Download Transgenic mice overexpressing hepatocyte

Carcinogenesis vol.27 no.8 pp.1547–1555, 2006
doi:10.1093/carcin/bgl003
Advance Access publication March 2, 2006
Transgenic mice overexpressing hepatocyte growth factor in the airways show
increased susceptibility to lung cancer
Laura P.Stabile1,4, Jennifer S.Lyker1,
Stephanie R.Land2,4, Sanja Dacic3,4,
Beth A.Zamboni2 and Jill M.Siegfried1,4,
1
Department of Pharmacology, 2Department of Biostatistics,
University of Pittsburgh, 3Department of Pathology and
4
Lung and Thoracic Malignancy Program, University of Pittsburgh
Cancer Institute, Pittsburgh, PA 15261, USA
To whom correspondence should be addressed at: The Hillman Cancer
Center, UPCI Research Pavilion, Suite 2.18, 5117 Centre Avenue, Pittsburgh,
PA 15213-1863, USA. Tel: +1 412 623 7769; Fax: +1 412 623 7768;
Email: [email protected]
Several studies have suggested a possible role of the
hepatocyte growth factor (HGF)/c-Met system in lung
tumor development and progression. Extent of expression
of both HGF and c-Met have been shown to be negative
prognostic indicators of survival and recurrence in
non-small-cell lung cancer, especially adenocarcinoma.
To further define a role for HGF in lung cancer development and growth, we have generated transgenic mice
that overexpress HGF in the airway epithelium. HGF
transgenic and wild-type mice were exposed to the
tobacco carcinogen, nitrosamine 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK), or saline control and
killed 10–38 weeks after exposure. Lungs were formalin
inflated, paraffin embedded and sectioned. It was verified
that the HGF transgene was expressed only in the lungs of
transgenic mice. The transgenic mouse lung histology
exhibited congestion in the alveolar spaces, excess production of blood vessels and a convoluted pattern of airways
with wide bifurcations. The number of lung tumors from
NNK-treated transgenic animals versus the number of lung
tumors from NNK-treated wild-type animals was significantly higher (P ¼ 0.0001, Poisson regression). The percentage of animals with tumors was 75% in the transgenic
group compared with 48.8% in the wild-type group. The
main effect was an increase in tumor multiplicity; average
size of tumors was not different between the groups.
Additionally, the tumors that arose in the transgenic
mice contained increased HGF protein compared with
tumors from the wild-type mice. These results indicate
that lung carcinogenesis induced by a tobacco carcinogen
is enhanced by expression of the HGF transgene. This
model recapitulates the phenotype of aggressive lung
adenocarcinoma that overexpresses HGF and will be useful
in evaluating antitumor agents that target either the HGF/
c-Met pathway or downstream effects such as angiogenesis
or invasion.
Abbreviations: CCSP, Clara cell secretory protein; hGH, human growth
hormone; HGF, hepatocyte growth factor; MDCK cells, Madin–Darby Canine
Kidney cells; NNK, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone; rHGF, recombinant HGF.
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Introduction
Lung cancer is currently the leading cause of cancer mortality
in both men and women in the US. The 5 year survival rate
for all stages of lung cancer combined is only 15% and for
late-stage disseminated disease is <5 % (1). Lung cancer
patients currently have few therapeutic options and new targeted approaches are needed to improve long-term survival.
Animal models remain pivotal to cancer drug discovery and
development, particularly in such a complex and inaccessible
organ as the lung. Novel preclinical models that mimic
aggressive lung cancer are necessary to test potential therapeutic targets for lung cancer treatment.
Several studies have suggested a role of hepatocyte growth
factor (HGF), also known as scatter factor, and its receptor,
c-Met, in the development and progression of many human
malignancies, including lung cancer (2–4). HGF is primarily
a paracrine factor produced by mesenchymal cells (5), particularly fibroblasts, although cancerous epithelial cells may
secrete HGF under some circumstances (6–9). Thus, HGF
can signal through both paracrine and autocrine mechanisms.
The c-Met receptor is expressed universally by both epithelial
and endothelial cells (5). The well-characterized activities of
the HGF/c-Met signaling pathway, including proliferation,
invasion, angiogenesis and anti-apoptotic effects, provide
examples of the different mechanisms by which this pathway
is involved in tumor development and progression.
We have demonstrated previously that the HGF/c-Met pathway is important for in vivo human lung tumor growth. In this
regard, we have reported a 3-fold stimulation of intraepithelial
tumor volume when recombinant HGF (rHGF) was injected
into human airway segments that contained lung adenocarcinoma cells implanted in scid mice (10). Additionally, the
number of tumors growing in the sub-mucosal spaces of the
airway xenografts increased from 11 to 67% when treated with
rHGF (10). In a separate study, lung adenocarcinoma HGF
protein levels were examined as a prognostic indicator in 56
patients (11). This analysis revealed that elevated HGF content
of tumors defined a subset of Stage I patients who recurred and
had shortened survival times. Furthermore, patients whose T
status was >1 and had elevated HGF rapidly recurred and died
from their disease (11). Thus, in non-small-cell lung cancer
patients, HGF could be a useful prognostic indicator for both
early and advanced stage patients. Higher HGF levels within
the tumor at the time of resection may be an indication that
more malignant cells have migrated to other sites, increasing
the probability of recurrence. Additionally, the HGF/c-Met
pathway has proven to be an effective therapeutic target for
lung cancer treatment both in vitro and in vivo (12,13).
To recapitulate the phenotype of aggressive lung cancer
containing elevated HGF, we created a transgenic mouse strain
that overexpresses human HGF in the airway epithelium under
control of the rat Clara cell secretory protein (CCSP) promoter.
Bell et al. (14) have demonstrated that this human HGF cDNA
The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 1547
L.P.Stabile et al.
sequence under control of the albumin promoter can promote
liver tumors in mice. CCSP is an abundant 10 kDa secreted
protein expressed in the non-ciliated airway epithelial (Clara)
cell of the conducting small airways (15,16). Clara cells are
believed to be the cells of origin for lung adenocarcinomas
(17). Clara cells also function as the progenitor cells of epithelial differentiation in the developing lung. The CCSP
promoter has been used previously to direct expression of
the human growth hormone (hGH) gene to the airways (18).
Normally, HGF in the lung is produced by cells of mesenchymal origin, mainly the stromal cells located in the submucosal areas just beneath the basement membrane of the
epithelial cell layer (19). HGF is normally produced at low
levels in the adult, but it is upregulated immediately following injury (20). Here we show that elevated production of
HGF by airway epithelial cells increased incidence of lung
tumor formation following exposure to a tobacco carcinogen.
This important preclinical model of human lung adenocarcinoma will be useful to develop and test antitumor agents
for lung cancer, especially those targeting HGF/c-Met signaling or downstream processes induced by phospho-c-Met such
as angiogenesis or invasion. Additionally, this animal model
may be useful for studying other diseases of the lung such as
pulmonary fibrosis.
Materials and methods
Reagents
The CCSP antibody was a kind gift from Dr Susan D.Reynolds, University of
Pittsburgh. HGF ELISA (enzyme-linked immunosorbent assay) kit, rHGF,
goat polyclonal anti-HGF antibody and mouse monoclonal anti-HGF antibody
were from R&D Systems (Minneapolis, MN). A549 lung tumor cells and
Madin–Darby Canine Kidney (MDCK) cells were from the American Type
Culture Collection (Manassas, VA). Nitrosamine 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) was from Toronto Research Chemicals,
North York, ON, Canada).
Construction of HGF transgene construct
A plasmid containing the HGF cDNA cloned into the pSPORT1 vector was
a kind gift from Dr Reza Zarnegar at the University of Pittsburgh (14). For the
promoter and downstream sequences, a plasmid containing the rat CCSP
promoter cloned into the BamHI site of p0GH was obtained from Dr Brian
Hackett at Washington University. The p0GH plasmid also contains the complete hGH gene (21). The 2.3 Kb CCSP promoter (2332 to +40) and a fragment of the hGH genomic sequences were isolated from this plasmid and
inserted into the pSPORT1 vector containing the HGF cDNA (Figure 1). A
1.6 Kb portion of the hGH polyadenylation site was included for proper processing of the mRNA to occur in transgenic mice and was inserted downstream
of the 30 untranslated sequences of the HGF cDNA. The vector was sequenced
and confirmed to be the correct sequence in the proper orientation at all
junctions.
Generation and identification of HGF transgenic mice
A 5.9 kb human HGF transgene fragment containing the CCSP promoter, HGF
cDNA and hGH polyadenylation site was excised from plasmid sequences by
digestion with the SphI restriction enzyme. The fragment was isolated from
an agarose gel and purified using the Qiaex II Gel Extraction Kit (Qiagen,
Valencia, CA) according to the manufacturers’ instructions. Microinjection of
the fusion gene into the FVB/N1 mouse strain was performed by the University
of Cincinnati Transgenic Mouse Facility.
Genomic DNA was isolated from mouse tail snips using the Easy-DNA
Isolation Kit (Invitrogen, Carlsbad, CA) according to the manufacturers’
instructions. Samples were screened for the HGF transgene using PCR with
the following conditions: 1X PCR buffer (Roche, Indianapolis IN), 2.5 mM
MgCl2 (Roche), 1 mM HGF forward primer (50 -TGGAGGGAGGCAATAGAAGGAG-30 ), 1 mM HGF reverse primer (50 -GCAGGGCTGGCAGGAGTTTG-30 ), 0.5 mM PCR nucleotide mix (Roche), 200 ng heat-treated
DNA and 2.5 U Amplitaq Gold (Applied Biosystems, Foster City, CA). Positive control for HGF was uncut HGF transgene vector; negative control was
water. Following PCR, products were separated on a 1% agarose gel and the
expected size of HGF transgene product was 540 bp. All DNA samples were
also screened for b-globin to ensure DNA integrity.
Isolation of conditioned medium and detection of secreted HGF
A549 cells were transiently transfected with the HGF transgene vector and
Lipofectamine 2000 (Invitrogen) according to the manufacturers’ instructions.
After 48 h, conditioned medium was isolated as described previously (22).
From each sample, 25 mg of protein was separated on a 10–20% SDS–tricine–
polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was immunoblotted with a 1:1000 dilution of monoclonal anti-human
HGF antibody (specific reactivity to human HGF) followed by a 1:1000 dilution of a horseradish peroxidase-congugated secondary antibody (Amersham,
Piscataway, NJ). Immunoreactive bands were visualized by SuperSignal West
Pico chemiluminescent substrate (Pierce, Rockford, IL) followed by exposure
to autoradiography film.
Scatter assay
MDCK cells were plated at a density of 1 · 104 cells/well in 6-well plates.
Tight colonies of cells were allowed to form for 3–5 days. Cells were serumdeprived overnight followed by addition of 50 ng/ml rHGF, conditioned media
from A549 transgene vector transfected cells, conditioned media from nontransfected A549 cells or no treatment. Amount of HGF in conditioned media
from A549 cells was quantitated by densitometry and the amount of media that
contained 50 ng/ml HGF was added to the wells. Cells were analyzed by
microscopy 24 h after the addition of experimental treatments for spindleshaped cells with pseudopodia-like protrusions, characteristics of scattered
cells.
RNA isolation and northern analysis
RNA was isolated from tissues or tumors from transgenic and wild-type control
animals by the guandinium thiocyanate method (23). For tumors, animals were
killed 40 weeks after a total of 24 mg NNK treatment to ensure presence of
large tumors. Individual tumors were microdisected from the surrounding lung
and placed in RNAlater (Ambion, Austin, TX). Total RNA (10 mg/lane) was
separated by size in a 1% denaturing agarose gel (0.66 M formaldehyde) and
transferred to GeneScreen membrane (Perkin Elmer, Boston, MA) using
a VacuGene XL blotting apparatus (Amersham). The transferred RNA was
UV-cross-linked to the membrane by two cycles on ‘autocrosslink’ (120 000
mj/cm2) using a Stratalinker UV illuminator (Stratagene, La Jolla, CA) and
hybridized with a random-prime labeled cDNA probe specific for the HGF
transgene (Figure 1). Probes were generated using the DECAprime II random
primed DNA labeling kit (Ambion) according to the manufacturers’ instructions. Hybridization and post-hybridization washes were as described previously (24,25). Radiographic signals on the washed filters were exposed to
a phosphor screen for 4 days. Blots were stripped and reprobed with a GAPDH
probe using DECAtemplate-GAPDH-mouse (Ambion).
Bronchioalveolar lavage and HGF quantification
A 22 gauge catheter was inserted into the trachea of killed mice and secured
with surgical suture. Two 800 ml aliquots of 0.9% saline were serially instilled
and withdrawn. The pooled sample was centrifuged at 3000 r.p.m. and the
supernatant was removed and concentrated using a Microcon YM-3 centrifugal
filter device (Millipore, Bedford, MA). The concentrated samples were then
analyzed using the Quantikine HGF Immunoassay Kit according to manufacturer’s instructions. Ten transgenic and ten wild-type littermate control mice
were used for the experiment. No cross-reactivity with mouse HGF was detected with either recombinant or endogenous mouse HGF in the ELISA assay
(data not shown).
Fig. 1. Structure of HGF transgene vector showing restriction sites used
for cloning and sizes of inserts. Solid black line represents the portion of
the vector that was used to generate the HGF probe for northern analysis.
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NNK carcinogenesis and histology
Mice were separated into groups by gender and HGF transgene status and given
two i.p. injections of 3 mg NNK (15 mg/ml) or 0.9% saline vehicle control over
Role of HGF in lung cancer
2 weeks. NNK is a potent mutagenic tobacco-specific carcinogen, which is
known to induce lung tumors in mice. All experiments using HGF transgenic
mice were performed using mice that were heterozygous for the transgene with
high copy number. Mice were monitored weekly and killed at 10–38 weeks
after the first injection. Lungs were inflated with 10% buffered formalin under
25 cm intra-alveolar pressure and removed. Tumors were counted under a dissecting microscope and measured using Motic Images 2000 software. Lungs
were paraffin embedded and sections were stained with hematoxylin and eosin
(H&E). All histological interpretation was performed under blinded experimental conditions by an expert lung pathologist. Animal care was in strict
compliance with the institutional guidelines established by the University of
Pittsburgh.
Immunohistochemical staining
Tissue samples were fixed in 10% buffered formalin. Tissues were paraffin
embedded, sliced and mounted on slides. Paraffin was removed from the slides
with xylenes, and slides were stained according to standard procedures. The
HGF and CCSP double staining (Figure 5) was performed using a 1:10 000
dilution of rabbit polyclonal anti-rat CCSP antibody and a 1:10 dilution of
goat polyclonal anti-human HGF antibody. The secondary antibodies were
biotinylated IgG antibodies specific for the primary antibodies followed by
horseradish peroxidase conjugated DAB/Ni chromogen (Vector Laboratories,
Burlingame, CA) for HGF visualization and alkaline phosphatase conjugated
New Fuschin chromogen (Vector) for CCSP visualization. Black staining was
considered positive for HGF whereas red staining was considered positive for
CCSP. Slides were counterstained with hematoxylin and analyzed using an
upright Leica Microscope with attached digital camera. Single staining was
performed for each antibody in addition to the double staining. Positive controls were placenta for HGF and rat lung for CCSP. Negative control staining
was done without the addition of primary antibody.
Primary antibody used for HGF single staining (Figure 8) was a 1:10
dilution of goat polyclonal anti-human HGF antibody (specific reactivity
with human HGF). The secondary antibody was a biotinylated IgG specific
for the primary antibody. Brown staining was considered positive. The positive
control was placenta and the negative control staining was done without the
addition of primary antibody.
Statistical analysis
Poisson regression analysis was used for analyses of the number of tumors in
the NNK carcinogenesis animal study. Logistic regression was used to test for
differences in the numbers of mice with tumors. A repeated measures analysis
was used for the tumor sizes in this experiment since there were multiple
tumors from some of the mice. Significance tests were performed with twosided significance level 0.05. The two sample Student’s t-test was used for
analysis in Figure 4. All values are expressed as mean ± SD.
Results
Construction of HGF transgene and verification of biological
activities
We have created the transgene vector as shown in Figure 1,
in which 2.3 Kb of the human HGF gene is under control
of the rat CCSP promoter. 1.6 Kb of the hGH 30 -untranslated
sequences containing the polyadenylation signal was also
included for proper processing of the transgene. The backbone
vector is pSPORT1. The transgene was sequenced and verified
to contain the correct sequences in the correct orientation.
When transiently transfected into A549 lung tumor cells
that normally do not express HGF, but will support transcription from the CCSP promoter because they express markers of
type II pneomocytes (17), the vector was expressed and A549
cells secreted HGF protein (Figure 2A). A normal lung
fibroblast (NLFB) cell line, which secretes HGF, was used
as a positive control.
The biological activity of the protein produced by the transgene was also tested using a scatter assay. Scattering of MDCK
cells by HGF is a well-documented assay to test the biological
activity of HGF. Treatment of MDCK cells with conditioned
media from A549 lung cancer cells transiently transfected with
the transgene (Figure 2E) can induce cell scattering of MDCK
cells similar to that observed with 50 ng/ml rHGF treatment
Fig. 2. Biological activities of the transgene in vitro. (A) A549 lung
tumor cells were transiently transfected with the transgene vector or control
empty vector or not transfected at all (none) and conditioned media was
isolated and analyzed by western blotting using a mouse monoclonal
anti-HGF antibody. NLFB were used as a positive control. (B) Scatter
assay of MDCK cells with no treatment (C) MDCK cells treated with
50 ng/ml rHGF (D) MDCK cells treated with conditioned media isolated
from control vector transfected A549 cells (E) MDCK cells treated with
conditioned media isolated from transgene vector transfected A549 cells.
Scale bars: 50 mm.
(Figure 2C) compared with no treatment (Figure 2B). The
conditioned medium from untransfected A549 cells cannot
induce cell scattering (Figure 2D). These results confirm the
ability of the transgene to be transcribed, secrete protein and
exert biological effects in vitro in cells that are permissive for
the CCSP promoter.
HGF is expressed in the lungs of transgenic animals
The transgene was sent to the University of Cincinnati Medical
Center for production of transgenic mice in the FVB/N1 mouse
strain, which is susceptible to lung tumorigenesis (26). Five
founder mice were generated containing between 1 and 25
copies of the transgene based on genomic Southern analysis
(data not shown). There were no apparent developmental
or behavioral differences observed between transgenic and
wild-type littermate control mice. In addition, there was no
difference observed in pulmonary function between the transgenic and wild-type control mice (data not shown). Furthermore, the transgenic mice (up to 1 year of age) did not develop
any dysplasia in the lung versus wild-type controls although
they showed evidence of a subtle Clara cell hyperplasia. The
transgenic mice were fertile and produced normal offspring.
Northern analysis with a human HGF specific probe shows
that in the HGF transgenic mice, the lung is the only tissue out
of the panel of nine tissues examined that expresses the 2.3 Kb
mRNA for the transgene (Figure 3). The endogenous mouse
HGF mRNA is over 6 Kb and was not detectable in the northern analysis. This probe should detect mouse HGF as well as
human HGF, if present. This reflects the low level of HGF
mRNA expression under normal circumstances. No tissues
from wild-type control mice expressed HGF transgene
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L.P.Stabile et al.
Fig. 3. HGF is only expressed in the lungs from transgenic mice. Total
RNA was isolated from tissues from a transgenic mouse and analyzed
using northern analysis with a probe specific for the HGF transgene.
Blots were stripped and reprobed using a GAPDH probe.
Fig. 4. Transgenic mice express more HGF and CCSP. (A),
Bronchioalveolar lavage was performed on 10 transgenic mice (HGF+) and
10 wild-type (WT) littermate controls and the levels of human HGF in the
bronchioalveolar lavage fluid were measured by ELISA. Results are
reported as the average ± SD. P < 0.0001, two-tailed unpaired
Student’s t-test. (B), Number of positive CCSP stained cells per 200 mm
length of five bronchioles per slide (3 slides/group) were counted manually
and the average ± SD. was reported. P ¼ 0.0103, two-tailed unpaired
Student’s t-test.
mRNA or endogenous mouse HGF (data not shown). GAPDH
was present in all samples indicating that the RNA was intact
from these tissues.
To confirm the northern analysis results, bronchioalveolar
lavage was performed on HGF transgenic mice (n ¼ 10) and
wild-type littermate controls (n ¼ 10) and the level of HGF in
the bronchioalveolar lavage fluid was determined by ELISA.
HGF is a secreted protein and can be detected in bronchioalveolar lavage fluid (27). Figure 4A shows that transgenic
animals secrete a significantly higher amount of HGF
(3.5-fold increase) into the airway lumenal space compared
with wild-type animals, which show barely detectable levels of
HGF (P < 0.0001). Furthermore, the number of CCSP positive
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cells in the small airways of HGF mice versus wild-type
mice was significantly increased by approximately 1.5-fold
(P ¼ 0.0103). Almost every cell in the bronchiole is expressing
CCSP in the transgenic mice whereas only a portion of the cells
are positive for CCSP in bronchioles from the wild-type mice.
These results confirm that the transgenic mice secrete HGF
protein in the airway lumen, as predicted by their genotype,
and suggest that in the transgenic mice there is subtle Clara cell
hyperplasia, possibly reflecting an autocrine response among
cells that support transcription of CCSP.
Based on the expected permissibility of the CCSP promoter,
most transgene expression should be located in the epithelial
cells of the small airways. In order to verify which cell types
in the lungs produce protein from the transgene, double
immunostaining was performed to detect protein expression
in the airway epithelial cells. In HGF transgenic mice, CCSP
immunostaining (red precipitate, Figure 5A) co-localized with
HGF immunostaining (black precipitate, Figure 5B) as shown
by serial sections of the same airway. CCSP/HGF double
immunostaining is shown in Figure 5C of representative
small airways from a transgenic mouse. The dominant black
precipitate, indicating HGF protein, is co-localized with the
red precipitate indicating CCSP (visible as a tinge of red
associated with the blackish/brown precipitate). Figure 5D
shows the CCSP/HGF double staining from a wild-type
mouse lung, demonstrating only CCSP staining (red color)
in these airways with little or no HGF production (black
color), as expected. These results confirm that the HGF transgene expression is found in the small airway Clara cells as
expected in the transgenic animals only. Using an antibody that
can detect both human and mouse HGF, the non-transgenic
airway still showed almost no detectable staining (data not
shown). c-Met expression was expressed at low levels uniformly in the normal Clara cells with no detectable difference
between HGF transgenic and wild-type animals (data not
shown).
Distinct differences in mouse lung histology
The transgenic mouse lung histology shows distinct alterations
(Figure 6), although lung function in these animals is not
impaired (data not shown). Based on H&E staining, the
lungs from transgenic mice (4–5 months old) (Figure 6D–F)
were congested in the alveolar spaces compared with wild-type
controls (72 versus <5%). There was excess production of
blood vessels in 38% of the transgenic animals compared
with <5% of wild-type littermates (arrows, Figure 6E). There
was also a convoluted pattern of airways that included wide
bifurcations, and the airways did not have the normal tapering
pattern compared with wild-type controls (Figure 6A–C). The
ratio of airway width measured 125 mm above the branch point
to 125 mm below the branch point was 2.36 ± 0.23 versus
1.26 ± 0.09 in wild-type (n ¼ 8) versus transgenic animals
(n ¼ 8), respectively (P < 0.0001). This suggests that there was
a problem with the development of branching, possibly
because of unregulated HGF production. HGF is a known
factor involved in branching in the developing lung (28).
HGF transgenic mice are more susceptible to NNK induced
lung carcinogenesis
The HGF transgenic mice and wild-type littermate control
mice were exposed to 6 mg of the tobacco carcinogen,
NNK, which shows selective uptake into the airway epithelium
and produces lung adenocarcinomas in mice (29). Based on the
Role of HGF in lung cancer
Fig. 5. HGF is localized within the Clara cells. (A), Representative lung tissue section showing CCSP immunostaining (red color) of transgenic mouse
bronchiole. (B), Representative lung tissue section showing HGF single immunostaining (black color) of transgenic mouse bronchiole. (C), Double staining
of HGF (black) and CCSP (red) in transgenic lung bronchiole. Inset is a 1.5-fold high-powered view of double staining in epithelium. (D) double staining of
HGF (black) and CCSP (red) in wild-type littermate control lung bronchiole. Scale bar: 50 mm.
Fig. 6. Effects of HGF transgenic expression on baseline lung histology. (A–C), H&E staining of sections of whole mouse lung preparations from
representative wild-type littermate controls and (D–F), from 4-month-old HGF transgenic mice. Scale bars: 100 mm. Arrows indicated areas of increased
blood vessel production.
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L.P.Stabile et al.
at 30 and 38 weeks were adenocarcinomas and 38% were
adenomas.
We have also altered this protocol to decrease the time
to tumor formation. Four times the amount of NNK was
administered to the animals for a total dose of 24 mg NNK.
the animals were killed 13 weeks after the first injection, and
the number of tumors from NNK treated transgenic animals
(mean 1.58, median 2, range 0–4, n ¼12) versus NNK treated
wild-type animals (mean 0.67, median 1, range 0–2, n ¼12)
was significantly higher (P ¼ 0.04, Poisson regression
analysis).
Fig. 7. Tumor growth in transgenic mice. The number of mice/treatment
group, wild-type control (WT) and HGF transgenic (HGF) treated with
either saline control (C) or NNK (N), and time point from two separate
experiments. The open bars show the number of mice with no detectable
tumors while the solid gray bars represent the number of animals with at
least one tumor. The number of mice with two tumors is the striped gray
bar and the number of mice with three or more tumors is the striped
black bar.
combined data from H&E staining on serial sections and gross
tumor visualization, the tumor number, size and histology were
compared between the treatment groups. Figure 7 represents
the combined data from two separate experiments and shows
the number of mice per treatment group and time point. The
white bars show the number of mice with no detectable tumors
whereas the gray shaded bars represent the number of animals
with one tumor. The number of mice with two tumors is
the striped gray bar and the number of mice with three or
more tumors is the striped black bar. Statistical analysis
demonstrates that the total number of tumors from NNK
treated transgenic animals (mean 1.64, median 1, range 0–7,
n ¼ 44) versus the number of tumors from NNK treated
non-transgenic animals (mean 0.70, median 0, range 0–4,
n ¼ 43) differs significantly (P ¼ 0.0001, Poisson regression
analysis). There was no difference in tumor number between
males and females. The percentage of animals with at least one
tumor was 75% (33/44) in the NNK treated transgenic group
compared with 48.8% (21/43) in the NNK treated wild-type
group and this was statistically significant (P ¼ 0.013, logistic
regression). The main effect was an increase in tumor multiplicity in HGF transgenic versus wild-type mice. At the later
two time points, there was also considerable tumor formation
with NNK in wild-type animals.
The size of tumors was also compared with repeated measures modeling analysis and the median or mean size was not
significantly different between HGF transgenic and wild-type
controls (P ¼ 0.87). However, the range of tumor size in the
transgenic group at 30 weeks was 0.148–6.654 mm2 whereas
the range in the wild-type group was 0.654–2.498 mm2, indicating that at least some of the tumors grew larger in the transgenic animals. At 10 and 20 weeks, 100% of the tumors were
papillary adenomas in both transgenic and wild-type animals.
At 30 and 38 weeks, 75% of all tumors in HGF transgenic mice
were adenocarcinomas and the remaining smaller tumors
(25%) were adenomas. In wild-type mice, 62% of all tumors
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Tumors that arise in transgenic mice contain HGF protein
We have further shown that the lung tumors that arise in
the transgenic mice contain high amounts of human HGF
protein (Figure 8). Immunostaining with a human HGF specific
antibody shows much increased HGF protein content in lung
tumors arising from transgenic (Figure 8A and B) compared
with wild-type mice (Figure 8C), suggesting that tumorigenesis is associated with functional transgene. All photographs
were taken of areas on the section demonstrating the highest
staining. Immunostaining with an antibody that detects mouse,
rat and human HGF shows the same staining pattern (data not
shown) indicating that the endogenous mouse HGF is barely
detectable in these tumors. The HGF staining pattern in the
tumors is a combination of membrane and cytoplasmic staining
suggesting internal production of HGF in at least some malignant cells. Additionally, a northern blot analysis of RNA
isolated from tumors from transgenic animals show the
2.3 Kb HGF transgene RNA whereas tumors from wild-type
animals do not (Figure 8D). This strongly suggests that in the
transgenic animals, the tumors arose from airway epithelial
cells that express the HGF transgene. It is also possible that
some tumors arose from cells that bound HGF secreted by the
transgene. This model may mimic lung tumorigenesis induced
by a tobacco carcinogen that results in a tumor having a high
HGF content. c-Met protein expression was observed in tumors
from both transgenic and wild-type mice with uniformly low
expression and no differences based on size of tumors (data not
shown). CCSP immunostaining was negative in the tumors
from both HGF transgenic and wild-type mice (data not
shown). The lack of CCSP protein expression in lung tumors
is consistent with other animal models that use the CCSP
promoter for transgene expression. It appears that endogenous
CCSP protein expression is down-modulated during the
process of carcinogenesis (30). Some HGF protein expression
in the tumors could also be arising from other areas of the lung,
not necessarily only from within malignant cells in the tumor.
Surfactant apoprotein C, an alveolar type II cell marker that
may also show some expression in small airways, was also
expressed in 81% of the tumors regardless of HGF genotype,
tumor location or size of tumor. Another way to characterize
whether the tumors arise from alveolar Type II cells or from
Clara cells is to examine whether tumors show a solid or
a papillary phenotype, respectively. In our cases, we observed
43% solid tumors in the pulmonary parenchyma and 57%
papillary tumors associated with the airways. There was no
difference in transgenic versus non-transgenic animals in this
phenotype. Our results suggest that the tumors in this animal
model may arise from both Clara cells and alveolar Type II
cells, or from a cell that shows lineage with both, consistent
with other reports (31,32).
Role of HGF in lung cancer
Fig. 8. HGF is expressed in the tumors from transgenic mice. Sections of tumors from transgenic mice (A and B) and from a non-transgenic mouse (C)
were immunostained using a mouse monoclonal anti-HGF antibody. Scale bar: 25 mm. Inset is a 1.5-fold high-powered view of the HGF staining. (D),
Northern blot analysis of RNA isolated from tumors from two transgenic (+) and two wild-type control animals (). Blots were probed with an HGF
transgene specific probe and stripped and reprobed for GAPDH.
Discussion
HGF transgenic mice with overexpression in the lung have
been created and verified to be the expected genotype, and
to show phenotypic evidence of HGF expression and expression of the c-Met protein in the airway allowing them to
respond to HGF. Airway alterations were present in the transgenic animals, which included congestion, wide bifurcations
and increased blood vessels. The main effect in tumor formation between transgenic and wild-type mice was an increase in
tumor multiplicity; average tumor size was not different but the
range of tumor size was much wider in the transgenic mice.
Tumors arising after NNK exposure expressed c-Met and so
have the capacity of responding to HGF. These results suggest
that lung tumors induced by a tobacco carcinogen are promoted by expression of the HGF transgene, possibly through
both greater proliferation of individually mutated cells or by
migration of tumor cells to other areas of the lung where
additional tumors form. This is similar to a common type of
clinical recurrence when a new tumor nodule arises, either in
the ipsilateral or contralateral lung. The results also suggest
that the increased NNK carcinogenicity is related to increased
Clara cell numbers, since the Clara cell is believed to be a cell
of origin for papillary adenocarcinoma.
Phenotypically, tumors arose in this animal model that had
characteristics of both Clara cells and Type II pneumocytes.
In this regard, two types of tumor phenotypes were present,
papillary and solid, and some tumors expressed the alveolar
Type II cell specific marker, SPC, whereas CCSP expression in
most tumors was not detectable. These results are consistent
with other transgenic mouse models utilizing the CCSP promoter (31,32). Since there are different lung adenocarcinoma
features in human lung disease, including bronchioalveolar
carcinoma, adenocarcinoma with bronchioalveolar features
and pure adenocarcinoma, this animal model is representative
of human lung adenocarcinoma disease.
Increased HGF and/or c-Met expression by human tumor
cells have been associated with high tumor stage and poor
prognosis in lung cancer. For example, in non-small-cell
lung cancer patients, HGF is a useful prognostic indicator
for both early and advanced disease (11). Additionally, in
non-small-cell lung carcinoma cell lines and primary tumors,
c-Met overexpression correlated with a higher tumor stage and
worse outcome than those without overexpression (33). Coexpression of HGF and c-Met has been reported in approximately
50% of lung adenocarcinomas and this coexpression has been
reported to correlate with poor prognosis (34,35). Furthermore,
the survival rate for non-small-cell lung cancer patients with
both intratumoral positive c-Met expression and stromal positive HGF expression was significantly lower than that for
patients with only HGF or c-Met positive tumors as well as
that for patients with both HGF and c-Met negative tumors
(36). The strong correlation between HGF and c-Met expression and patient survival clearly supports the hypothesis that
the HGF/c-Met pathway plays an important role in the pathogenesis of human cancers. These studies examined protein
levels in tumors by immunoblotting or immunohistochemistry.
Recently, HGF levels present in serum from breast cancer
patients (37) and small-cell lung cancer patients (38) were
measured by ELISA and were demonstrated to correlate
with more advanced disease. HGF can potentially act as
a serum tumor biomarker for different human cancers and
possibly as a predictor for response to clinical therapy.
The results presented here are consistent with other animal
models of HGF expression. In this regard, production of transgenic mice that express HGF under the control of promoters
targeting gene expression to specific organs has resulted in
tumor formation only in the organs where the genes are
expressed. For example, transgenic mice have been produced
that express HGF in the liver under the control of the albumin
promoter (14) and these mice develop hepatocellular carcinomas. Transgenic mice have also been made that express HGF
1553
L.P.Stabile et al.
under the control of the metallothionein (MT) promoter. In this
case, tissues where tumors were found are also those where the
transgene expression is high including the mammary gland,
muscle and skin (39) and diethylnitrosamine, an environmental carcinogen, has been shown to dramatically increase
HGF-mediated tumorigenesis in the livers of MT–HGF transgenic mice versus wild-type controls (40). In another interesting model, MT–HGF transgenic mice in a scid background
were demonstrated to have increased growth of subcutaneous
xenografts from c-Met expressing cancer cell lines versus
wild-type mice (41). Renal tubular hyperplasia has been reported in transgenic mice expressing HGF in the kidney (42).
These results suggest that HGF can alter growth processes
in many tissues and is an important oncogenic pathway in
general. Overexpression in these tissues results in either spontaneous tumor formation or enhanced sensitivity to carcinogens. Recently, Frankel et al. (43) demonstrated that transgenic
mice, over 12 months of age, that overexpress the human
insulin-like growth factor-IA in the airways, show increased
premalignant changes in the alveolar epithelium compared
with wild-type littermate controls. Our model is the first animal
model showing that growth factor modulation can increase
lung cancer malignancies due to a tobacco carcinogen.
Because of the overwhelming evidence for the role of the
HGF/c-Met pathway in the pathogenesis of human cancers,
therapeutic inhibitors that target this pathway are being
developed. A recent report described a small molecule inhibitor, PHA-665752, which shows >50-fold selectivity for the
c-Met kinase compared with other tyrosine kinases and can
inhibit the formation of phospho-c-Met in vitro and in vivo
(44). Another small molecule inhibitor, SU11274, was reported to inhibit c-Met auto-phosphorylation and downstream
signaling in non-small-cell lung cancer (45). However, these
compounds have shown solubility problems and may not be
feasible for clinical development. Recently, a single blocking
anti-HGF antibody, L2G7, was shown to inhibit various biological activities induced by HGF in vitro and had profound
antitumor effects on intracranial glioma xenografts (46). This
single antibody approach is superior to other described neutralizing antibodies that target HGF but require a combination
of at least three distinct antibodies binding to different epitopes
for inhibition of HGF activity (47). Other potential inhibitors
of this pathway include the HGF kringle variant antagonists
such as the extensively studied NK4 (48), c-Met ribozyme
(49,50), c-Met antisense (51), c-Met dominant negative (52)
and decoy receptors (53) and c-Met peptide antagonists
(53,54). Collectively, these studies show that the HGF/
c-Met pathway is an attractive target for therapy and could
be clinically useful for many human cancers, especially lung.
These experiments have demonstrated the ability of upregulated HGF expression in the lungs to promote tumorigenesis
and tumor progression, and have corroborated our observations
that lung cancer patients with elevated HGF have more
aggressive disease (11). Although we did not find an increase
in the rate of spontaneous tumor formation in this model, the
differences observed in the carcinogen-induced model were
significant. This animal model recapitulates the phenotype
of aggressive lung adenocarcinoma that overexpresses HGF
and will be useful to test inhibitors of the HGF/c-Met signaling
pathway as well as other novel therapeutic inhibitors for lung
cancer such as those targeting angiogenesis, invasion or cell
migration. We have recently determined that the HGF/c-Met
pathway can activate the enzyme cyclooxygenase 2 (COX-2)
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via the MAP kinase pathway in non-small-cell lung cancer cell
lines in vitro, which can result in upregulation of important
pathways that are controlled by prostaglandins. Thus, this
animal model can also be used to test inhibitors of the
COX-2 pathway alone and in combination with HGF targeted
inhibitors.
Acknowledgments
This work was supported by a grant CA79882-04 awarded to J.M.S. from the
National Cancer Institute. We would like to acknowledge Dr Susan Reynolds
from the University of Pittsburgh for the CCSP antibody, Dr Brian Hackett for
the p0GH plasmid, Dr Reza Zarnegar for the HGF cDNA and the University of
Cincinnati Transgenic Mouse Facility for preparation of the transgenic mice.
L.P.S. was supported by a Career Development Award from the Lung Cancer
SPORE grant P50 CA9045440 awarded to J.M.S.
Conflict of Interest Statement: None declared.
References
1. American Cancer Society. (2004) Cancer facts and figures. In Surveillance
Research. American Cancer Society, Atlanta, GA, pp. 1–60.
2. Nakamura,T., Nawa,K. and Icihara,A. (1984) Partial purification and
characterization of hepatocyte growth factor from serum of
hepatectomized rats. Biochem. Biophys. Res. Commun., 12, 1450–1459.
3. Weidner,K.M., Behrens,J., Vandekerckhove,J. and Birchmeier,W. (1990)
Scatter factor: molecular characteristics and effect on the invasiveness
of epithelial cells. J. Cell. Biol., 11, 2097–2108.
4. Rong,S., Bodescot,M., Blair,D., Dunn,J., Nakamura,T., Mizuno,K.,
Park,M., Chan,A., Aaronson,S. and Vande Woude,G.F. (1992)
Tumorigenicity of the met proto-oncogene and the gene for hepatocyte
growth factor. Mol. Cell. Biol., 12, 5152–5158.
5. Rosen,E.M., Meromsky,L., Setter,E., Vinter,D.W. and Goldberg,I.D.
(1990) Smooth muscle-derived factor stimulates mobility of human
tumor cells. Invasion Metastasis, 10, 49–64.
6. Tsao,M.S., Zhu,H., Giaid,A., Viallet,J., Nakamura,T. and Park,M. (1993)
Hepatocyte growth factor/scatter factor is an autocrine factor for human
normal bronchial epithelial and lung carcinoma cells. Cell Growth Differ.,
4, 571–579.
7. Yoshinaga,Y., Matsuno,Y., Fujita,S., Nakamura,T., Kikuchi,M.,
Shimosato,Y. and Hirohashi,S. (1993) Human lung cancer cell line
producing hepatocyte growth factor/scatter factor. Jpn. J. Cancer Res.,
84, 1150–1158.
8. Tokunou,M., Niki,T., Eguchi,K. et al. (2001) c-MET expression in
myofibroblasts: role in autocrine activation and prognostic significance
in lung adenocarcinoma. Am. J. Pathol., 158, 1451–1463.
9. Yi,S. and Tsao,M.S. (2000) Activation of hepatocyte growth factor-met
autocrine loop enhances tumorigenicity in a human lung adenocarcinoma
cell line. Neoplasia, 2, 226–234.
10. Pilewski,J.M., Bumbalo,T., Davis,A.G. and Siegfried,J.M. (2001)
Hepatocyte growth factor promotes tumor growth in a novel in vivo
model of human lung cancer. Am. J. Respir. Mol. Cell. Biol., 24, 556–562.
11. Siegfried,J.M., Luketich,J.D, Stabile,L.P., Christie,N. and Land,S.R.
(2004) Elevated hepatocyte growth factor level correlates with poor
outcome in early and late stage adenocarcinoma of the lung. Chest, 125,
116S–119S.
12. Stabile,L.P., Lyker,J.S., Huang,L. and Siegfried,J.M. (2004) Inhibition of
human non-small cell lung tumors by a c-Met antisense/U6 expression
plasmid strategy. Gene Ther., 11, 325–335.
13. Maulik,G., Kijima,T., Ma,P.C., Ghosh,S.K., Lin,J., Shapiro,G.I.,
Schaefer,E., Tibaldi,E., Johnson,B.E. and Salgia,R. (2002) Modulation
of the c-Met/hepatocyte growth factor pathway in small cell lung
cancer. Clin. Cancer Res., 8, 620–627.
14. Bell,A., Chen,Q., DeFrances,M.C., Micalopoulos,G.K. and Zarnegar,R.
(1999) The five amino-acid-deleted isoform of hepatocyte growth factor
promotes carcinogenesis in transgenic mice. Oncogene, 18, 887–895.
15. Beier,H.M., Kirchner,C. and Mootz,M. (1978) Uteroglobin-like antigen
in the pulmonary epithelium and secretion of the lung. Cell Tissue Res.,
190, 15–25.
16. Krishnan,R.S. and Daniel,J.C.,Jr (1967) ‘Blastokinin’: inducer and
regulator of blastocyst development in the rabbit uterus. Science, 158,
490–492.
Role of HGF in lung cancer
17. Singh,G. and Katyal,S.L. (1997) Clara cells and clara cell 10 kDa protein
(CC10). Am. J. Respir. Cell Mol. Biol., 17, 141–143.
18. Hackett,B. and Gitlin,J.D. (1992) Cell specific expression of a Clara cell
secretory protein-human growth hormone gene in the bronchiolar
epithelium of transgenic mice. Proc. Natl Acad. Sci. USA, 89, 9079–9083.
19. Singh-Kaw,P, Zarnegar,R. and Siegfried,J.M. (1995) Stimulatory effects of
hepatocyte growth factor on normal and neoplastic human bronchial
epithelial cells. Am. J. Physiol., 268, L1012–L1020.
20. Yanagita,K., Nagaike,M., Ishibashi,H., Niho,Y., Matsumoto,K. and
Nakamura,T. (1992) Lung may have an endocrine function producing
hepatocyte growth factor in response to injury to distal organs.
Biochem. Biophys. Res. Commun., 182, 802–809.
21. Selden,R.F., Howie,K.B., Rowe,M.E., Goodman,H.M. and Moore,D.D.
(1986) Human growth hormone as a reporter gene in regulation studies
employing transient gene expression. Mol. Cell. Biol., 6, 3173–3179.
22. Stabile,L.P.,
Gaither
Davis,A.L.,
Gubish,C.T.,
Hopkins,T.M.,
Luketich,J.D., Christie,N., Finkelstein,S. and Siegfried,J.M. (2002)
Human non-small cell lung tumors and cells derived from normal lung
express both estrogen receptor a and b and show biological responses to
estrogen. Cancer Res., 62, 2141–2150.
23. Chomczynski,P. and Sacchi,N. (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem., 162, 156–9.
24. Church,G.M. and Gilbert,W. (1984) Genomic sequencing. Proc. Natl Acad.
Sci. USA, 81, 1991–1995.
25. Amasino,R.M. (1986) Acceleration of nucleic acid hybridization rate by
polyethylene glycol. Anal. Biochem., 152, 304–307.
26. Mahler,J.F., Stokes,W., Mann,P.C., Takaoka,M. and Maronpot,R.R. (1996)
Spontaneous lesions in aging FVB/N mice. Toxicol. Pathol., 24, 710–716.
27. Yamanouchi,H., Fujita,J., Yoshinouchi,T., Hojo,S., Kamei,T., Yamadori,I.,
Ohtsuki,Y., Ueda,N. and Takahara,J. (1998) Measurement of hepatocyte
growth factor in serum and bronchoalveolar lavage fluid in patients with
pulmonary fibrosis. Respir. Med., 92, 273–278.
28. Ohmichi,H., Koshimizu,U., Matsumoto,K. and Nakamura,T. (1998)
Hepatocyte growth factor (HGF) acts as a mesenchyme-derived
morphogenic factor during fetal lung development. Development, 125,
1315–1324.
29. Belinsky,S.A., Devereux,T.R., Maronpot,R.R., Stoner,G.D. and
Anderson,M. (1989) Relationship between the formation of
promutagenic adducts and the activation of the K-ras proto-oncogene in
lung tumors from A/J mice treated with nitrosamines. Cancer Res., 49,
5305–5311.
30. Tchou-Wong,K.M., Jiang,Y., Yee,H., LaRosa,J., Lee,T.C., Pellicer,A.,
Jagirdar,J., Gordon,T., Goldberg,J.D. and Rom,W.N. (2002) Lungspecific expression of dominant-negative mutant p53 in transgenic mice
increases spontaneous and benzo(a)pyrene-induced lung cancer. Am. J.
Respir. Cell Mol. Biol., 27, 186–193.
31. Lee,J.J., McGarry,M.P., Farmer,S.C. et al. (1997) Interleukin-5 expression
in the lung epithelium of transgenic mice leads to pulmonary changes
pathognomic of asthma. J. Exp. Med., 185, 2143–2156.
32. Dhami,R., Zay,K., Gilks,B., Porter,S., Wright,J.L. and Churg,A. (1999)
Pulmonary epithelial expression of human alpha1-antitrypsin in
transgenic mice results in delivery of alpha1-antitrypsin protein to the
interstitium. J. Mol. Med., 77, 377–385.
33. Ichimura,E., Maeshima,A., Nakajima,T. and Nakamura,T. (1996)
Expression of c-met/HGF receptor in human non-small cell lung
carcinomas in vitro and in vivo and its prognostic significance. Jpn. J.
Cancer Res., 87, 1063–1069.
34. Harvey,P., Warn,A., Newman,P., Perry,L.J., Ball,R.Y. and Warn,R.M.
(1996) Immunoreactivity for hepatocyte growth factor and its receptor,
met, in human lung carcinomas and malignant mesothelioma. J. Pathol.,
180, 389–394.
35. Takanami,I., Tanana,F., Hashizume,T., Kikuchi,K., Yamamoto,Y.,
Yamamoto,T. and Kodaira,S. (1996) Hepatocyte growth factor and
c-Met/hepatocyte growth factor receptor in pulmonary adenocarcinomas:
an evaluation of their expression as prognostic markers. Oncology, 53,
392–397.
36. Masuya,D., Huang,C., Liu,D., Nakashima,T., Kameyama,K., Haba,R.,
Ueno,M. and Yokomise,H. (2004) The tumour-stromal interaction
between intratumoral c-Met and stromal hepatocyte growth factor
associated with tumour growth and prognosis in non-small cell lung
cancer patients. Br. J. Cancer, 90, 1555–1562.
37. Sheen-Chen,S.-M., Liu,Y.-W., Eng,H-L. and Chou,F.-F. (2005) Serum
levels of hepatocyte growth factor in patients with breast cancer.
Cancer Epidemiol. Biomarkers Prev., 14, 715–717.
38. Bharti,A., Ma,P.C., Maulik,G., Singh,R., Khan,E. and Skarin,A.T. (2004)
Haptoglobin alpha-subunit and hepatocyte growth factor can potentially
serve as serum tumor biomarkers in small cell lung cancer. Anticancer Res.,
24, 1031–1038.
39. Takayama,H., LaRochelle,W., Sharp,R., Otsuka,T., Kriebel,P., Anver,M.,
Aaronson,S.A. and Merlino,G. (1997) Diverse tumorigenesis associated
with aberrant development in mice overexpressing hepatocyte growth
factor/scatter factor. Proc. Natl Acad. Sci. USA, 94, 701–706.
40. Horiguchi,N., Takayama,H., Toyoda,M., Otsuka,T., Fukusato,T.,
Merlino,G., Takagi,H. and Mori,M. (2002) Hepatocyte growth factor
promotes hepatocarcinogenesis through c-Met autocrine activation
and enhanced angiogenesis in transgenic mice treated with
diethylnitrosamine. Oncogene, 21, 1791–1799.
41. Zhang,Y.W., Su,Y., Lanning,N., Gustafson,M., Shinomiya,N., Zhao,P.,
Cao,B., Tsarfaty,G., Wang,L.M., Hay,R. and Vande Woude,G.F. (2005)
Enhanced growth of human met-expressing xenografts in a new strain of
immunocompromised mice transgenic for human hepatocyte growth factor/
scatter factor. Oncogene, 24, 101–106.
42. Takayama,H., LaRochelle,W., Sabnis,G.S., Otsuka,T. and Merlino,G.
(1997) Renal tubular hyperplasia, polycystic disease, and
glomerulosclerosis in transgenic mice overexpressing hepatocyte growth
factor/scatter factor. Lab Invest., 77, 131–138.
43. Frankel,S.K., Moats-Staats,B.M., Cool,C.D., Wynes,M.W., Stiles,A.D. and
Riches,D.W. (2005) Human insulin-like growth factor-IA expression
in transgenic mice promotes adenomatous hyperplasia but not
pulmonary fibrosis. Am. J. Physiol. Lung Cell Mol. Physiol., 288,
L805–L812.
44. Christensen,J.G., Schreck,R., Burrows,J. et al. (2003) A selective small
molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes
in vitro and exhibits cytoreductive antitumor activity in vivo. Cancer Res.,
63, 7345–7355.
45. Ma,P.C., Jagadeeswaran,R., Jagadeesh,S. et al. (2005) Functional
expression and mutations of c-Met and its therapeutic inhibition with
SU11274 and small interfering RNA in non-small cell lung cancer.
Cancer Res., 65, 1479–1488.
46. Kim,K.J., Wang,L, Su,Y.-C., Gillespie,G.Y., Salhotra,A., Lal,B. and
Laterra,J. (2006) Systemic anti-HGF monoclonal antibody therapy
induces regression of intracranial glioma xenografts. Clin. Cancer Res.,
12, 1292–1298.
47. Cao,B., Su,Y., Oskarsson,M., Zhao,P., Kort,E.J., Fisher,R.J., Wang,L.M.
and Vande Woude,G.F. (2001) Neutralizing monoclonal antibodies to
hepatocyte growth factor/scatter factor (HGF/SF) display antitumor
activity in animal models. Proc. Natl Acad. Sci. USA, 98, 7443–7448.
48. Matsumoto,K. and Nakamura,T. (2003) NK4 (HGF-antagonist/
angiogenesis inhibitor) in cancer biology and therapeutics. Cancer Sci.,
94, 321–327.
49. Abounader,R., Ranganathan,S., Lal,B., Fielding,K., Book,A., Dietz,H.,
Burger,P. and Laterra,J. (1999) Reversion of human gliobastoma
malignancy by U1 small nuclear RNA/ribozyme targeting of scatter
factor/hepatocyte growth factor and c-met expression. J. Natl Cancer
Inst., 91, 1548–1556.
50. Herynk,M.H., Stoeltzing,O., Reinmuth,N., Parikh,N.U., Abounader,R.,
Laterra,J., Radinsky,R., Ellis,L.M. and Gallick,G.E. (2003) Downregulation of c-Met inhibits growth in the liver of human colorectal
carcinoma cells. Cancer Res., 63, 2990–2996.
51. Kaplan,O., Firon,M., Vivi,A., Navon,G. and Tsarfaty,I. (2000) HGF/SF
activates glycolysis and oxidative phosphorylation in DA3 murine
mammary cancer cells. Neoplasia, 2, 365–377.
52. Michieli,P., Mazzone,M., Basilico,C., Cavassa,S., Sottile,A., Naldini,L.
and Comoglio,P.M. (2004) Targeting the tumor and its
microenvironment by a dual-function decoy Met receptor. Cancer Cell,
6, 61–73.
53. Bardelli,A., Longati,P., Gramaglia,D., Basilico,C., Tamagnone,L.,
Giordano,S., Ballinari,D., Michieli,P. and Comoglio,P.M. (1998)
Uncoupling signal transducers from oncogenic MET mutants abrogates
cell transformation and inhibits invasive growth. Proc. Natl Acad. Sci.
USA, 95, 14379–14383.
54. Atabey,N., Gao,Y., Yao,Z.J., Breckenridge,D., Soon,L., Soriano,J.V.,
Burke,T.R.,Jr and Bottaro,D.P. (2001) Potent blockade of hepatocyte
growth factor-stimulated cell motility, matrix invasion and branching
morphogenesis by antagonists of Grb2 Src homology 2 domain
interactions. J. Biol. Chem., 276, 14308–14314.
Received November 9, 2005; revised January 31, 2006;
accepted February 25, 2006
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