterodimers
mediating adhesion to various extracellular matrix (ECM)
components. Integrin ligand binding leads to clustering of
various adaptor proteins and kinases that together regulate actin
polymerization and downstream signalling to MAP kinases
(Gahmberg et al., 2009; Guo and Giancotti, 2004; Schwartz and
Assoian, 2001). As integrins are important regulators of cell
proliferation and motility, their dysregulation can be associated
with inflammation or tumour progression (Desgrosellier and
Cheresh, 2010; Shattil et al., 2010). For example, studies with
conditional knock-out mouse models of breast carcinomas suggest
that b1 integrins have an important role in primary tumour growth
(White et al., 2004). Also, high expression levels of b1 integrins
have been shown to correlate with poor prognosis in breast cancer
(Yao et al., 2007), and signalling by b1 integrin might induce
dormant cancer cells to form proliferative metastasis in vivo
(Barkan et al., 2010). Furthermore, a recently described tumour
suppressor protein SCAI suppresses cell invasion by repressing b1-
integrin transcription and expression (Brandt et al., 2009).
Integrin activity (i.e. the ability to bind ligands) is strictly
controlled by intracellular signals and this has broad biological
importance in adhesion-dependent events in cancer (Desgrosellier
and Cheresh, 2010; Shattil et al., 2010). In leukaemia for example,
b2 integrin and b1 integrin can be constitutively activated, which
contributes to altered leukocyte trafficking and adhesion to stromal
components (Chen et al., 2008; Fierro et al., 2008).
The best-characterized regulatory step of integrin activation
involves binding of cytoplasmic regulators, such as talin-1 and -2
and kindlin-1, -2 and -3 (also known as FERMT1, 2 and 3), to the
cytoplasmic domains of integrins (Moser et al., 2009; Shattil et al.,
2010). Talins are known to stabilize the active open conformation
of integrins and also strengthen the integrin connection to the actin
cytoskeleton, which brings about the clustering of multiple actin-
binding proteins to adhesion sites (Moser et al., 2009; Shattil et al.,
2010). These focal contacts can mature into focal adhesions, which
can constitute an interactome of more than 150 components [www.
adhesome.org (Zaidel-Bar et al., 2007)]. Given the complexity of
cell adhesion machinery and the fact that there is even some
controversy about the well-characterized final steps of integrin
activation (binding of cytosolic proteins to the b-tail) (Shattil et al.,
2010), we set out to search for b1-integrin activity regulators. We
employed our recently developed cell spot microarray (CSMA)
technique (Rantala et al., 2010; Rantala et al., 2011) to perform an
unbiased druggable genome-wide RNA interference (RNAi)
screen. Our study identified new regulators of b1-integrin activity
in 12 different cell lines. In addition, we identified a set of
previously unknown integrin regulators, which might contribute to
cancer cell invasion in vitro and in vivo. Thus, these data
underscore the role of b1 integrins and their upstream regulators as
possible therapeutic targets.
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Fig. 1. See next page for legend.
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ResultsA druggable genome-wide RNAi screen for regulators of
integrin activity
Specific monoclonal antibodies (12G10 and 9EG7), which
distinguish active forms of b1 integrins (Byron et al., 2009),
were used in combination with a neutral integrin antibody (anti-
a2 integrin; this was chosen because a2b1 integrin is an
abundantly expressed integrin in these cell lines, and using an
a-subunit-recognizing antibody circumvents possible issues with
overlapping binding sites between different b1 antibodies) to
detect RNAi-induced changes in integrin activity in cancer cells
grown over a Matrigel matrix (Fig. 1A). Because the b1-integrin
antibodies could influence integrin conformation in live cells, it
was important to use fixed cells when using the antibodies as
indicators of siRNA effects on receptor activity. To validate our
experimental approach we assayed the effect of silencing the
genes encoding talin-1 and -2 in CSMA spots (supplementary
material Fig. S1A,B). Silencing of the talin-2 gene (using
the CSMA platform) significantly (P,0.05) reduced integrin
activity (9EG7 staining) in vertebral-cancer of the prostate
(VCaP) cells (supplementary material Fig. S1A,B). The silencing
efficiency and staining specificity on the CSMA were further
confirmed by silencing b1-integrin and a2-integrin subunits
(supplementary material Fig. S1C).
We assayed the VCaP prostate cancer cell line with the
druggable genome RNAi library (Qiagen v1.0) targeting 4910
human genes with two individual siRNA constructs per gene.
Fixed cells were analyzed for 9EG7 and 12G10 antibody staining
in separate experiments, each consisting of 9820 cell spots on a
single plate with each position containing a single individual
siRNA. Of these, significant changes with z-score standardized
values of ,–2 or .+2 were found in 4.5 and 4.4% of the siRNAs
for VCaP cells (9EG7 and 12G10, respectively; Fig. 1B;
supplementary material Fig. S1D). Importantly, z-scores for all
replicate control siRNA (n5235) transfections were between –2
and +2 (Fig. 1B; supplementary material Fig. S1D, yellow and
orange spots). Furthermore, RNAi effects on active integrin andphalloidin intensities showed high correlation co-efficiency
(supplementary material Fig. S1D,E), which was anticipatedbecause integrins are known to signal to the actin cytoskeletonand stimulate de novo actin polymerization or actin stress-fibre
bundling (DeMali et al., 2003).
Ingenuity pathway analysis of the genes involved in theregulation of b1-integrin activity indicated G-protein-coupled
receptor (GPCR) signalling as the major pathway influencing b1-integrin activity in the screen (Fig. 1C). This was not surprisingbecause the GPCR activation by various cytokines and peptide
hormones is one of the most well-characterized pathways in integrinactivity regulation (Laudanna and Alon, 2006; Li et al., 2010).Interestingly, many signalling pathways that are involved in theregulation of inflammation (e.g. GPCR, NF-kB, RANK, interleukin,
arthritis, glucocorticoid, PI3K–AKT and retinoid acid receptor)were identified as important regulators of integrin activity in thescreen. This suggests that inflammatory signalling could be coupled
to regulation of b1-integrin activity in several different ways.
Secondary screen validation of the integrin regulators
For validation and follow-up studies we selected 50 genes thatsignificantly either activated (25) or inactivated (25) b1 integrins
[median of the 9EG7 and 12G10 z-scores (n54) less than –1.0 ormore than +1.0; Fig. 1D; supplementary material Table S1 for z-scores and Table S2 for gene annotations]. Four new siRNAsfor each gene were applied for the secondary validation
(supplementary material Table S3) in order to ascertain thespecificity of the effects. Each siRNA was printed in duplicate onthe secondary CSMAs and screened for 9EG7 and 12G10
binding. Together these resulted in 16 technical replicates and/orobservations per gene. The VCaP secondary screen showed that34% of the primary screen hits could be consistently reproduced
when a more stringent criterion was applied (two additionalsiRNA oligonucleotides scoring for each gene in duplicate;Fig. 2A; supplementary material Table S4). Silencing of five outof the 50 genes (ERCC1, CDK5R1, HDAC4, LCK, COL9A1) in
the secondary screen resulted in an opposite effect on integrinactivity to that in the primary screen, highlighting the importanceof the secondary validation screen with more siRNAs.
To investigate the generality of our findings from VCaP cells,we silenced the 50 genes in seven other prostatic cell lines (PC3,ALVA31, 22RV1, MDAPCA2a, RWPE1, primary epithelial
prostate cells and primary stromal prostate cells) and in four non-prostatic cell lines (lung cancers NCI-H460, A549 and coloncancers HCT-116, SW-480; z-scores in supplementary material
Table S4). Importantly, most of the reproducible VCaP hitsiRNAs induced similar effects on integrin activity in many ofthe other cell types. The best correlation between cell types was
seen between prostate cancer cell lines PC3 and ALVA31(R250.48). As a comparison, the lung carcinoma cell lines NCI-H460 and A549 had a correlation of R250.36. Some of the poor
correlation between VCaP and other prostate cancer cell lines(PC3, ALVA31, 22RV1, MDAPCA2a) could be due to the factthat VCaPs are androgen sensitive whereas the rest are androgeninsensitive (van Bokhoven et al., 2003).
From integration of these data, consistent regulators of integrinactivity could be identified. Altogether, silencing of 13 genes
resulted in downregulation and silencing of 10 genes involvedin upregulation of b1-integrin activity in at least four cell lines(gene IDs highlighted in green for downregulation or red for
Fig. 1. Results of the primary screens for b1-integrin activity regulators.
(A) In high-density cell spot microarrays (CSMA) cells were silenced on
Matrigel spots containing siRNA and transfection reagent during 48 hours
incubation. The example image shows VCaP cells on arrays with antibody
stainings for a2 integrin (ITGA2), active b1-integrin epitope (9EG7) and F-
actin (phalloidin). The array was scanned with a Tecan laser scanner (large
panel) and further imaged using fluorescence microscopy (enlarged images).
(B) The druggable genome of 4910 genes with two siRNAs per target
(Qiagen), together with controls (AllStars negative, n5118; GFP, n5117)
was silenced in VCaP cell line as above and stained for a2 integrin, active b1
integrin (9EG7) and F-actin (phalloidin). Shown are z-scores for 9EG7
staining intensities normalized to total a2 integrin signal (dark blue for
druggable genome siRNAs, yellow for AllStars negative and orange for GFP
siRNAs) and phalloidin also normalized to a2 integrin (light blue) from each
spot. Druggable genome siRNAs with z-scores of ,–2 or .+2 are indicated
with green and red, respectively. (C) The genes with z-scores of ,–2 or .+2
from the screens where either 9EG7 or 12G10 antibodies (both for active b1
integrin) were subjected to ingenuity pathway analysis (IPA). The top portion
of the canonical pathways are displayed as a bar chart. The canonical
pathways that are involved in this analysis are shown along the y-axis. As the
default, the x-axis shows the –log of P-value, which was calculated using a
right-tailed Fisher’s exact test. (D) The z-score median for each gene as
analyzed from all the knockdowns in the 9EG7 and 12G10 primary screens
(n54). The 50 genes listed (25 up and 25 down) were chosen for
secondary validation.
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Fig. 2. See next page for legend.
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upregulation in Fig. 2A). Silencing of EPS15 resulted in the most
frequent integrin inactivation among the different cell lines.
Silencing of MAST2, by contrast, induced integrin activation
most often in the cell lines analyzed. Other high-scoring integrin-
activity-regulating siRNAs targeted ATRN, ERCC1 and DHRS4
(all inactivating) and HDAC4, INPP1 and MSX1 (all activating).
Many of these 23 regulators of integrin activity (including
EPS15 and MAST2) have not, to our knowledge, been identified
previously as being involved in the regulation of integrin
activity.
The integrin activity state can also be analyzed by measuring
the integrin–ligand binding of living cells in suspension, although
this situation can be very different from the cell spot microarray
approach, in which cells are adhering to the ligand over a period
of 2 days. To compare our CSMA screen results with a
conventional ligand-binding assay, we silenced the hits of the
PC3 cells and measured the binding to the soluble fibronectin
fragment (repeats 7–10). Importantly, most of the siRNAs
affecting 12G10 or 9EG7 staining in PC3 cells also influenced
binding of the monomeric fibronectin fragment to live cells in a
flow cytometric analysis, further confirming the effects of
the siRNAs on integrin activity (supplementary material Fig.
S2A,B). In addition to fibronectin binding, we analyzed the levels
of total cell surface b1 integrin and 9EG7 epitope in suspended
and parformaldehyde-fixed PC3 cells after treating the cells with
a set of integrin-inactivating and integrin-activating siRNAs
(supplementary material Fig. S2C). Silencing of CD9, EPS15 and
the talin-1 and -2 genes all reduced the surface-exposed 9EG7
epitope, whereas silencing of MASTL, MAST2 and MMP8
increased it. Measurement of the total surface b1 integrin
showed that knockdown of talin-1 and -2 also reduce the total
b1 integrin in PC3 cells, but of the other knockdowns only
siMASTL resulted in changes in surface b1 integrin by reducing it
slightly. However, this approach did not show changes in 9EG7
binding after silencing of the AKT3 or HDAC4.
The expression of the 50 genes was also analyzed from
Affymetrix HG-U133A gene expression microarray analyses of
four prostatic cell lines (VCaP, 22-RV1, MDA-PCA2a, PC3) and
A549 lung carcinoma cells (supplementary material Table S5).
From these data it is obvious that the expression levels of the
genes identified as regulators of integrin activity can vary
substantially between cell lines and this is likely to influence the
effects that specific siRNAs have in each given cell line. It is
important to note that some known integrin regulators, such asC1B1, kindlins and filamins (Shattil et al., 2010), were notincluded in the predefined commercial library used in this study.
Therefore, these genes were not identified in the screen. Inaddition, redundancy between protein family members (such astalin-1 and -2) and gene expression profiles in the cell lines usedmight have resulted in the absence of some previously described
candidates from the highest scoring hits.
The phenotypes and subcellular localization of active b1integrin in siRNA-treated cells were further analyzed with
confocal microscopy. In VCaPs, silencing of ALDH4A1 andPRKG2 decreased 12G10 staining, whereas silencing of MAST2
and HDAC4 prominently increased it (Fig. 2B). A highermagnification of VCaP cells on CSMA spots revealed that the
active integrin was also localized to small focal contacts on celledges (Fig. 2C). Increased active-b1-integrin staining at celledges and protrusions was also detected in 22RV1 cells after
treatment with activating siRNAs (HIP1, IFNA1, MSX1), andthe staining was reduced upon silencing of ATRN, EPS15 andCACNA1I (supplementary material Fig. S3A). Images of PC3
cells on ECM coating (supplementary material Fig. S3B) orcells grown on plastic (supplementary material Fig. S3C)revealed that in these cells 12G10 staining was highly
vesicular with no apparent focal contacts or focal adhesions.However, also in these cells b1-integrin inactivating siRNAs(PFKFB2, AKT3, CD9, DHRS4, PFTK1, EZH2) and activatingsiRNAs (MYCL1, MSX1, NCAM1, IRS2, MAST2, LCK) resulted
in marked changes in 12G10 staining, thus verifying thescreening data from the cell spot arrays. We also confirmedthe silencing efficiency of siRNAs for 20 different genes in the
PC3 cell line by quantitative Taqman RT-PCR (supplementarymaterial Fig. S4A).
CD9 and MMP8 associate with b1 integrins and regulateintegrin activity
Next we chose one activating (CD9) and one inactivating(MMP8) gene for further validation. We found that CD9 protein
is present in PC3 cells and the individual siRNAs used in thescreen readily silenced CD9 (Fig. 3A). In addition, silencing ofCD9 was verified using immunofluorescence staining of CD9and confocal microscopy imaging (supplementary material Fig.
S4B,C). Next we analyzed soluble ligand (fibronectin fragment)binding to PC3 cells subjected to sequential transfection of a CD9
siRNA that targets the 39 UTR (siCD9_7) or control siRNA
followed by cDNA encoding GFP–CD9. Silencing of CD9
inhibited b1-integrin ligand binding by 3162%. Conversely,expression of GFP–CD9 in these cells as well as control-siRNA-
treated cells strongly induced binding of soluble ligand tothe cells (Fig. 3B,C). In addition, we found using reciprocalimmunoprecipitations that CD9 and b1 integrin associate in PC3
cells (Fig. 3D). This is consistent with earlier reports of integrin–CD9 complexes in other cell types (Berditchevski et al., 1996).
MMP8 protein was also expressed in PC3 cells and was detectedboth in the conditioned medium of the cells as well as in full-cell
lysates (Fig. 3E), and MMP8 protein levels were downregulated incells transfected with MMP8 siRNA (Fig. 3E). Interestingly, wefound in reciprocal immunoprecipitations that MMP8 also
associates with b1 integrin (Fig. 3F). Because MMP8 is secretedinto the medium (Tanaka et al., 2007), we hypothesized thatMMP8 could inactivate integrins by binding to the receptor on the
Fig. 2. Secondary screen validation of integrin activity regulators in
multiple cell lines. (A) The colour map shows the results from 50 selected
genes with their corresponding median z-scores from the primary VCaP
screens and the secondary validation screens with 11 additional other cell
lines. The numbers in the left column indicate the median z-score of four
siRNAs (2612G10+269EG7) in the primary screen (see Fig. 1D). siRNAs
inducing downregulation or upregulation of b1-integrin activity in at least
four cell lines are indicated with coloured gene IDs in the right column
together with the total number of siRNAs affecting 9EG7 or 12G10 binding
(z-score ,–1 or .+1) in all of the cell lines studied (numbers in black on the
right column). Top-scoring genes were also ranked on the frequency of
siRNA effects on integrin activity across the 12 cell lines (RANK column:
green, inactivating; red, activating). (B) Confocal microscopy images of
VCaP cells on array spots, showing maximum intensity z-projections of a2
integrin (ITGA2), 12G10, phalloidin and DAPI staining. Intensity histograms
were drawn from a2 (green) and 12G10 (red) stainings (right). Scale bars:
20 mm. (C) Higher magnification of control-siRNA-silenced VCaP cells with
12G10 and phalloidin stainings. Scale bar: 20 mm.
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plasma membrane. To test this we analysed soluble ligand binding
of MMP8 siRNA-treated or control-siRNA-treated PC3 cells
exposed to purified recombinant MMP8 for 1 hour. Silencing of
MMP8 significantly (P50.002) induced b1-integrin ligand
binding and this was fully reversed by ectopic recombinant
MMP8 in the medium (Fig. 3G). Taken together, these data
confirm the regulatory effects of CD9 and MMP8 on integrin
activity identified in our screen and demonstrate that both of these
proteins associate with integrins in prostate cancer cells.
Integrin activity positively associates with cell invasion
in vitro
Invasion into matrix is integrin dependent in most cell lines
(Friedl and Wolf, 2003). However, limited data exist on the role
of integrin activity in cell invasion. To investigate this we
silenced 32 genes with replicates and analyzed by time-lapse
microscopy the invasive growth of PC3 cells in Matrigel. These
transfections included the identified b1-integrin inactivating and
activating siRNAs together with siRNAs with no apparent effect
Fig. 3. CD9 and MMP8 associate with b1 integrin. (A) PC3 cells were silenced with two different siRNAs against CD9, and CD9 protein was blotted from
lysates. (B) siRNA targeting the 39-UTR of CD9 (CD9_7; Qiagen) and cDNA encoding GFP–CD9 were used for rescue of fibronectin-647 binding in PC3 cells,
and some of the rescue experiment cells were used to analyse the protein levels by western blotting (C). (D) Immunoprecipitations (IPs) from PC3 lysates showed
co-precipitation of endogenous b1 integrin with CD9. (E) Western blotting was used to analyse the expression of MMP8 in PC3 cell lysate and in 2-day-
conditioned PC3 medium, and the knockdown effect of MMP8 was analyzed from cell lysates. (F) b1 integrin co-precipitated with CD9, but not control (Ctrl)
antibody in immunoprecipitations using PC3 cell lysates. (G) Recombinant purified MMP8 (rMMP8) or vehicle was added to the growth medium of PC3 cells to
rescue the MMP8 knockdown effect in soluble fibronectin-647 binding as measured by FACS analysis. Values are means 6 s.e.m., n53; **P,0.01, ***P,0.005.
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on integrin activity, as well as talin-1 and -2 as a positive control
(Fig. 4A). Silencing of 19 genes significantly (P,0.05) affected
the invasive growth in Matrigel compared with the effect of the
control siRNA (Fig. 4A–C). Out of these 19 genes, ten were our
newly characterized b1-integrin regulators, and for eight of these
ten genes there was a positive correlation between integrin
activity and invasion. These effects were not detected in cells
cultured on plastic, suggesting that regulation of invasive growth
is distinct from regulation of cell proliferation in two-
dimensional conditions (Fig. 4D). Talin-1 and -2 siRNAs and
Fig. 4. b1-integrin activity associates with cancer cell invasion. (A) PC3 cells were treated with the indicated siRNAs (24 hours; AllStars negative5ctrl) and
were overlaid with 25% Matrigel and cell growth and motility was followed for 160 hours with Incucyte live microscopy. Shown are invasive growth area (surface
area occupied by the cells) values after 160 hours relative to starting point (means 6 s.e.m., n56; *P,0.05). Inactivating siRNAs are indicated in green and
activating siRNAs are in red. (B) Examples of the invasive growth area curves of knockdowns with the indicated siRNAs from 4A (fold change). (C) Images of
invasive growth of the cells in A. (D) PC3 cells were treated with the indicated siRNAs (24 hours; AllStars negative5ctrl) growing on plastic were followed for
160 hours with Incucyte microscopy. Blue columns shown the cell growth area after 160 hours on plastic relative to the starting point (values are means 6 s.e.m.,
n56). The dashed line indicates the invasive three-dimensional growth of PC3 cells transfected with the corresponding siRNAs shown in A.
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Fig. 5. b1-integrin regulators alter cell invasion in Matrigel. (A) Maximum z-projections of representative PC3 invasion assays with 5 mg/ml of the indicated
antibodies (Mab13 is an b1-integrin-blocking antibody). z5100 mm; x–y57756775 mm. Values are mean invasive areas 6 s.e.m., n53; *P,0.05. (B) PC3
invasion assay of cells treated with the indicated siRNAs for 24 hours prior to the start of the assay. (C) Quantification of the invasion assays in B (mean invasive
areas 6 s.e.m., n56; *P,0.05). (D) Invasiveness of ALVA31 cells after treatment of cells with the indicated siRNAs. (Top panels) Cell morphology in the bottom
confocal plane after 5 days of invasion (phalloidin staining). (Bottom panels) Invasive areas as in A. The arrow on the left indicates direction of invasion (mean
invasive areas 6 s.e.m., n53; *P50.009). (E) Invasiveness of CD9-silenced 22RV1 cells compared with the control cells (mean invasive areas 6 s.e.m., n53;
*P50.003). Arrow indicates direction of invasion. (F) Still images from time-lapse movies (supplementary material Movies 1, 2) of control and CD9-silenced
PC3 cells in 25% Matrigel. Right hand panels show similarly treated cells fixed and stained for 12G10 and DAPI. Scale bars: 5 mm.
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five of our inactivating siRNAs inhibited invasiveness, whereas
siRNAs for three activating candidates increased invasive
growth. Intriguingly, two genes (MSX1, EPS15) out of the ten
newly found regulators in our assay perturbed the correlation
between the b1-integrin activity and invasion. Of these two
genes, the product of MSX1, a homeodomain transcription factor,
regulates various developmental genes and, for example, in
neuroblastoma can stimulate the expression of various canonical
Wnt signalling inhibitor genes (Revet et al., 2010). EPS15,
however, is an adaptor protein for clathrin and is involved in the
Fig. 6. MMP8 and CD9 regulate b1-integrin activity and lung invasion of breast cancer cells. (A,B) MMP8-silenced, CD9-silenced or control-siRNA-treated
MDA-MB-231 cells plated on fibronectin and stained as indicated. Shown are representative images and quantification of 9EG7 staining intensities (means 6
s.e.m., 48–52 cells, *P,0.05). Scale bars: 10 mm. (C) Cells were transfected with the indicated siRNAs and mRNA levels were analysed using Taqman
quantitative PCR with specific primers and probes. (D) CD9-silenced (green), MMP8-silenced (green) or control-siRNA-treated (red) MDA-MB-231 cells were
pre-labelled with fluorescent cell trackers and intravenously injected (56105 green and 56105 red cells together) into mice. After 48 hours the cells remaining in
the vasculature were flushed out with PBS perfusion. Cells from one lung per mouse were isolated and the fluorescence quantified, and the other lung was
processed for sectioning and counterstained with DAPI (representative sections from both experiments are shown). Results are expressed as means 6 s.e.m.
percentage of specified cells of all cells isolated (n510 mice; *P50.05). Scale bars: 50 mm.
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endocytosis and downregulation of different receptor tyrosine
kinases (Puri et al., 2005). The perturbation of the correlationbetween integrin activity and invasion for MSX1 and EPS15
could be due to these other functions elicited by the gene
products.
The data of invasive growth were further validated with three-dimensional Matrigel invasion assays, in which cells invade thegel in a b1-integrin-dependent manner (Fig. 5A). The silencing
of the genes encoding talin-1 and -2, CD9 and AKT3 againinhibited the invasiveness (Fig. 5B,C; supplementary materialFig. S5), and another b1-integrin activity inhibiting siRNA
against COL9A1, which did not show an effect on invasivegrowth, decreased the invasiveness of PC3 cells in three-dimensional matrix (supplementary material Fig. S5).Conversely, b1-integrin stimulatory siRNAs against LCK and
MMP8 induced invasiveness correlating well with the invasivegrowth assays (Fig. 5B,C). Silencing of CD9 inhibited invasionof ALVA31 and 22-RV1 cells also (Fig. 5D,E). CD9 is a
tetraspanin family protein, which has been shown to associatewith integrins on the membrane (Berditchevski, 2001). Usinghigher-resolution time-lapse imaging we observed that the cells
in which CD9 was silenced were unable to protrude intoMatrigel, but instead circulated in a non-polarized fashion(Fig. 5F; supplementary material Movies 1, 2). These cells also
contained lower levels of active b1 integrin, as determined byimmunofluorescence imaging of the same cells with 12G10antibody (Fig. 5F). These results further highlight the fact thatb1-integrin activity regulators identified in this screen, such as
CD9, can function as regulators of cell invasion in vitro.
MMP8 and CD9 regulate cancer cell extravasation in vivo
The observed positive association between integrin activity and
invasion in vitro prompted us to investigate whether some of theidentified integrin regulators could also regulate the invasion oftumour cells from the blood stream into the lung parenchyma.
Our attention was drawn to two genes (MMP8; an integrin-activating siRNA, and CD9; an integrin-inactivating siRNA)with apparently opposing effects on integrin activity and in
vitro invasiveness. MDA-MB-231 (ATCC) breast cancer cellsextravasate from the blood stream efficiently (Bos et al., 2009;Chabottaux et al., 2009; Gupta et al., 2007). Because this cell linewas not included in the screens, we silenced CD9 and MMP8 in
these cells and analyzed changes in integrin activity by stainingwith 9EG7 (Fig. 6A,B). Consistent with data from the other celllines, silencing of CD9 (77% silencing, as measured by qRT-
PCR) inhibited, and silencing of MMP8 (92% silencing)increased the intensity of staining for the active epitope of b1integrin.
To visualize the early events of lung metastasis, we inoculated
mice with an equal number of control-siRNA-silenced andMMP8- or CD9-silenced cells (Fig. 6C). Within 2 days ofentering the circulation, tumour cells could be detected in the
lungs. Importantly, CD9-silenced cells (green in Fig. 6)extravasated from the lung vasculature significantly (P50.03)less than control cells (red), whereas MMP8-silenced cells
(green) entered the lungs significantly (P50.04) moreefficiently than control cells (Fig. 6D). This was not due todifferences in proliferation, as proliferation rates of CD9-silenced
and control cells were equal and silencing of MMP8, in fact,slightly reduced the proliferation (supplementary material Fig.S6). These data demonstrate that genes identified in this screen,
such as CD9 and MMP8, are involved in biologically importantprocesses, for example, the control of cancer cell extravasationfrom the blood stream, in vivo.
DiscussionIn this study we performed a cell-microarray-based functionalgenetic screen with 12 different cell lines to identify regulators ofb1 integrin in human cancer cells. We found a substantial number
of previously unidentified proteins and pathways that contributedirectly or indirectly to modulation of cell-matrix interactions.
Many of the hits, such as the highest scoring b1-integrin-positive (EPS15, ATRN and ERCC1) and b1-integrin-negative(MAST2, HDAC4 and INPP1) regulators have not been
previously linked with integrin activity modulation. This isinteresting because EPS15 is a well-characterized coat adaptorprotein functioning in endocytosis of receptor tyrosine kinases
(RTKs), such as EGFR, through clathrin-coated pits (Puri et al.,2005). It is possible that silencing of EPS15 influences integrinactivity by altering the cross-talk between integrins and RTKs
or by influencing clathrin-mediated endocytosis of integrinsthemselves. Alternatively, EPS15 could physically associate withintegrins and influence their active conformation through this
mechanism, as EPS15 was recently identified, using proteomics,as a putative integrin-binding protein (Humphries et al., 2009).MAST2, by contrast, is a poorly studied microtubule-associatedserine-threonine kinase. Interestingly, it contains a PDZ domain
(Lumeng et al., 1999; Prehaud et al., 2010), which could interactwith integrins and influence their function similarly to ZO-1binding to a5 integrin through its PDZ domains (Tuomi et al.,
2009). Furthermore, MAST2 has been shown to phosphorylateand stabilize the tumour suppressor PTEN, which could influenceintegrin activity by upregulation of the PI3K–AKT pathway
(Valiente et al., 2005). However, some of the 23 consistentregulators of b1-integrin activity in our screens have been linkedto integrins or cell migration. For example, PFTK1, which was apositive regulator of b1-integrin activity in our assays, has also
been shown to increase migration and perturb focal adhesiondynamics in two other RNAi screens (Simpson et al., 2008;Winograd-Katz et al., 2009). In addition, HDAC4 and NCBP2
were also recently identified in a proteomic screen as componentsof the integrin interactome (Humphries et al., 2009).
We demonstrate here that, in prostate cancer cells, CD9 associateswith b1 integrins. We also verify with loss-of-function and
overexpression experiments that CD9 regulates integrin binding toa soluble ligand in these cells. The integrin-binding CD9 tetraspaninprotein has been shown to increase cell motility and b1-integrin
activity when ectopically expressed in Chinese hamster ovarian(CHO) cells (Kotha et al., 2008). In agreement with our findings,CD9 has been shown to be positively associated with cancer cellinvasion in vivo, especially during the process of extravasation
through the endothelium (Hori et al., 2004; Sauer et al., 2003).
Taken together, our data demonstrate that regulation ofintegrin activity is highly complex and could be regulated notonly by integrin binding proteins such as talins and kindlins but
also by numerous other signalling pathways that can influenceintegrin function by indirect mechanisms or directly by physicalinteractions with integrins.
An interesting finding of this study was that the ingenuity
pathway analysis of the genes involved in the regulation of b1-integrin activity yielded signalling pathways generally implicatedin inflammation. In line with this, one of the negative regulators
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of integrin activity, MMP8 has been shown to have a protectiverole against inflammation in arthritis and lung fibrosis bysuppressing the expression or availability of inflammatory
players such as IL-1b and IL-10 (Garcia et al., 2010; Garcia-Prieto et al., 2010). In addition, a reduction of the plasma levelsof MMP8 has been associated with higher lymph node and distant
metastasis in inflammatory breast cancer patients (Decock et al.,2008). Thus, the mechanistic connection of inflammation andintegrin activity regulation in cancer would be an interesting
topic for future studies. These studies are in accordance with ourfindings where we show that MMP8 is a negative regulator ofcancer cell invasion in vitro and in vivo, and that this could bedue to its role in the suppression of b1-integrin activity.
Interestingly, we find that MMP8 associates with b1 integrinsin prostate cancer cells and that exposure of cells to recombinantMMP8 protein is sufficient to rescue the increased b1-integrin
activity in MMP8-silenced cells. These data suggest a previouslyunknown mechanism, by which MMP8 binding to b1 integrins onthe plasma membrane negatively regulates their activity.
The siRNA effects were most similar among the hormone-
insensitive TP53 mutant prostate cancer cell lines PC3, ALVA-31, 22-RV1 and MDA-PCA-2a. Several siRNAs with prominenteffects on integrin activity in prostatic cell lines had limited
effects in the two lung cancer cell lines and in HCT-116 coloncancer cells with wild-type TP53 (Fig. 2A). This is interestingbecause these differences could be linked to the accelerated
endosomal trafficking of integrins, which drives cell invasion andmetastasis downstream of mutant TP53 (Muller et al., 2009;Selivanova and Ivaska, 2009). On the basis of the plausiblefunctional link between integrin activity and trafficking (Caswell
et al., 2009), the genes identified in this screen could influenceboth processes and thus be crucial for invasion of cancer cells,depending on their TP53 status. In conclusion, these data suggest
that regulation of adhesion receptor function can be highly cell-context dependent.
b1 integrins have been investigated for more than two decades,and so far only relatively few activators and inhibitors of their
function have been found. Here, in a functional screen, weidentified 13 activators and 10 inhibitors of b1-integrinregulation in four or more of the cell lines tested. We
employed two different in vitro invasion assays and showedthat there is a positive link between b1-integrin activity and cellinvasion. Furthermore, we demonstrated that the in vitro invasioneffects could be reproduced in vivo in a lung extravasation assay
by silencing of two different targets, CD9 and MMP8. Becauseour screen was performed with the druggable genome library,these pathways and proteins might be relevant in the
development of new approaches to control dysregulatedintegrin activity in cancer.
Materials and MethodsCSMA
The cell spot microarrays (CSMA) used for the analysis were as follows. AnsiRNA library with two individual siRNAs for 4910 target genes (Qiagendruggable genome library v1.0) and 220 replicate negative control samples of twocontrol siRNAs were used for CSMA printing. The library was prepared with aHamilton STAR liquid handling robot (Hamilton Robotics) by mixing (for eachsample) 5 ml 1.67 mM siRNA (Qiagen) diluted in OptiMEM-I (Gibco) with 0.8 mlsiLentFect (Bio-Rad) transfection reagent and 0.2 ml OptiMEM I. Arrays wereprinted on untreated polystyrene microplates with four rectangular wells (Nunc)using a Genetix Qarray2 (Genetix Ltd) microarray printer with 200 mm solid tippins (PointTechnologies). For the CSMA experiments with all the described celllines, cells were grown to 80% confluency on 10 cm culture dishes anddissociated with HyQtase (HyClone) treatment for 5 minutes, suspended in
culture medium and dispersed on the arrays as a uniform cell suspension. To eacharray well 36106 cells in 4.5 ml of medium were added and allowed to adhere at
37 C for 20 minutes. Non-adhered cells were washed off and 4.5 ml of freshculture medium was added. Cells were then transfected by taking up the siRNAfrom the spot on arrays for 48 hours. Cells were fixed with 2% paraformaldehyde(PFAH), permeabilized with 0.15% Triton X-100 in PBS for 15 minutes and
stained with antibodies for integrin b1, integrin a2 and for F-actin withphalloidin–Alexa-Fluor-555 and Syto60. Primary array analysis was performedusing laser microarray scanning (Tecan LS400). Cumulative fluorescence
intensities for measured channels per spot were normalized using pinnormalization (Array ProAnalyzer v4.5) and used to calculate the ratio ofintegrin-b1 and F-actin staining against integrin-a2 counter staining. A z-scorestandardization was used to evaluate the significance of the measured signal
ratios against the global array mean and standard deviation. Microscopy of thearrays was used to validate the results.
Antibodies
Integrin-b1 antibodies 12G10 and 9EG7 were from Abcam and BD Pharmingen,
respectively. K20 total integrin-b1 antibody was from Beckman Coulter. Inhibitoryintegrin-b1 antibody Mab13 was from BD Biosciences. The neutral integrin-a2-recognizing antibody, AB1936, was from Millipore. Anti-talin-1/2 (clone 8d4) was
from Sigma. Anti-CD9 was a generous gift from Francisco Sanchez-Madrid(CNIC, Madrid). Anti-MMP8 antibody MAB3316 was from Chemicon.
Western blotting and immunoprecipitations
Cells were lysed in lysis buffer (40 mM Hepes–NaOH, 75 mM NaCl, 2 mMEDTA, 1% NP-40 supplemented with protease and phosphatase inhibitors).
Lysates were subjected to immunoprecipitation using the indicated antibodies at+4 C overnight. Protein-G beads (GE Healthcare) in lysis buffer were added andincubated for 1 h at 4 C. Beads were washed with wash buffer [20 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 1% NP-40] and suspended into loading buffer. Sampleswere separated in SDS-PAGE and analyzed using western blotting.
Fluorescence-activated cell sorting with labelled fibronectin fragment and
K20 or 9EG7 antibodies
PC3 cells were transfected with 20 nM siRNAs using siLentFect (Bio-Rad) in 48-
well plates for 48 hours. The binding of Alexa-Fluor-647-labeled fibronectinrepeat 7–10 (FN) to cells (Montanez et al., 2008) was determined by fluorescence-activated cell sorting (FACS). Cells were incubated in Tyrodes buffer at 37 C for20 minutes with 100 mg/ml Alexa-Fluor-647-FN, washed with cold Tyrodes buffer
and analyzed using a FACSarray (BD Biosciences) high-throughput flowcytometer. A total of 5000 cells from each sample well were measured and ageometric median value from each sample population was used for analysis of
fibronectin binding. The binding of K20 and 9EG7 antibodies (1 mg/ml each 1hour +4 C) to cells was carried out in Tyrodes buffer after fixation with 4%PFAH–PBS. Surface-bound antibodies were probed with Alexa-Fluor-conjugatedsecondary antibodies (Invitrogen; 1:400) for 1 hour and analyzed by FACS. For the
rescue experiments, PC3 cells were sequentially transfected with CD9 or controlsiRNA and 24 hours later with cDNA encoding GFP–CD9 [a kind gift fromFrancisco Sanchez-Madrid (Barreiro et al., 2008)] or empty plasmid and analyzed
for fibronectin binding as described above. Alternatively, PC3 cells weretransfected with MMP8 siRNA or control siRNA and 48 hours later the mediumwas changed to fresh medium to remove MMP8. 16 hours later the cells wereexposed to 5 mg/ml recombinant MMP8 protein (R&D) or buffer alone and
analyzed for fibronectin binding.
Taqman qPCR
Taqman quantitative real-time PCR (qRT-PCR) analysis was performed with anApplied Biosystems 7900HT instrument using specific primers designed by theUniversal Probe Library Assay Design Center (Roche Applied Biosciences). The
expression of specific mRNAs was determined relative to GAPDH mRNA levels.
Microscopy
Images were acquired either directly from the CSMA platform or frommicroscopic cell chambers (Ibidi, Germany). Confocal images were taken usinga Zeiss Axiovert 200M microscope with a Yokogawa CSU22 spinning disc
confocal unit and a Zeiss Plan-Neofluar 636 oil/1.4 NA objective. Z-stacks with 1Airy unit optical slices were acquired with a step size of 0.3 mm. The maximumintensity projections were created with SlideBook 4.2.0.7 software and NIHImageJ. Time-lapse phase-contrast images were taken with a Zeiss inverted wide-
field microscope equipped with a heated chamber (37 C) and CO2 controller(4.8%) and with an LD Plan-Neofluar 406/0.6 NA Korr objective (6 frames/hour). For invasive growth assays, Incucyte HD (Essen Bioscience, UK) imaging
was used. QuickTime movies from time-lapse experiments were created with NIHImageJ software.
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Invasion assays
For invasive growth assays 0.5 ml siRNA (5 mM stock), 0.8 ml Hiperfect (Qiagen),10 ml Optimem (Gibco) and 79 ml PC3 cell medium (see above) were mixed in thewells of 96-well plates. After 15 minutes incubation, 1500 cells were added toeach well. Cells were grown for 24 hours, medium was changed to Matrigel-medium mixture (25% Matrigel) or cell were supplemented with medium andallowed to proliferate in two dimensions. Images were acquired each hour for160 hours using Incucyte HD (Essen Bioscience, UK). Images were analyzedusing ImageJ software.
For three-dimensional invasion assays, 7500 cells were applied onto 15-well m-Slide angiogenesis chambers (Ibidi, Germany) and left to adhere for 6 hours, Themedium was then replaced with 10 ml of Matrigel-medium mixture (33% Matrigel,serum-free medium) and overlaid with 50 ml 2% serum-containing medium afterpolymerization. Cells were allowed to invade upwards for 5 days by replacing 5 mlof the medium with fresh 10% serum-containing medium each day. Cells werefixed with 30 ml 3.7% PFAH (15 minutes), permeabilized with 0.3% Triton X-100in PBS (15 minutes) and stained with Hoechst 33432 or with phalloidin-555 inPBS with 1% BSA. Confocal images were taken by using a Zeiss Axiovert 200Mmicroscope with a Yokogawa CSU22 spinning disc confocal unit and a Zeiss 206objective. Z-stacks of 2 mm optical slices were acquired with a step size of 2 mm,and the maximum intensity projections were created with SlideBook 4.2.0.7software and NIH ImageJ.
Lung extravasation assay
Female athymic nude mice (Hsd:Athymic Nude-nu; Harlan Scandinavia, Allerod,Denmark), aged between 4 and 6 weeks, were used for the xenograft studies. Theexperimental procedures were approved by the local ethical committees. Micewere anesthetized with ketamine (Pfizer) and xylazine (Bayer). TransientlysiRNA-silenced MDA-MB-231 cells were stained with live cell dyes [AllStarsnegative control siRNA (Qiagen) stained red with CMTPX (Invitrogen); siMMP8or siCD9 stained green with CMFDA (Invitrogen)] according to manufacturer’sinstructions. Cells were harvested, suspended in 50 ml PBS (56105 each), mixedand injected (control and siCD9 or control and siMMP8) into the lateral tail vein ofmice (n510). The mice were anesthetised 48 hours post-injection and thepulmonary vasculature was perfused with PBS through the right ventricle(2 minutes) and blood was allowed to escape by a small incision in the leftatrium. Animals were killed and cells were harvested from one lung per animalwith collagenase XI treatment for 1 hour at 37 C, washed with PBS, and thefluorescence analyzed using a ScanR automated microscope. The other lung wasprocessed as frozen sections and stained with DAPI.
Statistical analysis
All statistical analyses were performed using Student’s t-test. P,0.05 wasconsidered significant.
AcknowledgementsWe thank H. Marttila, J. Siivonen, L. Lahtinen and P. Laasola fortheir excellent technical assistance. This study has been supportedby Academy of Finland, ERC Starting Grant, Sigrid JuseliusFoundation, EMBO YIP and Finnish Cancer Organizations. A.Arjonen and T. Pellinen have been supported by Turku GraduateSchool of Biomedical Sciences. The authors declare no conflict ofinterest.
FundingThis study was supported by the Academy of Finland [J.I. and O.K.];a European Research Council Starting Grant [to J.I.]; the SigridJuselius Foundation [J.I.]; the European Molecular BiologyOrganisation Young Investigator Programme [J.I.]; and FinnishCancer Organizations [J.I. and O.K.]; and Turku Graduate School ofBiomedical Sciences [A.A. and T.P.].
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.090704/-/DC1
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