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Tumor Biology |
Lombardi Cancer Center, Department of Biochemistry, Georgetown University Medical Center, Washington, DC 20007 [C. K. T., X-Q. G., M. E. L.], and Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [D. K. M., A. J. W.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSBiological Effect of EGFRvIII...DISCUSSIONREFERENCES |
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To delineate the biological significance of EGFRvIII in human
breast cancer, we expressed EGFRvIII in the MCF-7 human breast cancer
cell line. Expression of EGFRvIII in MCF-7 cells produced a
constitutively activated EGFRvIII receptor. Expression of EGFRvIII in
MCF-7 cells also elevated ErbB-2 phosphorylation, presumably through
heterodimerization and cross-talk. These MCF-7/EGFRvIII transfectants
exhibited an 
3-fold increase in colony formation in 1% serum with
no significant effect observed at higher percentages of serum. A
similar result was also seen in anchorage-dependent assays.
Furthermore, EGFRvIII expression significantly enhanced tumorigenicity
of MCF-7 cells in athymic nude mice with P < 0.001. Collectively, these results provide the first evidence
that EGFRvIII could play a pivotal role in human breast cancer
progression.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSBiological Effect of EGFRvIII...DISCUSSIONREFERENCES |
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, amphiregulin, BTC, and
heparin-binding EGF-like growth factors (HB-EGF; Ref. 10
).
Overexpression of EGFR in human malignancy has been extensively
studied, where it has become increasingly apparent that alterations of
the EGFR gene may be as important as amplification
toward the oncogenic effects (11
, 12)
. A spontaneous
rearrangement within the EGFR gene termed EGFRvIII was first
identified in primary human glioblastoma tumors (13, 14, 15, 16)
.
This type III EGF deletion-mutant receptor (EGFRvIII) is characterized
by the deletion of exons 2–7 in the EGFR mRNA and correspond to cDNA
nucleotides 275-1075, which encode amino acids 6–276, presumably
through alternative splicing or rearrangements (13, 14, 15)
.
Deletion of 801 bp within the extracellular domain of the
EGFR gene causes an in-frame truncation of the normal EGFR
protein, resulting in a 145-kDa receptor. A number of functional
differences between EGFRvIII and normal EGFR have been characterized.
The EGFRvIII molecule appears to be unregulated by EGF or TGF-
(17, 18, 19)
. However, EGFRvIII is constitutively
activated. Overexpression of EGFRvIII in NIH3T3 and NR6 cells results
in transformed morphology, enhanced growth, and tumorigenicity in
athymic mice (17
, 20)
. Recent reports demonstrated that
the EGFRvIII is also frequently detected in other human cancers,
including breast, ovarian, lung, and medulloblastoma tumors (21
, 22)
, but has not been detected in normal adult tissue (21
, 23)
. The frequent expression of this EGFRvIII in various tumors
types suggests a strong selective advantage conferred upon tumor cells
in vivo (24
, 25)
. However, the tumorigenic
potential of EGFRvIII in breast cancer cells has not yet been explored.
Understanding the function and biology of EGFRvIII will have important
implications in the prognosis and treatment of breast cancer. In this report, we describe the biological consequences resulting from the expression of EGFRvIII in a murine, hematopoietic, IL-3-dependent cell line lacking endogenous EGF-family receptors, as well as in a breast cancer cell line (MCF-7). In this comprehensive study, our data provide evidence that overexpression of EGFRvIII is capable of transforming a nontumorigenic, IL-3-dependent murine hematopoietic cell line (32D) into an IL-3-independent, ligand-independent, and highly tumorigenic phenotype in athymic nude mice. Such profound transforming activity has not been observed in any homo- or heterodimers of wild-type ErbB-family receptors in this system. Transformation ability of EGFRvIII is dependent upon the level of EGFRvIII expression. We also demonstrated clearly that expressing a relatively low level of EGFRvIII in human breast cancer cells significantly enhanced its tumorigenicity compared with untransfected cells. These results are agreement with those observed with NIH3T3, NR6 cells, and U87 MG where EGFRvIII is a potent oncoprotein with ligand-independent transforming activity (18 , 20 , 24) . Our results provide the evidence for the pivotal role of EGFRvIII in human breast cancer progression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSBiological Effect of EGFRvIII...DISCUSSIONREFERENCES |
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Transfection.
32D derivative cells (1 x 107)
were used for transfection. Ten µg of the full-length EGFRvIII cDNA
were electroporated into 32D cells at 250 V, using a Bio-Rad
electroporation system. Derivatives transfected with constructs
expressing EGFRvIII receptors were selected by supplementation with 750
µg of G418 in the medium.
For MCF-7 Cells, 1 x 106 cells and 10–15 µg of plasmid DNA were used for each transfection. Transfections were performed using the Calcium Phosphate Transfection System (Life Technologies, Rockville, MD), according to the manufacturer’s protocol. The cells were then selected in a growth medium containing appropriate amounts of Geneticin (G418 sulfate; Life Technologies, Inc.).
Autophosphorylation of ErbB-Family Receptors.
Prior to cell lysis, the cells were serum starved overnight at
37°C. After incubation, cells were then treated with 100 ng/ml of
neuregulin (R & D Systems) or 100 ng/ml of BTC (R & D Systems) for 5
min at 37°C. After a 5-min incubation, cells were lysed in a
HEPES-lysis buffer, and the cell debris was pelleted by centrifugation
(27)
.
The lysates were immunoprecipitated with either anti-EGFR (Ab-1; NeoMarkers, Union City, CA) or anti-ErbB-2 (Ab-3; Oncogene Science, Uniondale, NY) overnight at 4°C with gentle agitation. Protein A-Sepharose (50 µl; Pharmacia, Piscataway, NJ) was then added to the immunocomplex and incubated at 4°C for 1 h. Immunoprecipitates were then separated by SDS-PAGE and transferred to nitrocellulose. Bound proteins were immunoblotted with anti-phosphotyrosine monoclonal antibody PY20 (UBI, Lake Placid, NY), followed by blotting with 0.5 µg/ml of secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected by an enhanced chemiluminescence reagent (ECL; Amersham Corp.).
Fluorescence-activated Cell Sorter (FACStar) Analysis.
Cells (1 x 106) cells were
harvested and then stained for 1 h with anti-EGFRvIII antibody at
4°C. Stained cells were then washed with cold PBS. A secondary
FITC-antirabbit antibody, was added and the EGFRvIII level was
quantitated by flow cytometry.
Anchorage-dependent Growth Assays.
Cells were harvested using trypsin, and 1500 cells/well were plated in
24-well plates (Costar). Three independent assays were performed in
triplicate. Cells were counted in a Coulter Counter (Coulter
Electronics Ltd., Hialeah, FL) on day 1 (the following day), day 3, and
day 7. Values indicate the mean of triplicate determinations ± SD.
Anchorage-independent Growth Assays.
A bottom layer of 1 ml of IMEM containing 0.6% agar and 10% FCS was
prepared in 35-mm tissue culture dishes. After the bottom layer
solidified, cells (10,000/dish) were then added in a 0.8-ml top layer
containing 0.4% Bacto Agar and 5% FCS. Three independent assays were
performed, and all samples were prepared in triplicate. Cells were
incubated for 12 days at 37°C. Colonies larger than 60 µm were
counted in a cell colony counter (Ommias 3600; Imaging Products
International, Inc., Charley, VA).
In Vivo Studies.
Ovariectomized athymic nude mice were inoculated s.c. with either
32D/wt, 32D/EGFR, 32D/EGFRvIII P5, or MCF-7/wt, MCF-7/vector, as well
as EGFRvIII transfected clone, MCF-7/EGFRvIII C4 in the presence of
estrogen source (0.72 mg). The slow-release pellets (60-day release)
were implanted s.c. into the cervical scapular space. Tumor growth was
monitored twice weekly for 3–6 weeks. Tumor size was measured twice
weekly and calculated by measuring tumor volume (length x width x thickness). When tumors reached up to 2
cm in diameter, mice were sacrificed.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSBiological Effect of EGFRvIII...DISCUSSIONREFERENCES |
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We first transfected EGFRvIII cDNA into 32D cells. The resulting
transfectants were designated 32D/EGFRvIII. A number of the
32D/EGFRvIII clones were selected for biochemical and biological
characterization. The expressions of the cell surface EGFRvIII
receptors in these transfectants were quantitatively measured by flow
cytometric analysis (FACS) with an EGFRvIII-specific antibody. Three
pooled populations, 32D/EGFRvIII P5, 32D/EGFRvIII C2, and
32D/EGFRvIII C6, which express relatively high, medium, and low
levels of EGFRvIII, respectively, were selected for further
characterization (Fig. 1
A). The appropriate molecular mass of the EGFRvIII was
confirmed by Western blot analysis with a specific antibody, which
recognizes the intracellular domain of either the wild-type EGFR or
EGFRvIII receptors. Fig. 1B
shows the appropriate 145-kDa
protein expressed by 32D/EGFRvIII cells, whereas 32D/EGFR (wt) cells
expressed a 170-kDa protein. The EGFR transfected 32D cells are denoted
as 32D/EGFR.
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A). The addition of exogenous EGF and BTC both further
enhanced their growth (Fig. 2A
). However, TGF-, HB-EGF,
and amphiregulin had no significant effect in these EGFRvIII
transfectants. In contrast, the parental 32D cells absolutely required
IL-3 for growth and were unresponsive to any of the EGF-like ligand
treatment. The wild-type 32D/EGFR cells required either IL-3 or ligands
for EGFR for growth (Fig. 2A
). In the absence of IL-3,
32D/EGFR cells did not survive, despite the fact that FACS analysis
revealed that the EGF receptor expression level was similar to
EGFRvIII in 32D/EGFRvIII P5. However, the intermediate-
expressing transfectant (32D/EGFRvIII C2) sustained cell survival
in the absence of IL-3 but not growth, whereas EGF and BTC stimulated
low levels of mitogenic activity. In the low-expressing clone
32D/EGFRvIII C6, EGF had no significant biological effect (Fig. 2A
). Apparently, overexpression of EGFRvIII was able to
abrogate the IL-3 requirement for 32D cells in the absence of ligand.
In contrast, neither homo- nor heterodimers of the wild-type EGF-family
receptors have the same ability to grow in the absence of ligand
supplementation (29, 30, 31, 32)
. Furthermore, dose-response
analysis revealed 32D/EGFR cells to require 100 pg/ml of EGF to
stimulate a maximal level of proliferation, whereas 32D/EGFRvIII cells
required a 500-fold higher concentration (50 ng/ml) of EGF to further
stimulate the EGF-mediated proliferation (Fig. 2B
). Similar
effects were also observed in BTC-mediated proliferation.
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A). We further examined growth rate differences between the
short-term and long-term culture in the absence of IL-3, which
correlated with expression levels of EGFRvIII. FACS analysis revealed a
higher expression level of EGFRvIII in the long-term, IL-3-withdrawn
culture than the short-term, IL-3-withdrawn culture (Fig. 3B
). These results support the argument for an
IL-3-independent phenotype, which depends upon levels of EGFRvIII
expression. The differences between the EGFRvIII and the wild-type
EGF-family receptors indicated that EGFRvIII is a more potent mitogenic
oncoprotein.
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). Although the 32D/EGFR cells exhibited high levels of functional EGFR,
the parental 32D cells and 32D/EGFR cells remained nontumorigenic
during a 2-month observation period. Furthermore, none of the EGFR
family receptors, ectopically expressed singly or in pairwise
combinations in 32D cells, had a similar ability (Table 1)
. A
dose-dependent experiment was conducted to assess the minimal number of
32D/EGFRvIII P5 cells that would induce tumor growth in nude mice.
Strikingly, 100% of these mice produced tumors independent of the
number of cells injected (Table 2
). A distinguishing feature of this experiment was that mice given
1 x 106 cells required elicited a
longer latency period for tumor formation (10 days) as compared with
the one receiving 5 x 106 cells.
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Biological Effect of EGFRvIII in Human Breast Cancer Cells |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSBiological Effect of EGFRvIII...DISCUSSIONREFERENCES |
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). MCF-7/EGFRvIII transfectants from single clones and pooled population
clones appeared to exhibit similar expression levels of EGFRvIII. We
then evaluated the activation of EGFRvIII in MCF-7/EGFRvIII-transfected
cells. Autophosphorylation of EGFRvIII was assessed by
immunoprecipitation with an anti-EGFRvIII-specific antibody and Western
blot with an anti-phosphotyrosine-specific antibody. Fig. 5
shows that EGFRvIII was constitutively activated in a
ligand-independent manner in MCF-7/EGFRvIII cells. In addition, ErbB-2
phosphorylation was enhanced in EGFRvIII-transfected MCF-7 cells. These
results indicated that EGFRvIII could activate ErbB-2 kinase activity.
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). Induction of colony formation was independent of threshold colony
size. Moreover, EGF-like ligands increased proliferation substantially
(Fig. 7
). These data suggest that EGFRvIII could play an important role in
breast cancer progression. Constitutively autophosphorylated EGFRvIII
may contribute to enhanced proliferation of MCF-7/EGFRvIII cells.
Expression of EGFRvIII increases the spectrum and potency of
ligand-mediated proliferation in vitro.
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, MCF-7/EGFRvIII cells grew large tumors with a mean tumor size of
600 ± 125 mm3
within 6 weeks,
whereas tumors derived from the parental MCF-7 cells were smaller
(80 ± 40 mm3
). Tumors developed
by MCF-7/EGFRvIII cells were more than seven times larger than the
parental MCF-7 cells. Thus, EGFRvIII significantly enhances
tumorigenicity in breast cancer cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSBiological Effect of EGFRvIII...DISCUSSIONREFERENCES |
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). 32D/EGFRvIII P5 cells subject to long-term
culture conditions in the absence of IL-3 revealed further elevation of
EGFRvIII expression levels (Fig. 3
). These data further indicated
that the IL-3-independent phenotype is mediated by EGFRvIII. The level
of expression is a critical driving force for the IL-3-independent
phenotype. Moreover, 32D/EGFRvIII P5 formed large tumors in nude mice,
even when no exogenous EGF ligand was administered (Table 1)
. These
tumors progressed rapidly, necessitating sacrifice of all animals
within 1 month. In contrast, no tumors grew in the mice injected with
32D/EGFR cells, with low-expressing clone 32D/EGFRvIII C2 cells, or
with parental 32D cells after 2 months (Table 1)
. Cytological analysis
of 32D/EGFRvIII P5 xenografts showed these EGFRvIII tumors were
characteristic of high-grade tumors. High percentages of the tumor
cells appeared to be in mitosis (data not shown). This degree of
mitogenic activity and highly tumorigenic property of the
EGFRvIII-transfected cells has not been observed in any of the homo- or
heterodimers EGF-family receptors. These results suggest that the
EGFRvIII is an extremely potent oncoprotein and is capable of
contributing to malignant transformation, which is substantially
greater than any of the wild-type EGF-family receptors.
A recent report has demonstrated that 78% (21 of 27) of primary
poorly differentiated breast carcinomas express EGFRvIII
(21)
. EGFRvIII expression has not been detected in normal
breast tissues or in benign breast neoplasms (21
, 23)
.
Similar results were obtained in our tissue array experiments (data not
shown). However, the tumorigenicity of EGFRvIII in human breast cancer
cells has not yet been explored. The understanding of the function and
biology of EGFRvIII is important in the prognosis and treatment of
breast cancer. In this study, we explored the potential biological role
of EGFRvIII in breast cancer by transfecting the EGFRvIII cDNA into the
MCF-7 breast cancer cell line. These MCF-7/EGFRvIII cells exhibited a
3-fold increase in colony formation in 1% serum but no significant
effect at higher percentages of serum (Fig. 6
). The dependency of
colony formation on serum concentration and the reduction of serum
requirement for proliferation in MCF-7/EGFRvIII cells characterized
EGFRvIII-mediated enhancement of proliferation as ligand
independent. In addition, expression of EGFRvIII elevated ErbB-2
phosphorylation (Fig. 5
), presumably through heterodimerization and
cross-talk. These results suggested the possibility for EGFRvIII to
heterodimerize with other EGF-family receptors and to diversify its
signaling pathways. Furthermore, EGFRvIII expression dramatically
enhanced tumorigenicity of MCF-7/EGFRvIII cells with estrogen
supplement (Fig. 8
), although the expression levels of EGFRvIII was
relatively low. It suggested that EGFRvIII is a potent oncoprotein.
These results provide the first evidence that EGFRvIII could play a
role in human breast cancer progression.
Studies have shown EGFRvIII to be unregulated by extracellular ligands
(17
, 18
, 20)
. The deletions in EGFRvIII correspond to
subdomains I and II of the EGFR (27
, 33, 34, 35, 36)
. Although the
ligand-binding domain of the EGFR subdomain III (32)
is
preserved in the EGFRvIII oncoprotein, EGFRvIII fails to bind ligand in
NIH3T3 cells (17)
and has minimal low-affinity ligand
binding in some other system (20)
. Although TGF- cannot
bind to EGFRvIII when expressed in Chinese hamster ovary cells, these
cells had increased levels of DNA synthesis and the receptor was
constitutively phosphorylated (19)
. Similar
observations were seen in our EGFRvIII-transfected 32D cell system.
Dosage-dependent studies demonstrated requirement for a 500-fold
increase of EGF to stimulate the EGF-mediated proliferation in
32D/EGFRvIII P5 cells than in the 32D/EGFR cells (Fig. 2B
).
Our results confirm and extend the observations in NIH3T3 and U87 MG and Chinese hamster ovary cells, where cells expressing EGFRvIII proved to be ligand independent and highly tumorigenic (19 , 23 , 24 , 37) . The extreme aggressiveness of EGFRvIII in vivo suggested the potential role for its altered conformation to include the possibility to recruit different downstream effectors and to contribute to the potent mitogenic activities and tumorigenicity in vivo. It is also possible that EGFRvIII is able to interact with unknown ligands or dimerize with other receptor members in the family, thereby eliciting an amplification of biological effects. This could explain why expression of EGFRvIII, even at relatively low levels in MCF-7 cells, influences their phenotype in vivo. Moreover, the high prevalence of the EGFRvIII in a wide variety of human tumors suggests a preferential selection during the process of tumorigenesis attributable to some alteration or enhancement of the mitogenic properties of the EGFR. Taken together, the altered EGFRvIII conformation might be the essential feature responsible for its extremely potent transforming ability.
Two model systems (32D and MCF-7 cells), which we have reported here, indicate that transformation to a malignant phenotype in vivo requires high levels of EGFRvIII expression or coexpression with other family members, such as low levels of ErbB-2. Given the potency of EGFRvIII and detection of EGFRvIII expression in a high percentage of poorly differentiated breast carcinomas, it is possible for low levels of EGFRvIII expression to play a role in human breast cancer evolution.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant award (to
C. K. T.) from the Susan G. Komen Breast Cancer Foundation. The FACS
analysis data shown in Figs. 1
A, 3B, and
4 were supported in part by the Lombardi Cancer Center Flow Cytometry
Core Facility. Tables 1
2
and Fig. 8
xenografts were supported by
the Lombardi Cancer Center Animal Shared Resource Facility, USPHS Grant
P30-CA-51008. 
2 To whom requests for reprints should be
addressed, at Lombardi Cancer Center, Department of Biochemistry, E512
Research Building, 3970 Reservoir Road NW, Washington DC 20007-2197.
Phone: (202) 687-0361 (Office) or (202) 687-0342 (Lab); Fax:
(202) 687-7505; E-mail: Tangc{at}gunet.georgetown.edu
3 The abbreviations used are: EGFR, epidermal
growth factor receptor; TGF, transforming growth factor; BTC,
betacellulin; IMEM, Iscove’s modified Eagle’s medium; IL,
interleukin; FACS, fluorescence-activated cell sorter.
Received 11/ 5/99. Accepted 4/ 4/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSBiological Effect of EGFRvIII...DISCUSSIONREFERENCES |
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