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INSERM U482, Signal Transduction and Cellular Functions in Diabetes and Digestive Cancers, Hôpital Saint-Antoine, 75571 Paris Cedex 12, France;
* The Laboratory of Experimental Cancerology, Ghent University Hospital, B-9000 Ghent, Belgium;
Novo Nordisk, Bagsvaerd, DK-2880, Denmark;
Department of Pathology, University of Newcastle, Newcastle upon Tyne NE1 4LP, U.K.; and
§ GBMC, INSERM U184, Université Louis Pasteur, 67404, Illkirch Strasbourg Cedex, France
1Correspondence: INSERM Unit U482, Hôpital Saint-Antoine, 75571 Paris Cedex 12, France. E-mail: gespach{at}st-antoine.inserm.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: inflammation • pS2 • SP • ITF • PI3'-kinase • phospholipase C • rapamycin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Three members of this
family have been identified in humans: pS2 (trefoil factor 1: TFF1),
spasmolytic polypeptide (SP: TFF2), and intestinal trefoil factor (ITF:
TFF3). They share a highly conserved motif
(CX9CX9CX4CX9WCF),
comprised of six conserved cysteine residues responsible for the
trefoil motif formation. PS2 and ITF also form homodimers and
heterodimers with different degree of glycosylation, and these
modifications may be of functional significance (3)
. The
trefoil peptides are involved in mucosal healing processes and are
expressed at abnormal elevated levels in neoplastic diseases
(4
5
6
7
8
9
10
11
12
13
14)
. A wide range of human carcinomas and
gastrointestinal inflammatory malignancies, including peptic ulceration
and colitis, Crohn’s syndrome, pancreatitis, and biliary disease,
aberrantly express trefoil peptides (10
11
12)
. The trefoil
peptide pS2 is a breast cancer-associated secreted protein of 60 amino
acids that is transcriptionally induced by estrogens in the mammary
carcinoma cell line MCF-7 (13
, 14)
. In the antrum of the
normal stomach, pS2 is intimately associated with mucus secretions
(15
, 16)
and is regulated by a variety of factors
including estrogens, epidermal growth factor (EGF), tumor promoter TPA,
and the c-Ha-ras oncogene (17)
. PS2 expression is closely
associated with premalignant stages in human gastric carcinoma
(18)
and with the neuroendocrine differentiation of
prostate cancers, via an estrogen-independent pathway (11
, 12)
. In agreement, pS2 was negative in patients with benign
prostate hyperplasia, whereas 92% of patients at risk with prostate
cancer are positive for pS2 expression (19)
.
One of the early events in inflammation and wound healing is the
epithelial restitution of the digestive mucosa, which is tightly
related to cell proliferation, migration, and a risk/benefit balance
between anti-apoptotic and proapototic signals in response to mucosal
injury by cellular and DNA damaging agents such as cytokines, free
radicals, and nitric oxide. In animal models, trefoil peptides are
essential participants of mucosal repair in inflammation and wound
healing (5
6
7
, 9)
. They are considered as morphogen
regulatory peptides (20
, 21)
. Intestinal trefoil peptide
also confers colonic epithelial resistance to apoptosis, because
transgenic mice deficient in ITF showed an increase in colonocyte
apoptosis unaccompanied by changes in expression of receptor-related
(TNF-R/Fas) or stress-related (Bcl-family) cell death regulators
(22)
. However, our actual knowledge on the signaling
pathways activated by the putative TFF receptors or recognition sites
from the cell surface to the cytoplasm and nucleus is still very
incomplete. Recent studies designated the possible contribution of EGF
receptors, MAPK, and APC/E-cadherin/ß-catenins as downstream elements
involved in trefoil peptide signals, leading to decreased cell–cell
and cell–substratum adhesion (23
24
25
26)
.
In view of the critical role of the TFFs in the regulation of
epithelial cell migration and their possible action on signaling
pathways involved in cell adhesion and transformation, we investigated
the role TFFs play in regulating tumor progression and invasion. To
determine whether such an effect might be relevant to disease, we used
colonic and kidney epithelial cell lines at various stages of the
neoplastic transformation controlled by the src oncogene and the human
colonic cell line HCT8/S11 established from a sporadic tumor
(27
28
29)
. The signaling activity of pS2, SP, and ITF was
examined with reference to possible connections with several cellular
invasion pathways (30
31
32)
, namely, PI3'-K, Rho-like
G-proteins, and protein kinase C (PKC). Evidence is provided that TFFs
can regulate cell transformation and neoplastic progression in two
ways, by inducing cell scattering and invasion of basement membranes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and PCKß I isoenzymes that has no
effect on the atypical Ca2+-independent PKCs),
and GF109203X (abbreviated GF109), a PKC inhibitor with high
selectivity for PKC, ßI, ßII, 
, and 
isoenzymes were from
Calbiochem (Meudon, France). Forskolin (FK), pertussis toxin (PTx),
rapamycin, and phenylmethysulfonyl fluoride (PMSF) were from Sigma
(Saint-Quentin Fallavier, France). Collagen type I was from Upstate
Biotechnology (Lake Placid, N.Y.).
Cell lines and tissue samples from human gastrointestinal tumors
Parental Madin-Darby canine kidney epithelial cells MDCK were a
generous gift from Dr. J. Downward (Imperial Cancer Research Fund,
London, U.K.). MDCKts.src transformed by a temperature-sensitive mutant
of v-src (MDCKts.src, Cl2) and the MDCKts.src-p110DN cell line
(transfected with the dominant negative mutant p110 EcoS of PI3'-K)
were previously described (27
, 30)
. The human embryonic
kidney cell line HEK-293T and MDCKT23 cells expressing the mutant
G-proteins RhoAV14, RhoAN19, or Rac1V12 under the
tetracycline-repressible transactivator were generous gifts from Dr.
Silvio Gutkind (NIH, Bethesda, Md.) and Dr. J. Nelson
(33)
, respectively. Expression of V14RhoA and V12Rac1 was
induced in MDCKT23 cells by removing doxycycline (DOX, Sigma) for
16–18 h (V14RhoA and V12Rac1) or 40 h (N19RhoA) from the culture
media (33)
.
Human colorectal cell lines HCT-8/S11, HCT-8/S11/R1, PC/AA/C1, and
PCmsrc were routinely grown in 6 cm diameter Petri dishes, as described
previously (28
, 29)
. The PC/AA/C1 cell line was
established from a colonic adenoma in a patient with familial
adenomatous polyposis (FAP). This cell line is nontumorigenic and
exhibits a mucinous phenotype. After transfer of the activated c-src
oncogene, PCmsrc cells became tumorigenic in the athymic nude mice and
were invasive upon addition of HGF (28)
. Human breast
cancer cells MCF-7 were cultured as described (34)
.
Specimens from patients who had undergone surgery for gastric or
colonic cancers were obtained from the Center de Chirurgie Digestive
(Prof. R. Parc, Hôpital Saint-Antoine, Paris, France), as
described previously (35)
. Tissue samples from 0.5 to
3 g were snap-frozen in liquid nitrogen and stored at -80°C
until use. For each tumor, a frozen section was subjected to
histological analysis, to confirm the neoplastic origin of the sample.
Colonic adenocarcinomas (ADK) were sampled in the infiltrating area of
tumors. In each case, the corresponding control mucosa was dissected
out from the tumor and analyzed as paired control tissue with normal
histological architecture. Tissues for immunohistochemical analyses of
pS2 were fixed in neutral-buffered formalin and embedded in paraffin
wax.
Construction and validation of the pcDNA3-hpS2 expression plasmid
The hpS2 cDNA fragment (462 bp) was excised from the pGEM1
plasmid (13)
by EcoRI/BamHI
digestion and cloned in frame into the eukaryotic expression vector
pcDNA3 recombined with the neo-resistance gene (InVitrogen, Groningen,
The Netherlands). The new plasmid construct was named pcDNA3-hpS2.
After transforming into Escherichia coli (MC106/P3 strain,
InVitrogen), pcDNA3-hpS2 was prepared with mini-prep and identified by
an assay of restriction enzyme digestion. The structure and function of
pcDNA3-hpS2 was checked by direct DNA sequencing and expression in
HEK-293T cells. Transient transfection of HEK-293T cells in 10 cm
plates was performed using 2 µg of pcDNA3-hpS2 expression plasmid and
the LipofectAMINE Plus method (Gibco BRL, Cergy Pontoise, France),
according to the manufacturer’s protocol. After 6 h incubation at
37°C in a humidified atmosphere of 5% CO2 in
air, the transfection mixture was replaced with new medium. Transfected
cells were then cultured in standard conditions for an additional
48 h period, prior to assay for hpS2 expression by Western
blotting.
Stable transfection of colonic and kidney epithelial cells by the
hpS2 cDNA
MDCKts.src cells and HCT-8/S11 cells were stably transfected by
the human full-length hpS2 cDNA in the pcDNA3-hpS2 expression plasmid
using the LipofectAMINE Plus reagent. Control transfections were
performed using the empty vector pcDNA3. Cultures were selected in 1
mg/ml neomycin (Gibco BRL) for 2 wk and resistant colonies were
ring-cloned as individual colonies or pooled for hpS2 analysis by
immunoblotting and further functional characterization.
Preparation of recombinant trefoil factors
Human SP and ITF were produced in yeast and purified as
described (36
, 37)
, recombinant hpS2 produced in E.
coli was purified by affinity chromatography (38)
.
Western blot analyses and immunohistochemistry for hpS2
For immunoblotting, cultured cells were homogenized at 4°C in
RIPA buffer containing 0.1 mg/ml PMSF, 100 µM benzamidine, and 100 mM
Na3VO4 as protease inhibitors. Frozen tissues from human
gastrointestinal tumors were disrupted in ice-cold 10 mM Tris-HCl (pH
7.5) containing 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 50 mM
NaF, 5 mM EDTA, 1 mM EGTA, 0.2 mM orthovanadate, 0.2 mM PMSF, 10 mg/ml
each of leupeptin, and aprotinin. A Polytron apparatus was used, with
three bursts of 15 s. Insoluble material was removed by
centrifugation for 15 min at 4°C and 12,000 g. Proteins
were resolved in nonreducing conditions in 15% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to Hybond-P Extra membranes (Pharmacia Biotech, Buckinghamshire,
England). Membranes were blocked in Tris-buffered saline (TBS: 20 mM
Tris-HCl, pH 8, 150 mM NaCl) containing 5% dried skimmed milk. The
blots were then incubated in blocking solution for 1 h at 25°C
and overnight with the mAb p2802 (dilution: 1/100). The anti-pS2
monoclonal antibody (mAb p2802) raised against the carboxyl-terminal
domain of pS2 was previously described (39)
. Membranes
were washed in TBS containing 0.1% Tween 20, probed with a goat
anti-mouse immunoglobulin G polyclonal antibody (1/2000, Santa Cruz
Biotechnologies, Santa Cruz, Calif.), and revealed by enhanced
chemiluminescence Western detection (ECL, Amersham, Saclay, France).
Tissue sections from a sigmoid ADK (stage D), including the primary
colorectal tumor and adjacent mucosa, were stained for hpS2 with mAb
p2802 (1:40 dilution) and revealed by LSAB (linked streptavidin biotin)
and peroxidase.
Collagen invasion and cell scattering assays
For invasion of collagen gels by renal and intestinal epithelial
cells, Petri dishes were filled with 1.35 ml of neutralized type I
collagen and incubated overnight at 37°C to allow gelling. Cells were
harvested using Moscona buffer and trypsin/EDTA, and seeded on top of
the collagen gels. Cultures were incubated for 24 h at the
indicated temperature in the presence or absence of TFF alone or
combined with appropriate inhibitors of signal transduction pathways.
The depth of cell migration inside the gels was measured using an
inverted microscope (40)
. Invasive and superficial cells
were counted in 12 fields of 0.157 mm2. The
invasion index is the percentage of cells invading the gel over the
total number of cells.
For cell scattering, MDCKts.src Cl2 cells were seeded at 40°C in 24-well plates at the density of 2 x 104 cells and treated for 4 h with 0.1 µM each of hSP and hITF. The cell scattering was monitored under an inverted phase-contrast microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the trefoil peptides pS2, SP, and ITF (at 0.1 µM) and
scatter factor HGF (10 U/ml) are not competent for inducing cellular
invasion by the premalignant human colonic cell line PC/AA/C1
established from a polyp in a patient with FAP. As a positive control,
we show that leptin (100 ng/ml) promotes invasion in PC/AA/C1
epithelial cells, as described (41)
. Similar results are
obtained with HGF, leptin, and ITF in the nontumorigenic kidney
epithelial cell line MDCKT23 (Fig. 1B
). Because Rho-like
GTPases regulate cell adhesion and migration through reorganization of
the cytoskeleton, we investigated the possible contribution of RhoA in
the ability of ITF to induce invasiveness of collagen gels. To test
this hypothesis, MDCKT23 cells transfected by the dominant constitutive
form of RhoA (V14Rho) or the dominant negative form N19Rho were
incubated in the presence of ITF or scatter factor HGF. The ectopic
expression of these dominant forms of RhoA is controlled by the
tetracycline-repressible transactivator tTA, according to Jou and
Nelson (33)
. As depicted, MDCKT23-V14Rho cells are not
invasive in collagen type I gels (Fig. 1B
). We found that
ITF, but not HGF, cooperates with this constitutively active form of
RhoA in inducing cellular invasion. RhoA activation appears to be
necessary and sufficient for induction of the invasive phenotype by ITF
in MDCK cells. Such a cooperativity was not observed in MDCKT23 cells
transfected by the dominant negative construct N19Rho. In parallel
experiments, we checked (31)
that the dominant active form
of Rac1 was efficient for inducing a submaximal activation of
invasiveness in MDCKT23-V12Rac cells (invasion index: 7%) that is
wortmannin-sensitive (0.7% of invasive cells were counted in the
presence of this PI3'-K inhibitor).
|
Collaboration between TFFs and src for collagen invasion by colonic
and kidney epithelial cells
Activation of the src oncogene has been reported to be frequently
associated with premalignant colon adenomatous mucosa. C-Src kinase
activity is known to modulate cell spreading, migration, and invasion
in many cell types (28
, 42)
. Increased c-src protein
expression and activating mutations in the carboxyl-terminal regulatory
region of this nonreceptor tyrosine kinase are the critical molecular
defects involved in src activation and colon cancer progression
(43
, 44)
. Two steps of src activation take place during
colonic carcinogenesis: one in the polyps and primary tumors, and a
second, greater increase in liver metastases (45)
,
suggesting a potential role for src in local and distant invasion.
As shown in Fig. 2A
, activation of src in PCmsrc cells (28)
is not
sufficient to induce the invasive phenotype in collagen gels. This
invasive property was induced by the scatter factor HGF, the natural
ligand of the Met oncogene, a tyrosine kinase receptor that is
overexpressed and amplified during the progression of colorectal cancer
(46)
. The trefoil factor ITF (0.1 µM) mimicked the
HGF-induced invasiveness of PCmsrc cells, and this effect was abolished
by the PI3'-K inhibitor wortmannin (10 nM, WORT). Inhibiting RhoA by C3
exotransferase (C3T, 3 µg/ml), thereby converting it to a dominant
interfering form (47)
, leads to a loss of ITF-induced
invasion in PCmsrc cells. We conclude that the downstream activation of
Rho contributes to the invasive phenotype of PCmsrc cells induced by
TFFs, as suggested in Fig. 1
for the permissive interactions between
Rho and ITF signals in MDCK cells transfected with the activated form
of RhoA. These observations are consistent with RhoA and src playing a
critical role as a signal transducers in the multiple signaling
pathways emerging from focal adhesion complexes, cell–cell contacts,
and leading to the coordinated reorganization of the actin/myosin
network by growth factor receptors during cell migration and invasion.
C3T toxin treatment of MDCKts.src cells inhibited by 90% the invasion
of collagen gels induced by src activation at 35°C (data not shown),
suggesting that RhoA is implicated in the recruitment and activation of
src at the plasma membrane or is a downstream element in src signaling.
Rho-dependent regulation clustering of intercellular adhesion molecules
and muscarinic or EGF receptor trafficking and internalization in the
endosomal compartment were also found (48
, 49)
.
|
Because the PLC and PKC cascade has been shown to activate Rho
(50
, 51)
, we next investigated the effects of
pharmacological inhibitors of these signal transduction elements. As
shown in Fig. 2A
, the two PKC inhibitors GF109203X (GF109, 1
µM; Gö6976, 1 µM) as well as persistent activation and
down-regulation of PKC by 100 ng/ml TPA for 24 h prevented the
ITF-induced invasion in PCmsrc cells. In contrast, ITF-induced cellular
invasion was not reversed by PTx (100 ng/ml) or FK (10 µM). These
results imply that the signaling pathways activated by TFFs are not
connected with PTx-sensitive trimeric G-protein subunits, in contrast
to our previous data on inhibition of HGF- and leptin-induced
invasiveness by PTx in PCmsrc and MDCKts.src cells (30
, 41)
. Alternatively, we presented evidence that the ITF-induced
invasiveness of PCmsrc cells is insensitive to cAMP-inducing agents
that increase adenylate cyclase activity directly (forskolin) or
indirectly via the ADP-ribosylation and neutralization of the Gi/Go
subunits (pertussis toxin). Similar results were obtained with
MDCKts.src cells, where invasion was induced by HGF and ITF (Fig. 2B
). Likewise, wortmannin, C3T, and PKC inhibitors
neutralized ITF-induced invasion, whereas PTx or FK were ineffective.
We also checked that stable expression of the dominant negative mutant
PI3'-K in the MDCKts.src-p110DN cell line (30)
abolished
the ITF-induced invasion of collagen gels in this model (not shown).
Our data thus provide a new link between TFF-dependent signaling and
PI3'-K, PKC and Rho in the control of cellular invasion. PKC is
composed of a family of serine-threonine kinases that plays an
important role in tumor development and angiogenesis (52
, 53)
. As the PLC signaling pathway upstream PKC also regulates
endothelial cell motility (54)
, it is clear that
phospholipases C (PLC) are potentially involved in tumor invasion and
progression toward the metastatic cascade. Consequently, we next
examined whether invasiveness induced by trefoil peptides is sensitive
to the PLC inhibitor U-73122. Treatment of MDCKts.src cells with 1 µM
U-73122 completely abolished the invasion of collagen gels induced by
100 nM of each TFF: pS2, SP, and ITF (data not shown). This
pharmacological blockade is specific for the invasiveness induced by
the TFFs because U-73122 had no effect on HGF- and leptin-induced
invasion. Moreover, PLC isoforms ß, , and 
harbor pleckstrin
homology PH domains responsible for appropriate membrane targeting and
functionality regarding upstream signals activated by src and growth
factor receptors. Most important, binding of PLC to plasma membrane via
the PH domain is dependent on PI3'-K products such as PtdIns (3, 4,
5)-trisphosphate (55)
, suggesting interdependent
activation and function between these two enzymes for definition of the
invasive phenotype induced by TFFs in colonic and kidney epithelial
cells. We next investigated the effect of various concentrations of the
three trefoil peptides on invasion of collagen gels by PCmsrc and
MDCKts.src cells.
Dose-dependent cellular invasion in response to the trefoil
peptides
As shown in Fig. 3
, pS2, SP, and ITF dose-dependently stimulated invasion of collagen gels
by colonic PCmsrc cells (Fig. 3A
) and kidney epithelial
cells MDCKts.src (Fig. 3B
). In the two models, very similar
and parallel dose-response curves were observed for the three TFFs,
with the same extent of maximal effect. Significant stimulation of
invasion was observed in PCmsrc and MDCKts.src cells incubated in the
presence of 10 nM SP or ITF (P<001). Half-maximal
stimulation of invasion by SP and ITF was observed at similar
concentrations (EC50=20–40 nM). Maximal
stimulation induced by pS2, SP, and ITF represented a 10-fold increase
of invasiveness above control.
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Ectopic expression of full-length cDNA of human pS2 induced
invasiveness in colonic HCT8/S11 and kidney MDCKts.src cells
Since our results indicate that exogenously added TFF peptides
induce cellular invasion in our experimental models of src- and
RhoA-transformed colonic and kidney epithelial cells, we next
investigated the functional role of pS2, SP, and ITF on the invasive
phenotype of human colon cancer cells established from a sporadic
tumor. For this purpose, we engaged a study in colonic HCT8/S11 cells
and their -catenin-deficient counterparts, HCT8/S11R1 cells
(29)
. As shown in Fig. 4A
, HCT8/S11R1 cells do not invade collagen gels in response
to scatter factor, leptin, or any of the three TFFs tested. The
functionality of the cell–cell junctional proteins involving
E-cadherin and its associated cytoplasmic protein -catenin is
therefore a prerequisite for induction of the invasive phenotype in
HCT8/S11 cells vs. the S11R1 variant. Indeed, the three TFFs all
induced strong invasiveness in HCT8/S11 cells (Fig. 4B
) at
levels similar to that observed in src-transformed colonic and kidney
epithelial cells (Invasion index=8–10%), whereas HGF was ineffective.
The TFF-induced invasion was completely blocked by rapamycin (10 nM) in
HCT8/S11 cells. Rapamycin binds to FKBP12, this complex inactivates
mTOR/FRAP and thus interferes with the activation of the PI3'-K/Akt/p70
S6 k/BAD signaling cascade that controls translation, cell growth and
survival (56
, 57)
.
|
We next examined whether the constitutive overexpression of the pS2
protein, frequently observed in inflammatory conditions in the
gastrointestinal tract, breast, and prostate cancers, is associated
with induction of the invasive phenotype. We therefore expressed the
pS2 cDNA in the TFF-competent colonic HCT8/S11 and kidney MDCKts.src
cell lines. The pcDNA3-hpS2 expression plasmid was functional because
transiently transfected kidney HEK-293T.pS2 cells are positive for pS2
protein expression by SDS-PAGE and Western blot corresponding to
identification of a prominent signal at 
9.6 kDa (data not shown).
The same 9.6 kDa band was identified in two positive controls using a
human gastric tumor and the MCF-7 breast cancer cell line. This
expression vector was therefore used to establish constitutive
expression of the pS2 protein in HCT8/S11 cells and MDCKts.src cells.
Stably expressed transfectants were isolated by the selection using
G418. Immunoblotting analysis specified three pS2-positive cell lines
established from pcDNA3-hpS2-transfected HCT8/S11 cells (pool 1, clones
2 and 6), and three sublines were also isolated from pS2-transfected
MDCKts.src cells (pool 2, clones 2 and 5). Mock-transfected colonic and
kidney epithelial cells by using the empty pcDNA3 vector (pools 1 and
2) were negative for pS2 protein expression. Because pS2 is a secreted
protein, we checked by Western blot that the conditioned culture medium
by HCT/S11-pS2 (clone 2) and MDCKts.src-pS2 cells (clone 2) contained
detectable amounts of the TFF (data not shown). In contrast, pS2
protein was not detected in the culture medium conditioned by
nontransfected HCT8/S11 cells and mock-transfected MDCKts.src-pcDNA3
cells.
As shown in Fig. 5
, constitutive expression of the pS2 cDNA in HCT8/S11 cells (clone 2)
and MDCKts.src cells (clone 2) resulted in induction of the invasive
phenotype. Most important, the Rho inhibitor C3T failed to abolish the
pS2-induced cellular invasion in both cell lines, suggesting that the
persistent overexpression of pS2 may overcome or circumvent this
blockade. Another possibility is that constitutive and deregulated
expression of pS2 may initiate new invasion signals that are Rho
independent. This interesting observation forms the basis of our work
on new cellular invasion pathways and functions controlled by TFFs. In
contrast, we found that pS2 overexpression was still sensitive to
wortmannin (PI3'-K inhibitor), rapamycin, U-73122 (PLC inhibitor), was
also abolished by PKC inhibitors, and remained insensitive to PTx and
FK (Fig. 5A
, B
). Our data therefore suggest that the
constitutive overexpression of the pS2 protein during inflammatory
processes or neoplastic progression may have a pathophysiological
significance on autocrine migration, restitution, and invasive
properties of colonic and kidney mucosal cells.
|
Induction of cellular scattering by TFFs
Cell invasion is a major component of the complex multistep
process of metastasis that is the most pejorative event linked to the
development of fatal cancers. Acquisition of cell mobility and the
capacity to invade basement matrix membranes and adjacent tissues play
a central role in the adenoma-adenocarcinoma conversion and distant
metastasis. To address whether TFFs have a motogenic activity in
MDCKts.src cells, we examined cell scattering in MDCKts.src monolayer
cultures incubated for 4 h in the presence or absence of SP and
ITF. In control cultures, MDCKts.src cells incubated at 40°C formed
tight colonies of adjacent epithelioid cells whereas the addition of
100 nM SP or ITF resulted in inhibition of cell–cell contacts,
enhanced cellular motility, and induced scattering of the MDCKts.src
cells (Fig. 6
). On the other hand, HGF/scatter factor induced cell scattering, which
is consistent with previous reports (58)
.
|
Expression of pS2 in human colorectal tumors at various stages of
the neoplastic progression
Using the monoclonal antibody p2802, we next examined the
expression of the pS2 protein in human colorectal tumors and their
adjacent nontransformed, histologically normal mucosa. We observe in
Fig. 7A
that pS2 exhibited a single broad immunoreactive band on
SDS-PAGE, corresponding to 9.6 kDa. This band was detected in all
the samples examined, including colitis and early stages of the
cancerous progression (adenoma, stages A and B of the Dukes’
classification) and more advanced colonic tumors (C, D and liver
metastases). One exception was Sample 40, i.e., the liver parenchyma
(Liver) associated with a metastasis originating from a colonic tumor.
Accordingly, immunochemical staining of pS2 in a human sigmoid tumor
(stage D) revealed that expression of the pS2 protein is confined to
mucin-secreting epithelial cells in both the tumorous sample and its
corresponding adjacent control mucosa (Fig. 7B
). In this
control sample, typical colonic epithelial crypts showed a normal
architecture with marked pS2 accumulation restricted to the basal pole
of the epithelial cells, in close proximity to the nucleus.
Endothelial, inflammatory cells, and normal or tumor stroma were
negative in tumor and control samples.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A novel and important observation of the present study is the
observation that the three trefoil peptides pS2, SP, and ITF induce
invasion of collagen gels by kidney and colonic epithelial cells that
are already engaged in the cancerous progression. Trefoil peptides may
therefore exert a beneficial role on the restitution of injured
digestive epithelia (2
3
4
5
6
, 9
, 21)
or contribute to
neoplastic progression in the human colon, depending on the normal or
transformed status of the intestinal epithelial cells.
We have shown that TFFs are ineffective for inducing cellular invasion of premalignant colonic PC/AA/C1 and kidney MDCK epithelial cells and that src and Rho activation are required. The fact that TFFs need activated src/Rho signify that trefoil peptides do not activate these pathways efficiently, but need additional parallel and concomitant signals activating src/Rho. This condition is fulfilled by the frequent activation of src observed in early human colon cancers and can be also accomplished by RhoA activators such as lysophosphatidic acid and thrombin, two agents involved in inflammatory disorders, at the transition between preinvasive and invasive stages in the digestive tract.
To our knowledge, this is the first report that integrates the three
TFFs in a comparative investigation on their biological effects and
cell signaling in the context of the multistep process of
tumorigenesis. These results have important implications for further
studies on the involvement of TFFs as modulators of human digestive
cancer growth and metastasis because we clearly identified pS2 in
colitis, premalignant polyps, sporadic human colon cancers, and their
liver metastases. In the normal gastrointestinal tract, the TFFs are
expressed along the entire length of the gut, with pS2 and SP expressed
primarily by the stomach (39
, 59)
and ITF by the goblet
cells of small intestine and colon (60
, 61)
. No
significant staining of pS2 was observed in a variety of human
specimens from the normal colon, pancreas, liver, prostate, and kidney
(39)
. Mucosal ulceration in the duodenum and ileum induces
pS2 and SP expression in the surrounding mucosa. Additional studies
demonstrated intestinal pS2 expression in the ulcer-associated cell
lineage in Crohn’s disease (62)
. Numerous clinical and
experimental observations reveal that colonic cancer is
increasingly frequent in inflammatory bowel disease. Our data support
the notion that paracrine and autocrine TFF loops contribute to the
transformed phenotype in colonic mucosa during inflammatory conditions,
wound repair, and neoplastic progression. Furthermore, ITF also confers
resistance to apoptosis in premalignant IEC and cancerous HT-29
intestinal epithelial cells (22)
. Thus, TFFs apparently
are implicated in beneficial and in beneficial effects for the
gastrointestinal mucosal integrity during normal migration and healing
of the injured digestive mucosa. Additional adverse effects for
gastrointestinal TFFs should be considered in view of their ability to
promote cell scattering and invasion of basement membrane matrix
proteins during early human colon cancers initiated by activated src.
Our data suggest an important role for pS2, SP, and ITF in cancer
progression and provide a link between TFFs and signaling pathways
regulating the reorganization of the cytoskeleton, focal adhesion as
well as cellular motility and tumor invasion (30
31
32
, 63)
.
Nevertheless, the important question concerns the molecular identity of
the putative TFF binding sites or receptors involved in the initiation
of multiple pathways linked to cellular invasion and matrix
adhesion-dependent signaling and cell survival. Our data strongly
suggest that the three TFFs interact with the same recognition sites
mediating 1) both invasion and scattering, 2)
similar parallel dose-response curves, and 3) connection
with subsequent signaling cascades controlled by the same series of
pharmacological inhibitors. The challenge will be to characterize the
nature of these TFF recognition sites and their proximal downstream
signaling elements. In the present study, we identified several
signaling pathways playing a crucial role in the promotion of cellular
invasion by TFFs. These include src/RhoA, PI3'-K/Akt, and phospholipase
C/PKC, which are also activated by growth factor receptors, oncogenes,
and are connected with the rapamycin-sensitive TOR cascade, leading to
p70S6K activation. Targets of PI3'-K/Akt include BAD, an inhibitor of
cell survival (57)
. Identification of Cdc42 and Rac as
regulators of p70S6K (56)
provides further interplay and
controls between TFFs and signaling cascades involved in tumor
invasion, progression in the cell cycle, and apoptosis. Given the link
between PI3'-K and the Rho family G-proteins, we present evidence here
that MDCK cells are induced to invade collagen gels after activation of
Rac, not by activated Rho. Similarly, premalignant adenomatous PC/AA/C1
cells that harbor an activated Ras allele (28)
are not
invasive in the presence of TFFs and become invasive after activation
of src. Overexpression of pS2 cDNA confers constitutive induction of
the invasive phenotype in stably transfected colonic HCT8-S11-pS2 cells
that originate from a sporadic human colonic adenocarcinoma. Such a
constitutive overexpression is observed here in premalignant colon
adenoma, primary tumors, and their adjacent nontumorous mucosa. It is
therefore likely that early and late colonic tumors may initiate
distant induction of TFF in the histologically normal adjacent mucosa,
which reflects a local inflammatory situation. Our data argue for
autocrine, paracrine, and persistent deregulation of the TFF signals in
premalignant and tumorous digestive mucosa characterized by a loss of
mucosal polarity. The progressive accumulation of oncogenic defects,
combined with constitutive activation of TFF secretion and signals
during inflammatory bowel disease and neoplastic progression, can
therefore generate subsequent aberrant cellular dysfunctions in
initiated colonic epithelial cells.
As src activation is a frequent and early event in human colon cancer
progression, TFFs fulfill the criteria to be regarded as scatter
factors because of their involvement as modulators of cellular motility
and invasion. Our findings shed new light on the relationships between
trefoil factors and cancer progression as a general mechanism with
broad significance for solid tumors concerned by TFF expression. Our
data linking TFFs, cell invasion, and survival signaling pathways are
prerequisite for developing novel combination therapies against colon
cancer using new anti-invasion and -angiogenesis agents together with
classical anti-cancer drugs (35
, 64)
. Further studies will
bring more informations on TFF functions and signaling and how they
interact with epithelial cells to control cellular migration and
invasion during the renewal of the mucosa and progression of digestive
cancers.
|
ACKNOWLEDGMENTS |
|---|
Received for publication May 30, 2000.
Revision received July 31, 2000.
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