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Cancer Research Division (M.F.B., V.V., J.A.H., G.C.T.) and Chemistry Division I (P.J.C., F.P.-S.), Institut de Recherches Servier, Paris, France
Received September 23, 2002; accepted March 5, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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v integrins, in
the control of the survival of quiescent tumor cells is unclear. We used S
34961, a novel small molecule v integrin antagonist, to
investigate the role of integrin-signaling in the survival of populations of
quiescent human HT-29 and HCT 116 colon carcinoma cells. S 34961 at 1 µM
induced detachment, but cells retained viability, existing as clusters.
Nonligated 
-integrins may recruit and activate caspase-8 [J Cell
Biol 155:459–470, 2001]. However, congruent with the absence
of apoptosis, no activation of caspase-8 in these cells was detected after
incubation with S 34961. A rapid (2 h) change in conformation of the N
terminus of proapoptotic Bak was observed before detachment, together with a
decrease in phosphorylation of focal adhesion kinase (2 h) and subsequent (8
h) decreases in phosphorylation of extracellular signal-regulated kinase-1/2
and Akt. Together, these results suggested that although treatment with S
34961 has no effect on survival per se, it may reduce the survival threshold
of the tumor cells, with Bak in an activated state. Indeed, concomitant
incubation of S 34961 with 10 µM U-0126 (a mitogen-activated protein kinase
kinase inhibitor) was found to lead to apoptosis (at 24 h), whereas U-0126
alone had no effect. Together, these observations could guide the use of
combination therapy with integrin antagonists in the clinic.
Integrins are heterodimeric cell surface receptors that link structural and
functional components within the cell to the extracellular matrix (ECM), and
have been shown to play a major role in the regulation of cell survival.
Detachment of normal cells from the ECM rapidly induces apoptosis [anoikis
(i.e., detachment-induced cell death or suspension-induced apoptosis)] and is
a physiological process necessary for the control of normal tissue
architecture (Grossmann,
2002
). Recent reports have shown that unligated integrins, apart
from turning off survival signals, may also activate signals for apoptosis.
For example, caspase-8 activation has been proposed as an initiating event in
anoikis (Rytömaa et al.,
1999), and a recent study has shown that nonligated
-integrins may recruit and activate caspase-8
(Stupack et al., 2001).
Unlike nontransformed cells, many tumor cells seem not to depend on signals
from adhesion molecules such as the v integrins for survival
(Schwartz, 1997). However,
integrins are also implicated in cell migration, proliferation, and invasion;
tumor cells often express high levels of the vitronectin-binding integrins
such as v3 and
v5
(Clezardin, 1998;
Hood and Cheresh, 2002).
Although resistance of some tumor cells to anoikis is well documented, the
nature of intracellular events after loss of integrin ligation in tumors
remains poorly defined. We have sought to determine whether the absence of
integrin ligation to a natural substrate, after treatment with S 34961, a
novel and potent small molecule antagonist of v integrins
synthesized in our laboratory
(Perron-Sierra et al., 2002),
leads to a reduction in the survival threshold of quiescent anoikis-resistant
colon carcinoma cells.
One set of intracellular proteins that has been shown to play a pivotal
role in setting the cellular survival threshold is the Bcl-2 family. Some
members of this family, such as Bcl-XL and Bcl-2, promote cell
survival, whereas others, such as Bax, Bak, and the "BH3-only"
members Bid and Bmf, promote apoptosis
(Antonsson, 2001). The
proapoptotic functions of these proteins are regulated at several levels,
including transcription, proteolysis, phosphorylation, intracellular
translocation, protein-protein interactions, and conformational changes. In
the present study, S 34961 was found to have a marked effect on the N-terminal
exposure of Bak, suggesting a change in survival threshold of nonproliferating
tumor cells. In addition, S 34961 was seen to lead to reduced intracellular
signaling via both the PI3K/Akt and the Raf/mitogen-activated protein kinase
kinase/ERK1/2 pathways, both of which have been implicated in cell survival.
This decrease in survival threshold was demonstrated by the increased
sensitivity of the S 34961-treated quiescent colon carcinoma cells to the
mitogen-activated protein kinase kinase-1/2 inhibitor U-0126.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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v (BD Biosciences PharMingen, San Diego, CA),
mouse monoclonal anti-integrin 3 (Chemicon, Temecula, CA),
goat polyclonal anti-integrin 5 (Santa Cruz Biotechnology,
Santa Cruz, CA), mouse monoclonal anti-bromodeoxyuridine (BrdU)-FITC (BD
Biosciences PharMingen), rabbit polyclonal anti-Bak, residues 14 to 36 (BD
Biosciences PharMingen), rabbit polyclonal anti-Bak, residues 23 to 37
(Upstate Biotechnology, Lake Placid, NY), rabbit polyclonal anti-Bax, residues
11 to 30 (Santa Cruz Biotechnology), rabbit polyclonal anti-Bax, residues 1 to
171 (Santa Cruz Biotechnology). Rabbit polyclonal anti-focal adhesion kinase
(FAK) and anti-phospho-FAK were from Upstate Biotechnology, and rabbit
polyclonal anti-Akt, anti-phospho-Akt, anti-ERK1/2, anti-phospho-ERK1/2,
anti-PARP, anti-Bid, and mouse monoclonal anti-caspase-8 were from Cell
Signaling Technology (Beverly, MA). The anti-Fas monoclonal antibody (CH-11)
used to induce caspase-8 activation in cell culture was from Upstate
Biotechnology. The secondary antibodies used were peroxidase-conjugated
anti-mouse IgG, anti-rabbit IgG (Cell Signaling Technology), anti anti-goat
IgG (Santa Cruz Biotechnology) for Western blotting, Cy3-conjugated anti-mouse
IgG, and anti-rabbit IgG (Jackson ImmunoResearch, West Grove, Pennsylvania)
for immunofluorescence, and FITC-conjugated anti-rabbit IgG (Santa Cruz
Biotechnology) for flow cytometry.
Compounds. S 34961 was synthesized as described previously
(Perron-Sierra et al., 2002)
and solubilized at 10 mM in dimethyl sulfoxide. Staurosporine (Calbiochem, San
Diego, CA), U-0126 (Calbiochem), and wortmannin (Calbiochem) were dissolved at
10 mM in water. All compounds were aliquoted and stored at -20°C, and
diluted into the culture medium as required.
Models of Cell Quiescence. HT-29 and HCT 116 colon carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA), and were routinely passaged in RPMI 1640 medium supplemented with 10% decomplemented fetal calf serum (FCS), 2 mM l-glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin, and incubated at 37°C in 5% CO2/95% air. Media and supplements were from Invitrogen (Cergy-Pontoise, France), except for FCS, which was from Sigma. For the models of tumor cell quiescence, cells were seeded at a density of 25,000 cells per 35-mm tissue culture dish or per well of six-well tissue culture plates (Techno Plastic Products, Trasadingen, Switzerland) in 4 ml of RPMI containing 10% FCS. Two days later, the culture medium was removed; cells were washed once in phosphate-buffered saline (PBS), and 4 ml of serum-free RPMI containing 0.5% BSA (cell-culture tested; Sigma) was added. The subconfluent cell cultures were further maintained for a maximum of 12 days with no medium change.
MTT Cell Viability Assay. For the assay of cell viability, quiescent cell cultures were incubated with a solution of 1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma) in PBS for 4 h at 37°C. A 20% solution of SDS (Sigma) in 20 mM HCl was added, and cultures were incubated overnight at 37°C to solubilize the formazan metabolite. Global cell viability was estimated by measurement of optical density at 540 nm.
BrdU Incorporation. The proportion of cells in the cell cycle over a given 24-h period was measured by flow cytometric analysis of BrdU incorporation. Briefly, cells were incubated 24 h at 37°C in the presence of 10 µM BrdU (Sigma), detached by trypsin-EDTA (Invitrogen), and fixed in 70% ethanol at 4°C for at least 4 h. Samples were washed in PBS and incubated in 2 M HCl (30 min, 20°C) to denature DNA, then washed twice with PBS containing 0.5% Tween 20 and incubated with 20 µl of anti-BrdU-FITC for 45 min at 20°C. After washing with PBS, cells were incubated for 30 min at 20°C with 100 µg/ml RNase (Sigma) and 10 µg/ml propidium iodide (Sigma), and analyzed by flow cytometry. FITC and propidium iodide fluorescences were collected through 520- and 630-nm bandpass filters, respectively.
Western Blot Analysis. Whole-cell extracts were prepared with
radioimmunoprecipitation assay extraction buffer (150 mM NaCl, 50 mM Tris-HCl,
pH 7.8, 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholic acid) to which was
added protease [1% (v/v); Sigma] and phosphatase [1% (v/v); Sigma] inhibitor
cocktails. Protein concentration was determined by Bradford protein assay
(Bio-Rad Laboratories, Marnes la Coquette, France), and extracts were diluted
into Laemmli sample buffer (Bio-Rad) containing 5% (v/v)
-mercaptoethanol, heated for 3 min at 95°C, and resolved on
Tris-glycine gels (Novex, San Diego, CA). Biotinylated molecular mass
standards (Cell Signaling Technology) were included in all gels. Proteins were
transferred to nitrocellulose membranes (Hybond ECL; Amersham Biosciences,
Freiburg, Germany), which were blocked in PBS/0.1% Tween 20 (PBST) containing
5% milk, and probed at 4°C overnight with primary antibodies. Antibody
dilutions were: anti-integrin v, 1/250 (5% milk);
anti-integrin 3, 1/500 (5% milk); anti-integrin
5, 1/100 (5% milk); anti-Bak residues 23 to 37, 1/1000 (5%
milk); anti-Bax residues 11 to 30, 1/2000 (5% milk); anti-FAK and
anti-phospho-FAK, 1/1000 (5% milk); anti-Akt, anti-phospho-Akt, anti-ERK1/2
and anti-phospho-ERK1/2, 1/1000 (5% BSA); anti-caspase-8, 1/1000 (5% milk);
anti-Bid, 1/300 (5% milk); and anti-PARP, 1/1000 (5% BSA). Anti-mouse and
anti-rabbit peroxidase conjugated secondary antibodies were used at 1/3000
dilutions in PBST containing 5% milk (1 h, 20°C). Chemiluminescence
detection was performed using the ECL Plus Western blotting detection kit and
was recorded on ECL Plus hyperfilm (both from Amersham Biosciences).
Immunofluorescence Microscopy and Flow Cytometry. For immunofluorescence microscopy, cells in 35-mm culture dishes were fixed 5 min in 1% formaldehyde in PBS, washed in PBS, and incubated in primary antibody solution (600 µl) diluted in 500 µg/ml digitonin in PBS for 1 h at 20°C. Antibody dilutions were: anti-Bak residues 14 to 36, 1/500; anti-Bax residues 11 to 30, 1/100; and anti-Bax residues 1 to 171, 1/100. Cells were washed, then incubated with the relevant Cy3 conjugated secondary antibody for 1 h in PBS at 20°C at 1/200 dilution. Cells were washed, mounted in Vector-Shield (Vector Laboratories, Burlingame, CA) under a glass cover-slip, and images were captured using an Olympus upright fluorescent microscope. For flow cytometric analysis of Bak N-terminal exposure, cells were cultured and fixed as for microscopic analysis, collected as a single-cell suspension by incubation at 37°C in 20% (v/v) cell dissociation solution (Sigma) in trypsin-EDTA, washed in PBS, and incubated 1 h with 200 µl of anti-Bak (residues 14–36) diluted as above. Cells were washed, then incubated with FITC-conjugated secondary antibody for 1 h in PBS at 20°C at 1/10 dilution and analyzed by flow cytometry. FITC fluorescence was collected through a 520-nm bandpass filter.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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v integrins with
IC50 values in binding studies of 2 nM for
v3, 1 nM for
v5, and 4 µM for
IIb3 (compounds 1–2 in
Perron-Sierra et al., 2002).
Anoikis of human umbilical vein endothelial cells and cluster formation of
anoikis-resistant tumor cells occur at submicromolar doses of S 34961 on
tissue culture plastic in the presence or absence of adsorbed vitronectin (an
v integrin ligand) but not in the presence of adsorbed
collagens or laminin (unpublished observations).
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The v Integrin Antagonist S 34961 Induced Detachment
of Quiescent HT-29 and HCT 116 Cells from Tissue Culture Plastic, but Cells
Retained Viability, Existing as Clusters. Models of tumor cell quiescence
were developed by culturing HT-29 or HCT 116 colon carcinoma cells in
serum-free medium. After an initial 5-day period during which viable cell
number increased, there was a plateau phase of 7 days in which total cell
number remained constant, as indicated by MTT cell viability assay
(Fig. 2A). During this plateau
phase, cells were subconfluent (typically 70% confluence), and were thus not
contact-inhibited. Over a 24-h period, 77% of these HT-29 cells were
BrdU-negative, as opposed to 1% of cells in exponentially growing culture
(Fig. 2B). Similar data were
obtained for HCT 116 (data not shown). These results thus indicate that
although total viable cell numbers remained constant, a small proportion of
the cell population remained in the cell cycle. At all time points during the
plateau phase, approximately 5% of cells were apoptotic, as judged by
light-microscopic observation of nuclear fragmentation. This model may be
considered to be representative of solid tumors in which cells are often
proliferating at a slow rate, and in which many cells are quiescent. All
experiments described in this report used quiescent populations of HT-29 and
HCT 116 cells during the plateau phase from 7 to 11 days. Each cell line was
found to express all of the v, 3, and
5 integrin subunits in both the proliferating and quiescent
states (Fig. 2C). When
quiescent populations (day 7) of HT-29 or HCT 116 cells were incubated with 1
µM S 34961, the cells were seen to begin to detach from the tissue culture
plastic after 2 (HT-29) or 4 h (HCT 116), and to form free-floating cell
clusters within 24 h (Fig. 2, D and
E). The number of viable cells in these cultures was unchanged
even after 4 days of culture in continued presence of S 34961, as assessed by
MTT assay (data not shown).
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Detachment of Quiescent HT-29 and HCT 116 Cells after Incubation with S
34961 Was Preceded by Exposure of the N Terminus of Bak. Recent studies
have shown modulation of the N termini of the proapoptotic Bcl-2 family
members Bak and Bax after stress to various normal and tumor cell types
(Griffiths et al., 2001;
Makin et al., 2001;
Mandic et al., 2001). The
reversible activation of these proteins via exposure of their N termini
represents a critical event in the cell's sensing of damage, and was reported
to occur before the commitment to apoptosis. Antibodies to N-terminal epitopes
of both Bak and Bax were used to determine whether S 34961 antagonism of the
v3 and
v5 integrin-substrate interactions induced
changes in exposure of N-terminal epitopes in these Bcl-2 family proteins in
the quiescent HT-29 and HCT 116 cells. All data shown in this section are for
HT-29 cells; similar data were obtained for HCT 116 cells. A marked increase
in punctate Bak N-terminal staining with an antibody against residues 14 to 36
was observed in a general manner in the whole cell population within 2 h
(Fig. 3A). This result was
confirmed by flow cytometric analysis of fixed cells, where a global shift in
Bak N-terminal reactivity was seen after 2-h treatment with S 34961
(Fig. 3B). The exposure of the
N-terminal epitope of Bak was thus apparent before cell detachment occurred
and was also evident in the cell clusters after 24 h. The majority
(approximately 95%) of cells showing increased Bak staining had normal
nonfragmented nuclei. Staurosporine (1 µM, 4 h), used as a positive control
to induce apoptosis (Griffiths et al.,
2001), led to an increase in staining of only 
50% of cells.
The more punctate expression pattern here can be explained by the fact that
these staurosporine-treated cells are undergoing apoptosis (unlike the S
34961-treated cells) and have condensed mitochondria. These results were
confirmed by two further antibodies against residues 2 to 14 and 23 to 37 of
Bak. In contrast to these results for Bak, very few cells (< 1%) showed a
change in the N-terminal exposure of Bax (residues 11–30) in the
presence of 1 µM S 34961, although an increase in staining was observed
(approximately 20% of cells) when incubated with 1 µM staurosporine, used
as positive control (Fig. 3A).
The use of a further antibody directed against residues 1 to 21 confirmed this
result. Antibodies directed against the whole Bax protein (except C-terminal;
residues 1–171; Fig. 3A)
demonstrated no change in total protein staining, as did a BH3-proximal
epitope of Bax (residues 43–61; data not shown). Importantly, no changes
in protein levels of either Bak or Bax were detected by Western blotting
during this 24-h period (Fig.
3C) or during the subsequent 48 h (data not shown). Similarly, no
changes in the protein levels, by Western blotting, of the anti-apoptotic
Bcl-2 family members Bcl-2 and Bcl-XL were seen during the same time course
(data not shown).
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A Decrease in Phosphorylation of FAK and Subsequent Decreases in
Phosphorylation of ERK1/2 and Akt Were Observed after Incubation of Quiescent
Cells with S 34961. The tyrosine phosphorylation of the integrinlinked
FAK, and downstream Akt and ERK1/2, has an important role in adhesion-mediated
signal transduction (Schwartz and
Ginsberg, 2002). In the present study, phosphorylation of FAK was
seen to be reduced in quiescent populations of HT-29 cells after 2-h
incubation with 1 µM S 34961 and then to decrease progressively to 8 h
(Fig. 4, top left). Only a
slight change in total phosphorylation of FAK could be detected by Western
blot in the quiescent HCT 116 cells incubated for 24 h with 1 µM S 34961
(Fig. 4, top right). A decrease
in phosphorylation of Akt was observed after 8-h incubation with 1 µM S
34961 for both cell lines; phosphorylation of this protein became virtually
undetectable by 24 h (Fig. 4, middle). A similar decrease in phosphorylation of ERK1/2 was observed after
8-h incubation, although for these proteins, no further phosphorylation
decrease was observed at 24 h (Fig.
4, bottom). Total protein levels of all these proteins were
unchanged during the experiment, and phosphorylation levels of each in the
control cultures were identical at both the start and end of treatment.
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Incubation of Quiescent Cells with S 34961 Did Not Induce Caspase-8
Activation. A recent study using a number of cell lines has shown that
nonligated -integrins may recruit and activate caspase-8, hence
contributing to detachment-initiated apoptosis
(Stupack et al., 2001); these
authors went on to suggest that integrin antagonists might also induce
apoptotic signals via the same mechanism. We thus investigated the activation
of this caspase after antagonism of integrin-substrate interactions by 1 µM
S 34961. In quiescent HT-29 cells, no activated isoforms of caspase-8 and no
cleavage of the caspase-8 substrate Bid were detected
(Fig. 5, A and B). An anti-Fas
monoclonal antibody and staurosporine were used as positive controls for
caspase-8 activation. In agreement with the low percentage of cells with
apoptotic morphology in the quiescent HT-29 cultures, the activity of
downstream caspases (such as caspase-3) was very low, as evidenced by minimal
PARP cleavage (Fig. 6A,
top).
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Simultaneous Incubation of Quiescent HT-29 Cells with 1 µM S 34961 and 10 µM U-0126 Led to Caspase
Activation and Apoptosis, Whereas U-0126 Had No Effect Alone. Together, the above results show that incubation of the quiescent HT-29 and HCT 116 cells with 1 µM S 34961 led to activation of Bak, near-total inhibition of PI3K-mediated phosphorylation of Akt, and a reduction in the level of MEK1/2 phosphorylation of ERK1/2. Despite this reduction in intracellular signals for survival, no caspase activity was detected, and cells did not undergo apoptosis. To test the hypothesis that the S 34961-treated cells had nevertheless undergone a reduction in survival threshold, quiescent HT-29 cells were incubated simultaneously with 1 µM S 34961 and 10 µM U-0126, a MEK1/2 inhibitor. Whereas neither compound alone had an effect on survival, their combination led to a marked increase in apoptosis, as shown by high levels of PARP cleavage (Fig. 6, top) and a 52 ± 4% reduction in the surviving population of cells after 48 h (Fig. 6, bottom). Concurrent incubation with S 34961 and 500 nM wortmannin (a PI3K inhibitor) did not lead to apoptosis above control levels. The levels of phosphorylation of ERK1/2 and Akt in the presence of S 34961, U-0126, and wortmannin are also shown (Fig. 6, middle). HCT 116 cells, like HT-29 cells, were seen to be sensitive only to the association of 1 µM S 34961 and 10 µM U-0126, with a reduction in surviving cell numbers of 48 ± 5% at 48 h. No effect on survival was observed for U-0126 or wortmannin alone or the association of wortmannin with S 34961.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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v integrins are overexpressed in many tumor cell
lines and have been shown to be important for tumor cell migration and
invasion (Clezardin, 1998).
Although normal cells require integrin-mediated attachment to a solid ECM for
survival, many tumor cell lines have been described to survive prolonged
periods in suspension (Grossmann,
2002). However, the intracellular events occurring in these
anoikis-resistant tumor cells after abrogation of the survival-promoting
integrin-ECM interactions are poorly understood. In the present study,
quiescent cultures of colon carcinoma cells were used to assess the effect of
a novel small molecule v integrin antagonist, S 34961, on
tumor cell survival signaling. S 34961
(Fig. 1) has been shown to
antagonize v3 and
v5 binding to purified extracellular matrix
ligands in the nanomolar range
(Perron-Sierra et al., 2002).
The colon carcinoma cell lines used in this study, HT-29 and HCT 116, were
both found to express each of the v3 and
v5 integrins
(Fig. 2). When quiescent
populations of these cells were cultured in the presence of 1 µM S 34961,
cells rapidly became detached from the culture dishes and formed free-floating
clusters (Fig. 2). No obvious
effect on cell survival was seen, even after 4 days of culture.
The study of cell survival in the absence of substrate attachment typically
involves the suspension culture of cells on nonadhesive substrates such as
polyhydroxyethylmethacrylate (Gilmore et
al., 2000). Recent studies have reported a positive correlation
between the level of v integrin expression and apoptosis in
suspension culture in both normal and tumor cell lines
(Brassard et al., 1999;
Kozlova et al., 2001;
Stupack et al., 2001). Indeed,
despite the widespread belief that tumor cells are innately resistant to
anoikis, a number of tumor cell lines expressing high levels of
v integrins have been shown to undergo apoptosis when
detached from a solid substrate (Townsend
et al., 2000; Kozlova et al.,
2001; Stupack et al.,
2001; Lewis et al.,
2002). Thus, although the expression of v
integrins confers a survival and proliferation advantage to both normal and
tumor cells when adhering to a solid ECM, these same integrins may
down-regulate survival signals, or possibly transmit death signals, when
unligated. We were interested in how cells might respond to a small molecule
inhibitor of v integrin binding.
The integrin antagonist S 34961 was seen not to affect survival of
quiescent populations of HT-29 and HCT 116 cells in terms of viable cell
numbers, and the low levels of cleaved PARP in cell extracts confirmed that
activation of death-executing caspases, such as caspase-3, was unchanged in
these cell populations compared with control cultures
(Fig. 6). Caspase-8 activation
has been proposed as a death receptor-independent initiating event in
detachment-initiated apoptosis
(Rytömaa et al., 1999),
and a recent study by Stupack et al.
(2001) has shown that
nonligated -integrins may recruit and activate caspase-8. It was
suggested by Stupack et al.
(2001) that integrin
antagonists may induce similar events. We investigated the activation of this
caspase after antagonism of integrin-substrate interactions by 1 µM S
34961. In quiescent HT-29 and HCT 116 cells, no activated isoforms of
caspase-8 and no cleavage of the caspase-8 substrate Bid were detected
(Fig. 5). These results suggest
that S 34961 treatment may not be equivalent to the absence of an appropriate
ligand. However, it is important to note that Stupack et al.
(2001) did not detect
caspase-8 activation in anoikis-resistant HeLa cells, thus suggesting that
such caspase activation is not a general phenomenon.
Although the integrin antagonist S 34961 was seen not to affect survival
per se by initiating apoptosis, it was conceivable that this compound may
affect the balance of integrin survival and apoptosis signals. A number of
recent studies have pointed to the importance of changes in exposure of the
N-terminal of the proteins Bak and Bax in the initiation of apoptosis
(Griffiths et al., 2001;
Makin et al., 2001;
Mandic et al., 2001). These
N-terminal conformational changes in Bak and Bax occurred before commitment to
apoptosis and were reversible, representing an "activated state"
after the cell had sensed perturbation. In the present study, a rapid exposure
(2 h) of the N terminus of Bak was seen in virtually all cells after
incubation with S 34961 (Fig.
3). Importantly, this occurred before the cells became detached
from the culture dish. Detachment of both normal and tumor cells from
substrate has been shown to lead to the exposure of the N-terminal of Bax
(Gilmore et al., 2000;
Makin at al., 2001). In our
studies, a small number of S 34961-treated cells became positive for exposure
of Bax N terminus, but only after a delay of 24 h and specifically in cells
exhibiting apoptotic morphology. Thus Bax "activation" was
secondary to exposure of the Bak N-terminal and seemed to be downstream to
commitment to apoptosis. No changes in protein levels of the pro- or
antiapoptotic Bcl-2 proteins were seen, even after 3 days of culture with S
34961. Although integrin ligation has been shown to affect transcription of
Bcl-2 family proteins (Petitclerc et al.,
1999; Matter and Ruoslahti,
2001), our data clearly corroborate that of other studies, showing
that the role in damage sensing by these proteins, and in this case Bak, is
much more rapid than could be explained by changes in protein levels
(Griffiths et al., 2001,
Makin et al., 2001).
Very little is known about how cellular stress signals are relayed to Bak
or Bax, although a model has been suggested that involves activation by the
Bcl-2 family BH3-only proteins, such as Bad, Bid, and Bmf
(Cheng et al., 2001). Exactly
how these proteins become activated when integrin ligation is disrupted is not
clear. One upstream candidate in a signaling cascade from integrins is a
cleavage fragment of MEKK1. This protein was shown to be cleaved when cells
lose matrix contact (Cardone et al.,
1997), and the proapoptotic conformation of Bak was induced in a
MEKK1-dependent manner after DNA damage
(Mandic et al., 2001). We
detected no MEKK1 cleavage fragment after either 2- or 24-h S 34961 treatment
of the HT-29 and HCT 116 cell lines (data not shown). Thus, the process
whereby Bak becomes activated is not clear here, although its change in
conformation suggests that the survival threshold of the cell may be
compromised, a hypothesis we then went on to test.
A major upstream protein involved in adhesion-dependent cell survival
signaling is FAK (Panetti,
2002). In the present study, phosphorylation of FAK was seen to be
reduced after 2-h incubation of quiescent populations of HT-29 cells with 1
µM S 34961 and then to decrease progressively to 8 h
(Fig. 4). Interestingly, total
inhibition of phosphorylation of this protein was not observed, suggesting
either alternative phosphorylation from other signaling molecules (e.g.,
activated Src; Biscardi et al.,
1999) or that other FAK-linked integrins were present and
activated in the detached cells. Only a slight change in total phosphorylation
of FAK could be detected in HCT 116 cells incubated for 24 h with 1 µM S
34961, again possibly because of the presence of other integrinlinked
signaling pathways.
Adhesion-mediated cell survival via FAK or other signaling proteins has
been shown to involve primarily either ERK1/2 activation
(Finlay et al., 2000;
Le Gall et al., 2000;
Howe et al., 2002) or PI3K
signaling (Sonoda et al.,
2000; Maeshima et al.,
2002), apparently depending on the culture conditions and the cell
lines used. In our studies, a decrease in the PI3K-mediated phosphorylation of
Akt was observed after 8-h incubation with 1 µM S 34961 for both cell
lines, phosphorylation of this protein becoming virtually undetectable by 24 h
(Fig. 4). A recent study has
shown similar data, whereby the integrin antagonist Tumstatin was shown to
interact with v3 integrin to inhibit
activation of FAK, PI3K, and Akt in endothelial cells
(Maeshima et al., 2002). A
similar decrease in phosphorylation of ERK1/2 was observed after 8 h
incubation with S 34961, although for these proteins no further
phosphorylation decrease was observed after 24 h. Both the PI3K and ERK1/2
pathways have been shown to control pro- and antiapoptotic signals mediated by
the Bcl-2 family proteins, including translocation and change in conformation
of Bax (Boucher et al., 2000;
Gilmore et al., 2000). However,
in our experiments, the conformational change in Bak was seen well before
changes in phosphorylation of ERK1/2 and Akt (2 h as opposed to 8 h; Figs.
3 and
4), suggesting that these
signaling pathways were subsequent to this event.
Together, our results suggest that although treatment with S 34961 has no effect on caspase activation and survival per se, it may reduce the survival threshold of the tumor cells, with Bak in an activated state, a reduction in the level of ERK1/2 phosphorylation, and a near-total inhibition of PI3K-mediated phosphorylation of Akt. When quiescent HT-29 and HCT 116 cells were incubated concurrently with 1 µM S 34961 and the MEK1/2 inhibitor U-0126, marked apoptosis was seen, as evidenced by reductions in cell survival and high levels of PARP cleavage (Fig. 6). U-0126 had no effect on survival alone. This clearly demonstrates the markedly increased sensitivity of the S 34961-treated cells to inhibition of downstream survival signaling molecules. Concurrent incubation with S 34961 and 500 nM wortmannin (a PI3K inhibitor) did not lead to apoptosis. This agrees with the observation that 1 µM S 34961 alone caused a near-total inhibition of Akt phosphorylation in both cell lines.
In summary, S 34961 was seen to induce a two-step reduction in survival
threshold in HT-29 and HCT 116 colon carcinoma cells. The immediate detection
of cellular stress was signaled by the change in N-terminal exposure of Bak,
followed by a decrease in PI3K and ERK1/2 survival signals. This reduction in
survival threshold markedly increased the sensitivity of the HT-29 cells to a
MEK1/2 inhibitor. This suggests that strategies based on blocking
v integrin-mediated survival signals may represent a new
therapeutic approach to improve the response to colon cancer chemotherapy in
the clinic.
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Acknowledgements |
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Footnotes |
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Address correspondence to: Dr. Mike F. Burbridge, Cancer Research Division, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy sur Seine, Paris, France. E-mail: mike.burbridge{at}fr.netgrs.com
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