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Tumor Biology |
Friedrich Miescher Institute, CH-4002 Basel, Switzerland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-6 is a member of the IL-6-type cytokine family, which comprises OSM,
LIF, IL-11, ciliary neurotrophic factor, and cardiotrophin-1 (1
, 2)
. These peptides promote similar biological responses in
various tissues and cells. This redundancy in biological actions can be
explained at the molecular level because the different members of this
cytokine family share signaling molecules. Indeed, IL-6-type cytokines
bind to multimeric receptors comprising an 
chain, which confers
ligand specificity and a signal-transducing ß subunit (gp130) common
to all IL-6-type cytokines.
On ligand binding, the gp130-associated intracellular tyrosine kinases Jak1, Jak2, and Tyk2 become activated and phosphorylate the gp130 cytoplasmic tail on specific tyrosine residues. These phosphotyrosines serve as docking sites for signaling molecules, including STATs and molecules mediating activation of the Ras/MAPK pathway, such as the tyrosine phosphatase SHP-2 and the Shc adapter molecule. STATs, in turn, are phosphorylated on tyrosine residues, allowing their dimerization and translocation to the nucleus, where they regulate gene transcription. Shc and SHP-2 are also tyrosine phosphorylated by the Jaks, initially leading to the recruitment of adapter molecules, such as Grb2, and ultimately leading to the activation of MAPK. More recently, the multisubstrate docking molecule, Gab1, was shown to act as an adapter linking gp130 to both the MAPK and the PI3K pathways (3) .
Recent studies have shown that cytokine-induced activation of the Ras/MAPK pathway is, in some instances, dependent on cross-talk with ErbB receptor tyrosine kinases. Aberrant expression of members of the ErbB receptor tyrosine kinase family, which is observed in a variety of human tumors (4 , 5) , has been linked to abnormal cell growth and transformation. The ErbB family includes four members: (a) the EGF receptor/ErbB-1; (b) ErbB-2/Neu; (c) ErbB-3; and (d) ErbB-4. ErbB receptor activity is regulated by EGF-related peptides, which bind to ErbB-1 and ErbB-4, and neuregulins, which are ligands for ErbB-3 and ErbB-4. ErbB-2 has no known ligand, but it is the preferred dimerization partner of all ErbB receptors and plays a central role in ErbB signaling (6 , 7) . In prostate carcinoma cells, IL-6 appears to induce activation of ErbB-2 and ErbB-3 receptors, forming a gp130/ErbB-2/ErbB-3 complex and leading to MAPK activation and cell proliferation (8) . GH-induced activation of the Ras/MAPK pathway has also been shown to be dependent on the EGF receptor. In this case, a mechanism involving Jak2-mediated tyrosine phosphorylation of a Grb2 binding site in the cytoplasmic domain of the EGF receptor was proposed (9) .
IL-6-type cytokines have diverse actions on breast cancer cell lines,
including changes in morphology, decreased cell-cell association, and
inhibition of cell proliferation (10, 11, 12)
. Interestingly,
expression of gp130 and the -specific subunits of IL-6-type cytokine
receptors has been found by PCR analyses in a large majority of breast
cancer cell lines and in primary malignant breast tissue
(12)
. In addition, IL-6 and other IL-6-type cytokines are
expressed in many primary breast tumors (13, 14, 15)
. However,
IL-6 expression is reduced in invasive breast carcinoma relative to
normal mammary tissue and appears to be inversely associated with
histological tumor grade (15
, 16)
. Although little is
known about IL-6-induced signal transduction in breast cancer, these
observations suggest that this cytokine might be involved in regulating
the growth of these cancer cells.
We have investigated IL-6-induced signaling in the T47D breast carcinoma cell line. Our results show that IL-6-type cytokines inhibit proliferation and increase cell migration. We demonstrate that IL-6-induced growth inhibition is dependent on activation of the Jak/STAT pathway, whereas activation of the MAPK and PI3K pathways is required for IL-6-stimulated cell motility. Finally, we show that in T47D cells, contrary to what has been described in some prostate carcinoma cell lines, IL-6-induced MAPK activation does not involve ErbB-2 activation but depends on the cooperation of an EGF receptor autocrine loop.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For transient transfections, plasmids were introduced into the cells using Superfect transfection reagent (Qiagen). A HA-tagged MAPK (Erk2) construct (kindly provided by M. El-Shemerly and Y. Nagamine; Friedrich Miescher Institute, Basel, Switzerland) was cotransfected with a DN EGF receptor construct lacking 533 COOH-terminal amino acids (kindly provided by A. Ullrich; Max Planck Institut, Martinsried, Germany), a DN ErbB-2 construct (18) , or a DN Jak1 construct (kindly provided by O. Silvennoinen; University of Tampere, Tampere, Finland). Cells were allowed to grow for 24 h before starvation and cytokine treatment.
Cells were starved for 16 h in serum-free medium before treatment with recombinant human IL-6 [R&D Systems (Minneapolis, MN) or PeproTechEC (London, United Kingdom)], recombinant human OSM and LIF (PeproTechEC), heregulin ß (Neomarkers, Fremont, CA), or EGF (Sigma Chemical Co., St. Louis, MO) for 15 min. In some experiments, cells were pretreated for 90 min with the EGF receptor and ErbB-2 inhibitor PD153035 (4 µM), the EGF receptor inhibitor CGP59326 (3 µM; kindly provided by P. Traxler and D. Fabbro; Novartis, Basel, Switzerland), or antibodies to the EGF receptor (mAb 528 or R1; Santa Cruz Biotechnology, Santa Cruz, CA) before treatment with IL-6.
Immunoprecipitation and Western Blots.
Cells were solubilized in NP40 extraction buffer [50 mM
Tris (pH 7.5), 1 mM EGTA, 5 mM EDTA, 120
mM NaCl, 1% NP40, 2 mM sodium orthovanadate,
50 mM sodium fluoride, 20 mM
ß-glycerophosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin,
and 0.5 mM phenylmethylsulfonyl fluoride] for 5 min
on ice. The lysates were clarified by centrifugation at
15,000 x g for 15 min. For
immunoprecipitation, equal amounts of proteins were incubated with
specific antibodies for 1 h. Antibodies used were ErbB-2-specific
mAbs FWP51 and FSP77 (19)
; EGF receptor-specific mAbs R1
and 528; SHP-2, Jak1, Tyk2, STAT1, STAT3, and HA epitope-specific
polyclonal antibodies from Santa Cruz Biotechnology; gp130, Jak1, and
Jak2 polyclonal antibodies from Upstate Biotechnology, Inc.
(Lake Placid, NY); Shc polyclonal antibody from Transduction
Laboratories (Lexington, KY); STAT5a and STAT5b polyclonal antipeptide
antisera (20)
; and Gab1 rabbit antisera (kindly provided
by A. Ullrich). Immune complexes were collected with protein A-
or protein G-Sepharose (Sigma) and washed three times with lysis
buffer. Precipitated proteins were released by boiling in sample buffer
and subjected to SDS-PAGE. The proteins were blotted onto
polyvinylidene difluoride membranes (Boehringer Mannheim, Mannheim,
Germany). After blocking with 20% horse serum (Life Technologies,
Inc.) in 50 mM Tris (pH 7.5), 150
mM NaCl, and 0.05% Tween 20, filters were probed
with specific antibodies. Antibodies used for Western blotting were the
same as those described above, plus a phosphotyrosine-specific mAb
(21)
, anti-phospho-specific p44/42 Erk, anti-p44/42 Erk,
anti-phospho-specific Akt/PKB, and anti-Akt/PKB polyclonal antibodies
from New England Biolabs (Beverly, MA); EGF receptor polyclonal
antibody 1005 (Santa Cruz Biotechnology); and an anti-p85 polyclonal
antibody (Upstate Biotechnology, Inc.). Proteins were visualized
with peroxidase-coupled secondary antibodies using the enhanced
chemiluminescence detection system (Amersham Pharmacia Biotech, Little
Chalfont, United Kingdom).
Colony Growth Assay.
Cells plated onto 6 cm-dishes (1500 cells/dish) were allowed to attach
overnight in FCS-supplemented DMEM before the addition of cytokines.
After 10 days in culture, cells were fixed in a solution of
methanol:acetic acid (3:1) and stained with Giemsa stain. Colonies were
counted using an Artek 880 colony counter (Dynatech Laboratories).
Experiments were performed in triplicates, and data are presented as
mean ± SD.
Migration Assay.
Cell motility was tested in 8-µm-pore polycarbonate membrane
Transwell chambers (Costar, Cambridge, MA) essentially as described
previously (22)
. The underside of the polycarbonate
membranes was coated with 25 µg/ml rat tail collagen I (Boehringer
Mannheim). Cells were resuspended in DMEM supplemented with 1 mg/ml
BSA, and 75,000 cells were added to the top chamber of the Transwell
chambers. DMEM containing 1 mg/ml BSA and 100 ng/ml IL-6 or OSM
was added to the bottom chamber, and cells were allowed to migrate for
20 h. For some experiments, cells were incubated in the presence
of EGF receptor mAb 528 (10 µM), EGF receptor inhibitor
CGP59326 (3 µM), MAPK kinase inhibitors PD98059 (20
µM; New England Biolabs) or UO126 (50 µM;
Promega, Madison, WI), or PI3K inhibitor LY294002 (25 µM;
Calbiochem, San Diego, CA) for 90 min before testing cell
motility. Analysis of MAPK and PKB phosphorylation in parallel
experiments showed that IL-6-induced MAPK kinase and PI3K activities
were efficiently blocked using the concentrations of inhibitors
indicated above. Nonmigrated cells were scraped off the top of the
membrane. Migrated cells were fixed in 4% formaldehyde and stained in
1% crystal violet. Cells were counted under a microscope in five
different high-power fields in duplicate wells, in at least three
independent experiments.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The effect on the formation of large colonies was more
pronounced, indicating that IL-6 retards proliferation. Flow cytometry
analysis of T47D cells grown in the presence of IL-6 for 24 h
showed a reproducible 10–15% increase in the number of cells in the
G1 phase of the cell cycle (data not shown).
Whereas OSM was even more potent than IL-6 in inhibiting T47D cell
proliferation, LIF had a more moderate effect, as seen in the colony
growth assay (Fig. 1B)
or by cell cycle analysis (data not
shown).
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. We then investigated which particular Jak and STAT were
activated by IL-6. Although Jak1, Jak2, and Tyk2 were expressed in T47D
cells, only Jak1 was significantly tyrosine phosphorylated on IL-6
stimulation (Fig. 2A)
. Differential activation of the Jak
family members in different cell lines has been described previously
(23
, 24) . IL-6 also induced the phosphorylation of STAT3
and, to a lesser extent, STAT1 (Fig. 2B)
. Neither STAT5a nor
STAT5b, which can be activated in response to IL-6 in some cell lines,
was tyrosine phosphorylated in IL-6-stimulated T47D cells.
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. These cells are also known to express moderate levels of
all ErbB receptors. To evaluate the role of these receptors in IL-6
signaling, we tested the effect of ErbB-specific pharmacological
inhibitors on IL-6 induction of the MAPK and PI3K pathways. T47D cells
were pretreated with the EGF receptor-specific inhibitor CGP59326
(25)
or with the bispecific EGF receptor/ErbB-2 inhibitor
PD153035 (26)
before stimulation with IL-6, EGF, or
heregulin ß, a member of the neuregulin family. Heregulin ß signals
through ErbB-2-containing heterodimers, whereas EGF signaling involves
EGF receptor- and ErbB-2-containing dimers (7)
. As
expected, whereas both the CGP59326 and the PD153035 inhibitors
completely blocked EGF-induced MAPK phosphorylation, only the PD153035
inhibitor effectively inhibited heregulin ß-induced MAPK activation
(Fig. 3A)
. Interestingly, both inhibitors strongly
diminished IL-6-induced MAPK activation (Fig. 3A)
. The PI3K
pathway was affected by the ErbB inhibitors in the same manner (Fig. 3A)
. In contrast, IL-6-induced STAT3 phosphorylation was not
influenced by the presence of the ErbB inhibitors (Fig. 3B)
.
These results indicate that EGF receptor activity might be involved in
IL-6 induction of the MAPK and PI3K pathways, but not the STAT pathway.
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. This result confirms that IL-6 requires the presence of
active EGF receptor for full activation of MAPK. Interestingly, DN Jak1
also interfered with IL-6-induced MAPK activation (Fig. 3C)
,
indicating that Jak1 is required for MAPK activation in response to
IL-6.
ErbB-2 Is Not Required for IL-6-induced Activation of MAPK and
PI3K.
The above-mentioned results indicate that ErbB-2 activity is not
required for MAPK activation in response to IL-6. It is possible,
however, that ErbB-2 may still be used as a scaffold by IL-6 to
activate MAPK, in the same manner as the GH receptor uses EGF receptor
(9)
. To more definitely establish the role of ErbB-2 in
IL-6-induced MAPK activation, we have functionally inactivated the
ErbB-2 receptor through intracellular expression of an
ErbB-2specific single chain antibody, scFv5R, which traps ErbB-2
in the endoplasmic reticulum (17)
. T47D-5R cells expressed
levels of ErbB-2 similar to those of the control T47D cells (Fig. 4A)
, but intracellular retention of ErbB-2 resulted in faster
electrophoretic mobility because of impaired glycosylation
(17
, 27)
. Treatment of T47D cells with heregulin ß
strongly stimulated ErbB-2 tyrosine phosphorylation and activation of
intracellular signaling pathways, including the PI3K and MAPK pathways
(Fig. 4B)
. IL-6, although inducing MAPK and PKB
phosphorylation (Fig. 4B)
, did not significantly increase
ErbB-2 tyrosine phosphorylation (Fig. 4A)
. As shown
previously (27)
, T47D-5R cells treated with heregulin ß
displayed decreased ErbB-2 tyrosine phosphorylation and, consequently,
decreased MAPK and PI3K activation relative to control cells (Fig. 4)
.
Moreover, heregulin ß-stimulated proliferation of T47D-5R cells was
strongly impaired (data not shown). Despite the fact that ErbB-2 was
functionally inactive, IL-6 was still capable of activating the MAPK
and PI3K pathways to levels comparable to those in control cells.
Therefore, ErbB-2 does not appear to be involved in IL-6-induced
activation of the MAPK and PI3K pathways in the T47D cell line. Similar
results were obtained using the scFv-5R-expressing MCF7 and SKBr3
breast carcinoma cell lines, which express moderate and high levels of
ErbB-2, respectively (data not shown). Thus, in breast carcinoma cells,
ErbB-2 does not appear to be a critical element in IL-6-induced MAPK
activation.
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are expressed in T47D cells
(5)
.4
To test this hypothesis, T47D cells were pretreated with the EGF
receptor-specific mAb 528, which interferes with ligand binding to the
EGF receptor, before being stimulated with IL-6. As a control, cells
were pretreated with mAb R1, which also binds the EGF receptor
extracellular domain but does not interfere with ligand binding.
Treatment with mAb 528 decreased the basal level of MAPK activation,
indicating the presence of an EGF-like ligand/EGF receptor autocrine
loop (Fig. 5A)
. Interestingly, IL-6-induced MAPK activation was also
strongly decreased in the presence of the EGF receptor blocking
antibody (Fig. 5A)
. IL-6-induced PKB phosphorylation was
similarly decreased by the EGF receptor blocking antibody (data not
shown). This effect was due to the blocking ability of mAb 528 because
the control mAb R1 did not inhibit IL-6-induced MAPK phosphorylation
and even increased its activation slightly, probably through EGF
receptor clustering (Fig. 5B)
. Importantly, densitometric
analysis indicated that, when combined, IL-6 treatment and EGF receptor
autocrine activity (Fig. 5A
, Lane 5) stimulated
MAPK activity to a larger extent than would be predicted by the simple
addition of the effects of EGF receptor autocrine activity (Fig. 5
A, Lane 1) and IL-6 (Fig. 5A
, Lanes
6–8). Thus, in T47D breast cancer cells, IL-6 acts
synergistically with an EGF receptor autocrine loop to activate the
MAPK and PI3K pathways.
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, or the Shc-Grb2 association (data not shown). However,
inhibition of EGF receptor autocrine activity using the blocking
antibody mAb 528 or the EGF receptor-specific inhibitor CGP59326 did
result in decreased Shc phosphorylation and association with the EGF
receptor (Fig. 6A)
. Thus, Shc contributes to basal but not
IL-6-induced MAPK activation in T47D cells.
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. Interestingly, analysis
of coimmunoprecipitated proteins revealed that IL-6 induced the
formation of a complex of SHP-2-associated tyrosine-phosphorylated
molecules (Fig. 6
B, Lane 4) identified as Gab1 and the p85
subunit of PI3K. Similarly, on IL-6 stimulation, Gab1 associated with
SHP-2 and p85 (visible only at longer exposure of Fig. 6B
;
data not shown). It is noteworthy that although no
phosphotyrosine was detected on Gab1 or SHP-2, they were both
associated with the tyrosine-phosphorylated EGF receptor, even in the
absence of IL-6 (Fig. 6B
, Lanes 1 and
7). Pretreating the cells with mAb 528 or with the EGF
receptor-specific inhibitor CGP59326 resulted in decreased
phosphorylation of SHP-2 and Gab1 (Fig. 6B)
, and decreased
association with the other members of the immune complex,
including the EGF receptors (data not shown). Thus, IL-6-induced
activation of Gab1 and SHP-2 is largely dependent on EGF receptor
autocrine activation.
SHP-2 complexes were further examined. SHP-2 association with gp130 and
Gab1 was significantly increased in the presence of IL-6, whereas its
interaction with EGF receptor was not dependent on IL-6 (Fig. 6C)
. Thus, in the absence of IL-6 stimulation, SHP-2 and
Gab1 are present in a complex comprising the EGF receptor and gp130
(data not shown). On IL-6 treatment, there is increased tyrosine
phosphorylation and recruitment to gp130 of SHP-2 and Gab1 (Fig. 6C)
.
IL-6/OSM-induced Inhibition of T47D Cell Proliferation Is STAT3
Dependent.
The biological responses elicited by IL-6 treatment of T47D cells are
2-fold: (a) decreased proliferation; and (b)
increased cell motility, as shown below. In the following experiments,
we probed the contribution of the cytoplasmic pathways activated by
IL-6, namely, the Jak/STAT, MAPK, and PI3K pathways, to IL-6-mediated
biological responses.
To analyze the contribution of STAT3 activity, we generated T47D clones expressing a DN form of STAT3 (STAT3F) and selected clones expressing similar amounts of wild-type STAT3 (STAT3wt) as controls. STAT3F is mutated at tyrosine 705 and therefore cannot be phosphorylated on docking to gp130 (34) . OSM- and IL-6-induced STAT3 DNA binding activity to a specific probe was found to be strongly decreased in the T47D-STAT3F clones (data not shown). In contrast, OSM- and IL-6-induced MAPK activity was not significantly altered in T47D-STAT3F relative to T47D cells or T47D-STAT3wt cells (data not shown).
In a colony outgrowth assay, IL-6 and OSM inhibited the proliferation
of T47D-STAT3wt cells as efficiently as that of the parental T47D cells
(Fig. 7A)
. In comparison, T47D-STAT3F cells were less sensitive to
the effect of IL-6 or OSM. Indeed, the IL-6 inhibitory effect was about
75% less for T47D-STAT3F cells relative to control cells, and OSM
efficiency was reduced by more than 60% (Fig. 7A)
.
Furthermore, cell cycle analysis indicated that, contrary to what was
observed with T47D or T47D-STAT3wt cells, IL-6/OSM treatment did not
increase the percentage of T47D-STAT3F cells in the
G1 phase of the cell cycle (data not shown).
Thus, the inhibition of T47D cell proliferation induced by IL-6 and OSM
requires STAT3 activity. In contrast, inhibition of the EGF receptor or
the MAPK and PI3K pathways with specific inhibitors resulted in an
increased percentage of the cells in the G1 phase
of the cell cycle, showing that the MAPK and the PI3K pathways
positively affect proliferation (data not shown).
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. IL-6 and OSM effects on
cell migration were completely reversed in the presence of the MAPK
kinase inhibitors PD98059 (data not shown) and UO126 or in the presence
of the PI3K inhibitor LY294002 (Fig. 7B)
. Thus, IL-6-type
cytokines stimulate T47D cell migration through a MAPK- and
PI3K-dependent mechanism. Because IL-6-induced MAPK and PI3K activation
is dependent on EGF receptor activity, we evaluated the contribution of
the EGF receptor to T47D cell migration. IL-6-induced migration was
inhibited in the presence of the EGF receptor blocking antibody (mAb
528) or the specific inhibitor CGP59326 (Fig. 7C)
. Finally,
IL-6 and OSM stimulated migration of T47D-STAT3F to a similar extent as
control cells (Fig. 7D)
, indicating that cell migration is
independent of STAT3 activity. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have shown here that STAT3 plays a crucial role in IL-6/OSM-induced growth inhibition of T47D cells. Studies using DN forms of STATs demonstrated that growth inhibition of M1 leukemic cells and the A375 melanoma cells in response to IL-6-type cytokines is also dependent on STAT3 activation (34, 35, 36) . On the other hand, the mitogenic effect of IL-6 for some cell types was shown to involve SHP-2 and MAPK activation (34 , 37) . Thus, in some cells, the biological effect of IL-6 depends on the balance of a growth-inhibitory STAT3-dependent pathway and a growth-promoting MAPK/PI3K-dependent pathway (38) . Whereas we could not observe increased proliferation in STAT3F-expressing T47D cells, blocking MAPK/PI3K in IL-6-stimulated T47D cells did lead to a higher percentage of cells in the G1 phase of the cell cycle. However, such a simplistic model for IL-6 action, involving two main opposing pathways to control cell proliferation, may not apply to all cell systems. Indeed, several reports demonstrate that STAT3 itself can promote cell growth (39) and even behave as an oncogene when constitutively active (40) . These observations indicate that there must be as yet unknown cell-specific mechanisms that dictate the biological outcome to STAT3 activation.
In T47D cells, basal MAPK and PI3K signaling is due mainly to an EGF receptor autocrine loop, which is very likely kept active by one or more of the EGF-related peptides expressed in the cells. Whereas Shc is not activated on IL-6 treatment, low levels of Shc are associated with the constitutively active EGF receptor and are tyrosine phosphorylated and associated with Grb2 in the absence of exogenous stimuli. Furthermore, on treatment with the EGF receptor blocking antibody 528, we observed less Shc complexed to the EGF receptor and decreased Shc tyrosine phosphorylation and association to Grb2, which was concomitant with the decrease in MAPK activation. Thus, our results indicate that Shc mediates basal activation of MAPK.
In addition to Shc, SHP-2 and Gab1 are also present in the
autocrine-activated EGF receptor complex. However, in the absence of
IL-6, tyrosine phosphorylation of SHP-2 and Gab1 was not observed. This
indicates that the relatively low levels of EGF receptor activity in
T47D cells are sufficient for recruitment but not for phosphorylation
of the two molecules. On IL-6 stimulation, SHP-2 and Gab1 show
significantly increased levels of phosphorylation and increased
association with gp130, but not with the EGF receptor. Moreover, SHP-2
and Gab1 have been reported to be direct substrates for the Jaks
(33
, 41) , and expression of DN Jak1 blocked IL-6 induced
MAPK activation. Thus, it is likely that SHP-2 and Gab1 are
phosphorylated by Jak1. Whereas the EGF receptor might not be directly
responsible for their phosphorylation, it is intriguing that inhibition
of EGF receptor activity results in a diminished association of SHP-2
and Gab1 with the EGF receptor and in decreased IL-6-induced SHP-2 and
Gab1 tyrosine phosphorylation. We have also observed that gp130 is part
of the EGF receptor complex (data not shown). Taken together, we
envision that the EGF receptor, by recruiting SHP-2 and Gab1 to a
complex that includes gp130 and its associated Jak1, increases the
amount of SHP-2 and Gab1 locally available for phosphorylation by Jak1.
Tyrosine-phosphorylated Gab1 can then serve as a docking molecule for
PI3K (through p85), Grb2, and even SHP-2. SHP-2 associated with gp130
or Gab1 can interact via its phosphotyrosine with Grb2, leading to
activation of the Ras/MAPK pathway. In summary, we postulate that the
EGF receptor provides a scaffold for signaling molecules, making them
locally available for activation by IL-6. This, in turn, leads to
increased activation of downstream signaling pathways (Fig. 8)
.
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Other ErbB family members were previously shown to mediate the effects of IL-6 on the MAPK pathway. In LNCaP prostate carcinoma cells, IL-6-induced MAPK activation was dependent on transactivation of ErbB-2/ErbB-3 (8) . In T47D, MCF7, and SKBr3 breast carcinoma cells, IL-6 does not lead to activation of ErbB-2/ErbB-3, and the suppression of ErbB-2 activity does not affect IL-6-induced MAPK activation. However, in our hands, IL-6 also failed to stimulate ErbB-2 activity in prostate carcinoma cell lines,5 suggesting that further experimentation is required to clarify the role of ErbB-2 in IL-6 signaling.
IL-6 was previously shown to decrease cell-cell association and to stimulate scattering of breast carcinoma cells (10) . We show here that IL-6 and OSM act as chemoattractants for T47D breast carcinoma cells and that IL-6/OSM-induced migration is dependent on MAPK and PI3K activity. Whereas IL-6 itself induces a moderate increase in T47D cell basal migration, it also further stimulates T47D cell migration triggered by heregulin ß, a potent activator of breast cancer cell migration (43) , showing that IL-6 could be an important regulator of breast cancer cell progression toward an invasive phenotype.5
The fact that the modest activity of the EGF receptor in T47D cells is critical for IL-6-induced MAPK signaling and migration strengthens the emerging concept of cooperation between intracellular signaling pathways that individually might have minimal biological effects but in combination result in superadditive cellular responses. For instance, it was recently shown in LNCaP cells that paired combinations of EGF, IL-6, and agonists that elevate intracellular cyclic AMP result in potentiated elevation of MAPK activity (44) . IL-6 was also shown to synergize with low concentrations of nerve growth factor and to induce MAPK phosphorylation and neurite outgrowth in PC12 cells (45) .
The role of IL-6 in cancer progression is dependent on the balance of multiple pathways triggered simultaneously by the cytokine. However, concomitant stimulation of the cells by other endogenous or exogenous factors, even at low concentrations, may tip the balance toward one biological response, e.g., proliferation and antiapoptosis, or another, e.g., growth arrest and differentiation. Thus, a better understanding of IL-6 function in tumor cells will require examination of IL-6 cross-talk with other inhibitory or, as illustrated in this study, synergistic signaling pathways.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Novartis Research Foundation.
A. B. was supported in part by a grant from the Gertrud
Hagmann-Stiftung für Malignomforschung, Swiss Cancer League. 
2 To whom requests for reprints should be
addressed, at Friedrich Miescher Institute, P.O. Box 2543, CH-4002
Basel, Switzerland. Phone: 41-61-697-2069; Fax: 41-61-697-8102; E-mail: badache{at}fmi.ch
3 The abbreviations used are: IL, interleukin;
EGF, epidermal growth factor; GH, growth hormone; GPCR, G
protein-coupled receptor; LIF, leukemia inhibitory factor; mAb,
monoclonal antibody; MAPK, mitogen-activated protein kinase; OSM,
oncostatin M; PI3K, phosphatidylinositol 3'-kinase; PKB, protein kinase
B; Jak, Janus-activated kinase; STAT, signal transducers and activators
of transcription; HA, hemagglutinin; DN, dominant negative.
4 D. Salomon, personal communication.
Received 4/13/00. Accepted 11/ 1/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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N. Underhill-Day and J. K. Heath Oncostatin M (OSM) Cytostasis of Breast Tumor Cells: Characterization of an OSM Receptor {beta}-Specific Kernel. Cancer Res., November 15, 2006; 66(22): 10891 - 10901. [Abstract] [Full Text] [PDF]
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 C. J.F. de Wolf, R. M.J. Cupers, R. M. Bertina, and H. L. Vos Interleukin-6 Induction of Protein S Is Regulated Through Signal Transducer and Activator of Transcription 3 Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2168 - 2174. [Abstract] [Full Text] [PDF]
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 H. Takaki, Y. Minoda, K. Koga, G. Takaesu, A. Yoshimura, and T. Kobayashi TGF-beta1 suppresses IFN-gamma-induced NO production in macrophages by suppressing STAT1 activation and accelerating iNOS protein degradation. Genes Cells, August 1, 2006; 11(8): 871 - 882. [Abstract] [Full Text] [PDF]
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 H.-J. Chae, M.-S. Ha, D.-H. Yun, H.-O. Pae, H.-T. Chung, S.-W. Chae, Y.-K. Jung, and H.-R. Kim Mechanism of Cyclosporine-induced Overgrowth in Gingiva Journal of Dental Research, June 1, 2006; 85(6): 515 - 519. [Abstract] [Full Text] [PDF]
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 S. Ueno, S. Saito, T. Wada, K. Yamaguchi, M. Satoh, Y. Arai, and T. Miyagi Plasma Membrane-associated Sialidase Is Up-regulated in Renal Cell Carcinoma and Promotes Interleukin-6-induced Apoptosis Suppression and Cell Motility J. Biol. Chem., March 24, 2006; 281(12): 7756 - 7764. [Abstract] [Full Text] [PDF]
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 P. Igaz, R. Salvi, J.-P. Rey, M. Glauser, F. P. Pralong, and R. C. Gaillard Effects of Cytokines on Gonadotropin-Releasing Hormone (GnRH) Gene Expression in Primary Hypothalamic Neurons and in GnRH Neurons Immortalized Conditionally Endocrinology, February 1, 2006; 147(2): 1037 - 1043. [Abstract] [Full Text] [PDF]
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M. M. Queen, R. E. Ryan, R. G. Holzer, C. R. Keller-Peck, and C. L. Jorcyk Breast Cancer Cells Stimulate Neutrophils to Produce Oncostatin M: Potential Implications for Tumor Progression Cancer Res., October 1, 2005; 65(19): 8896 - 8904. [Abstract] [Full Text] [PDF]
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 N. E. Avissar, L. Toia, and H. C. Sax Epidermal Growth Factor and/or Growth Hormone Induce Differential, Side-Specific Signal Transduction Protein Phosphorylation in Enterocytes JPEN J Parenter Enteral Nutr, September 1, 2005; 29(5): 322 - 336. [Abstract] [Full Text] [PDF]
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 L. A. Hefler, C. Grimm, T. Lantzsch, D. Lampe, S. Leodolter, H. Koelbl, G. Heinze, A. Reinthaller, D. Tong-Cacsire, C. Tempfer, et al. Interleukin-1 and Interleukin-6 Gene Polymorphisms and the Risk of Breast Cancer in Caucasian Women Clin. Cancer Res., August 15, 2005; 11(16): 5718 - 5721. [Abstract] [Full Text] [PDF]
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 C. Weidt, B. Niggemann, W. Hatzmann, K. S. Zanker, and T. Dittmar Differential Effects of Culture Conditions on the Migration Pattern of Stromal Cell-Derived Factor-Stimulated Hematopoietic Stem Cells Stem Cells, November 1, 2004; 22(6): 890 - 896. [Abstract] [Full Text] [PDF]
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K. S. Selander, L. Li, L. Watson, M. Merrell, H. Dahmen, P. C. Heinrich, G. Muller-Newen, and K. W. Harris Inhibition of gp130 Signaling in Breast Cancer Blocks Constitutive Activation of Stat3 and Inhibits in Vivo Malignancy Cancer Res., October 1, 2004; 64(19): 6924 - 6933. [Abstract] [Full Text] [PDF]
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N. Schick, E. J. Oakeley, N. E. Hynes, and A. Badache TEL/ETV6 Is a Signal Transducer and Activator of Transcription 3 (Stat3)-induced Repressor of Stat3 Activity J. Biol. Chem., September 10, 2004; 279(37): 38787 - 38796. [Abstract] [Full Text] [PDF]
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 K. Mahtouk, M. Jourdan, J. De Vos, C. Hertogh, G. Fiol, E. Jourdan, J.-F. Rossi, and B. Klein An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti-IL-6 antibody-induced apoptosis Blood, March 1, 2004; 103(5): 1829 - 1837. [Abstract] [Full Text] [PDF]
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 V. Bliskovsky, E. S. Ramsay, J. Scott, W. DuBois, W. Shi, S. Zhang, X. Qian, D. R. Lowy, and B. A. Mock Frap, FKBP12 rapamycin-associated protein, is a candidate gene for the plasmacytoma resistance locus Pctr2 and can act as a tumor suppressor gene PNAS, December 9, 2003; 100(25): 14982 - 14987. [Abstract] [Full Text] [PDF]
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A. DeMichele, A.-M. Martin, R. Mick, P. Gor, L. Wray, M. Klein-Cabral, G. Athanasiadis, T. Colligan, E. Stadtmauer, and B. Weber Interleukin-6 -174G->C Polymorphism Is Associated with Improved Outcome in High-Risk Breast Cancer Cancer Res., November 15, 2003; 63(22): 8051 - 8056. [Abstract] [Full Text] [PDF]
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M. Trikha, R. Corringham, B. Klein, and J.-F. Rossi Targeted Anti-Interleukin-6 Monoclonal Antibody Therapy for Cancer: A Review of the Rationale and Clinical Evidence Clin. Cancer Res., October 15, 2003; 9(13): 4653 - 4665. [Abstract] [Full Text] [PDF]
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A. V. Lee, R. Schiff, X. Cui, D. Sachdev, D. Yee, A. P. Gilmore, C. H. Streuli, S. Oesterreich, and D. L. Hadsell New Mechanisms of Signal Transduction Inhibitor Action: Receptor Tyrosine Kinase Down-Regulation and Blockade of Signal Transactivation Clin. Cancer Res., January 1, 2003; 9(1): 516S - 523S. [Abstract] [Full Text] [PDF]
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 L. Zhao, J. J. Melenhorst, and L. Hennighausen Loss of Interleukin 6 Results in Delayed Mammary Gland Involution: A Possible Role for Mitogen-Activated Protein Kinase and Not Signal Transducer and Activator of Transcription 3 Mol. Endocrinol., December 1, 2002; 16(12): 2902 - 2912. [Abstract] [Full Text] [PDF]
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A. B. Motoyama, N. E. Hynes, and H. A. Lane The Efficacy of ErbB Receptor-targeted Anticancer Therapeutics Is Influenced by the Availability of Epidermal Growth Factor-related Peptides Cancer Res., June 1, 2002; 62(11): 3151 - 3158. [Abstract] [Full Text] [PDF]
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 J. A. Hutt and J. W. DeWille Oncostatin M Induces Growth Arrest of Mammary Epithelium via a CCAAT/enhancer-binding Protein {delta}-dependent Pathway Mol. Cancer Ther., June 1, 2002; 1(8): 601 - 610. [Abstract] [Full Text] [PDF]
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M. Alberich Jorda, S. E. Verbakel, P. J. M. Valk, Y. V. Vankan-Berkhoudt, M. Maccarrone, A. Finazzi-Agro, B. Lowenberg, and R. Delwel Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol Blood, April 15, 2002; 99(8): 2786 - 2793. [Abstract] [Full Text] [PDF]
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K. Horsch, M. D. Schaller, and N. E. Hynes The Protein Tyrosine Phosphatase-PEST Is Implicated in the Negative Regulation of Epidermal Growth Factor on PRL Signaling in Mammary Epithelial Cells Mol. Endocrinol., December 1, 2001; 15(12): 2182 - 2196. [Abstract] [Full Text] [PDF]
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