Award Number; DAMD17-00-1-0265
TITLE: Growth Factor Regulation of an Angiogenic Factor, the
Fibroblast Growth Factor-Binding Protein (FGF-BP), in Breast
PRINCIPAL INVESTIGATOR: Benjamin L. Kagan
CONTRACTING ORGANIZATION: Georgetown University Medical Center
Washington, DC 20057
REPORT DATE: August 2001
TYPE OF REPORT: Annual Summary
PREPARED FOR: U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012
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_August 2001 Annual Summary (01 Aug 00 - 31 Jul 01)
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Growth Factor Regulation of an Angiogenic Factor, the Fibroblast DAMD17-00-1-0265
Growth Factor Regulation of an Angiogenic Factor, the Fibroblast
Growth Factor-Binding Protein (FGF-BP), in Breast Cancer
Benj atnin L. Kagan
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)
Georgetown University Medical Center
Washington, DC 20057
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U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012
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A secreted carrier protein has been described which is able to bind to FGF-1 and FGF-
2 in a non-covalent, reversible manner. FGF-2 bound to this protein was not subject to
degradation and retained its mitogenic activity. This FGF-binding protein (FGF-BP) has
been studied extensively by our laboratory. FGF-BP is highly expressed in squamous cell
carcinomas (SCC) and EGF is able to increase the expression of FGF-BP in SCC derived cell
lines through PKC, MEK/ERK, and p38 MAPK signaling. We have found FGF-BP mRNA to be
expressed in two breast cancer cell lines (MDA-MB-468, MCF-7/ADR), by Northern
Analysis/Ribonuclease Protection. EGF treatment of MDA-MB-468 cells resulted in an
increase in FGF-BP mRNA expression in a time-dependent manner. EGF signaling occurs
primarily through the PKC, and p38 MAPK pathways. Finally, EGF induction of the FGF-BP
promoter is mediated through CCAAT/enhancer binding protein and AP-1 transcription factor
binding sites on the promoter.
14. SUBJECT TERMS
Growth Factors, Ribonuclease Protection, Transcription, DNA-Protein
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17. SECURITY CLASSIFICATION
18. SECURITY CLASSIFICATION
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Table of Contents
Table of Contents.3
Key Research Accomplishments.6
' ___ P.I.; Kasan. Beniamin L.
Annual report for Grant Number DAMD17-00-1-0265
August 1, 2000 to July 31, 2001
P.L: Benjamin L. Kagan
Title: Growth Factor Regulation of an Angiogenic Factor, the Fibroblast Growth Factor-
Binding Protein (FGF-BP), in Breast Cancer
Paracrine and autocrine growth factors have many functions, including a crucial role in inducing the
formation of new blood vessels in a healing wound, as well as in a growing tumor. Many studies have
demonstrated that a solid tumor mass cannot grow beyond a few millimeters in size without a sufficient supply
of blood to the tumor. Tumor blood vessels provide a pathway for tumor cells to metastasize to distal sites, as
well as a source of nourishment [1-4]. The most important and best-studied angiogenesis factors belong to the
family of fibroblast growth factors (FGFs) [5, 6]. FGF-1 and FGF-2 (aFGF and bFGF, respectively) are unique
in that their biological activities can be quenched by binding tightly to heparansulfate proteoglycan molecules in
the extracellular matrix [7-10]. Two alternate mechanisms of FGF-1 and FGF-2 activation have been theorized
as a result of a multitude of studies over the last decade. One mechanism involves the solubilization of FGF-2
from its storage site by heparanase digestion of the glycosaminoglycan portion of the cell attachment [11-14].
The second mechanism involves the binding of FGF to a secreted carrier protein delivering the activated FGF to
its target receptor. A secreted carrier protein has been described which is able to bind to FGF-1 and FGF-2 in a
non-covalent, reversible manner . FGF-2 bound to this protein was not subject to degradation and retained
its mitogenic activity . This FGF-binding protein (FGF-BP) has been studied extensively by our
Expression of FGF-BP in cell lines that express FGF-2 results in these cells having a tumorigenic and
angiogenic phenotype . FGF-BP transfected cells have been shown to release the protein into their media
along with FGF-2 in a non-covalently bound form; the released FGF-2 is now biologically active . FGF-
BP mRNA is expressed in SCC, colon, and breast tumor cell lines and primary tumor tissue . The role of
FGF-BP during tumor progression has been studied by our laboratory using skin carcinogenesis as a model for
epithelial cancers. We have shown that FGF-BP mRNA is upregulated in the skin during mouse development,
but drops to low levels in adult mouse skin. In both mouse and human skin, FGF-BP mRNA and protein levels
increase at least 3-fold upon treatment with PKC-activating TPA (12-0-tetradecanoylphorbol-l 3-acetate), and
increase further in DMBA/TPA induced papillomas and carcinomas .
The human FGF-BP promoter was recently isolated and cloned revealing positive and negative
regulatory elements within a 118 base pair region just upstream of the FGF-BP transcription start site (Figure
1). The phorbol ester TPA was then shown to upregulate FGF-BP transcription in MEl 80 squamous cell
carcinoma cells. This transcription was mediated through the activation of protein kinase C, and the Spl, AP-1,
and C/EBP positive regulatory elements in the FGF-BP promoter . Treatment of ME180 SCC cells resulted
in the upregulation of FGF-BP mRNA. Signal transduction was mediated through the EGFR, PKC, MEK/ERK.,
and p38 pathways, while transcription was mediated through the AP-1 and C/EBP regulatory elements in the
promoter . Finally, we have also shown that serum upregulates FGF-BP expression in MEl 80 cells,
predominantly through PKC and p38 signaling, while only through the C/EBP site on the FGF-BP promoter
. In my accepted proposal, I hypothesized that an angiogenic “funneling” effect exists in which
intracellular signals intiated by EGF and related ligands result in the activation of FGF through the
P.I.; Kaean. Beniamin L.
modulation of the FGF-BP gene. I planned to consider the relevance of this “funneling” effect with
respect to the breast cancer system.
We have found FGF-BP mRNA to be expressed in two breast cancer cell lines, and 4 out of 6 clinical
samples of human breast cancers, by Northern Analysis/Ribonuclease Protection, and RT-PCR, respectively.
We have also detected FGF-BP mRNA in the human and mouse mammaiy gland. This report summarizes
the flndings by Benjamin Kagan as PI of the funded research, testing the role of FGF-BP in human
breast cancer cell progression and its regulation by the epidermal growth factor.
Aim 1: To study the regulation of the FGF-BP mRNA. bv growth factors, in breast cancer.
Detection of endogenous FGF-BP mRNA in MCF-7/ADR and MDA-MB-468 human breast cancer
cell lines. Previously, we were able to show that FGF-BP mRNA was expressed in 9 out of 15 breast cancer
cell lines, by RT-PCR. To study the regulation of FGF-BP expression in breast cancer cell lines, we wanted to
use a quantitative method for detection of FGF-BP mRNA. A ribonuclease protection assay specific for human
FGF-BP was developed using a riboprobe derived from a pRC/CMV vector plasmid containing the FGF-BP
open reading frame . We were able to detect FGF-BP mRNA only in the MCF-7/ADR cell line, an
adriamycin resistant clone of the MCF-7 cell line, as well as the ME180 SCC cell line, which was used as a
positive control. Northern analysis was also used, screening a wider array of breast cancer cell lines. We were
able to detect expression of FGF-BP mRNA in both MCF-7/ADR and the MDA-MB-468 cell lines.
Expression of FGF-BP mRNA, as determined by RNase protection and Northern analysis, is summarized
in Table 1.
EGF regulation of endogenous FGF-BP in MDA-MB-468 cells. Studies have shown that the MDA-
MB-468 cell line overexpresses the EGFR as compared to MCF-7 breast cancer cells [22-24]. Biscardi et al.
 measure levels of EGFR to be 35 fold that of MCF-7 cells. Because the MDA-MB-468 cell line, like the
ME180 cell line, express high levels of the EGFR , we decided to test whether FGF-BP mRNA expressed in
these cells can be regulated by EGF and/or TPA. MDA-MB-468 cells were grown to 80% confluency, serum
starved for 24 hours, and treated with EGF for 1, 3, 6, or 24 hours. FGF-BP mRNA levels were analyzed by
Northern analysis, and we were able to observe that EGF induced FGF-BP upregulation at about 3-fold above
control, peaking at 6 hours of EGF treatment (Figure 2). The time-course of EGF induction of FGF-BP mRNA
in MDA-MB-468 cells was similar to that observed in the ME180 SCC cell line, suggesting similar mechanisms
of regulation . These data demonstrate that EGF can regulate FGF-BP in MDA-MB-468 cells, in a
similar manner to ME180 SCC cells
EGF induction of FGF-BP in MDA-MB-468 cells is mediated through PKC and p38 MAPS
signaling. EGF regulation of FGF-BP mRNA in ME-180 cells occurs through PKC, and the MEK/ERK and
p38 MARK signaling pathways . Serum, in contrast, mediates FGF-BP transcription through PKC and p38
MAPK signaling, but not MEK/ERK . To discern between the possible signaling pathways involved in
EGF induction of FGF-BP in MDA-MB-468 cells, we tested pharmacological inhibitors of signal transduction
at various concentrations for their affect on FGF-BP regulation. We found that treatment with the EGFR
tyrosine kinase inhibitor PDl53035 resulted in a significant concentration dependent inhibition of EGF
induction of FGF-BP mRNA (Figure 3). Therefore, as expected, EGFR tyrosine kinase activity is essential for
the EGF effect. To establish whether PKC activation was also required for the EGF effect on FGF-BP, we
treated MDA-MB-468 cells with the bisindoylmaleimide PKC inhibitor Ro 31-8220 . At concentrations of
1 pM and 10 pM, Ro 31-8220 was able to signficantly inhibit the EGF induction of FGF-BP (Figure 3). At
these concentrations Ro 31-8220 is also able to inhibit other kinases including the mitogen- and stress-activated
protein kinase-1 (MSKl) , therefore we tested whether the PKC-specific inhibitor calphostin C  could
also inhibit the EGF effect. Treatment with 100 nM calphostin C significantly reduced EGF-induced FGF-BP
P.I.: Kasati. Beniamin L.
mRNA expression by 50% (Figure 3). Taken together, these data suggest a role for PKC in the EGF induction
of FGF-BP in MDA-MB-468 cells.
To determine whether different MAP kinase pathways were also involved in the EGF effect on FGF-BP,
we used the MEKl/2 specific inhibitor U0126 and the p38 MAPK specific inhibitor SB202190 [28, 29].
Treatment with 1 |j,M and 10 pM U0126 did not significantly inhibit EGF induction (Figure 3). Although
20 pM U0126 significantly inhibited EGF induction of FGF-BP, the overall inhibition was only around 30% as
compared to the ability of U0126 to inhibit the EGF induction of FGF-BP in ME-180 cells by 70% . This
suggests a lesser role for the MEK/ERK pathway in the EGF effect in MDA-MB-468 cells. In contrast, as seen
in the ME-180 cells, treatment with increasing concentrations of the p38 MAPK inhibitor SB202190, resulted in
a concentration-dependent inhibition of EGF-induced FGF-BP mRNA expression ranging from 55% inhibition
at 5 pM to 80% inhibition at 20 pM. Furthermore, as described above, the bisindoylmaleimide Ro 31-8220 was
able to significantly inhibit EGF-induced FGF-BP mRNA expression at concentrations specific for PKC and
other kinases such as MSKl. MSKl has been shown to be activated by p38 MAPK phosporylation [26, 30].
Taken together, these data suggest that p38 MAPK plays a dominant role in the induction of FGF-BP by EGF in
Other intracellular targets for EGF receptor-induced intracellular signaling include members of the c-Src
protein tyrosine kinase family. c-Src family members interact with the EGFR at tyrosine residues via SH2
domains . MDA-MB-468 cells have been shown to express moderate levels of c-Src protein as compared
normal breast epithelium . Therefore, we used the c-Src family specific inhibitor PPl . Treatment
with PPl resulted in a maximal inhibition of EGF induction of FGF-BP of 20% only at the highest
concentration, 10 pM (Figure 3). Concentrations of 1 pM and 0.1 pM, also shown to inhibit s-Src family
members , had no effect. This suggests that c-Src family members do not play a role in the EGF effect.
Aim 2: To study the regulation of the human FGF-BP promoter in breast cancer cells.
EGF regulation of the FGF-BP promoter in MDA-MB-468 cells. As described above, EGF induces
the upregulation of FGF-BP in MDA-MB-468 breast cancer cells. To determine if this regulation occurred at
the transcriptional level, we tested whether EGF regulated the activity of FGF-BP promoter in MDA-MB-468
cells. As described above, various portions of the human FGF-BP promoter, full-length, mutated, or deleted,
have been cloned upstream of a luciferase reporter gene. These constructs have been used successfully to assess
the activity of the FGF-BP promoter in ME180 cells [19, 20, 33]. We were able to show that in MDA-MB-468
cells, treatment with EGF was able to induce the activity of the -1060/+62 and -118/+62 promoter constructs 4-
to 5-fold above basal (Figure 4). Deletion of either the AP-1 or the C/EBP, and not the Spl(b) site, reduced the
induction by EGF of the promoter constructs, suggesting the AP-1 and the C/EBP sites were necessary for EGF
induced FGF-BP transcription in this cell line. This observation is similar to what was observed in the ME 180
cells . Upon further investigation, cell-type specific differences were observed. Deletion of the AP-1 site
resulted in a statistically significant decrease in promoter basal activity, suggesting the AP-1 site is necessary
for basal activity. Deletion of the C/EBP site revealed a statistically significant increase in promoter basal
activity, suggesting differences in C/EBP binding to the site affecting both basal and EGF induced activity of
the FGF-BP promoter. These data show that EGF is able to induce the activity of the FGF-BP promoter in
MDA-MB-468 cells, through the AP-1 and C/EBP sites, as seen in ME180 cells. In addition, C/EBP
binding to the FGF-BP promoter may repress basal activity while enhancing promoter activity after EGF
ITT. Key Research Accomplishments
• Expression of FGF-BP mRNA was detected in both MCF-7/ADR and the MDA-MB-468 cell lines by
Northern analysis and RNase protection.
• EGF upregulates FGF-BP expression in MDA-MB-468 cells, in a similar manner to ME180 SCC cells.
This occurs predominantly through the PKC and p38 MAPK signaling pathways.
P.I.; Kaean. Beniamin L.
• EGF is able to induce the activity of the FGF-BP promoter in MDA-MB-468 cells, through the AP-1 and
C/EBP sites, as seen in ME180 cells. Deletion of the C/EBP site on the FGF-BP promoter results in a
significant increase in basal promoter activity.
IV. Reportable Outcomes
Manuscripts, abstracts, and publications produced as a result of this funded research:
Kagan BL, Harris VK, Coticchia CM, Ray R, Wellstein A and Riegel AT, Transcriptional regulation of a
binding protein for FGF (FGF-BP) through p38/SAPK2 signaling. In: Proceedings of the American Association
for Cancer Research, New Orleans, LA, March 24-March 28, 2001.
Kagan BL, Cabal-Manzano R, Stoica GE, Nguyen Q, Wellstein A, and Riegel AT, EGF-induced fibroblast
growth factor-binding protein (FGF-BP) expression in breast cancer is mediated through C/EBPp-regulated
transcription and p38 MAPK signaling. 2001 (manuscript in preparation)
We were able to observe FGF-BP expression in the MDA-MB-468 cell line. In this model we
demonstrated that EGF was able to upregulate FGF-BP transcription. This is important in the context of breast
cancer because expression of the EGFR has been inversely correlated with ER expression, and along with
expression of the EGFR family member HER2, the EGFR has been correlated with a poor prognosis for breast
cancer. FGF-BP expression, and its regulation by EGF in the MDA-MB-468 breast cancer cell line, may
suggest that FGF-BP plays a role in the expression of a more angiogenic phenotype in breast cancer.
As described above, deletion of the C/EBP site on the FGF-BP promoter resulted in a significant
increase in promoter basal activity. This suggests that a C/EBP factor binding to this site acts as a repressor.
Recently, a variant of C/EBPP, the liver enriched inhibitory protein (LIP), translated from the same mRNA as
the full length protein (also called liver enriched activating protein or LAP), has been described [34, 35]. LIP is
similar to LAP, except that it does not contain a transcactivating domain. The LIP-LAP dimer is able to bind to
its normal consensus site on a promoter, with greater affinity than LAP-LAP dimers, but is not able to promote
transcription, therefore acting as a dominant negative . LIP has also been found to be expressed in human
breast cancer samples that are both ER and PR negative . These data suggest the possibility that LIP
may be present on the FGF-BP promoter in the MDA-MB-468 cells under basal conditions acting as a
repressor of FGF-BP basal activity. When stimulated with EGF, the C/EBP dimer might change to a
LAP/LAP dimer and therefore enhance FGF-BP promoter activity. These hypotheses are currently under
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Figure 1. Regulatory region of the FGF-BP promoter
I " "" 'T
Table 1. Levels of FGF-BP, ER, and EGFR in human breast cancer cell lines, (adapted from Biscardi et al.,
Mol Carcinog2\:26\-212^ 1998)
P.I.: Kasan. Beniamin L.
Figure 2. EGF induction of FGF-BP mRNA in MDA-MB-468 cells.
Figure 3. Effect of signal transduction inhibitors on the EGF induction of FGF-BP in MDA-MB-468
cells. The following inhibitors were used: Calphostin C (PKC), PD 153035 (EGFR), U0126 (MEKl/2),
PPl (c-Src), Ro 31-8220 (PKC, MSKl), and SB202190 (p38/MAPK).