Malignant melanoma is the cancer with the most
rapid increase in incidence in the United States. Ultraviolet light and
deficiency of the p16ink4a gene are known factors that
predispose one to the development of malignant melanoma. The signal
transduction pathways that underlie the progression of melanoma from
their precursors, atypical nevi, are not well understood. We examined activation of the MAP kinase pathway in atypical nevi and melanoma cells and found that this pathway is activated in melanomas. To determine the functional significance of this activation, we introduced constitutively active MAP kinase kinase (MAPKK) into immortalized melanocytes. The introduction of this gene into melanocytes leads to
tumorigenesis in nude mice, activation of the angiogenic switch, and
increased production of the proangiogenic factor, vascular endothelial
growth factor (VEGF), and matrix metalloproteinases (MMPs).
Activation of MAP kinase signaling may be an important pathway involved
in melanoma transformation. Inhibition of MAP kinase signaling may be
useful in the prevention and treatment of melanoma.
 |
INTRODUCTION |
Malignant melanoma is a major cause of morbidity and mortality.
Melanomas often arise from precursor lesions called atypical nevi (1).
Although the prognosis of thin melanoma is excellent, prognosis
decreases with increased thickness of the lesions. The diminished
prognosis is due mainly to the well established tendency of melanoma to
metastasize. In addition, melanomas are highly resistant to most forms
of chemotherapy and radiation; therefore, cure of the disseminated
disease is uncommon. Thus, there is an urgent need to understand the
signal transduction pathways underlying transformation of melanocytes
to melanoma.
Several histologic and immunohistochemical markers have been shown to
correlate with prognosis. The first and still most widely used markers
of melanoma prognosis are Breslow thickness and Clark's levels (2, 3).
Breslow thickness measures the width of the lesion, and lesions >0.75
mm have a distinctly worse prognosis than thinner melanomas. Clark's
levels measure the depth of invasion of the melanoma from the
dermoepidermal junction to deeper layers of the dermis and subcutaneous
fat. Both of these markers are measures of the ability of melanoma
cells to grow three dimensionally (4, 5). More recently, other genes
have been shown to be expressed or repressed in different stages of
melanoma progression (6, 7). Telomerase is present in radial and
vertical growth melanomas but not in atypical nevi (8, 9). Various
integrins have been shown to be expressed in advanced melanomas and
correlate with decreased apoptosis and invasion (10, 11). A potent
angiogenesis factor, VEGF,1
is highly expressed in advanced melanoma. Finally, expression of the
small G protein rhoC is associated with increased metastatic ability of
melanoma (12, 13). However, the signal transduction pathways associated
with melanoma transformation are not well understood.
We have previously noted that an inverse correlation exists between the
expression of activated MAP kinase and the degree of malignancy in
tumors derived from endothelium (4, 14). Other investigators have
demonstrated a direct correlation between MAP kinase expression and the
degree of malignancy in other tumors (15). These findings suggest that
the role of MAP kinase in malignancy may be tissue specific. We
examined the expression of activated MAP kinase in human nevi and
melanomas and found increasing expression in malignant cells,
particularly in early (radial growth) melanoma (16). Furthermore, we
demonstrate that MAP kinase activation functionally contributes to the
development of melanoma, as the introduction of a constitutively active
MAPKK into melanocytes leads to transformation in vivo. The
activation of MAP kinase results in the activation of AP-1 but not the
activation of NF
B. Inhibition of MAP kinase signaling (17) may
provide a therapeutic strategy for the prevention and treatment of melanoma.
 |
MATERIALS AND METHODS |
Immunohistochemistry--
Paraffin-fixed sections of nevi and
melanomas were stained with an antibody specific for phosphorylated MAP
kinase according to the procedure of Arbiser et al.
(18).
Generation of Cell Lines--
L10BIOBR cells are an immortalized
murine melanocyte cell line that proliferates best in the presence of
exogenous phorbol ester (G. P. Dotto, Cutaneous Biology Research
Center, Harvard Medical School, Charlestown, MA) (19). These cells were
maintained in F-10 nutrient mixture (Ham's medium) with 7% horse
serum. L10BIOBR cells were infected with retroviruses encoding green
fluorescent protein (pDIVA-GFP) (A. Kowalczyk, Emory University) or a
constitutively active MAPKK mutant, LIDA/MANA (C. Marshall, Institute
of Cancer Research, London). Both vectors encode puromycin resistance,
and cells were selected in 2 µg/ml puromycin and pooled (20). GFP expression was confirmed by fluorescence microscopy, and expression of
the active MAP kinase kinase gene was confirmed by performing Western
blot analysis with an antibody specific to phosphorylated MAP kinase.
Protein extracts were prepared as described by Arbiser and co-workers
(21). Expression of the constitutively active MAPKK was confirmed using
antibody 177 to rabbit MAPKK (C. Marshall, London).
Western Blotting--
Cells were lysed in lysis buffer
containing 20 mM Tris HCl (pH 7.5), 150 mM
NaCl, 1% (v/v) Triton X-100, 10% glycerol, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM benzamidine, 1 mM phenylmethylsulfonylfluoride, and 1 mM
Na3VO4.
Protein concentration was determined by the Bradford assay using bovine
serum albumin as a standard. Samples were treated with Laemmli
sample buffer and heated to 90 °C for 5 min prior to SDS-PAGE
(National Diagnostics, Atlanta, GA) and transfer to nitrocellulose
membranes. The membranes were then blocked with 5% nonfat dry milk in
Tris-buffered saline with Tween 20 and subsequently incubated
with the appropriate antibody for immunoblotting. The anti-Erk2
monoclonal antibody was from Santa Cruz Biotechnology, and
anti-phospho-MAPK, phospho-c-Jun (Ser73), phospho S6
ribosomal protein (Ser235-Ser236), phospho S6
ribosomal protein (Ser240-Ser244), and phospho
p70 S6 kinase (Thr421-Ser424) antibodies were
from Cell Signaling Technologies (Beverly, MA).
Assays of Matrix Metalloproteinase Bioactivity--
Cells were
grown to ~75% confluence in Dulbecco's Modified Eagle Medium
supplemented with 10% fetal calf serum. After washing with
phosphate-buffered saline, the medium was replaced with Cellgro serum-free medium (Mediatech, Herndon, VA). Substrate gel
electrophoresis (zymography) was conducted as described by us
previously (14). Briefly, Type 1 gelatin was added at a concentration
of 1 mg/ml to the standard Laemmli acrylamide polymerization mixture
(22). Conditioned media from equivalent numbers of cells were diluted 3:1 with sample buffer (10% SDS, 4% sucrose, 0.25 M Tris,
pH 6.8, and 0.1% bromphenol blue) and electrophoresed as described
previously. At the end of electrophoresis, gels were rinsed in 2.5%
Triton X-100 for 30 min and then incubated overnight in substrate
buffer (50 mM Tris, pH 8, 5 mM
CaCl2, 0.02% NaN3). The gels were stained with
0.5% Coomassie Blue R-250 in acetic acid/isopropyl
alcohol/H20 (1:3:6) and destained in the same buffer in
which the Coomassie Blue was prepared. The densitometry of destained
areas was quantified using a Datascopy GS Plus scanner connected to
Macintosh II computer with Macimage software (Xerox Imaging Systems).
Assay of Tissue Inhibitor of Matrix Metalloproteinase
Bioactivity--
TIMP bioactivity was quantified using conditioned
media from equal numbers of L10BIOBR cells expressing active MAPKK or
GFP vector control. A solid collagen film assay was performed using C14-labeled collagen as described by us previously
(14).
In Vivo Tumorigenesis--
One million cells were injected
subcutaneously into 4-5 week old male nude mice (Charles River) in the
presence of a small quantity of trypan blue to mark the inoculation
site. Tumors were excised after one month, fixed in formalin, and
subjected to histologic and in situ hybridization. Tumor
volume was calculated according to the formula (w2 × l)0.52, where w represents the shortest dimension (14).
In Situ Hybridization for Histone H3--
In situ
hybridization was performed on 4 mm-thick sections of formalin-fixed,
paraffin-embedded tissue. Details of in situ hybridization
have been reported previously (18, 23, 24). The emulsion was developed
with Kodak D19 developer, and the slides were counterstained with
hematoxylin (23, 25).
RT-PCR for VEGF--
Total RNA was isolated using TRI Reagent
(Sigma). RT-PCR was done with Promega Accession RT-PCR kit. Primers
used were as follows: actin (728 bp), forward 5'-AAG ATG ACC CAG ATC
ATG TTT GAG AC-3' and reverse 5'-CTG CTT GCT GAT CCA CAT CTG CTG G-3'; VEGF(445 bp), forward 5'-TCA TGC GGA TCA AAC CTC ACC AA-3' and reverse
5'-TCT CGC CCT CCG GAC CCA AAG T-3'. Reactions were performed in an
Eppendorf master cycler. One PCR cycle at 45 Co for 45 min
and 94 Co for 2 min followed by forty PCR cycles under
standard conditions with an annealing temperature of 60 °C were
performed.
-actin mRNA was used as a reference message to
normalize the content of total RNA. VEGF expression was calculated as
the relative expression ratio to that of
-actin. All reactions were
carried out in triplicate. Quantification was determined by triplicate
repeats (26). Curcumin used for AP-1 inhibition studies was obtained
from Sigma and prepared as a stock solution in Me2SO
at 10 mg/ml.
Signal Transduction Array--
The protein array was
accomplished to compare the transcription factors involved in
L10BIOBRMAPKK and L10BIOBRGFP cells. Nuclear extract was isolated from
these cells using the Panomics nuclear extraction kit (catalog number
AY2002), and the array was performed using TranSignalTM protein/DNA
arrays. In brief, DNA/protein hybridization was carried out according
to the manufacturer's instructions. The gel area was excised
containing the protein/DNA complex, and the protein/DNA complex was
extracted using the extraction buffer and finally incubated with 6 µl
of gel extraction beads at room temperature for 10 min. The mixture was
centrifuged at 10,000 rpm for 30 s to pellet out the beads. The
beads were washed, and the supernatant was removed. The bound probes
were eluted by resuspending the pellet in 50 µl of dH2O and incubated
at room temperature for 10 min with vortexing for two to three times
during incubation.
Detection was done after the addition of 20 ml of 1× blocking buffer
to each membrane and incubation at room temperature for 15 min with
gentle shaking. 20 µl of streptavidin-horseradish peroxidase
conjugate was added directly to 20 ml of 1× blocking buffer, and
continued shaking at room temperature was conducted for 15 min. Each
membrane was washed three times at room temperature with 20 ml of 1×
wash buffer each 8 min followed by the exposure of 20 ml of 1×
detection buffer to each membrane and incubation at room temperature
for 5 min. 2 ml of a substrate working solution containing equal
volumes of the luminol enhancer and peroxide solution was added, and
the membranes were incubated at room temperature for 5 min and then
exposed using Kodak BioMax film.
 |
RESULTS |
Active MAPK Is Found in Melanoma but Not in Benign Nevi--
To
determine whether phosphorylated (active) MAP kinase is activated in
malignant melanoma, we performed immunohistochemical analysis of both
benign melanocytic nevi and malignant melanoma according to the method
of Arbiser et al. (18). We have previously demonstrated the
specificity of this antibody using melanoma cell lysates (16, 18). Very
little staining was observed in benign melanocytic nevi, but prominent
nuclear staining was observed in human melanoma tumors (Fig.
1).

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Fig. 1.
Immunohistochemical analysis of nevi and
melanoma. The top panel represents
immunohistochemistry for phospho MAPK on an atypical nevus, and
the bottom panel represents an invasive melanoma
(40×).
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Introduction of Active MAPKK into Immortalized
Melanocytes--
The L10BIOBR cell line is an immortalized murine
melanocyte cell line that has been used to assess tumorigenesis. To
determine whether the activation of MAP kinase signaling results in
tumorigenesis, we infected these cells with retroviruses encoding
constitutively active MAPKK or GFP as a vector control. The
introduction of active MAPKK led to an increase in total (endogenous
and retrovirally transduced) MAPKK protein as assessed by Western blot
(Fig. 2A). To determine
whether the introduced MAPKK is functional, we examined levels of
active (phosphorylated) MAPK in both cell lines. MAPK phosphorylation
is significantly increased in L10BIOBR cells expressing active MAPKK
(Fig. 2A). Melanocytes expressing active MAPKK showed increased refractility and elongated shape compared with GFP-transduced control cells (Fig. 2B).

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Fig. 2.
A, analysis of phospho MAP kinase
and total MAP kinase expression in immortalized (L10BIOBR-GFP)
( ) and transformed melanocytes (L10BIOBR-MAPKK)
(+). The bands at the top represent an immunoblot
with an antibody specific for phosphorylated MAPK, the gel in the
middle represents total MAPKK protein showing overexpression
of the retroviral transgene, and the gel at the bottom
represents an immunoblot with an antibody for total MAPK. B,
introduction of MAPKK into L10BIOBR cells induces a transformed
morphology. The left panel shows vector control
(GFP)-transduced melanocytes, whereas the right
panel shows MAPKK-expressing melanocytes.
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|
Melanocytes Expressing Activated MAPKK Are Tumorigenic in
Vivo--
To determine whether overexpression of MAPKK causes
malignant transformation in melanocytes, one million cells of either
vector control or MAPKK expressing L10BIOBR cells were injected
subcutaneously into nude mice. Overexpression of MAPKK leads to
malignant transformation in vivo, whereas progressive tumor
growth was not observed in vector expressing cells (Fig.
3).

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Fig. 3.
MAPKK activation causes transformation
in vivo. The left panel
shows mice containing MAPKK-induced tumor (mouse on the
left) and GFP control tumor (mouse on the right).
The right panel shows that introduction of MAPKK
into L10BIOBR cells leads to a significant difference in tumor volume
at 1 month post inoculation (p < 0.05).
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Introduction of Active MAPKK Induces the Angiogenic
Switch--
Part of the action of dominant oncogenes is to activate
the angiogenic switch toward cells that produce angiogenesis
stimulators. We examined the production of VEGF mRNA by RT-PCR and
found ~5-fold induction by MAPKK (Fig.
4A).

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Fig. 4.
A, effect of MAPKK
activation on VEGF expression. The top panel
demonstrates a representative RT-PCR analysis for VEGF in
MAPKK-expressing and control L10BIOBR melanocytes. The left
lane is a molecular weight standard, the middle
lane represents RNA from MAPKK-expressing melanocytes, and
the right lane represents RNA from control
melanocytes. RNA samples were analyzed for -actin expression as a
loading control. B, effect of MAPKK activation on MMP
bioactivity The left lane represents triplicate samples of
conditioned media from L10BIOBR-GFP (vector control) cells, and the
right lane represents conditioned media from
active MAPKK-expressing L10BIOBR cells. The lower
band at 65 kDa indicates gelatinolysis induced by MMP-2, and
the upper band at 92 kDa indicates gelatinolysis
induced by MMP-9. C, effect of MAPKK on TIMP bioactivity.
The left lane represents triplicate samples of
conditioned media from L10BIOBR-GFP (vector control) cells, and the
right lane represents conditioned media from
active MAPKK-expressing L10BIOBR cells. The inhibitory activity of
conditioned media from MAPKK-expressing cells was significantly higher
than that of control GFP cells.
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|
Substrate gel electrophoresis using gelatin as the substrate indicated
that the introduction of MAPKK into melanocytes resulted in an increase
in the gelatinase activity of MMP-9 (~92 kDa) and MMP-2 (~65 kDa)
in comparison to control cells (Fig. 4A). Surprisingly, when
this same conditioned media was tested in a radiometric enzyme assay
for TIMP activity, we found that the MAPKK cells produced significantly
higher levels of MMP inhibitory activity than did the control
cells (Fig. 4C).
Analysis of Tumor Dormancy and Vascularity--
In situ
hybridization of vector control and MAPKK-expressing tumors for the
proliferation-associated marker histone H3 revealed little expression
of this marker in vector control dormant tumors but diffuse expression
in MAPKK-expressing L10BIOBR tumors (Fig. 5, A-D). These results are
similar to what we have observed with immortalized and transformed
endothelial cells in that both three-dimensional growth and
increased angiogenesis are required for tumor growth in vivo
(14). MAPKK-overexpressing melanoma tumors also demonstrate a high
level of vascularity consistent with elevated expression of VEGF.
MAPKK-expressing tumors also were notable for a high level of
vascularity compared with GFP vector controls (Fig. 5, E and
F).

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Fig. 5.
Histologic, in situ
hybridization studies and neovascularity of L10BIOBR GFP and
MAPKK tumors. Panels A and C show
40× views of L10BIOBR GFP tumors, and panels B
and D show 40× views of L10BIOBR MAPKK tumors,
respectively. Panels C (L10BIOBR GFP) and
D (L10BIOBR MAPKK), show histone H3 in situ
hybridization. Note the diffuse H3 mRNA expression in the L10BIOBR
MAPKK tumor (panel D), whereas H3 mRNA expression is
nearly absent in the vector control tumors (panel
C). In addition, note the numerous blood vessels
present in the L10BIOBR MAPKK (panel F) tumors compared with
the GFP vector controls (panel E).
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Effect of MAP Kinase Activation on AP-1 and NF
B--
Both AP-1
and NF
B have been reported to be potential downstream mediators of
MAP kinase signaling (27-29). To determine which pathway is downstream
of MAPKK in melanocytes overexpressing MAPKK, we performed a Panomics
assay, which measures relative activation of transcription factors. In
this assay, we observed activation of AP-1 but not NF
B (data not
shown). We further demonstrated that activation of MAP kinase kinase in
melanocytes led to phosphorylation of the AP-1 transcription factor
c-Jun (Fig 6A). Finally, MAP kinase activation led to increased phosphorylation of p70 S6 kinase and
its downstream target, S6 ribosomal protein (Fig 6B).
Increased AP-1 activation was functionally important in melanocytes
overexpressing MAP kinase kinase, because inhibition of c-Jun with the
small molecular weight inhibitor curcumin (30-33) led to decreased
production of VEGF mRNA in a dose-dependent manner (Fig
7). Curcumin has little effect on MMP
activity in MAPKK-transformed L10BIOBR melanocytes, suggesting that a
c-Jun-independent pathway mediates MAPKK activation of MMP activity
(data not shown). We did not observe any effect of MAPKK on
hypoxia-inducible factor-1
(HIF-1
) activation either (data not
shown). Consistent with our findings that there is a lack of activation
of NF
B in MAPKK-transformed L10BIOBR melanocytes, treatment of these
cells with NF
B inhibitors (SN50, hypoestoxide) had no significant
effect on VEGF mRNA levels (data not shown).

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Fig. 6.
Constitutive activation of MAP kinase results
in activation of c-jun. Lane
1 represents lysates from L10BIOBR GFP vector control cells,
and lane 2 represents lysates from L10BIOBR
MAPKK-expressing cells. Western analysis demonstrates phosphorylation
(activation) of c-jun in response to constitutive MAPK
activation.
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Fig. 7.
Inhibition of AP-1 c-jun
activation results in the inhibition of VEGF mRNA
levels. L10BIOBR MAPKK cells were treated with the
AP-1/c-jun inhibitor curcumin, and VEGF mRNA was assayed
by RT-PCR. Treatment of cells with curcumin led to a
dose-dependent decrease in VEGF mRNA levels. The
top panel represents VEGF mRNA, and the
bottom panel represents -actin as a loading control.
Lane 1 represents cells treated with vehicle
control, lane 2 represents 5 µM
curcumin, lane 3 represents 10 µM
curcumin, lane 4 represents 15 µM curcumin,
and lane 5 represents 20 µM
curcumin.
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Effect of MAP Kinase Activation on Phosphorylation of p70 S6 Kinase
and S6--
Activation of p70S6 kinase and S6 by phosphorylation has
been implicated in the regulation of VEGF mRNA and protein
synthesis (29, 34). To determine whether the activation of MAP kinase activated p70S6 kinase and S6, Western blot analysis using
phospho-specific antibodies was performed. The activation of MAP kinase
led to constitutive activation of p70S6 kinase and S6 (Fig.
8).

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Fig. 8.
Constitutive activation of MAP kinase results
in activation of 70S6 kinase and S6. Left
lane represents lysates from L10BIOBR GFP vector control
cells, and right lane represents lysates from L10BIOBR MAPKK
expressing cells. Western analysis demonstrates constitutive
phosphorylation (activation) of 70 S6 kinase and S6 in response to
constitutive MAPK activation.
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 |
DISCUSSION |
Melanoma is a common malignant tumor with a rapidly increasing
incidence. Although early melanoma is curable through surgical excision, the prognosis of advanced melanoma is dismal. Therefore, knowledge of the signal transduction pathways involved in melanoma genesis is crucial for the future treatment of melanoma. Most melanomas
are thought to arise in precursors termed atypical nevi, and transition
zones have been observed in atypical nevi, which show frank malignant
changes (35). Melanomas progress to a early stage called radial growth
melanoma, which is characterized by the growth of tumors along the
dermoepidermal junction, but without invasion. Further genetic changes
convert melanoma into an invasive tumor capable of three-dimensional
growth, increased angiogenesis, and metastasis (14, 36-38). Our
findings in this model closely resemble the changes observed in human
early radial growth melanoma (16) in which MAP kinase is activated and
VEGF is induced. Other investigators have implicated NF
B in melanoma
cells, but these studies have involved cell lines known to have
aggressive behavior (39, 40). A comparison of vector control
melanocytes with melanocytes overexpressing activated MAP kinase kinase
reveals preferential activation of AP-1 rather than NF
B by MAPKK. In this system, MAPKK activation results in the activation of c-Jun and
the induction of p70S6 kinase, resulting in the induction of VEGF. This
pathway has also been described as a response to the angiogenic factor
insulin-like growth factor 1 (IGF-1), which is also capable of causing
MAP kinase activation and the subsequent induction of p70S6 kinase,
hypoxia-inducible factor-1
, and VEGF (29,34). The data presented
here support a stepwise progression, with activation of MAP kinase/AP-1
signaling being an initial step seen in early melanoma, followed by
activation of NF
B associated with increased aggressive behavior
(39-41).
Genetic changes commonly associated with melanoma include the loss of
the tumor suppressor p16ink4a and the increased expression of the
p16ink4a-suppressing gene Id-1 (42). Later changes in melanoma include activation of ras and the loss of the tumor
suppressor PTEN (43, 44). Transgenic experiments have demonstrated a synergy between oncogenic ras and p16ink4a loss
in the development of melanoma, and maintenance of melanoma is
dependent upon the continuous presence of activated ras
(45). However the pathways downstream of ras necessary for
melanoma growth in vivo are not known. The activation of
ras or the loss of PTEN is capable of activating several
signal transduction pathways, including MAP kinase signaling
(46-50).
The introduction of MAPKK into melanocytes resulted in an increase in
the levels of the gelatinase MMP-2. Interestingly, we also detected
significantly higher levels of TIMP activity in this same conditioned
media. Because the radiometric enzyme assay that was used to determine
the MMP inhibitory activity measures levels of "free" TIMP
activity, these results may suggest that the net proteolytic balance in
these cells is shifted in favor of MMP inhibition. An inhibition of MMP
by TIMP may account in part for the poorly invasive growth of the
MAPKK-transformed tumor (51, 52).
Controversy exists over the role of MAP kinase in tumorigenesis (53).
Initial studies have shown that MAP kinase activation can lead to the
transformation of NIH3T3 cells but also leads to differentiation of
PC12 cells (47, 55). We have previously demonstrated that inhibition of
MAPK signaling with a dominant negative MAPKK leads to induction of MMP
and does not inhibit tumorigenesis in a murine model of angiosarcoma
(21). Consistent with our findings in mice, we have also demonstrated
that the expression of activated MAPK decreases with increasing
malignancy in human endothelial tumors (18). Thus, the role of MAP
kinase activation in promoting tumorigenesis may be dependent upon the tissue and tumor suppressor context of a given tumor (4, 56). We felt
that MAPK was a strong candidate in melanoma, because activated MAP
kinase is expressed in melanoma but not benign nevi (54, 57).
Our results suggest that inhibition of MAP kinase may be a therapeutic
target in melanoma. In addition, immunohistochemical analysis of a
given tumor type with antibodies specific for active MAP kinase may
help determine whether this gene is involved in the angiogenic switch
in a given tumor type and help guide therapy.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M212929200
The abbreviations used are:
VEGF, vascular
endothelial growth factor;
MAP, mitogen-activated protein;
MAPK, MAP
kinase;
MAPKK, MAPK kinase;
AP-1, activator protein 1;
NF
B, nuclear
factor
B;
TIMP, tissue inhibitor of matrix metalloproteinase;
GFP, green fluorescent protein;
RT, reverse transcription;
MMP, matrix
metalloproteinase.
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