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From 1 St. Erik’s Eye Hospital, Stockholm, Sweden; 2 Cellular and Molecular Tumor Pathology, Department of Oncology and Pathology, Karolinska Hospital, Stockholm, Sweden; the 3 Department of Pathology, Regina Elena Cancer Institute, Rome, Italy; and the 4 Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Paraffin material from 36 clinicopathologically well characterized
cases of primary uveal melanomas (18 of which had metastasized to the
liver) with more than 15 years’ follow-up was used for
immunohistochemical analysis. In the experimental studies, three uveal
melanoma cell lines (OCM-1, OCM-3, and 92-1) were used. The expression
level of IGF-1R in the cell lines was modulated by glycosylation
inhibitors, and the IGF-1R was neutralized with the antibody 
IR-3.
Expression of IGF-1R was assayed by Western blot analysis and
immunohistochemistry. Cell growth and survival were analyzed by cell
counting, thymidine incorporation, and viability assays.
RESULTS. Western blot analysis and immunohistochemistry confirmed that IGF-1R is expressed in uveal melanoma. Although 10 of 18 patients who died of metastasizing disease showed high IGF-1R expression, only 5 of 18 tumors from patients who survived for 15 years or more after enucleation exhibited a high IGF-1R expression. Kaplan-Meier analysis showed a significant association (P = 0.035) between a high IGF-1R expression and death due to metastatic uveal melanoma. Using in vitro experimental models, we found that inhibition of the IGF-1R activity (tyrosine phosphorylation) was associated with a drastic decrease in uveal melanoma cell viability.
CONCLUSIONS. These data suggest an important role of IGF-1R in uveal melanoma. The significant association between high IGF-1R expression and death due to metastatic disease may be explained by the fact that IGF-1 is mainly produced in the liver, which is the preferential site for uveal melanoma metastases. These data also point to the possibility of therapeutically interfering with IGF-1R, which appears to be expressed preferentially in uveal melanomas that appear to follow an aggressive clinical course.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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That uveal melanoma shows early and preferential dissemination to the liver raises the possibility that hepatic environmental factors are important for the growth, survival, and progression of this malignancy. Such favorable conditions may involve growth factors produced in the liver—for example, hepatocyte growth factor (HGF; scatter factor). HGF exerts its biological effects through binding to the plasma membrane receptor c-Met. An interesting study by Hendrix et al.4 has shown that uveal melanoma cells with metastatic phenotype express c-Met.
Another growth factor mainly produced in the liver is insulin-like
growth factor (IGF)-1, which binds to the IGF-1 receptor (IGF-1R),
which is a heterotetrameric plasma membrane glycoprotein composed of
two -subunits (130 kDa each) and two ß-subunits (96 kDa each)
linked by disulfide bonds.5
6
Ligand binding to the IGF-1R
induces activation of the intrinsic tyrosine kinase of the ß-subunit,
leading to phosphorylation of IGF-1R and several intracellular
proteins, including the family of insulin receptor substrates
(IRSs)—for example, IRS-1.7
This leads to activation of
Ras, mitogen-activated protein kinase, and phosphoinositol-3-kinase
(i.e., events involved in the mitogenic cascade).5
In an
increasing number of malignant cell types, IGF-1R has been shown
essential for tumorigenesis as well as for the establishment and
maintenance of a transformed phenotype.5
7
Furthermore,
protection from apoptotic cell death has been ascribed to
IGF-1R.5
7
Previously, we have shown that N-linked glycosylation is critical for the translocation of IGF-1R from the endoplasmic reticulum to the cell surface.8 9 10 11 Treatment with tunicamycin (TM), which is a specific inhibitor of N-linked glycosylation, or lovastatin, which is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, both downregulate IGF-1R at the cell surface.8 9 10 11 The inhibitory effect of lovastatin on N-linked glycosylation is explained by a decreased synthesis of dolichyl phosphate, which acts as a carrier of oligosaccharides in the assembly of glycoproteins.12 Growth arrest and apoptosis follows the decreased cell surface expression of IGF-1R.10 13 14
The purpose of this study was to evaluate the expression of IGF-1R in uveal melanoma regarding its correlation with clinical behavior and regarding its role in growth and survival of uveal melanoma cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IR-3)
was purchased from Oncogene Science (Manhasset, NY). Lovastatin was
obtained from Merck, Sharp & Dohme (Rahway, NJ) and was converted to
its sodium salt by incubation in 0.1 M NaOH at 50°C for 2 hours. All
other chemicals, unless stated otherwise, were from Sigma (St. Louis,
MO).
Tumor Material
Paraffin blocks from 36
patients(14 females and 22 males), derived from surgically removed primary
uveal melanomas, were used for immunohistochemistry (IHC; Table 1
).
The ages of the patients varied between 23 and 87 years with a
mean age of 61 years at diagnosis. Eighteen patients died of
tumor-related causes, 3 died of other causes, and 15 were still alive
at the time of follow-up. The follow-up time was 15 years or more
(Table 1)
.
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Cell Culture
Three cell lines obtained from human primary uveal melanomas
(OCM-1, OCM-3, and 92-1) were used. OCM-1 and OCM-3 were kindly
provided by June Kan-Mitchell (Wayne State University, Detroit,
MI). Cell line 92-1 was established in one of our laboratories (Leiden
University Medical Center, Leiden, The Netherlands). The OCM-1 cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with10% fetal bovine serum (FBS), and the OCM-3 and 92-1
cells in RPMI 1640 supplemented with 10% FBS and 3 mM
L-glutamine.
Cells were grown in monolayers in tissue culture flasks, maintained in a 95% air-5% CO2 atmosphere at 37°C in a humidified incubator. For experimental purposes, the cells were cultured in 35- or 60-mm plastic Petri dishes. Cells were seeded at a density of 3000 to 5000 cells/cm2, and experiments were initiated when cells had reached subconfluence.
Immunoprecipitation and Western Blot Analysis
Preparation of cell membranes was performed essentially as
described elsewhere.15
In brief, cells were harvested and
homogenized in a buffer containing 0.32 M sucrose, 1 mM
taurodeoxycholic acid, 2 mM MgCl2, 1 mM EDTA, 25
mM benzamidine, 1 µg/mL bacitracin, 2 mM phenylmethylsulfonyl
fluoride, 10 µg/mL aprotinine, 10 µg/mL soybean trypsin inhibitor,
and 10 µg/mL leupeptin. After a 10-minute centrifugation at
600g (4°C) the pellet (containing unbroken cells, nuclei,
and cytoskeleton) was discarded. The supernatant was then centrifuged
at 17,300g for 30 minutes. The resultant pellet, containing
cell membranes, was used for Western blot analysis.
For immunoprecipitation, cell lysates were obtained using PBSTDS buffer
(100 mL 1x PBS with 10 mL 100% Triton X-100, 5 g sodium
deoxycholate, and 1 g SDS in 100 mL deionized water), containing
the aforementioned protease inhibitors. An equal amount of protein from
each sample was immunoprecipitated with 15 µL protein G plus-agarose
and 1 µg IR-3. After a 24-hour incubation at 4°C on a rocking
platform, the immunoprecipitates were collected by centrifugation in a
microcentrifuge at 2500 rpm for 15 minutes. The supernatant was
discarded, and the pellet was washed four times with 1 mL PBSTDS. The
material was then dissolved in sample buffer for SDS-PAGE. Protein
samples (from plasma membrane preparations or total cell lysates) were
dissolved in a sample buffer containing 0.0625 M Tris-HCl (pH 6.8),
20% glycerol, 2% SDS, bromophenol blue, and dithiothreitol. Sample
amounts obtained from 100 µg cell protein were analyzed by SDS-PAGE
with a 4% stacking gel and a 10% separation gel, essentially
according to the protocol of Laemmli.16
Molecular weight
markers (Bio-Rad, Richmond, CA) were run simultaneously. For Western
blot analysis, the proteins were transferred overnight to
nitrocellulose membranes (Amersham, Buckinghamshire, UK) and then
blocked for 1 hour at room temperature in a solution of 5% (wt/vol)
skimmed milk powder and 0.02% (wt/vol) Tween 20 or 1% (wt/vol)
skimmed milk powder and 1% bovine serum albumin (immunoprecipitation)
in PBS (pH 7.5). Incubation with the primary antibody, against the
-subunit of IGF-1R (N-20), was performed for 1 hour at room
temperature, followed by three washes with PBS and incubation with a
biotinylated secondary antibody (Amersham) for 1 hour. After a
15-minute incubation with streptavidin-labeled horse peroxidase,
detection was performed using enhanced chemiluminescence with
autoradiographic film (Hyperfilm-ECL; Amersham)
Cell Growth Assay
To determine DNA synthesis, cells were cultured in 35-mm dishes
and, after the experimental conditions, labeled with [3H]thymidine (1
µCi/mL, 5 Ci/mmol) for 4 hours. The acid-precipitable material was
then taken for scintillation counting as described
elsewhere.8
Cell proliferation was measured by determining
the number of cells attached to the plastic surface of duplicate 35-mm
dishes. This was performed by microscopic counting of all cells in
ink-marked areas on the dish bottom. By repeating the counting after
specified time intervals, changes in the number of attached cells could
be observed.10
Cell Viability Assay
A cell proliferation kit (Kit II) was purchased from Roche
Diagnostic GmbH (Mannheim, Germany). The test is based on a
colorimetric change of the yellow tetrazolium salt XTT into orange
formazan dye by the respiratory chain of viable cells.17
Cells seeded at a concentration of 5000/well in 100 µL medium in a
96-well plate were treated with different drugs in specified
concentrations. After 24 hours, cells were incubated according to the
manufacturer’s protocol with the XTT labeling mixture. After 4 hours,
the formazan dye was quantified using a scanning multiwell
spectrophotometer with a 495-nm filter. The absorbance is directly
correlated with the number of viable cells. To draw the standard
absorbance curve, we used untreated cells seeded at concentrations from
1,000 to 10,000 cells/well with an increasing rate of 1,000 cells/well.
All standards and experiments were performed in triplicate.
Immunohistochemistry
Immunostaining was performed using the standard avidin-biotin
complex technique (ABC Elite Standard Kit, cat. PK-6100; Vector
Laboratories, Burlingame, CA). Deparaffinized, rehydrated sections (two
adjacent sections for each specimen) were pretreated with microwaves
for 10 minutes in 0.1 M citrate buffer at pH 6.0 (IGF-1R) or digested
with 0.05% pronase for 15 minutes (CD68). Before immunostaining, the
endogenous peroxidase activity was blocked by hydrogen peroxidase
dissolved in methanol (3% hydrogen peroxide in methanol, 1:5 volume)
for 30 minutes. Sections were then rinsed in and incubated with
blocking serum (1% bovine serum albumin) for 20 minutes followed by
incubation with the primary antibodies diluted 1:1000 (IGF-1R) and 1:50
(CD68). The incubation was performed overnight at 4°C. A biotinylated
anti-rabbit or anti-mouse IgG was used as a secondary antibody and
followed by the ABC complex. The peroxide reaction was developed using
3,3'-diaminobenzidine tetrahydrochloride (DAB; 0.6 mg/mL with 0.03%
hydrogen peroxide) for 6 minutes. Counterstaining was performed by
Mayer’s hematoxylin. Tris-buffered saline (pH 7.6) was used for
rinsing between the different steps. Placenta tissue was used as the
positive control for the polyclonal antibody against IGF-1R. In
addition, a control peptide against N-20 (SC712P) was used to secure
the staining results. All the slides were coded and analyzed in a
blinded fashion.
After tissue processing, all cells displaying distinct immunoreactivity
were considered positive, irrespective of staining intensity. We
classified the results of IGF-1R stainings as zero when less than 15%,
low when 15% to 50%, and high when more than 50% of melanoma cells
were positive according to an arbitrary scale: 0, negative; 1, low
expression; and 2, high expression. At a later stage, and without
knowledge of the initial result, the same observer repeated the
assessment for each of the 36 uveal melanomas. These specimens were
also assessed by an independent observer using the same grading system.
The interobserver reproducibility using the 
test was 0.75 (95%
confidence interval [CI]0.54–0.95) and = 0.95 for
intraobserver reproducibility (95% CI 0.85–1.05). Both observers were
masked to results from earlier assessments and to survival data.
Determination of Protein Content
Protein content of cell lysates was determined by a dye-binding
assay18
with a reagent purchased from Bio-Rad. Bovine
serum albumin was used as a standard.
Statistical Analysis
Survival data without loss to follow-up were obtained, according
to the tenets of the Declaration of Helsinki, for all patients with
uveal melanoma from the Swedish National Causes of Death Registry. Time
from the date of surgery to death or the end of 1996 was considered
censored if the patient was alive at the end of 1996 or had died of any
other than a melanoma-related cause. The log-rank test was used to
assess survival differences. Calculations were computer based
(Statistica; StatSoft, Tulsa, OK; and MedCalc; MedCalc Software,
Mariakerke, Belgium).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit of IGF-1R.8
Eight of the cases resulted in, to a variable extent, a positive signal
(Fig. 1) . Thus, in three of the uveal melanoma cases as well as in normal human
fibroblasts (Fig. 1
, lane 1), there was no detectable expression of
IGF-1R. These 11 samples were obtained from recent surgical cases
(follow-up, <2 years) and could not be used for association with
clinical outcome. Therefore, we investigated the expression of IGF-1R
by IHC on paraffin-embedded surgical specimens of 36 cases of primary
uveal melanoma with long follow-up. Positive immunoreactivity was found
in 34 of the cases. The expression level regarding intensity of
immunoreactivity was scored: no expression, low expression, and high
expression, as described in Materials and Methods. An additional
immunostaining using the macrophage marker CD68 was also performed on
adjacent sections from all specimens. Macrophages usually represented
less than 10% of cells within the core of the tumors. Figure 2A
shows photomicrographs of immunostaining for IGF-1R and CD68 in a case
of uveal melanoma.
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10 mm), which is an established prognostic parameter for
uveal melanoma,3
and survival is shown. There was a clear
trend for decreased survival in tumors of 10 mm or more, but this did
not reach statistical significance in this study sample
(P = 0.157).
In Vitro Assays
In the second part of the study, we investigated the expression of
IGF-1R in the three uveal melanoma cell lines OCM-1, OCM-3, and 92-1
and sought to determine how modulation of IGF-1R expression affects
cell growth and survival. Each cell line was treated with the IGF-1R
blocking antibody IR-3 (1 µg/mL), which has been shown to be
specific for IGF-1R,19
or with lovastatin (10 µM) or TM
(5 µg/mL) for 4 hours. None of these agents caused any decrease in
cell viability after 4 hours (data not shown). The choice of the
concentrations of TM, lovastatin, and IR-3 in this study was based
on previous dose–response experiments (data not shown). In Figure 3
, we show that the basal level of IGF-1R expression at the cell surface,
as detected by Western blot analysis of the -subunit, was almost
similar in the three cell lines. As expected from previous studies on
other cell types,8
9
10
11
the expression of IGF-1R at the
cell surface was decreased after treatment with lovastatin and TM in
all cell lines, but not after prior treatment with the monoclonal
antibody IR-3. Consequently, lovastatin and TM reduced
ligand-mediated activation of the 96-kDa ß-subunit of IGF-1R in all
three uveal melanoma cell lines, whereas IR-3 produced a strong
effect only in OCM-1 and OCM-3 cells (Fig. 3)
. The 92-1 cells responded
to treatment with IR-3 with a moderate decrease in IGF-1R
phosphorylation. Differences in modulating IGF-1R phosphorylation using
a blocking monoclonal antibody could be explained by different
concentrations of the receptor at the cell surface. However, the
concentration of IR-3 used was found to be optimal. Therefore, we
believe that IR-3 binds to all cell surface receptors, but for some
other reason it does not block ß-subunit phosphorylation completely
in the 92-1 cell line. Although IR-3 mostly has been reported to
block IGF-1R activation, exceptions have been
demonstrated.20
21
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IR-3 treatments on cell and DNA
replication and cell survival are demonstrated. As shown in Figure 4A
,
the basal proliferative rate differed between the three uveal melanoma
cell lines. During a 72-hour period, the OCM-1 control cells had
increased fivefold in cell number, whereas 92-1 and OCM-3 had increased
only 2- to 2.5-fold. All cell lines responded quickly to incubation
with IR-3, lovastatin, or TM (Fig. 4A)
and after 24 to 72 hours cell
numbers decreased. Consistent with the lower inhibitory effect of
IR-3 phosphorylation of 92-1, the decrease in 92-1 cell
proliferation was also less after IR-3 incubation than with
lovastatin and TM treatments. A 24-hour treatment with IR-3 led to a
60% decrease in DNA synthesis in this cell line (Fig. 4B)
, whereas
cell viability was only slightly (15%) reduced (Fig. 4C)
. Regarding
OCM-1 cells, a drastic reduction of total cell number was observed
after TM treatment (Fig. 4A)
. This was correlated with a drastic
decrease in both DNA synthesis and cell viability. Whereas cell
replication of OCM-1 cells was efficiently blocked by lovastatin and
IR-3, there was only a moderate or no, respectively, loss of cells
(Fig. 4A) . However, using the viability assay, it was confirmed that
almost all OCM-1 cells had died after the treatment with these two
agents (Fig. 4C)
.
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IR-3 reduced the number of
OCM-3 cells with similar kinetics, and after 72 hours there was a total
cell loss. Although the cell number was decreased by only 20% to 25%
after a 24-hour treatment, the OCM-3 cell viability was reduced by as
much as 75% to 85% (Fig. 4C)
. The decrease in DNA synthesis differed
somewhat between the three agents. IR-3 caused a 40% decrease,
compared with 70% to 80% for TM and lovastatin. In Figure 5 the level of tyrosine phosphorylation at 4 hours is correlated with DNA synthesis and cell viability. The values are obtained from Figures 3 and 4 . As can be seen, there is an especially strong correlation between IGF-1R phosphorylation and cell viability.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In several cell types, IGF-1R has been shown to play a pivotal role in cell cycle regulation, differentiation, apoptosis protection, and cell transformation.7 26 27 28 29 30 31 32 33 Furthermore, a high expression has been shown to be correlated with a poor clinical outcome in certain cancers. Xie et al.34 recently demonstrated that metastatic dissemination of synovial sarcoma, a highly malignant soft tissue tumor, was significantly correlated with a high expression of IGF-1R in the localized tumor, as assayed by both RT-PCR and Western blot analysis. Egan et al.35 have reported the importance of IGF-1 and its serum binding protein, IGFBP-3, both of which were found to independently predict metastasis in choroidal melanoma. However, as has been concluded by Baserga,36 IGF-1 (or IGF-2) can stimulate the growth of tumors, but it is the receptor that defines the outcome and the presence of an IGF-1R is obligatory for malignant transformation.36 To our knowledge, the present study is the first one to investigate the expression and role of IGF-1R in uveal melanoma.
From the present study we can conclude that IGF-1R is variably expressed in primary uveal melanoma and that (based on our selected sample) a high expression of IGF-1R is associated with a decreased survival in this disease. Although it is possible that some macrophages showed positive IGF-1R immunostaining, such macrophages were usually confined to the peripheral part of the tumor. Furthermore, most macrophages could be detected by their morphologic appearance. Our study was exploratory, and the patients were selected to represent one of two extremes (i.e., those who died of uveal melanoma or those who survived 15 years or more). In this respect, our data may be biased and would need confirmation by a larger study of consecutive patients, by means of multivariate analysis.
From our study we can also conclude that growth and survival of uveal
melanoma cell lines are strongly dependent on IGF-1R expression and
activation. Specifically, we have shown that inhibition of N-linked
glycosylation induced by treatment with TM and lovastatin caused
decreased IGF-1R expression and consequently decreased tyrosine
phosphorylation. Subsequently, this induced growth arrest and cell
death in the three investigated uveal melanoma cell lines. In this way,
treatment with TM and lovastatin simulate the effect of growth factor
depletion, which in itself induces apoptosis in tumor
cells.37
It could be argued that lovastatin and TM kill
the cells by other mechanisms than by downregulating the IGF-1R.
However, as shown in this study and in others,10
11
14
the
kinetics of the growth inhibition and cell death correlate well with
the effects obtained by the decrease in IGF-1R tyrosine
phosphorylation, using the IGF-1R blocking monoclonal antibody IR-3.
Furthermore, it has been shown that addition of high concentrations of
IGF-1 to cells treated with glycosylation inhibitors can temporarily
prevent growth inhibition and apoptosis and that this effect
can be abrogated by IR-3.10
Similar results have been
obtained in other cell lines.38
These data suggest that
downregulation of IGF-1R at the cell surface is an important mechanism
by which N-linked glycosylation inhibitors decrease cell growth and
survival. Taken together, our results raise the possibility of
targeting IGF-1R as a form of treatment in patients with uveal
melanoma.
The in vitro results may also support our immunohistochemical data, because we demonstrated a significant association between IGF-1R activity and cell proliferation and cell survival of uveal melanoma cells (see Fig. 5 ). Deregulated cell growth and suppression of apoptosis represent fundamental properties for propelling the tumor cell and its progeny into uncontrolled expansion and invasion.39 Therefore, our experimental data may reflect an association between the level of IGF-1R and the malignant phenotype of uveal melanoma.
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Footnotes |
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Submitted for publication February 12, 2001; revised September 4, 2001; accepted September 14, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Olle Larsson, CCK R8:04, Department of Oncology and Pathology, Karolinska Hospital, S-171 76 Stockholm, Sweden; olle.larsson{at}onkpat.ki.se.
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