[CANCER RESEARCH 60, 490 – 498, January 15, 2000]
Oncogenes and Tumor Angiogenesis: Differential Modes of Vascular Endothelial
Growth Factor Up-Regulation in ras-transformed Epithelial Cells
and Fibroblasts1
Janusz Rak,2 Yoshihiro Mitsuhashi,2 Cap Sheehan,2 Ami Tamir, Alicia Viloria-Petit, Jorge Filmus, Sam J. Mansour,
Natalie G. Ahn, and Robert S. Kerbel3
Division of Cancer Biology Research, Sunnybrook Health Science Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M4N 3M5 Canada
[J. R., Y. M., C. S., A. T., A. V-P., J. F., R. S. K.], and Department of Molecular, Cellular and Developmental Biology and Howard Hughes Medical Institute Department of
Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 30303 [S. J. M., N. G. A.]
ABSTRACT
A possible link between oncogenes and tumor angiogenesis has been
implicated by the finding that expression of various oncogenes, particu-
larly mutant ras, can lead to a marked induction of a potent paracrine
stimulator of angiogenesis, vascular endothelial growth factor (VEGF).
We sought to determine how oncogenic ras induction of VEGF is mediated
at the molecular level and whether the mechanisms involved differ fun-
damentally between transformed epithelial cells and fibroblasts. Our
results suggest that in a subline (called RAS-3) of immortalized nontu-
morigenic rat intestinal epithelial cells (IEC-18) that acquired a tumori-
genic phenotype upon transfection of mutant ras, up-regulation of VEGF
occurs in the absence of an autocrine growth factor circuit. The expression
of VEGF mRNA and protein by RAS-3 cells was strongly suppressed in
the presence of LY294002, an inhibitor of phosphatidylinositol 3ⴕ-kinase,
but remained largely unaffected in the same cells treated with an inhibitor
(PD98059) of mitogen-activated protein/extracellular signal-regulated
kinase kinase 1 (MKK/MEK-1). This is consistent with the observation
that overexpression of a constitutively activated mutant of MEK-1 (⌬N3/
S222D) in the parental IEC-18 cells did not result in up-regulation of
VEGF production. The impact of mutant ras on VEGF expression was
also significantly amplified at high cell density, conditions under which
RAS-3 cells became less sensitive to LY294002-induced VEGF down-
regulation.
In marked contrast to cells of epithelial origin, ras-transformed murine
fibroblasts (3T3RAS) up-regulated VEGF in a manner that was strongly
inhibitable by MEK-1 blockade (i.e. treatment with PD98059), whereas
these cells were relatively unaffected by treatment with the phosphati-
dylinositol 3ⴕ-kinase inhibitor LY294002. In addition, VEGF was up-
regulated by 2–3-fold in NIH3T3 cells overexpressing mutant MEK-1.
Collectively, the data suggest that the stimulatory effect of mutant ras on
VEGF expression is executed in a nonautocrine and cell type-dependent
manner and that it can be significantly exacerbated by physiological/
environmental influences such as high cell density.
INTRODUCTION
Historically, the major functional emphasis on oncogenes as con-
tributors to tumor development has been on their impact on promoting
aberrant cellular mitogenesis. However, we and Grugel et al. (1, 2)
first suggested that oncogenes, such as mutant ras, may also have an
important impact on tumor formation and growth through an indirect
mechanism, namely, by driving tumor angiogenesis. Thus, transfec-
tion of mutant ras oncogenes into immortalized nontumorigenic ro-
dent fibroblasts (2) or epithelial cells (1) resulted in a strong induction
Received 8/2/99; accepted 11/12/99.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by grants from the Medical Research Council of Canada and the United
States NIH, (CA-41233) to R. S.K.
2
J. R., Y. M., and C. S. contributed equally to this work.
3
To whom requests for reprints should be addressed, at Sunnybrook Health Sciences
Center, S-218 Research Building, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5
Canada. Phone: (416) 480-5711; Fax: (416) 480-5703.
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of VEGF,4 also known as vascular permeability factor. VEGF is a
ubiquitous and potent stimulator of tumor angiogenesis with no
known direct (autocrine) tumor cell growth-promoting activity (3).
We also found that genetic disruption of the single mutant K-ras allele
in two different human colorectal carcinoma cell lines (4) was asso-
ciated with a significant suppression of VEGF expression (1) and loss
of tumorigenic competence (1, 4). The latter effect could be dupli-
cated by antisense-mediated down-regulation of VEGF in K-ras-
expressing human colon cancer cells (5). Moreover, embryonic stem
cells (6) in which the VEGF gene has been disrupted have grossly
impaired tumor-forming capacity, even after transfection with onco-
genic ras (7), as do VEGF⫺/⫺ mouse embryo fibroblasts harboring
an activated ras oncogene (8). Collectively, these studies suggest that
VEGF expression may be a necessary but insufficient mediator (5, 9)
of the tumorigenic function of mutant ras oncogenes. These results
also appear to be consistent with in vivo studies in which the expres-
sion of a ras oncogene is conditionally switched off in growing
tumors (10).
The exact mechanisms by which mutant ras exerts this impact on
VEGF expression are not well understood. Our desire to study this
relationship was based, in part, on the prediction that it may be
possible to identify new targets for inhibitors of oncoprotein signal
transduction that could obliterate tumor angiogenesis, albeit in an
indirect manner. In this regard, we showed that ras farnesyltransferase
inhibitors (Ras FTIs) can block VEGF expression in ras-transformed
cells (1), a result that was recently confirmed in ras-transformed
human keratinocyte (HaCAT) cell line (11). Thus, signal transduction
inhibitors such as ras FTIs may bring about a part of their antitumor
activity by inhibiting expression of proangiogenic growth factors such
as VEGF (1, 12, 13), an effect that could contribute to the drug’s
overall antitumor efficacy in vivo, but not in vitro.
One of the best-characterized signaling pathways directly activated
by ras involves a cascade of interactions comprising Raf-1, MAPK
kinase, or MEK, and its substrates, MAPKs, also known as ERK1 and
ERK2. Expression of constitutively active mutants of Raf-1 or MEK
in mouse fibroblasts can result in oncogenic transformation resem-
bling that induced by oncogenic ras itself (14). This linear epistasis of
different oncogenic proteins has been interpreted as evidence that the
raf/MEK/MAPK cascade plays a central role in ras-driven tumorigen-
esis (15). However, more recent studies, have suggested that a number
of other ras-activated pathways contribute significantly to oncogenic
cellular transformation (15). These include the activity of the multi-
molecular complex containing rac-1 and NADPH-oxidase, which is
thought to be responsible for the generation of high levels of ROIs and
4
The abbreviations used are: VEGF, vascular endothelial growth factor; MAPK,
mitogen-activated protein kinase; TSP-1, thrombospondin 1; TGF, transforming growth
factor; EGFR, epidermal growth factor receptor; ROI, reactive oxygen intermediates;
NAC, N-acetylcysteine; FBS, fetal bovine serum; PI3K, phosphatidylinositol 3⬘-kinase;
MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; ERK, ex-
tracellular signal-regulated kinase; MBP, myelin basic protein; FTI, farnesyl transferase
inhibitor; NDGA, nordihydroguaiaretic acid; L-NAME, L-Nc-nitroarginine methyl ester.
 ONCOGENES AND TUMOR ANGIOGENESIS
for stimulation of gene transcription and mitogenesis in mutant H-ras-
transformed fibroblasts (16). Further emphasis on pathways other than
ras/raf/MAPK has become apparent with the observation that activity
of PI3K is required for ras-dependent transformation and that the
catalytic subunit of this enzyme (p110) can synergize with activated
Raf-1 (17, 18). Moreover, it has been shown that constitutive activa-
tion of the Raf-1/MAPK cascade fails to recapitulate the transforming
effect of mutant ras on morphology, anchorage-independent growth in
soft agar, and tumorigenicity in vivo in rodent epithelial cells (19). In
such cells, unlike in fibroblasts (20), many features of malignant
transformation can be mediated in an indirect, autocrine fashion by
exuberant, ras-dependent overproduction of TGF-␣ (19, 21, 22). In
this respect, it is noteworthy that TGF-␣ itself is a strong inducer of
VEGF production via activation of the EGFR (13, 23–25).
Both oncogenic and certain physiological/regulatory influences can
affect VEGF expression at multiple levels. This includes regulation of
gene transcription (26 –29), mRNA stability (30 –33), the rate of
mRNA translation (34 –37), secretory pathways, and alternative splic-
ing, as well as by expression of and heterodimerization with other
members of the same growth factor family, e.g., placenta growth
factor (38). In theory, each of these levels of regulation could be open
to influences by mutant ras.
The purpose of the present study was to examine the contribution to
VEGF up-regulation of some of the possible signaling cascades trig-
gered by mutant H-ras and to determine whether they may differ in
relative importance between epithelial and fibroblastic cells. Our
results show that the mode of VEGF induction by activated ras is cell
type specific and can be largely dissociated from the respective impact
of this oncogene on cellular mitogenesis.
MATERIALS AND METHODS
Cells and Culture Conditions. The origin of cell lines used and their
growth requirements have been described elsewhere in detail (1, 14). Briefly,
immortalized rat intestinal epithelial cells (IEC-18) and their derived transfec- perpendicular diameters from which the tumor volume was calculated accord-
tants were cultured in ␣-MEM supplemented with glucose, insulin, and 5%
FBS. NIH3T3 mouse fibroblasts and their sublines were maintained in DMEM
with 10% FBS (1). The mutant H-ras-transformed NIH3T3 cell line (3T3RAS)
consists of a pool of over 100 clones transfected with the expression vector
(pSV2Ras3) that encodes V12 mutant of human H-ras proto-oncogene. The
same vector was used to generate the IEC-18-derived series of H-ras trans-
fectants. All transfections were carried out by using lipofectin reagent accord-
ing to the recommendations of the manufacturer (Life Technologies, Inc.,
Gaithersburg, MD). The cell lines NIH3T3/MKK1/wt (cl D1, wtMEK) and
NIH3T3/MKK1/⌬N3/S222D (cl F1 mutant MEK-1) are NIH3T3 transfectants
overexpressing either wild-type (CMV-MKK1/wt) or a modified (CMV-
MKK1/⌬N3/S222D or “11A/55B”), constitutively active form of MEK-1,
respectively. These cell lines were generated and characterized previously by
P. A. and N. A. (14). Confluence was varied by plating different numbers of
cells at least 24 h before further manipulations. Cultures less than 50%
confluent were considered sparse, whereas cultures with more than 90%
confluence were assumed to be dense. All the assays were performed in either
ExCell300 medium or DMEM with supplements, as indicated.
Inhibitors and Treatments. The specific MEK-1 inhibitor PD98059 was
initially supplied as a gift by Parke-Davis Pharmaceuticals (Ann Arbor, MI)
and subsequently purchased from commercial sources (New England Biolabs,
Beverly, MA). PD98059, which inhibits Raf-1-dependent activation of MEK-1
(39) and constitutive activity of mutant MEK-1 (40), was used at concentra-
tions ranging between 1.5 and 50 ␮M, which were made up in the assay
medium from concentrated (20 mM) stocks containing DMSO as a vehicle.
LY294002 (Biomol, Plymouth Meeting, PA), an inhibitor of PI3K, was used
at concentrations between 1 and 20 ␮M and was typically applied for 1–24 h
in different experiments. Other DMSO-soluble inhibitors (also purchased from
Biomol) were prepared and tested in a similar manner against DMSO-treated
controls at the following working concentrations: sulindac sulfide, 100 ␮M;
okadaic acid, 20 nM; genistein, 100 ␮M; calphostin C, 1 ␮M; GF109203X, 40
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␮M; BAY 11-7085, 20 ␮M, PP1, 20 ␮M; and rapamycin, 550 nM. Likewise,
NDGA (Calbiochem-Novabiochem Corp. La Jolla, CA) from Larrea divari-
cata was diluted in culture medium to a concentration of 20 ␮M from a
concentrated stock prepared in DMSO. NAC was purchased from Calbiochem
and used at the concentrations indicated, which were prepared from fresh 0.5
M aqueous stock solutions and adjusted to the physiological pH with 1N NaOH.
L-NAME (Biomol) was used at the working concentration of 10 ␮M, prepared
from aqueous concentrated stock. The monoclonal neutralizing antibody that
recognizes both human and rat EGFR (mR3), a generous gift of Dr. Tania
Crombet (Centre for Molecular Immunology, Havana, Cuba), was used at a
concentration of 50 ␮g/ml, and was previously demonstrated to obliterate
EGFR-dependent expression of VEGF in A431 cells,5 similar to results ob-
tained using a different monoclonal anti-EGFR antibody, i.e., C225 (13). Most
treatments were performed in the same assay medium (DMEM and 1% FBS),
unless otherwise indicated. Conditioned medium was obtained by incubating
cells with the growth medium or the assay medium for at least 24 h; then the
medium was collected, spun, passed through a 0.22 ␮m filter, and assayed or
used for further treatments.
Northern Blotting. Polyadenylated mRNA was prepared by SDS oli-
godeoxythymidylic acid method as described previously (1). Total RNA
was prepared by using Trizol reagent (Life Technologies, Inc., Grand
Island, NY) according to the manufacturer’s recommendations. RNA was
resolved on 1% agarose gel and transferred to a Hybond N⫹ (Amersham
Canada Limited, Oakville, Ontario, Canada) hybridization membrane. The
hybridizations with 32P-labeled probes were carried out at 65°C after which
the blots were washed while monitoring their radioactivity. The VEGF/
vascular permeability factor probe spanning the 200-bp sequence common
for all VEGF isoforms was a generous gift of Dr. Brygida Berse and Dr.
Harold Dvorak (Beth Israel Hospital, Boston, MA). The 1.29-kb TSP-1
probe was prepared from pGEM2 vector encoding human throm-
bospondin-1 (American Type Culture Collection, Rockville, MD). To
ensure equal loading, the gels were stained with ethidium bromide and
photographed, and/or the membranes were subsequently probed for glyc-
eraldehyde-3-phosphate dehydrogenase or for 28S ribosomal RNA.
Tumorigenicity Assay. Cells were injected s.c. into nude mice in equal
numbers, which, for different experiments, varied between 1 ⫻ 106 and
5 ⫻ 106 cells/injection. Tumor growth was monitored by measuring two
ing to the protocol published previously (1, 13). Absence of a measurable
tumor 3–10 months after injection was considered as “no take.”
VEGF Protein Quantitation. Production of VEGF was quantitated by an
ELISA (R&D Systems Inc., Minneapolis, MN) specific for either human or
mouse VEGF protein, as described by the supplier. The latter assay can also
detect rat VEGF. The results were expressed as either an absolute concentra-
tion of VEGF (in pg/ml; i.e. VEGF production in pg/ml/106 cells/time of
conditioning; 6 – 48 h) or as a percentage of change compared to the respective
controls.
Mitogenesis Assays. The [3H]thymidine incorporation assay was con-
ducted as described previously (1). Briefly, cells were plated at desired
confluence (usually 5–10 ⫻ 103 cells/well) in the 96-well plate in 0.1 ml of
medium and incubated with or without treatment for different lengths of time
(6 –18 h). The last 2 h of the incubation were conducted in the presence of 2
␮Ci of [3H]thymidine (Amersham) per well. The plates were frozen and
thawed, and radioactive DNA was harvested onto the filter paper. The [3H]thy-
midine incorporation was quantitated by using the BetaPlate (Pharmacia)
scintillation counter.
MAPK Assay. The treatment was carried out as indicated. The cells were
lysed in buffer containing 50 mM Tris (pH 7.3), 2% NP40, 5 mM sodium
orthovanadate, 5 mM EDTA, 5 mM sodium P-nitrophenyl phosphate, 5 mM
sodium fluoride, 50 mM sodium chloride, 50 ␮g/ml aprotinin, and 50 ␮g/ml
leupeptin. MAPK (ERK2) was immunoprecipitated from the equivalent of
3 ⫻ 106 cells by overnight incubation with 1 ␮g of the anti-ERK2 antibody
(Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with
protein A-Sepharose. The precipitates were washed and incubated at 30°C for
30 min with 20 ␮l of kinase buffer containing 30 mM Tris (pH 7.3), 20 mM
magnesium chloride, 2 mM manganese chloride, 10 ␮M ATP, 10 ␮Ci of
5
A. Viloria-Petit, T. Crombet, and R. S. Kerbel, unpublished observation.
 ONCOGENES AND TUMOR ANGIOGENESIS

[␥-32P]ATP, and 10 ␮g of MBP. The reaction was terminated by the addition

of 20 ␮l of 2⫻ sample buffer containing SDS. The samples were then resolved
by 15% SDS-PAGE and transferred onto the Immobilon P membrane (Mili-
pore, Bedford, MA). The radioactivity built into MBP was visualized by
autoradiography and quantitated by using a PhosphorImager scanner, whereas
equal loading was confirmed by either staining the membranes with Naphtol
blue black or probing them with the same anti-ERK2 antibody. All reagents
were purchased from Sigma (St. Louis, MO) unless otherwise indicated.
Data Analysis. All results shown are representative of at least two inde-
pendent reproducible experiments. The error bars represent SDs between
replicate determinations within each experiment.

RESULTS

Inverse Relationship between Expression of VEGF and TSP-1

in H-ras-transformed Intestinal Epithelial Cells. Transfection of a
mutant H-ras oncogene into the immortal but nontumorigenic rat
intestinal epithelial cell line IEC-18 resulted in cellular transformation
and expression of tumorigenic and angiogenic properties (1, 41).
Whereas it is generally believed that sustained tumor angiogenesis
occurs as a result of a shift in balance between various stimulatory and
inhibitory influences (42, 43), it is not clear whether different trans-
forming oncogenes produce a unique pattern or common pattern of
such molecular changes. In this regard, we observed that in IEC-18
cells, the impact of mutant ras on expression of angiogenesis regula-
tors such as VEGF (an angiogenesis stimulator) and TSP-1 (an en-
dogenous angiogenesis inhibitor) is different from that of the v-src
oncogene (Fig. 1A), although both oncogenes produce a similar type
of cellular transformation (44). Thus, VEGF mRNA was up-regulated
severalfold in both H-ras (RAS-3 and RAS-4) and v-src (SRC-3 and
SRC-4) transfectants (Fig. 1A), in agreement with results obtained
previously by us and others (1, 45– 47). In contrast, levels of TSP-1
transcript followed an oncogene-specific pattern of expression, i.e., it
became virtually nondetectable in all stable cell lines transfected with
oncogenic ras, whereas clones harboring v-src retained considerable
levels of TSP-1 expression (Fig. 1A). Despite its known angiogenesis
inhibitory properties in fibroblasts (48, 49), expression of TSP-1 did
not obliterate the tumorigenic capacity of VEGF-producing v-src
transfectants of IEC-18 cells, all of which formed aggressive and
highly angiogenic tumor outgrowths upon injection of the cells into
nude mice (Fig. 1B). Tumors also formed after injection of H-ras
transfectants, but not after injection of parental IEC-18 cells, or their
two independent clonal derivatives, IEC-18/4A and IEC-18/4B (en-
gineered to express TGF-␣). It is noteworthy that differential levels of
TSP-1 expression correlated with and could potentially account for
somewhat delayed tumor take in the case of v-src-transformed IEC-18
cells as compared to their counterparts harboring oncogenic ras (Fig.
1B). In this context, however, up-regulation of VEGF seems to be an
angiogenesis-related change that is more closely associated with on-
cogenic transformation of epithelial cells.
Up-Regulation of VEGF in ras-transformed Epithelial Cells Is
Not Mediated by an Autocrine Growth Factor Stimulation. It has
been demonstrated recently that in rodent epithelial cells, many char-
acteristics of ras-induced transformation are mediated by up-regula-
tion of and autocrine response to TGF-␣ (21, 22). Because this growth
factor is also known as a potent inducer of VEGF expression (25), it
is reasonable to anticipate that it could be involved in the mechanism
of ras-induced VEGF up-regulation. Furthermore, we noticed that
expression of VEGF mRNA (Fig. 2A) and protein (data not shown) is
considerably elevated with increasing cell density, but only in ras-
transformed IEC-18 cells (RAS-3), and not in the nontumorigenic
parental IEC-18 counterparts (Fig. 2A). An explanation for this find-
ing could lie in the existence of a growth factor-mediated autocrine
stimulation (50) possibly involving an oncogenic ras-induced growth
492
Fig. 1. Differential patterns of VEGF and TSP-1 mRNA expression in epithelial cells
transformed with oncogenic forms of ras or src. A, simultaneous up-regulation of VEGF
and down-regulation of TSP-1 in sublines of IEC-18 cells expressing mutant H-ras
(RAS-3, RAS-4; left panel). Up-regulation of VEGF without a change in TSP-1 levels in
v-src-expressing IEC-18 cells (SRC-3, SRC-4; right panel). Polyadenylated RNA prepa-
ration was analyzed as described in “Materials and Methods.” B, relative tumor-forming
capacity of various IEC-18 sublines on s.c. injection (2 ⫻ 106 cells were injected per
mouse, five mice per group). Tumor take was 100% for both H-ras or v-src-transformed
cell lines (RAS-3, RAS-4, SRC-3, and SRC-4), respectively. No tumors were observed in
the case of IEC-18 parental cells or two clones derived thereof by transfection with a
human TGF-␣ expression vector (21) for up to 5 months, at which point the experiment
was terminated.
factor (e.g., TGF-␣). However, there are several facts that argue
against such an interpretation. First, addition of the mR3 neutralizing
anti-EGFR antibody, which blocks binding of TGF-␣ to the rat EGFR,
did not diminish VEGF expression by RAS-3 cells (Fig. 2B), nor did
direct neutralization of the growth factor itself (data not shown).
Furthermore, treatment of RAS-3 cells with genistein, a tyrosine
kinase inhibitor also known to block EGFR activity, failed to abrogate
VEGF up-regulation; in fact, it led to a marked increase in expression
of this angiogenic growth factor (Fig. 2C; Table 1). Because inhibition
of phosphatase activity by okadaic acid treatment brought about the
opposite effect (Table 1), it is possible that the impact of mutant ras
on VEGF expression is, in fact, negatively regulated by events in-
volving tyrosine phosphorylation. Finally, addition of the RAS-3-
conditioned medium to parental IEC-18 cells did not result in any
detectable transfer of VEGF up-regulation (Fig. 2D). Interestingly, a
slight increase in VEGF mRNA was observed in RAS-3 cells in the
presence of their own conditioned medium as compared to the situ-
ation when the cells were exposed to control culture medium or
IEC-18-conditioned medium (Fig. 2D). This observation suggests that
RAS-3 cells may display some degree of hypersensitivity to their own

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 ONCOGENES AND TUMOR ANGIOGENESIS

cells expressing wild-type MEK-1 (wt-4.4) or to the parental (IEC-18)

population (Fig. 3A). Treatment of these various sublines as well as
ras-transformed IEC-18 cells (RAS-3) with a pharmacological inhib-
itor of MEK-1 activity (PD98059) did not result in abrogation of
VEGF secretion into the condition medium (Fig. 3A). Despite only a
slight reduction in expression of VEGF protein and mRNA in RAS-3
cells treated with PD98059 (Fig. 3, A and D, respectively), this drug
was highly effective in suppressing both MAPK enzymatic activity
and cellular mitogenesis (Fig. 3, B and C, respectively). This is
illustrated by an up to 6-fold reduction in MAPK-dependent phos-
phorylation of MBP within 1 h of the addition of PD98059 (Fig. 3B).
In the presence of the drug (50 ␮M), this suppression of MAPK
continued for at least 18 h (Fig. 3B), with a commensurate time-
dependent decrease in the rate of DNA synthesis (Fig. 3C). Again,
these obvious manifestations of PD98059 bioactivity stand in marked
contrast to the unremarkable effect of this inhibitor on ras-dependent
up-regulation of VEGF.
Up-Regulation of VEGF by Mutant H-ras in Epithelial Cells Is
Diminished on Inhibition of PI3K Activity. PI3K has been recently
shown to mediate many aspects of oncogenic ras-dependent transfor-
mation (17). This enzyme is constitutively activated in IEC-18 cells
lines harboring mutant H-ras (55). We therefore decided to examine
VEGF expression in RAS-3 cells in the presence or absence of a
potent PI3K inhibitor known as LY294002 (56). Indeed, the drug
profoundly down-regulated VEGF expression at both the mRNA (Fig.
3D) and protein (Fig. 4) levels. Interestingly, the combined effect of
PD98059 and LY294002 was greater than that of the latter drug alone,
suggesting that both respective pathways may show some degree of
cooperation in regulation of VEGF (Fig. 3D). Although secretion of
VEGF by RAS-3 cells into their conditioned medium could be largely
inhibited in the presence of LY294002, the magnitude of this effect
was significantly modified by external influences such as cell density
(Fig. 4). Thus, in sparse cell cultures, the effect of PI3K inhibition was
very pronounced, leading to reduction of VEGF levels by approxi-
mately 4 –10-fold (at 4 and 20 ␮M LY294002, respectively), whereas
in parallel confluent cultures, the corresponding values were only 5%
and 60% inhibition (Fig. 4). This observation suggests that whereas
oncogenic ras-induced up-regulation of VEGF expression is in itself
Fig. 2. Evidence for nonautocrine mechanism of VEGF up-regulation in epithelial cells dependent on PI3K activity, its cooperation with factors regulated by
harbouring mutant H-ras. A, up-regulation of VEGF at high cell density as a function of
ras-transformation. Parental IEC-18 or H-ras transformed (RAS-3) cells were grown as
cell density (see Fig. 2A) is not. The addition of higher serum
either dense, (confluence ⬃90%) or sparse (semiconfluent, 30 –50%) cultures, and total concentrations (10%) masked the resistance of confluent cells to
RNA (25 ␮g/lane) was probed for VEGF expression. Selective, density-dependent up- LY294002 treatment.
regulation of VEGF by RAS-3 cells was also confirmed by protein analysis (data not
shown). B, Ras-dependent up-regulation of VEGF mRNA is unresponsive to treatment
with neutralizing antibody to EGFR (mR3) at 50 ␮g/ml. C, tyrosine kinase inhibitor,
genistein (100 ␮M), is unable to down-regulate VEGF in RAS-3 cells (see Table 1). D,
transfer of conditioned medium from RAS-3 cells failed to up-regulate VEGF in parental Table 1 Interference of pharmacological signal transduction inhibitors with ras-
IEC-18 cells. Growth medium was incubated with respective cells lines for 24 h (CM dependent expression of VEGF in epithelial and fibroblastic cells
IEC-18, CM RAS-3) and added to cultures of either IEC-18 or RAS-3 cells for an VEGF mRNA (%
additional 24 h. Control medium (Con) was incubated in tissue culture dishes in the control)
absence of cells.
Inhibitor Targeta RAS-3 3T3RAS
Sulindac sulfide COX-2b 525% 250%
autocrine growth factors, to which IEC-18 cells are unresponsive. L-NAME NOS 121% 120%
However, taken together, these results argue for a more direct (non- NDGA LOX NT 122%
Okadaic acid Protein phosphatases 50% 124%
autocrine) linkage between expression of mutant ras and up-regula- Genistein Tyrosine kinases 659% 173%
tion of VEGF in transformed intestinal epithelial cells. Calphostin C Protein kinase C 155% 186%
Dissociation of MEK/MAPK Activity from Up-Regulation of GF109203X Protein kinase C 168% 106%
BAY 11-7085 Nuclear factor ␬B NT 124%
VEGF in H-ras-transformed Epithelial Cells. Because the raf/ PP1 src tyrosine kinases 103% 101%
MEK/MAPK pathway is thought to play a central role in oncogenic Rapamycin p70/S6K 116% 106%
NAC ROIs 97% 77%
ras-dependent transformation (14, 51–54), it was logical to examine a
Cells were treated for 9 h with the respective inhibitors or vehicle in DMEM
its potential contribution to the up-regulation of VEGF expression in containing 1% FBS. Expression of VEGF transcript in total RNA preparation was
an epithelial cell context. However, several independent clones (11A- quantitated by PhosphorImager scanner, normalized to the abundance of 28S RNA, and
4.2, 11A-2.3, and 11A-2.2) of IEC-18 cells engineered to overexpress expressed as a percentage of normalized VEGF expression in cultures incubated with
DMSO (in all cases except NAC and L-NAME, which was dissolved in medium directly.)
the constitutively active mutant of MEK-1 (⌬N3/S222D) did not b
COX-2, cyclooxygenase-2; NOS, nitric oxide synthase; LOX, lipooxygenase; NT,
display any appreciable increase in VEGF production as compared to not tested.
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 ONCOGENES AND TUMOR ANGIOGENESIS

Fig. 3. Up-regulation of VEGF in H-ras-transformed epithelial cells is dependent on PI3K activity and dissociable from regulation of MEK/MAPK and mitogenesis. A, absence of
VEGF up-regulation in IEC-18 cells expressing constitutively activated, mutant MEK-1 (MEK-1 exog.) at levels comparable to endogenous enzyme (MEK-1 end.). Similar low levels
of VEGF were detectable in conditioned medium of all clonal sublines of IEC-18 cells expressing mutant MEK-1 (11A-4.2, 11A-2.3, 11A2.2), wild-type MEK-1 (wt-4.4), or parental
IEC-18 cells. Mutant ras-dependent up-regulation of VEGF was not abrogated by treatment of RAS-3 cells with the MEK-1 inhibitor (PD98059), even at the maximal concentration
(50 ␮M). B, suppression of MAPK activity (MAPK-dependent MBP phosphorylation) during treatment of RAS-3 cells with PD98059 (50 ␮M). C, inhibition of DNA synthesis and
mitogenesis by PD98059 treatment of RAS-3 cells. D, down-regulation of VEGF mRNA expression in RAS-3 cells treated with inhibitor of PI3K (LY294002, 20 ␮M, LY). PD98059
(PD) was unable to cause a significant change in VEGF mRNA unless combined with LY294002 (PD ⫹ LY).

ipate in the activation of rac-1, triggering this signaling cascade (57).

Interestingly, ROIs have been directly implicated in up-regulation of
VEGF via increased mRNA stability (58). Taken together, these
observations suggest that PI3K-dependent up-regulation of VEGF by
mutant ras may be mediated by ROIs. However, comparative analysis
of VEGF mRNA half-life in RAS-3 and IEC-18 cells did not reveal
any significant differences (Fig. 5). In addition, treatment of RAS-3
cells with the antioxidant NAC did not obliterate expression of VEGF
mRNA (Fig. 6C), nor could we detect such an inhibitory effect in

Fig. 4. Dose-dependent down-regulation of VEGF protein production in ras-trans-

formed epithelial cells treated with PI3K inhibitor (LY294002). Culturing the RAS-3 cells
under high density conditions (dense) blunted the effect of LY294002 as compared to the
cells in the log phase of growth (sparse). Addition of 10% FBS modified both cell
density-dependent and -independent responses of RAS-3 cells to LY294002.

In Epithelial Cells, ROIs Do Not Mediate Up-Regulation of

VEGF Transcript by Mutant ras but Are Required for Secretion
of VEGF Protein. Mutant ras is known to exert its transforming
activity in part through activation of the multimolecular complexes
containing rac-1 and NADPH-oxidase (16), which in turn stimulate
the production of ROIs. It has been postulated that PI3K may partic-
494
Fig. 5. Similar VEGF mRNA half-lives in ras-transformed (RAS-3) and nontrans-
formed (IEC-18) epithelial cells. Semiconfluent cell cultures were treated with actinomy-
cin D (10 ␮g/ml), and total RNA was analyzed for VEGF transcript at the time points
indicated. The intensity of the specific VEGF signal was quantitated by PhosphorImager,
normalized to 28S ribosomal RNA (normalized volume), and expressed as a fraction of
the VEGF mRNA detectable at the time of drug addition.

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 ONCOGENES AND TUMOR ANGIOGENESIS

Fig. 6. Expression of VEGF by epithelial cells in the presence of antioxidant NAC. A, expression of endogenous (rat) VEGF protein by IEC-18 transformed with both oncogenic
H-ras and v-src (left panel) is inhibitable by NAC treatment (6 h) in a dose-dependent manner. In a subline of IEC-18 cells engineered to overexpress exogenous (human) VEGF121
(18V9) in an oncogene-independent constitutive manner, low concentrations of NAC (up to 2.5 mM) cause a precipitous dose-dependent decrease in VEGF immunoreactivity, whereas
at higher drug concentrations (2.5–20 mM), VEGF levels remain unaffected (right panel). Absence of VEGF signal from H-ras or v-src-transformed IEC-18 cells in this panel (human
VEGF) is due to species specificity of the ELISA detection system. B, VEGF mRNA expression in RAS-3 cells is unaffected by NAC treatment (20 mM), despite changes at the protein
level.

various other tumor cell lines harboring a mutant ras oncogene (data
not shown). Because the effect of H-ras on VEGF expression in
IEC-18 cells is primarily exerted at the mRNA (transcriptional) level,
we interpret these results as an indication that signals transmitted by
ROIs are not directly involved in this regulation. However, analysis of
conditioned media obtained from IEC-18 variants transfected with
mutant ras (RAS-3) or v-src (SRC-3) revealed that NAC treatment
can almost completely block the expression of VEGF protein (Fig.
6A). A somewhat similar blockade was observed when human
VEGF121 was expressed in IEC-18 cells (clone 18V9) from a heter-
ologous, constitutively active, viral (cytomegalovirus) promoter, fur-
ther suggesting the involvement of a posttranscriptional mechanism.
There were puzzling differences in the profiles of VEGF inhibitory
activity of NAC in the context of oncogene-driven, endogenous pro-
duction (rat VEGF) vis-à-vis expression of exogenous growth factor
in 18V9 cells (human VEGF). In the former case, the degree of
suppression increased steadily with increasing NAC concentration
and was nearly complete at 20 mM, whereas in 18V9 cells, the effect
was incomplete and biphasic, with a plateau beginning as low as at 2.5
mM of the antioxidant. The source of this discrepancy and the overall
mechanism of VEGF inhibition by antioxidants remain to be eluci-
dated.
Up-Regulation of VEGF in H-ras-transformed Fibroblasts Is
Mediated by the MEK/MAPK Pathway. Studies on malignant
transformation of mouse fibroblasts expressing a mutant ras oncogene
led to the notion that the raf/MEK/MAPK signaling pathway plays a
central and dominant role in mediating the transformed (tumorigenic)
phenotype (14, 15). Furthermore, it is well established that expression
of oncogenic mutants of ras, raf (2), and MEK-1 (59) in rodent
fibroblast result in strong up-regulation of VEGF (59). This is clearly
different from the results shown above, which were obtained using
epithelial cells (see Fig. 3D). We decided to study this apparent
discrepancy by testing ras-transformed NIH3T3 cells under identical
conditions and using approaches similar to those employed in our
analysis of ras-transformed epithelial (IEC-18) cells. Indeed, we
found that constitutive MAPK activity detected in NIH3T3 fibroblasts
495
harboring mutant H-ras (3T3RAS) could be blocked in a profound
and lasting manner by treatment with the MEK-1 inhibitor (Fig. 7A).
Again, as in the case of RAS-3 epithelial cells, this effect was
accompanied by a gradual inhibition of cellular mitogenesis, as meas-
ured by the rate of [3H]thymidine incorporation into the DNA of
asynchronously growing cells (Fig. 7B). However, in 3T3RAS fibro-
blasts, unlike their epithelial (RAS-3) counterparts, the antiprolifera-
tive effect of the MEK-1 inhibition was also accompanied by down-
regulation of VEGF mRNA expression (Fig. 7C). Furthermore, in the
case of 3T3RAS fibroblasts, inhibition of PI3K by treatment with
LY294002 did not lead to abrogation of VEGF expression (Fig. 7C)
as it so obviously did in RAS-3 cells (Fig. 3D). Combination of
LY294002 and PD98059 treatments were only slightly more effective
than treatment with the MEK-1 inhibitor alone (Fig. 7C). Finally,
expression of VEGF protein was elevated by 2–3-fold in transformed
and tumorigenic NIH3T3 fibroblasts (14) expressing a constitutively
activated mutant of MEK-1 (⌬N3/S222D) as compared to cells trans-
fected with the wild-type enzyme (MEK-1/wt). As expected, this
up-regulation was completely abrogated by treatment with the MEK-1
inhibitor PD98059 (Fig. 7D).
Cellular transformation under the influence of oncogenic ras is
associated with a multitude of pleiotrophic changes (15), each of
which can have phenotypic consequences and potentially (directly or
indirectly) impact VEGF expression (38). To explore some of these
possibilities, we tested the effect of various signal transduction inhib-
itors on expression of VEGF mRNA by RAS-3 epithelial and
3T3RAS fibroblast (Table 1). With the exception of the Ras FTI
(L-739,749) tested previously (1), MEK-1 inhibitor (PD98059), PI3K
inhibitor (LY294002), and dexamethasone (60), no remarkable VEGF
inhibition was observed on targeting such cellular activities as cy-
clooxygenase-2 (sulindac), lipooxygenase (NDGA), nitric oxide syn-
thase (L-NAME), protein kinase C (calphostin C; GFX), S6 kinase
(rapamycin), src-like kinases (PP1) or nuclear factor ␬B (BAY 11-
7085). This suggests that these activities are nonessential for onco-
genic ras-dependent VEGF up-regulation under standard testing con-
ditions (1% FBS) but obviously does not rule out participation of the

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 ONCOGENES AND TUMOR ANGIOGENESIS

Fig. 7. MEK/MAPK dependence of VEGF ex-

pression and mitogenesis in ras-transformed fibro-
blasts. A, suppression of MAPK activity in mutant
ras-transformed NIH3T3 cells (3T3RAS) in the
presence of MEK-1 inhibitor (PD98059, 50 ␮M);
B, time-dependent inhibition of DNA synthesis in
3T3RAS cells in the presence of PD98059; C,
inhibition of VEGF mRNA expression in 3T3RAS
cells treated with PD98059 but not with
LY294002; D, up-regulation of VEGF protein ex-
pression in NIH3T3 cells transformed with consti-
tutively active mutant of MEK-1 [MEK-1/mut
(⌬N3/S222D)] as compared to the cells expressing
wild-type MEK-1 (MEK-1/wt). This up-regulation
was abrogated by the addition of PD98059.

respective target proteins in the control of VEGF expression under
conditions in which there is a combined influence of ras and external
physiological/environmental stimuli such as growth factors, cell-cell
contacts, or hypoxia.
DISCUSSION
Our results should help resolve some conflicting findings regarding
the mechanisms of ras oncogene-induced up-regulation of VEGF
because they show that significant differences can be obtained, de-
pending on whether transformed epithelial or fibroblastic cells are
used for experimental analysis. Thus, just as the raf/MAPK pathway
seems to be dominant in oncogenic (morphological) transformation of
fibroblasts but not epithelial cells (19), the same appears to be the case
for ras-mediated up-regulation of VEGF. Moreover, the PI3K path-
way, which has been implicated in ras-mediated oncogenic transfor-
mation of epithelial cells in vitro or in vivo, was found in the present
study to contribute to VEGF up-regulation in transformed intestinal
epithelial cells, but in a much less conspicuous way, if at all, in
fibroblasts.
The linkage between expression of mutant ras oncogenes and
up-regulation of VEGF has now been demonstrated in a number of
systems with rather remarkable consistency (1, 2, 29, 49, 61– 63);
however, as stated above, there has been no widely accepted molec-
ular mechanism to account for this effect. For example, Grugel et al.
(2) pointed out that VEGF up-regulation occurs in fibroblasts trans-
formed by either mutant ras or v-raf, suggesting that by virtue of
epistatic hierarchy, the entire raf/MEK/MAPK module is involved (2).
This is consistent with the observation described recently by Milanini
496
et al. (59), who reported that VEGF transcript is up-regulated in
hamster fibroblasts transfected with activated MEK-1. However, there
have been at least two reasons for doubts being raised regarding a
universal and dominant role of the raf/MEK/MAPK pathway in the
up-regulation of VEGF by oncogenic ras. First, as discussed above,
there is mounting evidence that although this pathway is critical for
transformation of rodent fibroblasts, the same is not the case for
epithelial cells (19), which are the cellular progenitors of the vast
majority of cancers in man. Thus, a similar difference might be
extrapolated with respect to the proangiogenic mechanisms of ras
oncogene-induced transformation in general, including up-regulation
of VEGF. Second, experiments with immortalized human endothelial
cells transfected with an activated H-ras oncogene suggest that ele-
vated expression of VEGF mRNA, at least in this case, can be
mitigated by the presence of the PI3K inhibitor wortmannin (64).
Interestingly, wortmannin was more effective in restricting VEGF
up-regulation under the influence of hypoxia than by mutant H-ras
itself (64). This is consistent with earlier studies by Mazure et al., who
reported that mutant ras amplifies the stimulatory effects of hypoxia
on VEGF gene transcription in a manner that is dependent on the
respective activities of PI3K, its downstream target-protein kinase B
(PKB/Akt), and hypoxia inducible factor 1 (HIF-1) (29, 65). In this
study and in others, the effect of hypoxia was clearly separable from
the activity of the raf/MEK/MAPK cascade (29, 59), although some
recent reports seem to indicate otherwise (45, 66).
The results we have obtained with ras-transformed epithelial cells
differ fundamentally from those that we and others have obtained
using fibroblasts. Thus, even under normoxic conditions, the effect of

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 ONCOGENES AND TUMOR ANGIOGENESIS
mutant ras on VEGF expression in transformed epithelial cells was
markedly reduced on inhibition of PI3K activity and was resistant to
inhibition of MEK-1. The antithetical responses to the latter treatment
were not due to the differential potency of PD98059 in fibroblasts and
epithelial cells because MAPK activity and mitogenesis were mark-
edly inhibited in both cell types by the treatment with the drug. Our
transfection experiments also indicate that whereas mutant MEK-1,
which is known to readily transform rodent fibroblasts (14, 54), can
also induce an increase in VEGF mRNA levels (59) and protein
production in these cells (compare Fig. 7), it clearly fails to do so in
IEC-18 epithelial cells. However, although our experiments suggest a
lack of autonomous VEGF stimulating function of MEK-1 in IEC-18
cells, a degree of cooperation between this enzyme and PI3K signal-
ing can be inferred from the cumulative effect of combined LY294002
and PD98059 treatment.
It is intriguing that the expression of VEGF mRNA did not always
correspond to the VEGF protein levels in the conditioned medium of
various transformed and nontransformed cell lines. Notably, treatment
with NAC, an antioxidant and inhibitor of ras signaling via ROIs (16),
had only a slight effect on expression VEGF mRNA in 3T3RAS cells
and had no effect on their epithelial counterparts (RAS-3), whereas it
profoundly inhibited VEGF protein secretion. This result suggests a
requirement for ROIs at posttranscriptional levels of VEGF regulation
that may or may not be influenced by oncogenic transformation. There
are several possible examples of how such regulation might be ex-
erted, e.g., by alteration of VEGF gene translation through function of
internal ribosomal entry sites (36) or translational activity of the
eIF-4E protein (34). Whatever the mechanism of its activity, NAC can
serve as a prototype of a specific signal transduction inhibitor that has
been used extensively in the clinic (67) and deserves further explo-
ration in terms of its potential as a possible angiogenesis inhibitor.
Finally, it is important to keep in mind that the proangiogenic
phenotype induced by oncogenic ras is clearly not restricted to up-
regulation of VEGF. Whereas the latter factor is at the center of
angiogenesis regulation in many systems (5– 8, 68), there are growing
numbers of reports suggesting the involvement of other oncogene-
(and tumor suppressor gene)-dependent changes in expression of
angiogenesis stimulators, inhibitors, and modulators (12, 42, 60).
However, with few exceptions (49, 64, 69), these studies have focused
on a single growth factor and a single tumor-associated genetic
alteration (1, 48, 70). In this regard, we simultaneously examined the
expression of VEGF and TSP-1 in the context of a single epithelial
model system and under influence of two different transforming
oncogenes, H-ras and v-src. This led us to the realization that the
profile of proangiogenic changes expressed by tumor cells may, to
some extent, be oncogene specific. An example of this is the lack of
TSP-1 down-regulation in v-src but not in H-ras-transformed IEC-18
cells. Again, this pattern and its angiogenic consequences in vivo may
vary between different cell types harboring similar oncogenic changes
(49, 71).
In summary, an important practical implication of our results is that
up-regulation of VEGF, which is a crucial and common element of the
ras-dependent angiogenic phenotype (1, 5, 7, 8), is executed in a
tissue/cell type-specific manner. Although in this context, targeting
mutant ras itself (or a ras-related signaling modules) can, in principle,
be viewed as a form of de facto antiangiogenic therapy, development
of such similar approaches using signal transduction inhibitors should
not rest on the mere presence of mutant ras (or other analogous
genetic changes) in the target tumor cell population. For example,
whereas an inhibitor of ras may block VEGF production and hence
possibly, tumor angiogenesis in ras-transformed epithelial cells or
fibroblasts, the same cannot be said of signal transduction inhibitors
that act further downstream of ras. Thus, inhibitors of MAPK and
497
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Research.
PI3K may have quite different effects with respect to blocking ras-
induced VEGF expression, depending on the cellular origin of the
tumor. Hence, improved therapeutic opportunities may lie in a more
detailed understanding of the molecular linkage between specific
oncogenic changes and the resultant angiogenic phenotypes in spe-
cific types of cancer cells.
ACKNOWLEDGMENTS
The excellent secretarial assistance of Lynda Woodcock is gratefully ac-
knowledged.
REFERENCES
1. Rak, J., Mitsuhashi, Y., Bayko, L., Filmus, J., Sasazuki, T., and Kerbel, R. S. Mutant
ras oncogenes up-regulate VEGF/VPF expression: implications for induction and
inhibition of tumor angiogenesis. Cancer Res., 55: 4575– 4580, 1995.
2. Grugel, S., Finkenzeller, G., Weindel, K., Barleon, B., and Marme, D. Both v-Ha-ras
and v-raf stimulate expression of the vascular endothelial growth factor in NIH 3T3
cells. J. Biol. Chem., 270: 25915–25919, 1995.
3. Dvorak, H. F., Brown, L. F., Detmar, M., and Dvorak, A. M. Review: vascular
permeability factor/vascular endothelial growth factor, microvascular hyperperme-
ability, and angiogenesis. Am. J. Pathol., 146: 1029 –1039, 1995.
4. Shirasawa, S., Furuse, M., Yokoyama, N., and Sasazuki, T. Altered growth of human
colon cancer cell lines disrupted at activated Ki-ras. Science (Washington DC), 260:
85– 88, 1993.
5. Okada, F., Rak, J., St. Croix, B., Lieubeau, B., Kaya, M., Roncari, L., Sasazuki, S.,
and Kerbel, R. S. Impact of oncogenes on tumor angiogenesis: mutant K-ras up-
regulation of VEGF/VPF is necessary but not sufficient for tumorigenicity of human
colorectal carcinoma cells. Proc. Natl. Acad. Sci. USA, 95: 3609 –3614, 1998.
6. Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O’Shea, K. S., Powell-
Braxton, L., Hillan, K. J., and Moore, M. W. Heterozygous embryonic lethality
induced by targeted inactivation of the VEGF gene. Nature (Lond.), 380: 439 – 442,
1996.
7. Shi, Y. P., and Ferrara, N. Oncogenic ras fails to restore an in vivo tumorigenic
phenotype in embryonic stem cells lacking vascular endothelial growth factor
(VEGF). Biochem. Biophys. Res. Commun., 254: 480 – 483, 1999.
8. Grunstein, J., Roberts, W. G., Mathieu-Costello, O., Hanahan, D., and Johnson, R. S.
Tumor-derived expression of vascular endothelial growth factor is a critical factor in
tumor expansion and vascular function. Cancer Res., 59: 1592–1598, 1999.
9. Rak, J., Mitsuhashi, Y., Sheehan, C., Krestow, J. K., Florenes, V. A., Filmus, J., and
Kerbel, R. S. Collateral expression of proangiogenic and tumorigenic properties in
intestinal epithelial cell variants selected for resistance to anoikis. Neoplasia, 1:
23–30, 1999.
10. Chin, L., Tam, A., Pomerantz, J., Wong, M., Holash, J., Bardeesy, N., Shen, Q.,
O’Hagan, R., Pantginis, J., Zhou, H., Horner, J. W., Cordon-Cardo, C., Yancopoulos,
G. D., and DePinho, R. A. Essential role for oncogenic Ras in tumour maintenance.
Nature (Lond.), 400: 468 – 472, 1999.
11. Charvat, S., Duchesne, M., Parvaz, P., Chignol, M. C., Schmitt, D., and Serres, M.
The up-regulation of vascular endothelial growth factor in mutated Ha-ras HaCaT cell
lines is reduced by a farnesyl transferase inhibitor. Anticancer Res., 19: 557–561,
1999.
12. Kerbel, R. S., Viloria-Petit, A. M., Okada, F., and Rak, J. W. Establishing a link
between oncogenes and tumor angiogenesis. Mol. Med., 4: 286 –295, 1998.
13. Viloria-Petit, A. M., Rak, J., Hung, M-C., Rockwell, P., Goldstein, N., and Kerbel,
R. S. Neutralizing antibodies against EGF and ErbB-2/neu receptor tyrosine kinases
down-regulate VEGF production by tumor cells in vitro and in vivo: angiogenic
implications for signal transduction therapy of solid tumors. Am. J. Pathol., 151:
1523–1530, 1997.
14. Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukasawa,
K., Woude, G. F. V., and Ahn, N. G. Transformation of mammalian cells by
constitutively active MAP kinase kinase. Science (Washington DC), 265: 966 –970,
1994.
15. Khosravi-Far, R., Campbell, S., Rossman, K. L., and Der, C. J. Increasing complexity
of Ras signal transduction: involvement of Rho family proteins. Adv. Cancer Res.,
72: 57–107, 1998.
16. Irani, K., Xia, Y., Zweier, J. L., Sollott, S. J., Der, C. J., Fearon, E. R., Sundaresan,
M., Finkel, T., and Goldschmidt-Clermont, P. J. Mitogenic signaling mediated by
oxidants in Ras-transformed fibroblasts. Science (Washington DC), 275: 1649 –1652,
1997.
17. Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry,
M. J., Waterfield, M. D., and Downward, J. Phosphatidylinositol-3-OH kinase as a
direct target of Ras. Nature (Lond.), 370: 527–532, 1994.
18. Rodriguez-Viciana, P., Warne, P. H., Khwaja, A., Marte, B. M., Pappin, D., Das, P.,
Waterfield, M. D., Ridley, A., and Downward, J. Role of phosphoinositide 3-OH
kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell, 89:
457– 467, 1997.
19. Oldham, S. M., Clark, G. J., Gangarosa, L. M., Coffey, R. J., Jr., and Der, C. J.
Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation
of RIE-1 epithelial cells. Proc. Natl. Acad. Sci. USA, 93: 6924 – 6928, 1996.
 ONCOGENES AND TUMOR ANGIOGENESIS
20. McKay, I. A., Malone, P., Marshall, C. J., and Hall, A. Malignant transformation of
murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional
epidermal growth factor receptor. Mol. Cell. Biol., 6: 3382–3387, 1986.
21. Filmus, J., Shi, W., and Spencer, T. Role of transforming growth factor ␣ (TGF-␣) in
the transformation of ras-transfected rat intestinal epithelial cells. Oncogene, 8:
1017–1022, 1993.
22. Gangarosa, L. M., Sizemore, N., Graves-Deal, R., Oldham, S. M., Der, C. J., and
Coffey, R. J. A raf-independent epidermal growth factor receptor autocrine loop is
necessary for Ras transformation of rat intestinal epithelial cells. J. Biol. Chem., 272:
18926 –18931, 1997.
23. Goldman, C. K., Kim, J., Wong, W. L., King, V., Brock, T., and Gillespie, G. Y.
Epidermal growth factor stimulates vascular endothelial growth factor production by
human malignant glioma cells: a model of glioblastoma multiforme pathophysiology.
Mol. Biol. Cell, 4: 121–133, 1993.
24. Ciardiello, F., Damiano, V., Bianco, R., Bianco, C., Fontanini, G., De Laurentiis, M.,
De Placido, S., Mendelsohn, J., Bianco, A. R., and Tortora, G. Antitumor activity of
combined blockade of epidermal growth factor receptor and protein kinase A. J. Natl.
Cancer Inst., 88: 1770 –1776, 1996.
25. Gille, J., Swerlick, R. A., and Caughman, S. W. Transforming growth factor-␣-
induced transcriptional activation of the vascular permeability factor (VPF/VEGF)
gene requires AP-2-dependent DNA binding and transactivation. EMBO J., 16:
750 –759, 1997.
26. Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., and Fiddes, J. C.
The human gene for vascular endothelial growth factor. J. Biol. Chem., 266: 11947–
11954, 1991.
27. Shima, D. T., Kuroki, M., Deutsch, U., Ng, Y. S., Adamis, A. P., and D’Amore, P. A.
The mouse gene for vascular endothelial growth factor. Genomic structure, definition
of the transcriptional unit, and characterization of transcriptional and post-transcrip-
tional regulatory sequences. J. Biol. Chem., 271: 3877–3883, 1996.
28. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., and
Semenza, G. L. Activation of vascular endothelial growth factor gene transcription by
hypoxia-inducible factor 1. Mol. Cell. Biol., 16: 4604 – 4613, 1996.
29. Mazure, N. M., Chen, E. Y., Laderoute, K. R., and Giaccia, A. J. Induction of vascular
endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/
Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible
factor-1 transcriptional element. Blood, 90: 3322–3331, 1997.
30. Levy, A. P., Levy, N. S., and Goldberg, M. A. Post-transcriptional regulation of
vascular endothelial growth factor by hypoxia. J. Biol. Chem., 271: 2746 –2753,
1996.
31. Stein, I., Neeman, M., Shweiki, D., Itin, A., and Keshet, E. Stabilization of vascular
endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with
other ischemia-induced genes. Mol. Cell. Biol., 15: 5363–5368, 1995.
32. Levy, A. P., Levy, N. S., Iliopoulos, O., Jiang, C., Kaplin, W. G., Jr., and Goldberg,
M. A. Regulation of vascular endothelial growth factor by hypoxia and its modulation
by the von Hippel-Lindau tumor suppressor gene. Kidney Int., 51: 575–578, 1997.
33. Dibbens, J. A., Miller, D. L., Damert, A., Risau, W., Vadas, M. A., and Goodall, G. J.
Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the
cooperation of multiple RNA elements. Mol. Biol. Cell, 10: 907–919, 1999.
34. Kevil, C. G., De Benedetti, A., Payne, D. K., Coe, L. L., Laroux, F. S., and Alexander,
J. S. Translational regulation of vascular permeability factor by eukaryotic initiation
factor 4E: implications for tumor angiogenesis. Int. J. Cancer, 65: 785–790, 1996.
35. Stein, I., Itin, A., Einat, P., Skaliter, R., Grossman, Z., and Keshet, E. Translation of
vascular endothelial growth factor mRNA by internal ribosome entry: implications for
translation under hypoxia. Mol. Cell. Biol., 18: 3112–3119, 1998.
36. Akiri, G., Nahari, D., Finkelstein, Y., Le, S. Y., Elroy-Stein, O., and Levi, B. Z.
Regulation of vascular endothelial growth factor (VEGF) expression is mediated by
internal initiation of translation and alternative initiation of transcription. Oncogene,
17: 227–236, 1998.
37. Scott, P. A., Smith, K., Poulsom, R., De Benedetti, A., Bicknell, R., and Harris, A. L.
Differential expression of vascular endothelial growth factor mRNA versus protein
isoform expression in human breast cancer and relationship to eIF-4E. Br. J. Cancer,
77: 2120 –2128, 1998.
38. Ferrara, N., and Davis-Smyth, T. The biology of vascular endothelial growth factor.
Endocr. Rev., 18: 4 –25, 1997.
39. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. A synthetic
inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA,
92: 7686 –7689, 1995.
40. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. PD 098059 is
a specific inhibitor of the activation of mitogen-activated protein kinase kinase in
vitro and in vivo. J. Biol. Chem., 270: 27489 –27494, 1995.
41. Buick, R. N., Filmus, J., and Quaroni, A. Activated H-ras transforms rat intestinal
epithelial cells with expression of ␣-TGF. Exp. Cell Res., 170: 300 –309, 1987.
42. Bouck, N., Stellmach, V., and Hsu, S. C. How tumors become angiogenic. Adv.
Cancer Res., 69: 135–174, 1996.
43. Hanahan, D., and Folkman, J. Patterns and emerging mechanisms of the angiogenic
switch during tumorigenesis. Cell, 86: 353–364, 1996.
44. Jamal, H., Cano-Gauci, D. F., Buick, R. N., and Filmus, J. Activated ras and src
induce CD44 overexpression in rat intestinal epithelial cells. Oncogene, 9: 417– 423,
1994.
45. Mukhopadhyay, D., Tsiokas, L., Zhou, X-M., Foster, D., Brugge, J. S., and Sukhatme,
V. P. Hypoxic induction of human vascular endothelial growth factor expression
through c-Src activation. Nature (Lond.), 375: 577–581, 1995.
46. Ellis, L. M., Staley, C. A., Liu, W., Fleming, R. Y., Parikh, N. U., Bucana, C. D., and
Gallick, G. E. Down-regulation of vascular endothelial growth factor in a human
498
Downloaded from cancerres.aacrjournals.org on March 9, 2020. © 2000 American Association for Cancer
Research.
colon carcinoma cell line transfected with an antisense expression vector specific for
c-src. J. Biol. Chem., 273: 1052–1057, 1998.
47. Jiang, B. H., Agani, F., Passaniti, A., and Semenza, G. L. V-SRC induces expression
of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular
endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression.
Cancer Res., 57: 5328 –5335, 1997.
48. Dameron, K. M., Volpert, O. V., Tainsky, M. A., and Bouck, N. Control of angio-
genesis in fibroblasts by p53 regulation of thrombospondin-1. Science (Washington
DC), 265: 1582–1584, 1994.
49. Volpert, O. V., Dameron, K. M., and Bouck, N. Sequential development of an
angiogenic phenotype by human fibroblasts progressing to tumorigenicity. Oncogene,
14: 1495–1502, 1997.
50. Koura, A. N., Liu, W., Kitadai, Y., Singh, R. K., Radinsky, R., and Ellis, L. M.
Regulation of vascular endothelial growth factor expression in human colon carci-
noma cells by cell density. Cancer Res., 56: 3891–3894, 1996.
51. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. Activation of MAP kinase
kinase is necessary and sufficient for PC12 differentiation and for transformation of
NIH 3T3 cells. Cell, 77: 841– 852, 1994.
52. Leevers, S. J., Paterson, H. F., and Marshall, C. J. Requirement for ras in raf
activation is overcome by targeting raf to the plasma membrane. Nature (Lond.), 369:
411– 414, 1994.
53. Stokoe, D., Macdonald, S. G., Cadwallader, K., Symons, M., and Hancock, J. F.
Activation of Raf as a result of recruitment to the plasma membrane. Science
(Washington DC), 264: 1463–1467, 1994.
54. Brunet, A., Pages, G., and Pouyssegur, J. Constitutively active mutants of MAP
kinase kinase (MEK1) induce growth factor-relaxation and oncogenicity when ex-
pressed in fibroblasts. Oncogene, 9: 3379 –3387, 1994.
55. Rosen, K., Rak, J., Jin, J., Kerbel, R. S., Newman, M. J., and Filmus, J. Down-
regulation of Bak plays a critical role in the growth of the ras-induced malignant
phenotype in intestinal epithelial cells. Curr. Biol., 8: 1331–1334, 1998.
56. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. A specific inhibitor of
phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
(LY294002). J. Biol. Chem., 269: 5241–5248, 1994.
57. Pennisi, E. Superoxides relay Ras protein’s oncogenic message. Science (Washington
DC), 275: 1567–1568, 1997.
58. Kuroki, M., Voest, E. E., Amano, S., Beerepoot, L. V., Takashima, S., Tolentino, M.,
Kim, R. Y., Rohan, R. M., Colby, K. A., Yeo, K. T., and Adamis, A. P. Reactive
oxygen intermediates increase vascular endothelial growth factor expression in vitro
and in vivo. J. Clin. Investig., 98: 1667–1675, 1996.
59. Milanini, J., Vinals, F., Pouyssegur, J., and Pages, G. p42/p44 MAP kinase module
plays a key role in the transcriptional regulation of the vascular endothelial growth
factor gene in fibroblasts. J. Biol. Chem., 273: 18165–18172, 1998.
60. Rak, J., Filmus, J., Finkenzeller, G., Grugel, S., Marme, D., and Kerbel, R. S.
Oncogenes as inducers of tumor angiogenesis. Cancer Metastasis Rev., 14: 263–277,
1995.
61. Enholm, B., Paavonen, K., Ristimaki, A., Kumar, V., Gunji, Y., Klefstrom, J.,
Kivinen, L., Laiho, M., Olofsson, B., Joukov, V., Eriksson, U., and Alitalo, K.
Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum,
growth factors, oncoproteins and hypoxia. Oncogene, 14: 2475–2483, 1997.
62. Ogiso, Y., Hwang, Y. W., Shih, T. Y., and Kuzumaki, N. Biological activity of a
K-ras mutant that contains the 12R/59T/116Y mutations. Cancer Lett., 75: 19 –26,
1993.
63. Larcher, F., Robles, A. I., Duran, H., Murillas, R., Quintanilla, M., Cano, A., Conti,
C. J., and Jorcano, J. L. Up-regulation of vascular endothelial growth factor/vascular
permeability factor in mouse skin carcinogenesis correlates with malignant progres-
sion state and activated H-ras expression levels. Cancer Res., 56: 5391–5396, 1996.
64. Arbiser, J. L., Moses, M. A., Fernandez, C. A., Ghiso, N., Cao, Y., Klauber, N.,
Frank, D., Brownlee, M., Flynn, E., Parangi, S., Byers, H. R., and Folkman, J.
Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc. Natl.
Acad. Sci. USA, 94: 861– 866, 1997.
65. Mazure, N. M., Chen, E. Y., Yeh, P., Laderoute, K. R., and Giaccia, A. J. Oncogenic
transformation and hypoxia synergistically act to modulate vascular endothelial
growth factor expression. Cancer Res., 56: 3436 –3440, 1996.
66. Feldkamp, M. M., Lau, N., Rak, J., Kerbel, R. S., and Guha, A. Normoxic and
hypoxic regulation of vascular endothelial growth factor (VEGF) by astrocytoma cells
is mediated by Ras. Int. J. Cancer, 81: 118 –124, 1999.
67. Kelly, G. S. Clinical applications of N-acetylcysteine. Altern. Med. Rev., 3: 114 –127,
1998.
68. Hanahan, D. Signaling vascular morphogenesis and maintenance. Science (Washing-
ton DC), 277: 48 –50, 1997.
69. Perrotte, P., Matsumoto, T., Inoue, K., Kuniyasu, H., Eve, B. Y., Hicklin, D. J.,
Radinsky, R., and Dinney, C. P. Anti-epidermal growth factor receptor antibody C225
inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in
nude mice. Clin. Cancer Res., 5: 257–265, 1999.
70. Zabrenetzky, V., Harris, C. C., Steeg, P. S., and Roberts, D. D. Expression of the
extracellular matrix molecule thrombospondin inversely correlates with malignant
progression in melanoma, lung and breast carcinoma cell lines. Int. J. Cancer, 59:
191–195, 1994.
71. Slack, J. L., and Bornstein, P. Transformation by v-src causes transient induction
followed by repression of mouse thrombospondin-1. Cell Growth Differ., 5: 1373–
1380, 1994.
Oncogenes and Tumor Angiogenesis: Differential Modes of
Vascular Endothelial Growth Factor Up-Regulation in ras
-transformed Epithelial Cells and Fibroblasts
Janusz Rak, Yoshihiro Mitsuhashi, Cap Sheehan, et al.

Cancer Res 2000;60:490-498.

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