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Tumor Biology |
Expression in Head and Neck Squamous Carcinoma and Inhibition by Anti-Epidermal Growth Factor Receptor Treatments1
Laboratory of Oncology Research, Medical Oncology Service [J. Al., J. C-S., F. R., J. M. D. C., J. An., J. B.]; Centre de Transfusió i Banc de Teixits [S. S.]; Maxillofacial Surgery Service [G. R.]; Radiation Oncology Service [J. G.]; Epidemiology Service [J. R.]; and Universidad Autónoma de Barcelona [G. R., J. G., J. B.], Vall d’Hebron University Hospital, Barcelona 08035, Spain; Tenovus Cancer Research Center, Department of Pharmacology, Welsh School of Pharmacy, University of Cardiff, Cardiff, CF13XF United Kingdom [R. I. N.]; and The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030-4009 [J. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TGF-; P < 0.001), and HER2 (P = 0.066;
positive trend) correlated with activation of ERK1/2. In a multivariate
analysis, both TGF- (P < 0.0001) and
HER2 (P = 0.045) were independently
correlated with ERK1/2 activation. In turn, activation of ERK1/2 was
associated with a higher Ki-67 proliferative index
(P = 0.002). In EGF receptor-dependent
model cells (A431 and DiFi), a specific EGF receptor tyrosine kinase
inhibitor ("Iressa"; ZD1839) and a chimeric anti-EGF receptor
antibody ("Cetuximab"; C225) inhibited ERK 1/2 activation at
concentrations that inhibited autocrine cell proliferation. In patients
on treatment with C225, the activation of ERK1/2 in skin, an EGF
receptor-dependent tissue, was lower compared with control skin.
Parallel changes were seen in keratinocyte Ki67 proliferation indexes
in skin from C225-treated patients. Taken together, these studies
provide support for a role of activation of ERK1/2 in head and neck
squamous carcinoma and a correlation with EGF receptor/TGF-
expression. The inhibition of ERK1/2 activation in vitro
and in vivo by compounds targeting the EGF receptor
points to the interest of ERK1/2 as potential surrogate markers of
EGF-receptor signaling in clinical therapeutic studies. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence support the EGF receptor as a target for
therapy of head and neck carcinomas. EGF receptor and one of its
ligands, TGF-, are overexpressed in the majority of head and neck
tumors (Ref. 4
and references therein), and this
overexpression correlates with a poor clinical outcome. Furthermore,
inhibitors of receptor function such as MAbs and tyrosine kinase
inhibitors have antiproliferative effects on EGF receptor-expressing
cancer cells (5, 6, 7, 8)
. Recently (9, 10, 11)
,
clinical studies have demonstrated activity of anti-EGF receptor
therapies in patients with advanced malignancies, and studies are
currently under way with these agents in patients with head and neck
carcinomas.
As these agents move into the clinic, a better understanding of the EGF receptor-dependent pathways in vivo and their pattern of expression/activation would be of interest for at least two reasons. First, they may be of assistance in predicting the subset of EGF receptor-positive tumors that will benefit from therapy; second, downstream signaling transduction molecules may prove to be useful surrogate markers of complete receptor blockade. This latest point is particularly relevant with this novel type of agents because an optimal biological dose (i.e., a dose resulting in complete receptor inhibition) would be preferred to the maximally tolerated dose that is being used with conventional nontargeted chemotherapeutic agents (12) .
A major signaling route of EGF receptor is the Ras-Raf-MAPK pathway (2) . Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs (13) . The MAPKs ERK1 and ERK2 are activated by dual phosphorylation on a tyrosine and a threonine residue by dual specificity kinases. ERK1/2 subsequently regulate cell transcription and have been linked to cell proliferation, survival, and transformation in laboratory studies (13) . Elevated levels of MAPK activation in tumor tissues compared with their corresponding non-neoplastic tissues have been reported recently (14, 15, 16, 17, 18, 19, 20, 21) in several human tumors, although it remains uncharacterized in head and neck carcinomas.
In the present study, we have analyzed in a large series of head and
neck squamous carcinomas the expression of activated ERK1/2 and their
relationship with EGF receptor/TGF- expression and proliferation in
a clinical setting. Activated ERK1/2 were assessed by immunostaining
with an antibody specific for the dually phosphorylated and activated
MAPKs ERK1 and ERK2 (phospho-p44/42 MAPK; Ref. 22
). We
have found that activated ERK1/2 were present at different levels in
the majority of tumors, and expression levels were correlated with EGF
receptor/TGF- expression and with the tumor proliferative index. We
then explored whether the level of activated ERK1/2 expression could be
used as a marker of EGF-receptor function inhibition and
antiproliferative effects of anti-EGF receptor-targeted therapies.
Using two human tumor cell lines that have extensively been
characterized to be EGF receptor-dependent, we observed a marked
correlation between cell growth suppression and inhibition of ERK1/2
activation with two anti-EGF receptor agents currently in clinical
trials, the tyrosine kinase inhibitor ZD1839
(Iressa6
) and the MAb C225
(Cetuximab7
). In addition, we show in skin, a well-characterized EGF
receptor-dependent tissue (23, 24, 25, 26, 27)
, from patients treated
with C225 that the activation of ERK1/2 was lower compared with control
skin. This decrease in activated ERK1/2 was accompanied by a decrease
in keratinocyte proliferation. Taken together, these studies show that
activated ERK1/2 are present and correlated with EGF
receptor/TGF- in head and neck tumors and that they are
potential surrogate markers of EGF-receptor activation or inhibition
that should be further explored in the clinic.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and HER2 receptor), and
proliferative index (Ki-67). We also analyzed paired primary tumors and
relapsed tissue specimens from 19 patients of this series who suffered
a relapse. Overall, 120 specimens were analyzed from the 101 patients.
Demographic and treatment data had been recorded previously. Clinical
tumor staging was performed according to the TNM classification of
malignant tumors (28)
. Slides were reviewed for tumor
grade. We also studied 10 control skin samples (i.e., from patients not treated with EGF-receptor inhibitors) from our pathology archives and skin samples from four head and neck cancer patients participating in clinical trials with C225. Two biopsies from these C225-treated patients were from rashes that developed during treatment with C225 plus radiation therapy. The other two patients were on treatment with C225 plus cisplatin, and biopsies were from macroscopically normal skin. In one of them, a paired baseline (before C225 treatment) skin biopsy was also available. A separate written informed consent was obtained for these skin biopsies.
Compounds and Antibodies.
EGF receptor tyrosine kinase inhibitor ZD1839 (kindly provided by
AstraZeneca; Ref. 7
) and anti-EGF receptor MAb C225
(kindly provided by Imclone Systems; Ref. 5
) were used for
in vitro assays. The primary antibodies used were: rabbit
polyclonal phospho-p44/42 MAPK (Thr202/Tyr204) antibody to activated
ERK1/2 (New England BioLabs Inc., Beverly, MA), rabbit polyclonal
ERK1/2 antibody to total ERK1/2 (New England BioLabs Inc.), mouse
antibody to external domain of EGF receptor (BioGenex, San Ramon, CA),
mouse MAb Ab2 to TGF- (Oncogene Science, Cambridge, MA), mouse MAb
CB-11 to HER2 (BioGenex), and mouse MAb B126.1 to Ki-67 (Biomeda Corp.,
Foster City, CA). Two negative control rabbit polyclonal
immunoglobulins (BioGenex; Santa Cruz Biotechnology Inc., Santa Cruz,
CA) and a negative control mouse monoclonal immunoglobulin (BioGenex)
were also used.
Immunohistochemistry.
All of the specimens had been fixed in 10% buffered neutral formalin,
dehydrated, and paraffin embedded. Immunostaining was performed using
4-µm tissue sections placed on poly-L-lysine-coated glass
slides. After deparaffinization in xylene and graded alcohols, epitope
retrieval was performed. Target retrieval for activated ERK1/2,
TGF-, HER2, and Ki-67 was made in 10 mM EDTA buffer (pH
8) for 10 min in a microwave at 600 W. Epitope retrieval for EGF
receptor was made by pepsin digestion for 10 min. After epitope
retrieval, endogenous peroxidase was blocked by immersing the sections
in 0.03% hydrogen peroxide for 15 min. Slides were washed for 5 min
with PBS. Incubations with primary antibodies were made at room
temperature for 1 h at the following dilutions: activated ERK1/2
1:50, total ERK1/2 1:50, EGF receptor 1:30, TGF- 1:50, HER2
1:20, and Ki-67 1:1 (prediluted form). The peroxidase-labeled polymer
conjugated to goat antirabbit (activated ERK1/2 and total ERK1/2) or
antimouse (EGF receptor, TGF-, HER2, and Ki-67) method was used to
detect antigen-antibody reaction (DAKO EnVision+ System; DAKO
Corporation, Carpinteria, CA) for 30 min at room temperature. Sections
were then visualized with 3,3'-diaminobenzidine as a chromogen for 5
min and counterstained with Mayer’s hematoxylin. Slides were washed in
tap water, dehydrated, and mounted with glass coverslips. Positive
controls were sections of a tissue specimen previously found to be
positive for the antigen of interest. The negative controls consisted
of duplicate sections of the same specimens in which the primary
antibody had been excluded and replaced with PBS or negative control
immunoglobulins. The controls for activated and total ERK1/2 were
stained with the same amount of antihuman polyclonal rabbit IgG instead
of the primary antibodies. The controls for EGF receptor, TGF-,
HER2, and Ki-67 were stained with the same amount of antihuman
monoclonal mouse IgG instead of the primary antibodies. Representative
tumor sections were identified on a light microscope with an ocular
magnification of x25. To score a tumor cell as positive for a given
marker, complete membrane staining was required for EGF receptor and
HER2, cytoplasmic or membrane staining for TGF-, and nuclear
staining for activated ERK1/2 or Ki67. The percentage of stained tumor
cells was scored from these sections in 10 high-power fields (x400),
and the average percentage of tumor cell staining for each antibody was
calculated (by F. R.). Tumors with >1% of tumor cells staining for a
given marker were considered positive for such marker
(29)
. Grading of positivity ranged from a score of 1% to
100%. This scoring was used for statistical correlation analysis
between the studied markers and was not intended to provide a
clinical cutoff value. The same scoring system was used for skin
specimens. Scoring was blinded to the clinical data.
Cells and Monolayer Growth Assay.
Two human tumor cell lines that overexpress the EGF receptor, A431
vulvar squamous carcinoma cells and DiFi colon adenocarcinoma cells
(5
, 6)
, were used in this study. We confirmed the
overexpression of the EGF receptor in both A431 and DiFi cells by
immunocytochemistry, although we did not detect HER2 staining. Cells
were grown in monolayer culture with DMEM:Ham’s F-12 (1:1) with 10%
FBS at 37°C and 5% CO2. For monolayer growth
assay, cells were seeded in 6-well culture plates (model 3046; Falcon,
Lincoln Park, NJ) at 104
cells/cm2. The next day, cells were changed to
medium containing 0.5% FBS for 18 h, and ZD1839 or C225 was added
at various concentrations to the cultures. After 72 h, cells were
washed once with PBS, harvested with 0.1% trypsin-1 mM
EDTA in PBS, and counted with a Coulter counter.
Western Immunoblotting.
Western immunoblotting was performed as reported previously
(6)
with minor modifications. Cells were seeded in
parallel and under the same conditions as for monolayer growth assays.
The monolayers were then exposed to the indicated concentrations of
ZD1839 or C225. After 2 h, the medium was removed, the cells were
washed twice with cold PBS, and the monolayer was scraped into 1 ml of
ice-cold lysis buffer [50 mM HEPES (pH 7.0), 10%
glycerol, 1% Triton X-100, 5 mM EDTA, 1 mM
MgCl2, 25 mM NaF, 50 µg/ml
apronitin, 50 µg/ml leupeptin, 0.5 mM orthovanadate, and
1 mM phenylmethylsulfonyl fluoride]. The lysates were
transferred to a clean microfuge tube, placed on ice for 15 min, and
centrifuged for 10 min at 14,000 rpm. The supernatant was transferred
to a clean microfuge tube, and protein concentration was determined.
Protein extracts (50 µg) were boiled in Laemmli buffer 2x and
resolved on a 10% SDS-polyacrylamide gel, before transferring to a
nitrocellulose membrane. Membranes were blocked in Tris-buffered saline
[50 mM Tris-HCl (pH 7.4), 150 mM NaCl] with
5% nonfat milk overnight at 4°C and then incubated with antibodies
to total ERK1/2 (1:1000) or to activated ERK1/2 (1:1000) for
2 h at room temperature. Subsequently, membranes were washed three
times for 5 min each in the same solution and then incubated for 60–90
min with an antirabbit IgG horseradish peroxidase-conjugated secondary
antibody (Amersham Pharmacia Biotech, Freiburg, Germany). Subsequently,
membranes were vigorously washed four times for 5 min each in the same
solution, followed by a quick incubation using an ULTRA
SuperSignal chemiluminescence substrate (Pierce, Rockford,
Illinois).
Immunocytochemistry.
Cells were seeded on positive-charged slides placed in culture
plates under the same conditions used for monolayer growth assays and
Western blot assays. On the next day, cells were changed to medium
containing 0.5% FBS for 18 h, and then ZD1839 or C225 was added
at the indicated concentrations to the cultures for 2 h. After
2 h, the slides were removed from the culture plates, and cells on
the slides were fixed in 2% paraformaldehyde solution at 4°C
for 30 min and then treated with 1% BSA and 0.1% saponine to
permebealize cells at room temperature for 30 min. Nonspecific
protein reactivity was blocked with Protein Block (BioGenex) for 10
min, and endogenous peroxidase activity was blocked in hydrogen
peroxide solution at 0.01% for 10 min. The antibodies used were the
same as for the immunohistochemistry assays. Specimens were incubated
for 1 h at room temperature with the antibody to total ERK1/2
(dilution, 1:100) or to activated ERK1/2 (dilution, 1:100).
Immunodetection of antigens was made with Super Sensitive
Immunodetection System (BioGenex). To detect the binding of antibody to
antigen, slides were incubated with biotinylated
antirabbit-immunoglobulins for 20 min at room temperature and then with
horseradish peroxidase-labeled streptavidin complexes for 20 min at
room temperature. Slides were then visualized using
3–3'-diaminobenzidine as a chromogen for 1 min. Then, specimens were
dehydrated and coverslipped in nonaqueous permanent mounting media.
Positive and negative controls were included in each staining run.
Statistical Methods.
All of the statistical analyses were carried out using SPSS Data
Analysis Program version 9.0. A Kolmogorov-Smirnov test was applied to
determine whether a variable followed a normal distribution.
Pearson’s correlations were made between continuous variables
(i.e., percentage of squamous carcinoma cell staining) that
followed a normal distribution in our series of 101 tumors, and
nonparametric Spearman’s correlations were made when one or both of
the covariables did not follow a normal distribution. Activated ERK1/2,
EGF receptor, and Ki67 followed a normal distribution and TGF- did
not. In the case of HER2, because most of the tumors were zeros
(n = 82), correlation was calculated only for
those tumors that were HER2 positive (n = 19)
and followed a normal distribution. A multivariate regression analysis
that included all of the cases was planned to assess the independent
value of the association between all of the EGF receptor-signaling
members analyzed that may interact in vivo (i.e.,
EGF receptor, TGF-, and HER2) and activated ERK1/2, assessed on a
continuous scale. Paired samples were analyzed using the t
test for paired samples. The relationship between categorical variables
and continuous variables was analyzed by the Fisher’s exact test or
Mann-Whitney U test. All of the statistical tests were
conducted at the two-sided 0.05 level of significance.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At sampling, only 11 patients had received prior therapy, which
consisted of chemotherapy and radiation.
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; Ref. 19
). A subset (n = 20) of tumors was analyzed with an antibody to total ERK1/2.
Diffuse cytoplasmic staining was seen with this antibody to total
ERK1/2 in the majority of epithelial and stromal cells. Nuclear
staining for total ERK1/2 was observed in some tumor cells located in
areas that had tumor cells with nuclear staining for activated ERK1/2
in adjacent tissue slides (Fig. 1B)
. This differential
localization of total ERK1/2 staining, mainly in the cytoplasm if
phospho-ERK1/2-negative and nuclear if phospho-ERK1/2-positive, is most
likely attributable to the activation of ERK1/2 that involves their
translocation from the cytoplasm to the nucleus and represents further
proof of the specificity of the antibodies used (13)
.
Replacement of the primary antibody by the same amount of negative
control polyclonal rabbit IgGs (two negative controls from different
sources) did not show staining (Fig. 1, C and D)
.
|
. In head and neck squamous carcinoma cells, staining for activated
ERK1/2 was seen in the nucleus, and in general, a less intense
cytoplasmic staining was seen (Fig. 1A)
. Nuclear
staining was predominantly seen at the advancing margins of the tumor.
In 6 of the 101 head and neck carcinoma specimens analyzed, a frozen
tissue fragment of the same tumor specimen that had been
paraffin-embedded was available. A Western blot analysis of activated
ERK1/2 in those six samples showed a good relationship between the
scored percentage of positive tumor nuclei staining and Western blot
assay results (data not shown). Higher levels of activated ERK1/2,
measured by the percentage of tumor nuclei cell staining in every
specimen, were associated with a more advanced N stage
(N0–2 versus
N3; Mann-Whitney U test;
P = 0.048). Poorly differentiated tumors had
higher levels of activated ERK1/2 than did well- or moderately
differentiated tumors, but the difference did not have statistical
significance (P = 0.104). No trends for
associations were seen between activated ERK1/2 and T stage, sex, age,
or tumor site. In 19 patients who relapsed after initial therapy,
higher levels of activated ERK1/2 in the relapsed specimens were
detected as compared with their corresponding primary tumor specimens
(Fig. 1, E–F
; paired t test;
P = 0.021).
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, and the closely related HER2 receptor (Table 2)
. TGF- was
chosen because it is considered the predominant EGF receptor
ligand expressed in malignancy (30)
. HER2 was included
because this receptor also signals through ERK1/2 and is the preferred
partner for EGF receptor dimerization (2)
, although it is
detected in a limited subset of head and neck carcinomas (Ref.
31
; Table 2
). A significant relationship was observed
between activated ERK1/2 and EGF receptor (n = 101; Pearson, r = 0.21;
P = 0.037) and activated ERK1/2 and TGF-
(n = 101; Spearman, r = 0.37; P < 0.001; Fig. 2, A–B
). In the case of HER2, because most of the tumors were
negative (Table 2)
, the correlation between HER2 levels and ERK1/2
activation was calculated only for those tumors that were HER2 positive
(n = 19). This analysis showed a trend to a
positive association between HER2 and ERK1/2 activation levels
(Pearson, r = 0.43; P = 0.066; Fig. 2, A and C
). A linear
multivariate analysis that included all of the cases was made to assess
the independent value of the association between all of the EGF
receptor-signaling members analyzed that may interact in vivo (i.e., EGF receptor, TGF-, and HER2) and
activated ERK1/2. In this analysis, both TGF-
(P < 0.0001) and HER2
(P = 0.045) were independently associated
with activated ERK1/2. EGF receptor expression lost its statistical
significance in the multivariate analysis.
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and Fig. 2, D and F
; Ref.
32
). A significant correlation was observed between
activated ERK1/2 and Ki-67 staining (n = 101;
Pearson, r = 0.30; P = 0.002; Fig. 2D
). Further supporting the association,
the pattern of staining for both activated ERK1/2 and Ki-67 was
predominantly located in the periphery of the tumors, a finding that
may be related to the highest level of tumor proliferation at the edges
(Fig. 2, E and F)
. The EGF receptor
family-signaling members analyzed did not correlate with the
proliferative index.
EGF Receptor Tyrosine Kinase Inhibitor ZD1839 and Anti-EGF Receptor
MAb C225 Induce Suppression of ERK1/2 Activation at Doses Resulting in
Growth Inhibition.
To further define the possible role of activated ERK1/2 as a marker of
EGF receptor activation and of the antiproliferative effects of EGF
receptor-targeted therapies, a series of studies were performed
in vitro. For these studies, we used two human cell lines,
A431 and DiFi, that have high levels of EGF receptor expression and
that have been extensively characterized by us and others (5
, 6)
to be EGF receptor-dependent. In these model cells, we
analyzed the ability of the EGF receptor tyrosine kinase inhibitor
ZD1839 (7
, 9)
and of the chimeric anti-EGF receptor MAb
C225 (5
, 12)
to inhibit autocrine growth and activation of
ERK1/2. To evaluate whether a correlation exists between concentrations
of ZD1839 or C225 that inhibited growth and ERK1/2 activation, cell
cultures driven by endogenous (autocrine) ligand were incubated with
increasing concentrations of each compound. For Western blot assays,
cells were incubated in the presence of compound for 2 h and then
lysed. For proliferation assays, cells were incubated in the presence
of compound for 72 h and then counted. Both ZD1839 (Fig. 3, A and B)
and C225 (Fig. 3, C and D)
inhibited the growth of these cells at concentrations of
drug that can be achieved in patients. The effects on A431 cells and
DiFi cells were similar but not identical, because DiFi cells were more
sensitive to the growth inhibitory effects of the compounds analyzed
and the degree of inhibition of ERK1/2 activation was greater in DiFi
cells. Regardless of this, the concentrations of ZD1839 or C225 that
caused a decline in cell proliferation coincided with concentrations
that reduced or abolished endogenous ERK1/2 activation in both cell
lines, as assayed by Western blot with the activation state-specific
antibody (Fig. 3)
. These effects on ERK1/2 activation were unrelated to
down-regulation of total (phosphorylation state-independent) ERK1/2
proteins, because their levels did not decrease by treatment (Fig. 3)
.
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. Next, we studied in vitro whether
a differential cellular distribution of ERK1/2 exists as a function of
activation, as suggested from the observations in tumor samples. A
series of immunocytochemical analyses were performed using the A431
(Fig. 4A)
and DiFi cells (Fig. 4B)
under the same culture
conditions used for the proliferation and Western blot assays. We used
immunocytochemistry to provide subcellular localization of total and
activated ERK1/2 in situ. The cells were directly seeded in
immunocytochemistry slides placed on culture plates. This approach
avoids the steps of cell harvesting and centrifugation that would have
been needed if cells were grown directly in culture plates and that
might affect cell biology and morphology.
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The Anti-EGF Receptor MAb C225 Inhibits ERK1/2 Activation and
Proliferation in Patients.
Our observations in EGF receptor model cell lines indicated a tight
association between inhibition of growth by anti-EGF receptor compounds
(C225 and ZD1839) and inhibition of ERK1/2 activation, leading to the
concept that activated ERK1/2 might be a useful surrogate marker of
activity of these compounds. To assess whether an anti-EGF receptor
compound could also reduce ERK1/2 activation in patients, we assayed
activated ERK1/2 in skin specimens from controls versus
patients treated with C225. We chose to study skin because the basal
layer of the epidermis has high levels of EGF receptor expression and
the receptor plays a critical role in the regulation of epidermal
biology (23, 24, 25, 26, 27)
. Furthermore, C225 may cause skin
acneiform rashes, suggesting that this skin toxicity could be an
indirect indicator of receptor targeting by C225 (12)
, and
skin is easily accessible to biopsies.
We confirmed the expression of EGF receptor in epidermal and hair
follicle keratinocytes in all of the control and experimental (from
patients treated with C225) skin specimens analyzed and found a higher
level of EGF receptor expression in keratinocytes of the basal layer of
the epidermis (data not shown). Then, we characterized by
immunohistochemistry the expression of activated ERK1/2 in 10 normal
skin specimens using the same phospho-specific antibody to ERK1/2 that
we used for the studies performed in clinical head and neck tumors and
in tumor cells lines. In these control skin specimens (i.e.,
from patients not treated with an EGF receptor inhibitor), activated
ERK1/2 was mostly expressed in the nuclei of keratinocytes of the basal
layer of the epidermis and in the outer root sheath of the hair
follicles (Fig. 5, A and B)
, colocalizing with the population of
proliferating, undifferentiated keratinocytes, where the EGF receptor
is expressed at highest levels (Ref. 27
and data not
shown). The percentage of keratinocytes with nuclei staining to
activated ERK1/2 was scored in interfollicular epidermis in 10
high-power fields (x400). Hair follicles were qualitatively analyzed
when present, but no scoring was conducted because many samples lacked
them. In these 10 control samples, the percentage of basal
keratinocytes staining for activated ERK1/2 was 25 ± 7% (mean and SD).
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200
mg/m2 achieve sustained serum antibody
concentrations above 200 nmol/liter, a concentration high enough to
result in optimal antitumor activity in preclinical models. Therefore,
we should be able to demonstrate C225 inhibition of ERK1/2 activation
in these treated patients if the antibody inhibits EGF receptor
signaling in vivo. Two patients were on treatment with C225,
and radiation and skin biopsies were taken from skin rashes that
developed during therapy in nonirradiated areas. The other two patients
were on treatment with C225 plus cisplatin, and skin biopsies were
taken from macroscopically normal skin. In one of them, a baseline skin
biopsy was also available. Overall, in the four on-therapy biopsies,
there were low levels of activated ERK1/2 expression in keratinocytes
of the basal layer of the epidermis (Fig. 5C)
. The average
percentage of positive basal keratinocytes from the four C225-treated
patients was 5 ± 2% compared with an average
25 ± 7% positive basal keratinocytes from 10 control
specimens. There was also an evident reduction of activated ERK1/2 in
the outer root sheath of the hair follicle, as compared with the
expression seen in control skin specimens (Fig. 5D)
. In a
patient that underwent serial pre- and on-treatment skin biopsies, the
expression of activated ERK1/2 was clearly reduced during C225
treatment (i.e., 20% pre-C225 versus 3%
on-C225; Fig. 5
).
On the basis of the correlation between ERK1/2 activation and Ki67
proliferation index in our series of head and neck tumors and on the
association between growth inhibition and inhibition of activated
ERK1/2 in EGF receptor-dependent cell lines, we hypothesized that the
reduced levels of ERK1/2 activation observed in skin from patients
treated with C225 should be accompanied by a reduced proliferation
rate. To study this possibility, we scored the percentage of basal
keratinocytes with Ki67 staining in control versus
C225-treated skin biopsies. The Ki-67 staining colocalized with
activated ERK1/2; i.e., mainly in basal layers and in the
outer root sheath (Fig. 6, A and B)
. We observed a reduced keratinocyte
proliferation index in skin biopsies taken during C225 treatment
[n = 4; Ki67 proliferation index,
7 ± 4% (average and SD)] versus control
specimens [n = 10; Ki67 proliferation index,
32 ± 12% (average and SD); Fig. 6C
].
Inhibition of activated ERK1/2 was also seen in hair follicles (Fig. 6D)
. In the patient that underwent serial pre- and
on-treatment skin biopsies, keratinocyte proliferation was also clearly
reduced during C225 treatment (i.e., 25% pre-C225
versus 2% on-C225; Fig. 6
). This finding of a parallel
effect of C225 treatment in patients on ERK1/2 activation and on
proliferation in a human EGF receptor-dependent tissue further supports
the notion of a biological effect of C225 on receptor downstream
processes in vivo.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We chose to study ERK1/2 because of
its critical role in the EGF receptor downstream-signaling pathway
(2
, 13) , the known expression of total ERK1/2 in head and
neck tumors (33)
, and the availability of reagents to
study their level of activation in tumor samples at a cellular level.
The levels of expression of activated ERK1/2 correlated with a higher
nodal state and a higher proliferative rate and were increased in tumor
relapses. We found that the levels of activated ERK1/2 correlated with
EGF receptor, TGF-, and HER2 expression. In addition, inhibition of
ERK1/2 activation in EGF receptor-dependent model cell lines with two
different anti-EGF receptor therapies correlated tightly with growth
inhibition, supporting its potential role as a surrogate marker
in vivo of EGF receptor inhibition. We also showed that
activation of ERK1/2 was lower in skin, an EGF receptor-dependent
tissue (23, 24, 25, 26, 27)
, from patients treated with C225 compared
with control skin, and this decrease in activated ERK1/2 was
accompanied by a decrease in keratinocyte proliferation. In head and neck squamous carcinoma, overexpression of total ERK1/2 had been observed (33) , but the expression of the activated forms, which are necessary for EGF receptor-dependent signaling processes, was yet uncharacterized. We now show in a series of 101 primary head and neck squamous carcinomas that activated ERK1/2 is expressed in the nuclei of tumor cells in the majority of specimens, as determined by immunohistochemistry in paraffin-embedded specimens with an antibody that recognizes only the phosphorylated (activated) ERK1/2 (see "Results" and Ref. 22 ). This antibody allows the assessment of the activation state of ERK1/2 in situ and provides subcellular resolution of their nuclear translocation. We observed higher levels of ERK1/2 activation in tumors from patients with more advanced lymph node disease and in relapsed specimens compared with their corresponding primary tumors. These findings indicate that ERK1/2 are commonly activated in head and neck tumors and suggest they have a role in their malignant progression.
In our series of head and neck tumors, the levels of activated ERK1/2
were linked to the expression of EGF receptor-signaling members,
further supporting that the Ras-Raf-MAPK pathway is activated by the
EGF receptor in vivo. Increased levels of activated ERK1/2
were significantly correlated with the expression of the EGF receptor
and TGF-. In a multivariate analysis, TGF-, but not the EGF
receptor, independently associated with ERK1/2 activation, suggesting
that autocrine/paracrine stimulation of the EGF receptor by ligand may
be required for ERK1/2 activation. Perhaps, in head and neck tumors the
presence of TGF- may be required in addition to the almost universal
expression of the EGF receptor to activate the receptor tyrosine kinase
activity and drive critical downstream events, because it would be
predicted from laboratory studies showing that genetically induced
overexpression of EGF receptor ligands leads to cell transformation in
the presence of the receptor (3)
. Taking into
consideration that ERK1/2 may be activated by several pathways, the
significant correlations observed between EGF receptor
(P = 0.037) and TGF-
(P < 0.001) with activated ERK1/2 points to
an important role of the EGF receptor in activating ERK1/2 in many head
and neck tumors. The correlation coefficients (r) were less
than 0.5 (r = 0 would be lack of correlation;
r = 1 would be an absolute correlation in
every single case), suggesting that ERK1/2 may also be activated by
other receptors or downstream molecules in certain head and neck
tumors. One example suggested in the present study was HER2, which in a
multivariate analysis was associated with activated ERK1/2
independently of the EGF receptor and TGF-. In the case of HER2, as
expected (31)
, the number of HER2-overexpressing tumors
was low. However, in those tumors that overexpressed HER2, a trend to a
positive correlation between HER2 levels and ERK1/2 activation was
found. When included in a multivariate analysis, HER2 was independently
correlated with activation of ERK1/2. This finding may reflect that
HER2 receptor activation leads to ERK1/2 activation in head and neck
tumors or, alternatively, that HER2 availability for heterodimerization
with EGF receptor is rate-limiting for EGF-mediated signal transduction
and proliferation (34)
. However, the low rate of HER2
positivity detected in this series suggests that HER2 may play a role
in a limited number of head and neck cancers. It is possible that other
receptors or Ras mutations could lead to activation of ERK1/2, because
some specimens had high levels of activated ERK1/2 without high levels
of any of the EGF receptor family members analyzed. Conversely, in some
tumors with high levels of one or more of the EGF receptor family
members analyzed, the level of activated ERK1/2 was low or even
undetected. In such tumors, it is possible that other EGF
receptor-signaling transduction pathways, such as JAK/STAT or
phosphatidyilinositol-3-kinase, are preferred instead (2
, 35)
.
A few studies have addressed the expression of activated MAPK in
nonsquamous tumors and its potential clinical/biological correlations.
In prostate cancer, high levels of activated MAPK were observed in
high-grade and advanced-stage tumors, suggesting elevated Ras signaling
in advanced disease (19)
. In glial tumors, the presence of
activated MAPK was also reported, and among these tumors,
oligodendrogliomas showed an increase in the number of cells with MAPK
activation with malignant progression (20)
. In renal cell
carcinoma, MAPK activation correlated with MAPK kinase activation and
Raf-1 activation (16)
. In hepatocarcinomas, a relationship
was reported between ERK1/2 activation and the transcription factor
c-Fos and cyclin D1 levels (17)
. In breast cancer,
activated MAPK was associated with poor prognosis and decreased
sensitivity to endocrine therapy and with the expression of
phosphorylated-jun, a transcription factor activated by MAPK
(21)
. In this series of breast adenocarcinomas, activated
MAPK was correlated with the expression of EGF receptor, but not with
TGF-, which adds support, in a different histological tumor type, to
our present finding of a link between activation of MAPK and EGF
receptor expression in clinical tumors.
In the present study, we also report a significant correlation between activated ERK1/2 and the proliferative index, assessed by using Ki-67 as a marker. It is likely, however, that other pathways, such as the JAK/STAT or phosphatidyilinositol-3-kinase mentioned above (2 , 35) , also regulate proliferation in head and neck cancers. The pattern of staining for activated ERK1/2 and Ki-67 was predominantly at the tumor edges, suggesting that the EGF receptor may be predominantly activated in these areas. Preliminary experiments in a limited number of specimens with an antibody to the activated form of the EGF receptor suggest this might be the case.8
Additionally, our findings point to a potential role for activated
ERK1/2 as surrogate markers of EGF receptor activity that may be useful
to characterize in vivo the optimal biological dose of
anti-EGF receptor treatments. To give further support to this later
proposal, we studied in vitro the ability of the EGF
receptor tyrosine kinase inhibitor ZD1839 (7)
or the
chimeric anti-EGF receptor MAb C225 (5)
, currently in
clinical development (9, 10, 11)
, to affect growth and ERK1/2
activation in EGF receptor-dependent model cell lines. The results of
these studies showed that the concentrations of ZD1839 or C225 that
inhibited proliferation driven by endogenous (autocrine) ligand were
the same that inhibited ERK1/2 activation. These effects were seen at
drug concentrations that can be achieved in patients and that inhibit
EGF receptor phosphorylation in cultured cells (5
, 7)
. A
greater inhibition of ERK1/2 activation was seen in DiFi cells compared
with A431 cells, which is in agreement with the requirement of EGF
receptor activation for both cell cycle progression and prevention of
apoptosis in DiFi cells and a role of MAPK in survival (13
, 36)
. In concordance with our findings, in TGF-/HER2 bigenic
mice treated with the EGF receptor tyrosine kinase inhibitor AG1478,
regression of mammary tumors was associated with abolishment of
constitutive MAPK activity (37)
. We also analyzed in a
series of immunocytochemistry assays the effects of ZD1839 or C225 on
the cellular distribution of total and activated ERK1/2. In both A431
and DiFi cells, treatment with these anti-EGF receptor compounds
markedly decreased activated (phosphorylated) ERK1/2 staining and
shifted total (phosphorylation-independent) ERK1/2 staining to a mainly
cytoplasmic localization. These results suggested that treatment with
ZD1839 or C225 prevents phosphorylation of ERK1/2 and results in the
presence of nonphosphorylated ERK1/2 localized mainly in the cytoplasm.
We also studied whether an anti-EGF receptor compound could inhibit ERK1/2 activation in patients. This point was studied in a series of skin specimens, because skin is a well-characterized EGF receptor-dependent tissue (23, 24, 25, 26, 27) . These studies showed that in patients treated with C225 (n = 4) there are markedly reduced levels of ERK1/2 activation in skin compared with skin from control patients (n = 10). Low levels of activated ERK1/2 in skin were seen in patients treated with C225 and radiation or C225 and cisplatin. In a patient that underwent sequential pre- and on-C225 skin biopsies, an evident inhibition of activated ERK1/2 was observed during therapy. This decrease in activated ERK1/2 was accompanied by a decrease in keratinocyte proliferation. This finding of a parallel effect of C225 treatment in patients on ERK1/2 activation and on proliferation in a human EGF receptor-dependent tissue further supports the notion of a biological effect of C225 on receptor downstream processes in vivo. In a similar line of evidence, a preliminary analysis in skin biopsies from patients treated with ZD1839 showed a significant decline in the expression of activated ERK1/2 in keratinocytes during therapy (38) . We are extending these studies to a large set of skin biopsies from patients participating in ZD1839 Phase I trials to correlate the degree of ERK1/2 inhibition with clinically meaningful end points. The next challenge is to study activated ERK1/2 as a surrogate marker of EGF receptor inhibition in tumors from patients treated with anti-EGF receptor compounds. Such studies are currently ongoing based on the expression of activated ERK1/2 from our current series and the encouraging data obtained in nontumor skin biopsies post-C225 (current study) or post-ZD1839 therapy (38) .
In summary, the data reported here show the presence of activated
ERK1/2 in most primary head and neck squamous carcinomas. Elevated
levels of activated ERK1/2 were associated with advanced lymph node
metastasis and higher proliferation and were increased in relapses,
suggesting a role for activated ERK1/2 in the malignant progression of
these tumors. Our work also indicates that EGF receptor/TGF-
expression correlates with ERK1/2 activation in vivo.
Furthermore, two different anti-EGF receptor treatments inhibited
proliferation of EGF receptor-dependent model cell lines at
concentrations that inhibited ERK1/2 activation. We also found that
activation of ERK1/2 was lower in skin from patients treated with C225
compared with control skin, and this decrease in activated ERK1/2 was
accompanied by a decrease in keratinocyte proliferation. As a result,
we are incorporating assessment of ERK1/2 activation levels in pre- and
post-therapy tumor biopsies from patients treated with the anti-EGF
receptor tyrosine kinase inhibitor ZD1839 or with the MAb C225 to
provide evidence for successful inhibition of EGF receptor function
in vivo and to determine whether there is a correlation
between levels of activated ERK1/2 and response to therapy.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by Spanish Health Ministry
Grant "Fondo de Investigación Sanitaria" (99/0020-01; to
J. Al. and J. B.) and a Grant from Bristol-Myers Squibb Spain. 
2 J. A. and J. C-S. have contributed equally to
this work.
3 Dr. John Mendelsohn is on the Board of Directors
of ImClone Systems, Inc., and also holds stock options.<./>
4 To whom requests for reprints should be
addressed, at Medical Oncology Service, Vall d’Hebron University
Hospital, Paseo Vall d’Hebron 119–129. Barcelona 08035, Spain. Phone:
34-93-274-6077; Fax: 34-93-274-6059; E-mail: baselga{at}hg.vhebron.es
5 The abbreviations used are: EGF, epidermal
growth factor; MAPK, mitogen-activated kinase; ERK, extracellular
signal-regulated kinase; TGF, transforming growth factor; MAb,
monoclonal antibody; FBS, fetal bovine serum; T, primary tumor stage;
N, regional lymph node stage; TNM, Tumor-Node-Metastasis.
6 "Iressa" is the property of the AstraZeneca
group of companies.
7 "Cetuximab" is the property of Imclone
Systems Inc.
Received 7/17/00. Accepted 7/ 2/01.
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U. Vanhoefer, M. Tewes, F. Rojo, O. Dirsch, N. Schleucher, O. Rosen, J. Tillner, A. Kovar, A. H. Braun, T. Trarbach, et al. Phase I Study of the Humanized Antiepidermal Growth Factor Receptor Monoclonal Antibody EMD72000 in Patients With Advanced Solid Tumors That Express the Epidermal Growth Factor Receptor J. Clin. Oncol., January 1, 2004; 22(1): 175 - 184. [Abstract] [Full Text] [PDF]
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M. Nunes, C. Shi, and L. M. Greenberger Phosphorylation of extracellular signal-regulated kinase 1 and 2, protein kinase B, and signal transducer and activator of transcription 3 are differently inhibited by an epidermal growth factor receptor inhibitor, EKB-569, in tumor cells and normal human keratinocytes Mol. Cancer Ther., January 1, 2004; 3(1): 21 - 27. [Abstract] [Full Text]
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T. M. Grana, C. I. Sartor, and A. D. Cox Epidermal Growth Factor Receptor Autocrine Signaling in RIE-1 Cells Transformed by the Ras Oncogene Enhances Radiation Resistance Cancer Res., November 15, 2003; 63(22): 7807 - 7814. [Abstract] [Full Text] [PDF]
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 J. W. Mandell Phosphorylation State-Specific Antibodies: Applications in Investigative and Diagnostic Pathology Am. J. Pathol., November 1, 2003; 163(5): 1687 - 1698. [Abstract] [Full Text] [PDF]
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H. Niwa, A. L. Wentzel, M. Li, W. E. Gooding, V. W. Y. Lui, and J. R. Grandis Antitumor Effects of Epidermal Growth Factor Receptor Antisense Oligonucleotides in Combination with Docetaxel in Squamous Cell Carcinoma of the Head and Neck Clin. Cancer Res., October 15, 2003; 9(13): 5028 - 5035. [Abstract] [Full Text] [PDF]
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M. Lango, A. L. Wentzel, J. I. Song, S. Xi, D. E. Johnson, W. W. Lamph, L. Miller, and J. R. Grandis Responsiveness to the Retinoic Acid Receptor-selective Retinoid LGD1550 Correlates with Abrogation of Transforming Growth Factor {alpha}/Epidermal Growth Factor Receptor Autocrine Signaling in Head and Neck Squamous Carcinoma Cells Clin. Cancer Res., September 15, 2003; 9(11): 4205 - 4213. [Abstract] [Full Text] [PDF]
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 J. S. Kruger and K. B. Reddy Distinct Mechanisms Mediate the Initial and Sustained Phases of Cell Migration in Epidermal Growth Factor Receptor-Overexpressing Cells Mol. Cancer Res., September 1, 2003; 1(11): 801 - 809. [Abstract] [Full Text] [PDF]
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A. Handra-Luca, H. Bilal, J.-C. Bertrand, and P. Fouret Extra-Cellular Signal-Regulated ERK-1/ERK-2 Pathway Activation in Human Salivary Gland Mucoepidermoid Carcinoma: Association to Aggressive Tumor Behavior and Tumor Cell Proliferation Am. J. Pathol., September 1, 2003; 163(3): 957 - 967. [Abstract] [Full Text] [PDF]
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J. Mendelsohn and J. Baselga Status of Epidermal Growth Factor Receptor Antagonists in the Biology and Treatment of Cancer J. Clin. Oncol., July 15, 2003; 21(14): 2787 - 2799. [Abstract] [Full Text] [PDF]
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J. Baselga Skin as a Surrogate Tissue for Pharmacodynamic End Points: Is It Deep Enough? Clin. Cancer Res., July 1, 2003; 9(7): 2389 - 2390. [Full Text] [PDF]
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 V. Grunwald and M. Hidalgo Developing Inhibitors of the Epidermal Growth Factor Receptor for Cancer Treatment J Natl Cancer Inst, June 18, 2003; 95(12): 851 - 867. [Abstract] [Full Text] [PDF]
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M. L. Janmaat, F. A. E. Kruyt, J. A. Rodriguez, and G. Giaccone Response to Epidermal Growth Factor Receptor Inhibitors in Non-Small Cell Lung Cancer Cells: Limited Antiproliferative Effects and Absence of Apoptosis Associated with Persistent Activity of Extracellular Signal-regulated Kinase or Akt Kinase Pathways Clin. Cancer Res., June 1, 2003; 9(6): 2316 - 2326. [Abstract] [Full Text] [PDF]
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E. E. W. Cohen, F. Rosen, W. M. Stadler, W. Recant, K. Stenson, D. Huo, and E. E. Vokes Phase II Trial of ZD1839 in Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck J. Clin. Oncol., May 15, 2003; 21(10): 1980 - 1987. [Abstract] [Full Text] [PDF]
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C. L. Arteaga and J. Baselga Clinical Trial Design and End Points for Epidermal Growth Factor Receptor-targeted Therapies: Implications for Drug Development and Practice Clin. Cancer Res., May 1, 2003; 9(5): 1579 - 1589. [Full Text] [PDF]
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C. J. Barnes, R. Bagheri-Yarmand, M. Mandal, Z. Yang, G. L. Clayman, W. K. Hong, and R. Kumar Suppression of Epidermal Growth Factor Receptor, Mitogen-activated Protein Kinase, and Pak1 Pathways and Invasiveness of Human Cutaneous Squamous Cancer Cells by the Tyrosine Kinase Inhibitor ZD1839 (Iressa) Mol. Cancer Ther., April 1, 2003; 2(4): 345 - 351. [Abstract] [Full Text] [PDF]
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J. Anido, P. Matar, J. Albanell, M. Guzman, F. Rojo, J. Arribas, S. Averbuch, and J. Baselga ZD1839, a Specific Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor, Induces the Formation of Inactive EGFR/HER2 and EGFR/HER3 Heterodimers and Prevents Heregulin Signaling in HER2-overexpressing Breast Cancer Cells Clin. Cancer Res., April 1, 2003; 9(4): 1274 - 1283. [Abstract] [Full Text] [PDF]
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H. Liao, R. Bucala, and R. A. Mitchell Adhesion-dependent Signaling by Macrophage Migration Inhibitory Factor (MIF) J. Biol. Chem., January 3, 2003; 278(1): 76 - 81. [Abstract] [Full Text] [PDF]
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 N. Chegini and L. Kornberg Gonadotropin Releasing Hormone Analogue Therapy Alters Signal Transduction Pathways Involving Mitogen-Activated Protein and Focal Adhesion Kinases in Leiomyoma Reproductive Sciences, January 1, 2003; 10(1): 21 - 26. [Abstract] [PDF]
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A. Gschwind, N. Prenzel, and A. Ullrich Lysophosphatidic Acid-induced Squamous Cell Carcinoma Cell Proliferation and Motility Involves Epidermal Growth Factor Receptor Signal Transactivation Cancer Res., November 1, 2002; 62(21): 6329 - 6336. [Abstract] [Full Text] [PDF]
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J. Baselga, D. Rischin, M. Ranson, H. Calvert, E. Raymond, D.G. Kieback, S.B. Kaye, L. Gianni, A. Harris, T. Bjork, et al. Phase I Safety, Pharmacokinetic, and Pharmacodynamic Trial of ZD1839, a Selective Oral Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, in Patients With Five Selected Solid Tumor Types J. Clin. Oncol., November 1, 2002; 20(21): 4292 - 4302. [Abstract] [Full Text] [PDF]
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A. E. Wakeling, S. P. Guy, J. R. Woodburn, S. E. Ashton, B. J. Curry, A. J. Barker, and K. H. Gibson ZD1839 (Iressa): An Orally Active Inhibitor of Epidermal Growth Factor Signaling with Potential for Cancer Therapy Cancer Res., October 15, 2002; 62(20): 5749 - 5754. [Abstract] [Full Text] [PDF]
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J. B. Cordero, M. Cozzolino, Y. Lu, M. Vidal, E. Slatopolsky, P. D. Stahl, M. A. Barbieri, and A. Dusso 1,25-Dihydroxyvitamin D Down-regulates Cell Membrane Growth- and Nuclear Growth-promoting Signals by the Epidermal Growth Factor Receptor J. Biol. Chem., October 4, 2002; 277(41): 38965 - 38971. [Abstract] [Full Text] [PDF]
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P. A. Janne, M. L. Taffaro, R. Salgia, and B. E. Johnson Inhibition of Epidermal Growth Factor Receptor Signaling in Malignant Pleural Mesothelioma Cancer Res., September 15, 2002; 62(18): 5242 - 5247. [Abstract] [Full Text] [PDF]
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J. Mendelsohn Targeting the Epidermal Growth Factor Receptor for Cancer Therapy J. Clin. Oncol., September 15, 2002; 20(90001): 1s - 13. [Full Text] [PDF]
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 M. S. Iordanov, R. J. Choi, O. P. Ryabinina, T.-H. Dinh, R. K. Bright, and B. E. Magun The UV (Ribotoxic) Stress Response of Human Keratinocytes Involves the Unexpected Uncoupling of the Ras-Extracellular Signal-Regulated Kinase Signaling Cascade from the Activated Epidermal Growth Factor Receptor Mol. Cell. Biol., August 1, 2002; 22(15): 5380 - 5394. [Abstract] [Full Text] [PDF]
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