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Tumor Biology |
Departments of Internal Medicine I [G. v. W., S. K., B. O. B., G. A., T. S.] and Internal Medicine II [P. M. J.], University of Ulm, D-89081 Ulm, Germany; Department of Veterinary Medicine, Genzentrum, D-80539 Munich, Germany [A. H., E. W.]; Department of Internal Medicine/Division of Hepatology and Gastroenterology, Charite, Humboldt University of Berlin, D-13086 Berlin, Germany [B. W.]; and Department of Surgery, Martin Luther University, D-06099 Halle, Germany [H. D.]
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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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Neuropeptides (3) and polypeptide growth factors, such as nerve growth factor and fibroblast growth factor (4 , 5) , have been implicated in the regulation of neuroendocrine tumor cell growth. In addition, the presence of IGF-I3 and the IGF-I receptor has been reported in neuroendocrine tumors, such as midgut carcinoids (6 , 7) . However, the signaling pathways induced by IGF-I and its precise role for secretion and/or growth in human neuroendocrine tumors are largely unknown. IGF-I is a 70-amino acid peptide closely related to insulin that binds to distinct high affinity receptors with intrinsic tyrosine kinase activity. Upon binding to the IGF-I receptor, IGF-I stimulates cell cycle progression and growth in various cell lines (8, 9, 10) . In addition, IGF-I and the IGF-I receptor have been implicated in multistage carcinogenesis: overexpression of the IGF-I receptor or its ligand IGF-I causes abnormal growth, cellular transformation, inhibition of apoptosis, and spontaneous tumor formation in transgenic mice (11 , 12) . Furthermore, expression of the IGF-I receptor is crucial for tumorigenesis in athymic mice (10 , 13) .
Here, we report that human BON carcinoid cells express functionally active IGF-I receptors and secrete IGF-I. Exogenously added IGF-I stimulated PI3-kinase, p70s6k, and ERK activity in BON cells. The endogenously released IGF-I was found to be largely responsible for the high basal activity of p70s6k and ERK2 in serum-starved BON cells. Exogenously added IGF-I markedly increased chromogranin A secretion by BON cells by a PI3-kinase-dependent pathway. This kinase also mediated autocrine IGF-I secretion. Immunoneutralization of endogenously released IGF-I substantially reduced basal chromogranin A release. These data demonstrate, for the first time, the existence of an autocrine IGF-I loop regulating neuroendocrine secretion in a carcinoid tumor cell line. In addition, both exogenously added and endogenously released IGF-I stimulated growth of BON cells by a PI3-kinase, p70s6k, and MEK-1-dependent signaling pathway. At the level of the cell cycle, endogenously released IGF-I was found to selectively regulate the expression of cyclin D1 and p27kip1. Thus, neuroendocrine secretion and autonomous growth of human BON tumor cells are largely regulated by endogenously released IGF-I. Because the presence of IGF-I and the IGF-I receptor can be demonstrated in various neuroendocrine tumors, the IGF-I signaling pathway is a potential novel target for the treatment of hypersecretion syndromes and growth of these tumors.
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MATERIALS AND METHODS |
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125I-IGF-I Binding Assays.
IGF-I equilibrium-competition-inhibition binding studies were performed
as described previously (14)
. Briefly, BON cells
(1–2 x 106/dish) were washed
twice with PBS and once with binding buffer and then incubated for
1 h at room temperature with 125I-IGF-I (10
pM), and different concentrations of unlabeled IGF-I,
r3-IGF-I, and insulin (each at 10 pM to 0.1
µM). To assess whether binding of IGF-I changes during
cell proliferation, binding assays with
125I-IGF-I or 125I-rIGF-I
were performed in BON cells cultured for 24, 36, 48, and 72 h. At
the end of each experiment, cells were washed three times with PBS
containing 0.1% BSA. In all experiments, degradation of tracer was
less than 10%, thus excluding differences in binding caused by hormone
degradation. Cell bound and free intact activity were counted in an
automatic gamma counter (Berthold, Munich, Germany) with 70%
efficiency. Specific binding was determined by subtracting the amount
of 125I-IGF-I or
125I-rIGF-I unspecifically bound (<0.5%) in the
presence of 0.1 µM IGF-I and ranged between 5 and 10% in
all experiments. A computer-assisted curve fitting program was used to
determine the concentration of unlabeled peptide yielding a 50%
inhibition (IC50) of
125I-tracer binding (15)
. As
described by Scatchard (16)
, the ratio of bound to free
hormone was plotted as a function of total hormone, and the number of
binding sites per cell and binding affinity (Kd
values) were calculated.
Western Blotting.
BON cells were washed twice in serum-free DNM and incubated in
fresh serum-free medium for further 24 h. Cells were then treated
with factors as indicated in the legends to Figs. 3
4
5
6
and
lysed in 50 mM Tris-HCl, 5 mM EDTA, 100
mM NaCl, 40 mM ß-glycerophosphate, 50
mM NaF, 1 mM
Na3VO4, 1% Triton X-100, 1
mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10
µg/ml leupeptin, pH 7.6 (lysis buffer). For detection of the IGF-I
receptor in HEK 293, BON, COS, MiaPaCa-2, and LCC18 cells,
serum-starved cells were lysed in lysis buffer, and protein content was
determined. Proteins were subsequently extracted in 5x SDS-PAGE sample
buffer. Equal amounts of proteins were further analyzed by SDS-PAGE and
Western blotting with a polyclonal anti-IGF-I receptor antibody,
followed by ECL detection. For p70s6k mobility
shift assays and detection of cyclin D1, cyclin E, and
p27kip1, cells were treated as indicated in the
legends to Figs. 3
4
5
6
and lysed in SDS-PAGE sample buffer, and
samples were further analyzed by SDS-PAGE and Western blotting using
specific antisera to these proteins.
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-32P]ATP, and 20 µg of
phosphatidylinositol in 5 mM HEPES, pH 7.4, per condition
for 25 min at 25°C. Reactions were stopped by adding 100 µl of 1
M HCl and 200 µl of a mixture of
CHCl3 and methanol (1:1, v/v). Samples were mixed
by vortexing and subsequently briefly centrifuged. The
CHCl3 phase was further purified by mixing with
HCl/methanol (1:1, v/v). Finally the CHCl3 phase
was transferred into a new tube, dried under N2,
and subsequently run on TLC plates in a mixture containing
H2O, CHCl3, methanol,
acetone, and glacial acetic acid (16:30:26:30:24, v/v). TLC plates were
then developed by autoradiography. Autoradiographs were scanned using
the UMAX Vistascan program (version 3.1).
p70s6k and ERK2 Immune Complex Kinase Assays.
Serum-starved BON cells were incubated with IGF-I in the presence or
absence of rapamycin or PD 098059 as indicated in the legend to Fig. 3
.
Controls received an equivalent amount of solvent. Cells were then
lysed at 4°C in 1 ml of lysis buffer. Immunoprecipitations were
performed at 4°C using an anti-p70s6K antibody
or an anti-ERK2 antibody for 2 h, with protein A-agarose added for
the second hour. Immune complexes were washed three times in lysis
buffer and once with p70s6k kinase buffer (20
mM HEPES, pH 7.4, 10 mM
MgCl2, 1 mM DTT, and 10
mM ß-glycerophosphate) or ERK kinase buffer (15
mM MgCl2, 15 mM Tris-HCl,
pH 7.4). Kinase reactions were performed by resuspending the
protein A-Sepharose pellets in 25 µl of kinase assay mixture
containing the appropriate kinase buffer with 0.2 mM S6
peptide (RRRLSSLRA) or myelin basic protein, 20 µM ATP, 5
µCi/ml [-32P]ATP, 2 µM
cAMP-dependent protein kinase inhibitor peptide, and 100 nM
microcystin LR. Incubations were performed under linear assay
conditions at 30°C for 20 min and terminated by spotting 25 µl of
the supernatant onto Whatman p81 chromatography paper. Papers were
washed four times for 5 min in 0.5% o-phosphoric acid,
immersed in acetone, and dried before scintillation counting. The
average radioactivity of two blank samples containing no immune complex
was subtracted from the result of each sample.
Measurement of Chromogranin A.
To determine chromogranin A secretion, BON cells were incubated in
Krebs-Ringer-HEPES buffer for 2 h at 37°C. Cells were
subsequently treated with IGF-I for 25 min at 37°C in the absence or
presence of various inhibitors as indicated in the legend to Fig. 4
.
The supernatant was then aspirated and stored at –80°C until
assayed. Chromogranin A was subsequently determined in the supernatants
using a specific chromogranin A ELISA. Release is expressed as fold
stimulation above untreated controls.
Determination of IGF-I Secretion.
To characterize the autocrine release of IGF-I, cells were washed three
times in serum-free medium and incubated in serum-free medium for up to
7 days in the presence or absence of various compounds as indicated.
Medium was aspirated on specific days as indicated in the legend to
Figs. 2
3
4
and stored at –80°C until assayed. IGF-I concentration
was determined using a specific RIA. Values are expressed as ng of
IGF-I/ml of supernatant.
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Immunostaining of Cells and Cryostat Sections.
Serum-starved cultures of BON cells were washed with PBS and fixed in
4% formaldehyde at room temperature for 20 min. Cells were then
permeabilized with 0.2% Triton X-100 and stained with specific
anti-IGF-receptor or anti-IGF-I antibodies for 1 h followed by
detection with an Alexa-labeled secondary antibody. Samples were
further analyzed by confocal microscopy. Cryostat sections of
neuroendocrine tumor specimens obtained endoscopically or surgically
were fixed in 4% ethanol-free formaldehyde at room temperature for 30
min. Tissue sections were then permeabilized with 0.1% Triton X-100
for 10 min and preblocked with 2% BSA (w/v) for 2 h at room
temperature. Sections were subsequently incubated overnight in
2% BSA (w/v) with a monoclonal anti-IGF-I antibody or a polyclonal
anti-IGF-I receptor antibody. Tissues were then incubated with
Alexa-labeled antirabbit or antimouse antibodies and further
analyzed by immunofluorescence microscopy.
Growth Assay.
BON cells, 3 days postpassage, were washed in serum-free DNM,
trypsinized, and resuspended in serum-free DNM. Cells were plated at a
density of 1 x 104 cells in 1 ml
of serum-free DNM in the presence or absence of 20 ng/ml rapamycin in
duplicate. At the times indicated in the figure legends, cell number
was determined using a cell counting chamber.
Clonogenic Assay.
BON cells were washed, trypsinized, and resuspended in DNM. Cell number
was determined using a cell counting chamber. Cells (3 x 104) were mixed with serum-free DNM
containing 0.3% agarose in the presence or absence of rapamycin at the
concentrations indicated and layered over a solid base of 0.5% agarose
in serum-free DNM in the presence or absence of rapamycin at the same
concentrations in 33-mm dishes. The cultures were incubated in
humidified 5% CO2:95% air at 37°C for 14 days
and then stained with the vital stain nitroblue tetrazolium. Colonies
of >120 µm in diameter (20 cells) were counted using a microscope.
Statistical Analysis.
Cerenkov counts of the ERK and p70s6k immune
complex kinase assays (n = 6 for each
condition) were tested for normal distribution and extreme values. The
levels of significance were determined by Pearson correlation.
Materials.
The monoclonal antibody against Tyr(P) (clone 4G10), the monoclonal
anti-IGF-I antibody used for the immunoneutralization experiments, and
the polyclonal antibodies against IRS-2, the p85 subunit of PI3-kinase,
and the N-terminally directed anti-p70s6k
polyclonal antibody used to determine p70s6k
activity were obtained from Upstate Biotechnology, Inc. (Lake Placid,
NY). The polyclonal antibodies directed against the IGF-I receptor,
IRS-1, p70s6k, p27kip1,
cyclin D1, and cyclin E were from Santa Cruz Biotechnology (Santa Cruz,
CA). Peroxidase-coupled antirabbit IgG antibody was from Dianova
(Hamburg, Germany). The polyclonal anti-ERK2 antibody was the kind gift
of Dr. Jo van Lint (Katholieke Universiteit Leuven, Leuven, Belgium).
Rapamycin, LY 294002, and GF 109203X were from Calbiochem-Novabiochem
(Schwalbach, Germany). PD 098059 was from New England Biolabs
(Schwalbach, Germany). Protein A-Sepharose was obtained from Roche
Molecular Biochemicals (Mannheim, Germany). Human IGF-I, ECL
reagent, and [-32P]ATP were obtained from
Amersham Pharmacia Biotech (Freiburg, Germany). The Alexa
red-labeled and Alexa green-labeled antimouse and antirabbit IgG
antibodies were from Molecular Probes (Leiden, the Netherlands). The
ELISA kit to determine chromogranin A in the supernatants was from DAKO
Diagnostica GmbH (Hamburg, Germany). The RIA to determine IGF-I in the
supernatants was from Nichols Institute Diagnostics (San Juan,
CA). Nitrocellulose membranes and polyvinylidene difluoride
membranes for Western blotting were obtained from Millipore (Eschborn,
Germany). All other reagents were of the purest grade available.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, top panel). IGF-I receptor immunoreactivity was mainly
detected at the plasma membrane and around the nucleus (Fig. 1
A,
bottom panels).
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B, top panel), IGF-I competed for
125I-IGF-I binding well within the nanomolar
range (IC50, 0.72 ± 0.1
nM), whereas insulin showed a 100-fold lower
affinity (IC50, 71 ± 3
nM). Scatchard analysis (Fig. 1
B, bottom
panel) revealed a single class of IGF-I receptors (72700 ± 2000 sites/cell, Kd = 0.77 ± 0.1). As shown in Fig. 1
B, bottom
panel, the number of IGF-I receptors markedly increased during BON
cell proliferation, reaching a maximum after 48 h.
Interestingly, r3-IGF-I, which does not cross-bind with IGFBPs, showed
a slightly higher affinity (IC50, 0.4 ± 0.1 nM) than IGF-I (Fig. 1
B, top panel),
suggesting the existence of IGFBPs in the supernatant of BON cells. As
demonstrated by ligand blotting, only a single class of IGFBPs could be
detected in BON cell supernatants (Fig. 1
C, top panel). This
band was identified as IGFBP-2 by Western blotting (Fig. 1
C,
bottom panel). The amount of IGFBP-2 increased in the supernatant
of serum-starved cells with time, reaching a maximum 5 days after
plating of the cells.
To examine whether the increase in 125I-IGF-I binding with time could be attributable to cross-binding of 125I-IGF-I to IGFBPs, we performed binding assays using 125I-r3-IGF-I, which acts as a ligand for the IGF-I receptor but not for IGFBPs. These experiments revealed binding data very similar to those obtained with 125IGF-I: using 125I-r3-IGF-I, a 1.7-fold increase in IGF-I receptor number/cell was detected after 48 h of incubation (data not shown). Thus, the increase in 125I-IGF-I binding was not caused by increased binding to IGFBPs but was indeed the result of an increase in receptor numbers.
BON Cells Secrete IGF-I.
It has been speculated that IGF-I could act on neuroendocrine tumors by
an autocrine mechanism (6)
. Indeed, IGF-I could be
demonstrated in the cytoplasm of human BON cells by immunocytochemistry
using a specific anti-IGF-I antibody (Fig. 2
, left and middle panels). To examine
whether IGF-I was also secreted by BON cells, we determined the amount
of IGF-I in the supernatants of serum-starved BON cells at various
times. As shown in Fig. 2
, right panel, IGF-I concentrations
in the supernatant of serum-starved BON cells increased in a
time-dependent manner: a maximum concentration of 15 ng IGF-I/ml of
conditioned medium was obtained 7 days after plating of the cells.
These data suggest the existence of an autocrine loop involving IGF-I
in human BON cells. To substantiate these findings, we examined a panel
of various human neuroendocrine tumors by immunohistochemistry using
specific antibodies against IGF-I and the IGF-I receptor. As shown in
Table 1
, all but one neuroendocrine tumor exhibited positive staining for the
IGF-I receptor. IGF-I immunoreactivity was also detected in a large
proportion of tumor specimens either in the tumor itself and/or in the
surrounding connective tissue, suggesting an autocrine and/or paracrine
mechanism of action.
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A, left panel). Similar results were obtained with a
different PI3-kinase inhibitor, wortmannin (data not shown). The effect
of LY 294002 on PI3-kinase activation was selective, as treatment of
cells with the MEK-1 inhibitor PD 098059 did not block IGF-I-induced
PI3-kinase activation (data not shown). PI3-kinase is composed of two
subunits, a Mr 110,000 catalytic
subunit and a Mr 85,000 regulatory
subunit, which possesses two Src-homology 2 domains. Both of
these Src-homology 2 domains specifically associate with
tyrosine-phosphorylated IRS-1 and IRS-2 (19)
. To examine
which IRS would mediate PI3-kinase activation in response to IGF-I, we
determined the IRS-1- and IRS-2-associated PI3-kinase activity in BON
cells stimulated with IGF-I. Cells were treated with 100 ng/ml IGF-I
for 10 min and lysed. The lysates were immunoprecipitated with specific
anti-IRS-1 or anti-IRS-2 antibodies and further analyzed by PI3-kinase
assays. A 2- and 1.5-fold increase in PI3-kinase activity could be
detected in IRS-1 and IRS-2 immunoprecipitates, respectively (Fig. 3
A, middle and right panels). Thus, both adapter
proteins can directly mediate IGF-I-stimulated PI3-kinase
activation in human BON cells.
PI3-kinase regulates several downstream targets, one of which is
p70s6k (20)
. To determine whether
IGF-I-induced PI3-kinase activation was sufficient to trigger the
activation of downstream targets, we examined
p70s6k activation in response to IGF-I. As shown
in Fig. 3
B, stimulation of cells with IGF-I induced a
moderate but significant increase in p70s6k
activity in immune complex kinase assays (P < 0.02). IGF-I-stimulated p70s6k activity
could be prevented by treatment of cells with the PI3-kinase inhibitor
LY 294002, as well as the specific inhibitor of
p70s6k activation, rapamycin (21)
.
Rapamycin and LY 294002 also strikingly inhibited basal
p70s6k activity in BON cells, suggesting that
this kinase was constitutively active in BON cells. In contrast, the
MEK-1 inhibitor PD 098059 had no effect on basal and IGF-I-induced
p70s6k activity (Fig. 3
B, inset).
IGF-I also stimulated activation of ERK2 in human BON cells: as
shown in Fig. 3
C, 100 ng/ml IGF-I induced a moderate but
significant increase in ERK2 activity (P < 0.01). Similar results were obtained in ERK1 immune complex kinase
assays (data not shown). IGF-I-induced ERK2 activation could be
prevented by treatment of cells with the selective MEK-1 inhibitor PD
098059 (Ref. 22
; Fig. 3C
). PD 098059 also
reduced basal ERK2 activation by 40%, suggesting that ERK2 is also
constitutively active in these cells. Because IGF-I could activate
PI3-kinase in BON cells, we examined whether this kinase could be an
upstream regulator of ERK activation in BON cells. Treatment of cells
with the selective PI3-kinase inhibitor LY 294002 reduced basal
and IGF-I-stimulated ERK2 activation. Thus, IGF-I-stimulated ERK
activation requires the activation of MEK-1 and PI3-kinase (Fig. 3C
).
Constitutive Activation of ERK2 and p70s6k in
Human BON Cells Is Caused by Autocrine IGF-I Secretion.
As shown in Fig. 3
, A and B, both
p70s6k and ERK2 can be further activated by
exogenously added IGF-I but exhibit a high basal activity in
serum-starved BON cells (Fig. 3 and C
).
Therefore, we examined whether basal activation of these kinases in BON
cells could be attributable to stimulation by the endogenously released
IGF-I. Serum-starved BON cells were incubated with the
immunoneutralizing antibody directed against IGF-I, and subsequently,
p70s6k and ERK2 activities were determined.
Activation of p70s6k by mitogens can be
determined by the appearance of slower migrating forms in SDS-PAGE
attributable to phosphorylation of p70s6k
on Thr229, Thr389, and
Ser404, which are not basally phosphorylated in
quiescent cells (23)
. As shown in Fig. 3
D, left
panel, incubation of cells with the IGF-I-blocking antibody
reduced basal phosphorylation of p70s6k in BON
cells as demonstrated by the increased electrophoretic mobility of the
protein in SDS-PAGE. Incubation with the IGF-I-blocking antibody was
almost as efficient in inducing dephosphorylation of
p70s6k as treatment with the selective inhibitor
of p70s6k activation, rapamycin. Upon treatment
of cells with the IGF-I-blocking antibody basal kinase activity
of ERK2 was also decreased by 50% in immune complex kinase assays.
Indeed, inhibition of basal ERK2 activation by the IGF-I-blocking
antibody was comparable to the maximal inhibitory effect of the MEK-1
inhibitor PD 098059 in BON cells (Fig. 3
D, right panel).
Thus, endogenously released IGF-I is largely responsible for
constitutive activation of both the ERK cascade and
p70s6K in human BON cells.
Exogenously Added and Endogenously Released IGF-I Stimulate
Chromogranin A Release in Human BON Carcinoid Tumor Cells.
Carcinoid tumors are characterized by hypersecretion of various
neuroendocrine marker proteins such as chromogranin A
(24)
. There are few reports demonstrating that members of
the IGF family can regulate exocytosis in cultured cells (25
, 26)
. Having established a functionally active IGF-I signaling
pathway in BON cells, we examined whether IGF-I could stimulate
neuroendocrine secretion in these cells as judged by the secretion of
chromogranin A into the supernatant of cells. As expected, chromogranin
A could be detected in the cytoplasm of BON tumor cells by
immunocytochemistry (data not shown). Incubation of BON cells with 100
ng/ml IGF-I for 25 min led to a 5-fold increase in chromogranin A
release into the supernatant. Chromogranin A release in response to
IGF-I reached about 60% of the maximal secretory response induced by
PDB, a potent secretagogue for BON cells (Ref. 27
; Fig. 4
A, left panel).
If IGF-I plays a major role in the regulation of neuroendocrine
secretion in BON cells, endogenously released IGF-I should contribute
to basal chromogranin A secretion. Therefore, IGF-I in the supernatants
of cells was immunoneutralized by adding an IGF-I-blocking antibody. As
shown in Fig. 4
A, right panel, this treatment led to a
marked, 45% inhibition of basal chromogranin A secretion. Thus,
endogenously released IGF-I indeed regulates basal chromogranin A
secretion in human BON neuroendocrine tumor cells.
Chromogranin A Secretion by IGF-I Is Mediated by PI3-kinase.
Next, we examined which of the signaling pathways induced by IGF-I
could mediate chromogranin A secretion in BON cells. Activation of
PI3-kinase has been implicated in the regulation of exocytosis in mast
cells, neutrophils, and chromaffin cells
(28, 29, 30)
. Treatment of cells with the selective PI3-kinase
inhibitor LY 294002 completely blocked chromogranin A secretion in
response to IGF-I (Fig. 4B
). Activation of ERKs has also
been implicated in the regulation of secretion by some receptors
(31)
. Treatment of cells with the selective MEK-1
inhibitor PD 098059 reduced IGF-I-induced chromogranin A release by
about 30% (Fig. 4B
). Inhibition of
p70s6k activation using rapamycin reduced
chromogranin A secretion in response to IGF-I also by only 35%. IGF-I
has been demonstrated to activate phorbol ester-sensitive members of
the PKC family of serine threonine kinases (32)
. However,
GF 109203X treatment reduced chromogranin A secretion in response to
IGF-I only by about 35% (Fig. 4B
). In contrast,
chromogranin A release by PDB was completely abolished when cells were
treated with the selective PKC inhibitor GF 109203X but was only
reduced by about 20% in the presence of the PI3-kinase inhibitor LY
294002 (Fig. 4C
). These data demonstrate that IGF-I and
phorbol esters induce chromogranin A secretion by distinct signaling
pathways in BON cells.
Autocrine IGF-I Secretion Is Regulated by PI3-kinase.
To examine whether PI3-kinase could also mediate IGF-I secretion,
serum-starved BON cells were incubated in the presence or absence of LY
294002 for 80 h, and IGF-I concentrations in the supernatants were
determined. As shown in Fig. 4
D, endogenous IGF-I secretion
was completely blocked in the presence of the PI3-kinase inhibitor LY
294002. Thus, PI3-kinase regulates both autocrine IGF-I secretion and
neuroendocrine chromogranin A release in BON cells.
IGF-I Stimulates Anchorage-dependent and Anchorage-independent
Proliferation of Human BON Carcinoid Tumor Cells.
IGF-I and its receptor play an important role for the growth of various
cells, including tumor cells (10
, 33)
. However, its role
for anchorage-dependent and anchorage-independent growth of
neuroendocrine tumor cells is unknown. To examine whether IGF-I could
induce proliferation of BON cells, serum-starved cells were incubated
with 100 ng/ml IGF-I for various times, and cell numbers were
determined at the days indicated. IGF-I stimulated proliferation of BON
cells in a time-dependent manner: a maximum, 1.6-fold stimulation of
cell proliferation was obtained after 7 days of incubation with IGF-I
(Fig. 5
, A and B, top panels). The effect of IGF-I on
cell proliferation was concentration-dependent: half-maximum and
maximum effects were achieved at 50 and 100 ng/ml IGF-I (data not
shown).
Role of PI3-kinase, ERK, and p70s6k Activity for
Autonomous and IGF-I-stimulated Anchorage-dependent Growth of BON
Carcinoid Tumor Cells.
Next, we examined the role of PI3-kinase, p70s6k,
and ERK activation for basal and IGF-I-induced anchorage-dependent
growth of serum-starved human BON cells. Incubation of cells with
rapamycin or PD 098059 markedly inhibited basal (Fig. 5A
)
and IGF-I-stimulated (Fig. 5B
) anchorage-dependent
proliferation of BON cells, by about 70 and 65%, respectively.
Incubation of cells with the PI3-kinase inhibitor LY 294002 led to an
even more pronounced inhibition of basal and IGF-I-stimulated
proliferation of serum-starved BON cells; they were inhibited by 80 and
75%, respectively (Fig. 5 and B
). Thus,
activation of PI3-kinase, p70s6k, and the ERK
cascade are important mediators of autonomous and IGF-I-stimulated
anchorage-dependent growth of BON cells.
IGF-I Stimulates Anchorage-independent Growth of Human BON Cells.
Tumors and transformed cells are able to grow in an
anchorage-independent manner by forming colonies in agarose medium
(34)
. Inhibition of PI3-kinase,
p70s6k, and ERK activation using selective
inhibitors of these kinases resulted in the inhibition of basal colony
formation of BON cells in soft agar by 48, 43, and 25%, respectively
(Fig. 5C
). Exogenously added IGF-I further stimulated
anchorage-independent growth of BON cells in soft agar by 100%. Colony
formation in response to IGF-I was also markedly inhibited by about
70% when cells were treated with LY 294002 or rapamycin. In contrast,
treatment of cells with the MEK-1 inhibitor PD 098059 at concentrations
that substantially inhibited anchorage-dependent growth of BON cells in
response to IGF-I (Fig. 5B
) resulted in an only moderate,
25% reduction in IGF-I-stimulated colony formation (Fig. 5C
). This effect is unlikely to be caused by degradation of
the MEK-1 inhibitor during the course of the experiment, because PD
098059 markedly inhibited transforming growth factor 
-stimulated
colony formation of human pancreatic cancer cells under identical
experimental conditions (35)
. Thus, in contrast to
anchorage-dependent proliferation, autonomous and IGF-I-stimulated
anchorage-independent growth are largely dependent on
PI3-kinase/p70s6k activity and less dependent on
the activation of the ERK cascade.
Endogenously Released IGF-I Regulates Autonomous Growth of BON
Cells.
Our data demonstrate that endogenously released IGF-I regulates
constitutive activation of the ERK cascade and
p70s6K (Fig. 3D
) and that autonomous
proliferation of BON cells requires the activity of both kinases (Fig. 5A
). Thus, the interruption of the autocrine IGF-I loop
should inhibit the autonomous growth of BON cells in serum-free medium.
As shown in Fig. 6
A, treatment of BON cells with the IGF-I-blocking antibody
markedly inhibited proliferation of serum-starved BON cells by about
50%. The inhibitory effect of the antibody treatment was comparable to
the effect of a maximal efficient concentration of rapamycin and was
concentration dependent: a maximum effect was achieved at an antibody
concentration of 25 µg/ml (data not shown). The effect of the
IGF-I-blocking antibody on cell growth was selective. Incubation of
cells with a distinct monoclonal antibody directed against
tyrosine-phosphorylated proteins at the same concentration had no
effect on cell growth (Fig. 6B
). Thus, endogenously released
IGF-I is indeed a major mediator of autonomous proliferation of human
BON neuroendocrine tumor cells.
The Autocrine IGF-I Loop Regulates Constitutive Expression of
Cyclin D1 in Human BON Cells.
Growth factors regulate progression from G1 to S.
This process requires the expression of cyclins D and E, which modulate
the activities of the cyclin-dependent kinases (36)
, and
their respective inhibitors, such as p27kip1.
Cyclins and cyclin-related genes are amplified and/or overexpressed in
a major fraction of human tumors at a relatively early stage in the
carcinogenic process (37
, 38)
. To further elucidate the
role of the autocrine IGF-I loop for BON cell cycle progression, cyclin
D1, cyclin E, and p27kip1 expression was examined
in serum-starved BON cells in the presence or absence of the IGF-I
neutralizing antibody. Cyclin D1, cyclin E, and
p27kip1 were found to be constitutively expressed
in BON cells (Fig. 6B
). Upon incubation of cells with the
IGF-I-blocking antibody, expression of cyclin E was unchanged in
Western blots. In contrast, expression of cyclin D1 was markedly
reduced, and p27kip1 expression slightly
increased with time. Thus, the endogenously released IGF-I regulates
the level of cyclin D1 and p27kip1 expression in
human BON cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exogenously added IGF-I activated distinct signaling cascades in BON cells, such as PI3-kinase, p70s6k, and ERKs. Both IRS-1 and IRS-2 mediated IGF-I-stimulated PI3-kinase activation in these cells, in contrast to breast cancer cells, in which PI3-kinase activation in response to IGF-I is largely mediated by IRS-1 (42) . p70s6k and the ERKs exhibited a high level of basal activity in serum-starved BON cells that could be prevented by treatment of the cells with their respective selective inhibitors. The constitutive activation of certain kinases under serum-free conditions is increasingly appreciated as a mechanism leading to autonomous growth of tumor cells in culture. Therefore, these kinases constitute potential targets for tumor therapy (43, 44, 45, 46) . However, little is known about the underlying mechanisms regulating constitutive kinase activation. Our data demonstrate that treatment of serum-starved BON cells with an antibody that immunoneutralizes IGF-I led to a marked reduction in basal kinase activity of both p70s6k and ERK2 that was comparable to the effect of their respective selective kinase inhibitors. Thus, endogenously released IGF-I is largely responsible for the constitutive activation of p70s6k and the ERK cascade in serum-starved BON cells. Despite the fact that receptors for other growth factors have been reported in BON cells (2) , these data stress the importance of the autocrine IGF-I loop for the basal activity of certain signaling pathways in these human neuroendocrine tumor cells.
Human carcinoid tumor cells are characterized by strong secretory properties that are often debilitating for patients. Exogenously added IGF-I markedly increased secretion of chromogranin A, a marker protein for neuroendocrine secretion, in human BON cells. This is the first report demonstrating that the IGF-I receptor tyrosine kinase stimulates secretion in any tumor cell line. Interestingly, immunoneutralization of endogenously released IGF-I substantially reduced basal chromogranin A release by BON cells. Therefore, endogenously released IGF-I triggers neuroendocrine secretion in these cells. It has been suggested that the PI3-kinase-phosphorylated products of phosphatidylinositol play a role in the regulation of membrane trafficking along secretory pathways e.g., in chromaffin cells (30) . Indeed, IGF-I-induced chromogranin A secretion in BON cells was largely dependent on PI3-kinase activity and less dependent on the activation of the ERK cascade, mammalian target of rapamycin/p70s6k, or PKCs. Endogenous release of IGF-I into the supernatant of BON cells was also dependent on PI3-kinase activity. This suggests the existence of a positive feedback loop in BON cells in which IGF-I stimulates its own as well as neuroendocrine secretion via the activation of PI3-kinase. Therefore, by targeting either PI3-kinase or endogenously released IGF-I, both autocrine and neuroendocrine secretory pathways can be substantially blocked in BON cells.
IGF-I-induced mitogenesis has been linked to the activation of PI3-kinase and was found to be independent of the activation of the ERK cascade in certain tumor cells (47) . Another report states that neither activation of PI3-kinase nor activation of the ERK cascade is sufficient for IGF-I-induced mitogenesis and tumorigenesis (48) . In this study, we demonstrate that IGF-I is a potent growth factor for human BON neuroendocrine tumor cells stimulating anchorage-dependent and anchorage-independent proliferation. Anchorage-dependent proliferation of these cells in response to IGF-I was found to be largely dependent on PI3-kinase activation, but it also required the activation of a major downstream target of PI3-kinase, p70s6k, and the ERK cascade. Similarly, autonomous anchorage-dependent growth of BON cells required PI3-kinase, p70s6k, and ERK activity. In agreement with our results on constitutive kinase activation, autonomous growth of BON cells was inhibited by the IGF-I-blocking antibody to the same degree as by maximal efficient concentrations of the selective kinase inhibitors. Thus, endogenously released IGF-I is indeed the major regulator of autonomous growth of BON cells. Interestingly, IGF-I-induced anchorage-independent growth in soft agar was largely dependent on the PI3-kinase/p70s6k signaling pathway but less dependent on the activation of the ERK cascade. Thus, distinct signaling pathways mediate anchorage-dependent and anchorage-independent proliferation of BON cells in response to IGF-I.
Cyclins and cyclin-related genes are important regulators of cell cycle progression. Under physiological conditions, the expression of cyclins is tightly regulated. However, these proteins are amplified and/or overexpressed in a major fraction of human tumors at a relatively early stage in the carcinogenic process (37 , 38) , and overexpression of cyclin D1 in tumors can be associated with poor prognosis (49) . Here, for the first time, we provide evidence that constitutive expression of cyclin D1 in tumor cells can be induced by an autocrine IGF-I loop. In addition, immunoneutralization of endogenously released IGF-I led to a slight increase in the expression of the cyclin dependent kinase inhibitor p27kip1. Thus, the autocrine IGF-I loop targets two major regulators of the cell cycle to promote DNA synthesis, cyclin D1 and p27kip1. Interestingly, levels of other cyclins, such as cyclin E, remained unchanged, demonstrating that the effect of the neutralizing antibody was selective.
To prove that a secreted growth factor acts via an autocrine pathway, it is necessary to demonstrate the following points: (a) the ligand and its receptor are expressed by the cell; (b) the receptor mediates intracellular signals; and (c) interference with the ligand-receptor interaction specifically inhibits biological effects. The data presented in this paper fulfill all of these criteria and thereby establish the major importance of IGF-I and the IGF-I receptor for both secretion and growth of human BON neuroendocrine tumor cells. We also demonstrate the presence of IGF-I and its receptor in various human neuroendocrine tumors. IGF-I could hence be one of the major mediators of the so called hypersecretion syndromes leading to functional ("hormone-active") neuroendocrine tumor disease (24) . Treatment of patients with antibodies against the Her2/neu receptor tyrosine kinase is appreciated as a novel strategy for breast cancer therapy (50) . Thus, targeting IGF-I or the IGF-I receptor tyrosine kinase may constitute a novel therapeutic strategy for patients suffering from carcinoid tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Deutsche
Forschungsgemeinschaft Grant SFB 518/B3 (to T. S.) and the
Landesforschungsschwerpunkt Baden-Württemberg Grant
"Modulation von Wachstumsfaktoren als Therapieprinzip" (to
P. M. J). 
2 To whom requests for reprints should be
addressed, at Abteilung Innere Medizin I, Medizinische
Universitätsklinik Ulm, Robert Koch Strasse 8, D-89081 Ulm,
Germany. Phone: 49-731-50201; Fax: 49-731-5024302; E-mail: thomas.seufferlein{at}medizin.uni-ulm.de
3 The abbreviations used are: IGF, insulin-like
growth factor; DNM, Dulbecco’s-F12 nut mix; ERK, extracellular
signal-regulated kinase; IGFBP, IGF-binding protein; IGF-I, type 1
insulin-like growth factor; IRS, insulin receptor substrate; MEK,
mitogen-activated protein kinase kinase; PDB, phorbol 12,13-dibutyrate;
PKC, protein kinase C; PI3-kinase, phosphatidylinositol 3-kinase.
Received 4/13/00. Accepted 6/19/00.
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