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Award Number; DAMD17-00-1-0265 


TITLE: Growth Factor Regulation of an Angiogenic Factor, the 

Fibroblast Growth Factor-Binding Protein (FGF-BP), in Breast 


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 

DISTRIBUTION STATEMENT: Approved for Public Release; 

Distribution Unlimited 

The views, opinions and/or findings contained in this report are 
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_August 2001 Annual Summary (01 Aug 00 - 31 Jul 01) 


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 


Georgetown University Medical Center 
Washington, DC 20057 




U.S. Army Medical Research and Materiel Command 

Fort Detrick, Maryland 21702-5012 





Approved for Public Release; Distribution Unlimited 


13. Abstract (Maximum 200 Words) (abstract should contain no proprietary or confidential information) 

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. 


Growth Factors, Ribonuclease Protection, Transcription, DNA-Protein 
Interactions, Angiogenesis 















NSN 7540-01-280-5500 

Standard Form 298 (Rev. 2-89) 

Prescribed by ANSI Std. Z39-18 

Table of Contents 


SF 298.2 

Table of Contents.3 



Key Research Accomplishments.6 

Reportable Outcomes..7 





' ___ 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 

T. Introduction 

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 [15]. FGF-2 bound to this protein was not subject to degradation and retained 
its mitogenic activity [15]. 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 [16]. 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 [17]. FGF- 
BP mRNA is expressed in SCC, colon, and breast tumor cell lines and primary tumor tissue [16]. 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 [18]. 

II. Body 

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 [19]. 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 [20]. 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 
[21]. 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 [16]. 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. 
[24] 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 [22], 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 [20]. 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 [20]. Serum, in contrast, mediates FGF-BP transcription through PKC and p38 
MAPK signaling, but not MEK/ERK [21]. 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 [25]. 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) [26], therefore we tested whether the PKC-specific inhibitor calphostin C [27] 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% [20]. 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 
MDA-MB-468 cells. 

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 [31]. MDA-MB-468 cells have been shown to express moderate levels of c-Src protein as compared 
normal breast epithelium [24]. Therefore, we used the c-Src family specific inhibitor PPl [32]. 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 [32], 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 [20]. 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) 

V. Conclusions 

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 [35]. LIP has also been found to be expressed in human 
breast cancer samples that are both ER and PR negative [36]. 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) 

Cell Line 




























MDA-MB 231 




MDA-MB 468 












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).