Download ErbB4 increases the proliferation potential of human lung cancer

Int. J. Cancer: 119, 269–274 (2006)
' 2006 Wiley-Liss, Inc.
ErbB4 increases the proliferation potential of human lung cancer cells and its
blockage can be used as a target for anti-cancer therapy
Alex Starr1* , Joel Greif1* , Akiva Vexler2, Maia Ashkenazy-Voghera2, Valery Gladesh3, Chanan Rubin3, Gabriele Kerber3,
Sylvia Marmor4, Shahar Lev-Ari2, Moshe Inbar2, Yosef Yarden3 and Rami Ben-Yosef2
1
Lung and Allergy Institute, Tel-Aviv Sourasky Medical Center and Tel-Aviv University School of Medicine, Tel-Aviv, Israel
2
Division of Oncology, Tel-Aviv Sourasky Medical Center and Tel-Aviv University School of Medicine, Tel-Aviv, Israel
3
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel
4
Division of Pathology, Tel-Aviv Sourasky Medical Center and Tel-Aviv University School of Medicine, Tel-Aviv, Israel
Clinical and experimental data suggest that ErbB-4, a member of
the epidermal growth factor receptor family, may have a role in
cancer progression and response to treatment. We found recently,
using a retrospective clinical analysis, that expression of ErbB-4
receptor is correlated with metastatic potential and patient survival in non-small-cell lung cancer (NSCLC). The purpose of this
work was to correlate the expression of the ErbB-4 and lung cancer cells growth in vitro and in vivo and to determine the therapeutic potential of a monoclonal antibody to ErbB-4 against lung cancer. For this aim, we ectopically expressed ErbB-4 in a human
NSCLC cell line that did not express the ErbB-4 protein. Overexpression of ErbB-4 produced a constitutively activated ErbB-4 receptor. The transfected ErbB-4 positive clones showed an increased
cell proliferation in vitro and in vivo in comparison with parental
ErbB-4 negative cells and with the cells transfected by neomycin-resistant gene. A monoclonal antibody to ErbB-4 showed both an inhibitory effect on growth rate and an increasing apoptotic rate in
the cells expressing ErbB-4. The results of the current study provide
evidence that ErbB-4 plays a significant role in human lung cancer
and may serve as a molecular target for anticancer therapy.
' 2006 Wiley-Liss, Inc.
Key words: ErbB-4; lung cancer; molecular targeted therapy
Lung cancer is the leading cause of cancer death among both men
and women in the western world, and more lethal than breast, colorectal and prostate cancers combined together.1 The cure rate remains
<15% despite some recent advances in chemo- and radiotherapy.2
Progress in molecular biology has increased our understanding of
processes involved in oncogenesis, and thus has led to the development of targeted therapies against dominant oncogenes, which are
frequently overexpressed in tumor tissue because of gene amplification, increased chromosome copy number, dysregulated transcription
and other means.3 The ErbB (HER) family of oncogenes belongs to
those molecular targets, and its members are frequently overexpressed in different tumor tissues, including lung cancer.3–7
The ErbB gene family encodes for tyrosine kinase receptors that
play a role in the autocrine regulation of human lung cancer
growth.4–7 The Mr 180,000 ErbB-4 protein8 is the most recently
identified member of the ErbB receptor family that includes the
EGF-receptor (ErbB-1), ErbB-2 and ErbB-3. Cloned in 1993,
ErbB-4 shares extensive homology with ErbB-3 in its extracellular
domain, and has a 77–79% identity with the ErbB-1 and ErbB-2
within its cytoplasmic domain. Both ErbB-4 and ErbB-3 are activated by the neuregulin family of ligands,9,10 whereas ErbB-4 can
also be activated by betacellulin,11 heparin-binding EGF12 and epiregulin.13 The activated receptors interact with a variety of signaling
pathways that ultimately may lead to cell division, growth inhibition, differentiation or chemotaxis. ErbB-4 is expressed in many
adult and fetal tissues, including the lining epithelia of the gastrointestinal, urinary, reproductive and respiratory tracts, as well as the
skin, skeletal muscle, circulatory, endocrine and nervous systems.14
Recent reports demonstrated that ErbB-4 is frequently detected in
different human cancers, including breast, ovary and lung.14–16
ErbB-4 can stimulate cell proliferation. Kainulainen et al.17
studied two naturally occurring ErbB-4 cytoplasmatic isoforms
and demonstrated that neuregulin (NRG-1) stimulated the proliferPublication of the International Union Against Cancer
ation of cells expressing both isoforms, namely ErbB-4 CYT-1 or
ErbB-4 CYT-2. Russell et al.18 showed that stimulation of human
endothelial cells with a recombinant neuregulin induces cell proliferation, as well as rapid calcium fluxes and receptor tyrosine phosphorylation. Additionally, neuregulin provides marked angiogenic
response in vitro and in vivo. Further, evidence for proliferative and
tumorigenic properties of ErbB-4 was provided by Gilmour et al.,19
using ribozyme technology. They observed that down-regulation
of ErbB-4 in breast cancer cell lines expressing relatively high levels of ErbB-4 markedly reduces the ability of the cells to grow in
an anchorage-independent assay. Furthermore, ribozyme-mediated
down-regulation of ErbB-4 in these breast cancer cells inhibited
tumor formation in athymic nude mice. Overexpression of ErbB-4
in other tumor types suggests a strong selective advantage conferred
upon tumor cells in vivo.20,21 These data propose that ErbB-4 plays
a significant role in the proliferation of cells expressing high levels
of ErbB-4.
Several groups have evaluated the expression of ErbB-4 in
breast cancer patients22–29 and found that ErbB-4 is expressed at
moderate to low levels. Its expression correlates with a favorable
prognosis and this is in contradictory to ErbB-2 and ErbB-1 that
are correlated with a poor prognosis. In addition to mammary carcinoma, ErbB-4 expression has been noted in several other
tumors.30–37 These include carcinomas of the colon, prostate, lung,
ovary, pancreas, endometrium, bronchus, cervix, stomach and thyroid. Also, soft tissue sarcomas, astrocytoma and pediatric brain
tumors38–41 are reported to express ErbB-4. In ependymoma,41
ErbB-4 expression is quite high in about 75% of the cases. ErbB-2
expression was found in 30%, while the expression of ErbB-1 or
ErbB-3 is relatively low. Coexpression of ErbB-2 and ErbB-4
occurs in about 75% or more of these tumors and is correlated
with a high proliferative index and poorer survival outcome. Similar findings were reported in other tumors. Nevertheless, the role
of ErbB-4 in tumor progression is still undefined and further investigations are needed.42–44
The possible role of ErbB in cancer progression has led to an
extensive search for selective inhibitors. The results of a large
body of preclinical studies and the clinical trials thus far conducted
suggest that targeting the ErbB family could represent a contribution to cancer therapy. A variety of different approaches are currently being used for this purpose. Two therapeutic approaches have
been shown to be the most promising and are currently used to inhibit ErbB in clinical settings: (i) monoclonal antibodies (MAbs) to
prevent ligand binding and (ii) small molecule tyrosine kinase
inhibitors (TKIs) specific to ErbB tyrosine kinases, which inhibit autophosphorylation and downstream intracellular signaling. Among
the MAbs that gained wide clinical use is a MAb to ErbB-2 denoted
The first 2 authors contributed equally to this work.
*Correspondence to: Lung and Allergy Institute, Tel-Aviv Sourasky
Medical Center, 6 Weizmann Street, Tel-Aviv 64239, Israel.
Fax: 972-3-6925760. E-mail: [email protected]
Received 14 June 2005; Accepted after revision 24 November 2005
DOI 10.1002/ijc.21818
Published online 6 February 2006 in Wiley InterScience (www.interscience.
wiley.com).
270
STARR ET AL.
trastuzumab (Herceptin). It blocks signal transduction and cell
growth and combined with chemotherapy it produced a higher
response rate and a longer survival.45,46
The main purpose of the present study was to determine
whether ErbB-4 has a role in proliferation of non-small-cell lung
cancer (NSCLC) cells in vitro and in vivo, and whether ErbB-4
can be used as a target for anticancer therapy.
Materials and methods
Cell lines and culture conditions
Human large-cell cancer of lung H661 and human adenocarcinoma of lung H1299 were obtained from American Type Cell Collection (Rockville, MD). The cell lines were maintained in DMEM
supplemented with 10% heat-inactivated fetal bovine serum (FBS),
antibiotics, glutamine, 0.1 mM nonessential amino acids, 1.0 mM
sodium pyruvate (Biological Industries, Beit HaEmeq, Israel), and
grown in 5% CO2 at 37°C in a water-jacketed incubator with 100%
humidity. The cells were harvested by trypsin solution with 1–2 passages per week in a split ratio of 1:3–5. The 24-hr cell cultures were
used in all the experiments.
ErbB-4 gene transfection
H1299 cells that do not express ErbB-4 were transfected with
ErbB-4 pcDNA 3.1 using a calcium phosphate method. Semiconfluent cells were seeded at 5 3 105 cells per tissue culture plate
(60 mm), and 24 hr later, they were transfected with 10 lg of
ErbB-4 pcDNA 3.1 expression vector that contained a neomycinresistance gene. As control, the cells were transfected with a vector containing neomycin-resistance gene alone. The transfected resistant cells in both variants were selected in the medium containing
500 lg/ml G418. After 3–6 weeks, the colonies were expanded into
confluent cultures and then transferred into tissue culture plates
(35 mm). Each transfected cell clone was checked for ErbB-4 expression using immunohistochemistry, western blot and immunoprecipitation assays.
Antibodies
Polyclonal anti-ErbB-1, ErbB-2, ErbB-3 and ErbB-4 antibody were
purchased from Santa Cruz (CA). Monoclonal antibodies against
different epitopes of extracellular domain of ErbB-4 (MAb-3, clone
H.72.8 and MAb-1, clone H4.77.16) were purchased from NeoMarkers (Fremont, CA). Additionally, one of the monoclonal antibodies (MAb-3) was obtained from the hybridoma H.72.8 developed in the laboratory of Dr. Yarden.47 This antibody was purified
from the supernatant or mouse ascites using Protein-G-Sepharose
column 4B Fast Flow (Sigma, Israel). The same monoclonal antibodies were used for in vitro experiments.
Colorimetric in vitro cytotoxicity assay (XTT)
Cell density was evaluated by a colorimetric assay based on the
conversion of tetrazolium salt XTT to orange-colored formazan
compounds by means of cellular dehydrogenases. The advantage
of this assay (using 96 microwell plates) is the possibility of testing numerous arms of each experiment under the same conditions.
Typically, 200 ll of medium with a known number of cells from
exponentially growing culture were plated in 96 microwell flatbottomed plates. Following 24 hr in culture (to attach and to resume growth), MAbs were added in varying concentrations to
each of the 3 replicate wells. The cell growth was evaluated by
measuring the cell density during several days of incubation. The
effect of MAbs on cell survival was calculated by comparing the
density of intact cells and the cells exposed to different concentrations of MAbs for 72 hr. Cell density was determined by the XTT
assay as follows. A freshly prepared mixture of tetrazolium salt
XTT and an activation reagent (PMS) was added into each well.
Following 2 hr of incubation at 37°C, the plates were placed on a
mechanical plate shaker of a computerized automatic microwell
plate spectrophotometer and shaken for 1 min, after which the
optic density (OD) of the dye was read at 450 nm. The measurements were repeated following 4 and 6 hr of incubation. The time
point of the assay with the most optimal OD readings was chosen
to count the relative cell number. When more than one time point
fitted these criteria, the results of the different time points were
normalized and averaged.
Immunohistochemistry
For IHC analysis, the cells were centrifuged into a cell pellet that
was embedded in paraffin for immunohistochemical staining with
the ErbB-4-polyclonal (Santa Cruz, CA) or monoclonal (Neomarkers, CA) antibodies. Antigen retrieval was performed at 95°C
in citrate buffer pH 6.0, 6.4 M sodium citrate dehydrate and 1.6 M
citric acid monohydrate for 40 min. The slides were cooled at room
temperature for 20 min and washed 3 times of 3 min each with Tris
buffer pH 7.6, 0.15 M sodium chloride and 0.05 M Trizma HU.
They were peroxidase-blocked for 5 min and washed as above.
They were then incubated for 30 min with the primary antigen, followed by the secondary antigen (visualization reagent), followed by
the substrate–chromogen solution (3,30 -diaminobenzidine), and finally counter-stained with hematoxylin. Paraffin sections of normal
skeletal muscles known to express ErbB-4 were used as a positive
control. For a negative control, the primary antibodies were replaced with a nonspecific negative control antibody. Cell staining
was quantified in a blind manner by 2 independent investigators
(S.M. and A.S.) who graded them from 0 to 31, according to the
staining intensity and the percentage of stained cells.
Immunoprecipitation and western blot analyses
Cell lysates. Cells were grown in 10-cm tissue culture dishes,
washed briefly with ice-cold PBS and treated for 30 min on ice
with lysis buffer containing 50 mM HEPES pH 7.5, 150 mM
NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM EGTA,
10 mM NaF, 30 mM b-glycerol phosphate, 2 mM sodium orthovanadate, 2 mM PMSF and proteinase inhibitors (10 lg/ml aprotinin
and protein inhibitor cocktail). Cell lysates were cleared by brief
centrifugation (14,000 rpm, 1 min). Cleared lysates were kept at
280°C until use. Protein quantitation was performed using the
bicinchoninic/BCA (Pierce) protein assay using bovine serum albumin as a standard.
Immunoprecipitation. Fifteen microliters of beads (Protein
G-Sepharose, Sigma) for each sample (a ‘‘sample’’ is a lysate originating from one dish) were washed 3 times with HNTG buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Triton X-100
and 10% glycerol. Anti-ErbB-4 antibodies were coupled to the
beads for 1 hr to overnight at 4°C while shaking. Cell lysates were
added to the beads/Ab conjugates and incubated overnight at 4°C
while shaking. Immunoprecipitates were washed 3 times with HNTG
buffer, mixed with sample buffer, heated for 5 min at 95°C and subjected to SDS-PAGE electrophoresis (7.5%).
Western blot. After electrophoresis, the proteins were electrophoretically transferred to nitrocellulose membranes (1–2 hr, 4°C)
and then saturated overnight at room temperature in a blocking solution (TBST 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1%
Tween 20 with the addition of 5% low fat milk). The membranes
were then incubated for 1.5 hr at room temperature with the first
antibody (rabbit polyclonal IgG) diluted in TBST containing 2%
low fat milk. Filters were washed extensively with TBST solution
and incubated for 1 hr at room temperature with conjugated goat
anti rabbit IgG-HRP. Signals were detected by using the enhanced
chemiluminescence method (EZ-ECL, Biological Industries, Beit
Haemek, Israel).
Flow cytometry analysis of cell cycle and apoptosis
Cell cycle analysis was performed 2–3 times for each protocol
of treatment tested. The cells were trypsinized, rinsed twice with
ice-cold PBS (0.05 M, pH 5 7.4) and fixed in 70% ice-cold ethanol.
The fixed cells (106 cells/ml) were stored at 4°C until cell cycle
analysis. Before analysis, the cells were centrifuged and the pellet
ErbB-4 AND MOLECULAR TARGETED THERAPY
271
FIGURE 1 – Expression of ErbB oncoproteins in the H1299 parental
cell line and its clones transfected with ErbB-4 gene (immunoprecipitation and western blot analysis). Large cell carcinoma H661 cell line
with high expression of ErbB-4 was used as positive control. C-neo
cells contained the vehicle only, whereas c16 and c22 cells contained
plasmid with ErbB-4 gene. After immunoprecipitation with monoclonal ErbB-4 antibody, the cell lysates were processed by Western analysis and probed by polyclonal antibody.
was resuspended in 810 ll PBS after which RNAse-A (10 mg/ml,
90 ll) was added. After 30 min of incubation at 37°C, propidium
iodide (PI, 0.5 mg/ml, 90 ll) was added, and the samples were run
for additional 1 hr. FACScaliber (Becton Dickinson) was used for
flow cytometry. The percentages of the cells in the different phases
of the cell cycle were calculated using Cellquest software (Becton
Dickinson).
FIGURE 2 – Growth of H1299 cells in vitro. The cells were seeded
in 96 microwell plates in triplicates at the same density. The cell density was measured using XTT assay throughout 4 days of incubation.
The lines demonstrated exponential growth. Both 2 clones with ErbB4 expression (c16 and c22 – the clones of H1299 cells transfected with
ErbB-4 and neomycin resistance genes) manifested higher growth rate
in comparison with parental cells (par - parental line of H1299 cells)
and c-neo clone (the clone of H1299 cells transfected with neomycin
resistance gene only).
Animals
The 4- to 6-week-old male athymic CD-1 nude mice were obtained from the Harlan Animal Production Area (Jerusalem, Israel).
The animals were housed in a laminar flow cabinet under pathogen-free conditions in standard vinyl cages with air filter tops.
Cages, bedding and water were autoclaved before use. The local
Ethics Committee for Accreditation of Laboratory Animal Care
approved all facilities in accordance with the current regulations
and standards of the Israeli Ministry of Health.
Effect of ErbB-4 expression on NSCLC cell growth in vitro
The growth rate of H1299 cells (parental line), two ErbB-4 positive clones (c16 and c22) and a neomycin-resistant clone (c-neo)
was tested in vitro. The cells were seeded in 96 microwell plates,
and the numbers of living cells were determined by XTT assay during 4 days of cultivation. Both ErbB-4-positive clones demonstrated
a greater growth in comparison with parental cells and neomycin-resistant cells (Fig. 2). At day 4, the density of the ErbB-4-positive
cells was 2–3 times higher than the density of the parental cells.
Tumorigenicity assay
The harvested NSCLC tumor cells (3 3 106 cells in 0.2 ml
PBS) were injected subcutaneously into the animal’s flank area.
Each cell line was injected into 5 mice. The tumor size was measured twice weekly by digital caliper. The mean tumor size at each
time point was calculated from the pooled data.
Statistics
The results for each variant in the in vitro experiments with proliferation assay were represented as an average of 2–4 experiments, and each arm was typically performed in triplicate. The
mean values and standard errors were calculated for each point
from the pooled normalized data.
Results
ErbB1-4 expression in NSCLC cell lines
To evaluate in detail, a possible role of ErbB-4 in cell behavior,
the H1299 cell line that did not show ErbB-4 expression by IHC,
IP and Western analysis was transfected either by the ErbB-4 gene
in combination with the neomycin-resistance gene or by the neomycin-resistance gene alone. Two clones (c-16 and c-22), which
showed stable transfection and high expression of ErbB-4 receptor
revealed by IP (Fig. 1), were used in subsequent experiments. All
the clones transfected with the neomycin-resistance gene alone
were ErbB-4 negative (Fig. 1). Immunohistochemical staining for
ErbB-4 confirmed the results of biochemical analysis (not shown).
Western blot analysis for ErbB-1 and ErbB-3 showed approximately same expression in all studied cells; however, ErbB-2 was
expressed in c-16 only (Fig. 1)
Effect of ErbB-4 gene insertion on tumorigenicity of NSCLC cells
To determine the role of ErbB-4 overexpression in the tumorigenic capacity of human lung cancer cells in vivo, H1299 parental
cells and their ErbB-4 transfected clones (c16 and c22) were
implanted subcutaneously into the flank of athymic nude mice and
the tumor size was followed up during 2 months (Fig. 3). By day 57
after injection, the tumor generated by ErbB-4-expressive transfected
clones (c16 and c22) reached 14–18 mm in diameter, whereas
tumors produced by nonexpressive parental cells, as well as by the
c-neo clone, did not exceed 5 mm in diameter. Thus, ErbB-4 nonexpressive parental cells generated slow growing tumors, while the
highly ErbB-4 expressive clones generated rapidly growing tumors.
Effect of anti-ErbB-4 monoclonal antibodies on cell growth
in vitro
To test whether the ErbB-4 receptor might mediate growth
function, the blocking effect of ErbB-4 monoclonal antibody
(MAb-3) on H-661 (human large-cell carcinoma) and H1299 cell
lines (including c16, c22 and c-neo clones) was evaluated. The incubation of ErbB-4 positive H661 cells with MAb-3 resulted in dosedependent inhibition of cell survival: 30 lg/ml MAb-3 decreased
cell density up to 50% relative to untreated cells (Fig. 4). Similar
findings were found by using MAb-3 on ErbB-4-expressing c16 and
c22 clones of the H1299 cell line, whereas H1299 parental cells and
its neomycin-resistant clone (H1299-neo) were unaffected (Fig 4).
To investigate a possible mechanism of the inhibitory effect of
the blocking antibody on proliferation of ErbB-4 expressive cells, 2
mouse monoclonal antibodies—MAb-1 and MAb-3 were used.48
Both antibodies are directed to epitopes that map in the extracellular
domain of human ErbB-4. MAb-1 does not interfere with the receptor-heregulin binding but stimulates tyrosine kinase activity and internalization of ErbB-4. In opposite, MAb-3 strongly inhibits the bind-
272
STARR ET AL.
FIGURE 5 – MAb-induced apoptosis in ErbB-4 nonexpressive (parental
cells and c-neo) or ErbB-4 expressive (clones c16 and c22) H1299 cells.
The percentage of apoptotic cells (with DNA content less than in G1phase) was determined by FACS analysis using PI staining of the intact
cells (blue) and the cells incubated with 30 lg/ml MAb for 72 hr (brown).
FIGURE 3 – Growth of subcutaneous tumors generated by H1299
cells in nude mice. The cells (3 3 106) were injected subcutaneously
in flank of nude mice and tumor sizes were measured twice a week.
Each point represents the average data from 5 mice. (H1299p – parental line; H1299-c16 and H1299-c22 – the transfected clones of H1299
cells expressing high levels of ErbB-4; H1299-c-neo-the clone transfected with neomycin gene only.)
FIGURE 4 – Effect of anti-ErbB-4 monoclonal antibody on proliferation of ErbB-4 expressed (H661, H1299-c16 and H1299-c22) and
ErbB-4 non-expressed (H1299-parental and c-neo) cells in vitro. The
cells were exposed to different concentrations of MAbs for 72 hr and
the cell survival was measured by XTT assay.
ing of heregulin to ErbB-4. It has no effect on tyrosine kinase activity
or internalization of the receptor. As was demonstrated before, the
proliferation of H661 cells was inhibited by MAb-3 (Fig. 4) but
not by MAb-1 (data not shown). These results indicate that the
growth-inhibitory effect produced by MAb-3 in NSCLC cells was
mediated by the blocking of heregulin binding to ErbB-4.
Induction of apoptosis by anti-ErbB-4 monoclonal antibodies in
NSCLC cells
The distribution of the cells in the cell cycle and the extent of apoptosis were assessed by flow cytometry, following 72 hr incubation
of the cells with and without MAb-3 (30 lg/ml). In contrary to the
H1299 parental cell line and c-neo clone, the incubation of ErbB-4expressive clones (c16 and c22) with MAb-3 significantly increased
the percentage of cells with sub-diploid DNA content indicating
the development of apoptosis (Fig. 5). In the same time, the other
phases of the cell cycle were almost not affected by MAb-3 in all
cells tested (data not shown).
Discussion
ErbB-4 has a significant role in the growth and survival of different human tumors.48 We had earlier studied the correlation
between expression of ErbB-4 and prognosis in human lung cancer. Using immunohistochemistry, we analyzed ErbB-4 expression
in 80 patients with NSCLC who underwent surgery.49 The cumulative survival rate for patients with ErbB-4 protein overexpression was 16–22 months compared to 33–40 months in patients
without ErbB-4 over-expression (p < 0.002). Strong immunostaining was found in 78.1% of the tumors metastatic to lymph nodes,
whereas weak staining was detected in 80% of nonmetastatic
tumors, indicating a significant correlation between lymph node
involvement and ErbB-4 over-expression (p < 0.001). In another
clinical study, which considered response and survival of lung
cancer patients following gemcitabine and cis-platin chemotherapy, ErbB-4 was shown to have a possible role in the treatment
outcome.35 These findings suggested that ErbB-4 protein overexpression in NSCLC tumors may be a useful biological marker for
indicating a tendency to early lymph node metastasis, a poor clinical outcome and decreased response to chemo- and radiotherapy.
Therefore, down-regulation of ErbB-4 signaling might provide an
effective target for molecular cancer therapy.
We demonstrated that introducing ErbB-4 gene in the ErbB-4
negative cell line resulted in the stimulation of proliferation of
NSCLC cells in vitro, and in increasing the growth of human
NSCLC cells implanted subcutaneously into athymic nude mice.
In addition, specific monoclonal antibody to ErbB-4 (MAb-3) inhibited the cell growth and enhanced induction of apoptosis in
endogenously ErbB-4 expressed NSCLC cell line as well as in
H1299 transfected clones that demonstrated ErbB-4 expression.
The same antibodies were noneffective in the H1299 parental cell
line or in their clones not expressed ErbB-4. A number of mechanisms can be proposed as characterizing the effect of the ErbB-4
antibody on the growth and apoptotic rate of H1299 cells. The
direct mechanism is most probably the inhibition of ErbB-4 tyrosine kinase activation. The outcome of this blockade is reflected in
the disruption of a number of processes regulated by the ErbB-4
that include: (i) regulation of cell cycle progression, (ii) cell survival pathways, (iii) tumor cell invasion and (iv) angiogenesis.
Blockade of ErbB-4 signaling using antibodies against ErbB-4 have
been shown to inhibit receptor autophosphorylation in vitro.47
ErbB-4 AND MOLECULAR TARGETED THERAPY
Numerous articles have reported the influence of neuregulin/
heregulin on the growth of cell lines that endogenously express
ErbB-4. Transfection of ErbB-4 into 3T3 cell lines that do not normally express ErbB receptors has established that growth factor
activation of ErbB-4 in the absence of other ErbB receptors provokes a significant increase in cell proliferation.50 Also, this study
demonstrated that activation of ErbB-4 by neuregulin stimulated
cell proliferation and chemotaxis.
Other mechanisms of action of anti-ErbB-4 monoclonal antibody used in our experiments as well antibodies to other ErbB
receptors51 may be suggested:
a. Block of NRG binding to ErbB-4. The first step in signaling
cascade from ErbB-4 is binding of the specific ligand to extracellular domain of the receptor. To test this interaction, we examined
the effect of two mouse monoclonal antibodies—MAb-1 and
MAb-3 on proliferation of ErbB-4 expressing cells. Both MAbs
interact with the extracellular domain of human ErbB-4 but to different epitopes. In addition, MAb-3 strongly inhibits the binding
of heregulin to ErbB-4 while Mab-1 does not interfere with the receptor-heregulin binding. In our experiments, MAb-1 did not inhibit the proliferation of ErbB-4 positive NSCLC cells, whereas
MAb-3 demonstrated an inhibitory effect. These results indicate
that the growth-inhibitory effect of MAb-3 in lung cancer cells
was associated with the blocking of heregulin binding to ErbB-4.
b. Disturbances of ErbB-4 heterodimerization with other EGFR
members. Some investigators have evaluated ErbB-4 growth-stim-
273
ulating capacity by transfection of the receptors into the hematopoietic cell lines 32D (myeloid) or BaF3 (lymphoblastoid) that do
not exogenously express any ErbB family members. It is agreed
that by itself ErbB-4 has a weak capacity to support cell survival
in the presence of its cognate ligand. However, when coexpressed
with ErbB-2, most studies show that ErbB-4 more effectively
mediates cell survival in a manner that is dependent on the presence of an ErbB-4 ligand. The picture that emerges is that ErbB-4
can mediate a proliferative signal, but its potential to do so is significantly enhanced by the presence of ErbB-2. Several studies
reported cooperative effects of MAb combinations.48,52 Combining MAbs that engage distinct epitopes significantly accelerates
receptor degradation. In addition, MAb combinations are more
effective than single MAbs in inhibiting ErbB signaling in vitro
and tumorigenesis in animals.52
c. Influence on cell cycle and apoptotic death. The antitumor activity of ErbB-4 can be attributed to its influence on cell cycle and
apoptosis. In our experiments, we did not find the cell cycle deceleration but MAb-3 induced apoptosis.
In conclusion, our study demonstrates the proliferative and the
tumorigenic potential of ErbB-4 gene as well as antitumor activity
of anti-ErbB-4 MAb in in vitro NSCLC model. This treatment was
markedly effective against an established NSCLC cell line that
expressed the ErbB-4. The ability of ErbB-4 monoclonal therapy to
inhibit the growth of lung cancer cells in vitro suggests that the
ErbB-4 blockage by ErbB-4 MAb has potential therapeutic strategy
for anticancer therapy.
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