FGFR2 Signaling in Human Breast Cancer Cells

Institution: University of California, San Diego
Investigator(s): Daniel Donoghue, Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14IB-0065 Award: $100,000
Award Type: IDEA
Research Priorities
Etiology and Prevention>Etiology: the role of environment and lifestyle



Initial Award Abstract (2008)

FGFR2 is a receptor tyrosine kinase belonging to the Fibroblast Growth Factor Receptor (FGFR) family and, when activated, transfers a phosphate group (phosphorylates) to the amino acid tyrosine of target proteins. This leads to a cascade effect in which each phosphorylated protein is able to interact with another protein farther down in the pathway. When cells receive excessive signals to continue to divide or to escape normal cell death, cancer can be the result. Recently, two large studies independently identified FGFR2 as a major susceptibility locus for postmenopausal invasive breast cancer.

The recent genetic linkage studies identified single-nucleotide polymorphisms (SNPs) within FGFR2 as highly significant for increased risk. The essential hypothesis of our proposal is that one or more of these novel disease-associated polymorphisms creates a binding site for an estradiol-responsive transcription factor, resulting in altered FGFR2 expression or functional activity. This is based on a presumptive binding site for the estrogen receptor – an important transcription factor in mammary cells – that is created by one of the disease-associated SNPs. The changes in FGFR2 expression resulting from the variation of just a few nucleotides may be either qualitative or quantitative.

First, we will define the minimal FGFR2 sequence associated with one specific disease-associated SNP that is required for altered estrogen-dependent expression. These experiments will uncover effects of the sequence polymorphism on transcription factor binding, transcriptional activation, and alternate transcriptional start sites. As a second aim, we will examine FGFR2 expression in human breast cancer cells carrying disease-associated polymorphisms for either qualitative or quantitative changes in expression that are estradiol-dependent. These experiments will examine the expression of full-length FGFR2 in breast cancer cells, and will uncover estradiol-dependent changes in FGFR2 expression and kinase activation, alternative splicing, and the composition of FGFR2 signaling complexes. These experiments will reveal potentially important biological consequences – from the point of view of signaling – made possible by the disease-associated haplotype (i.e., combination of alleles that are transmitted together on the same chromosome) in FGFR2.

This research has the potential to open up a new line of study for breast cancer, since there is currently no published data confirming estradiol-dependent FGFR2 expression and any disease-associated polymorphisms.




Final Report (2010)

FGFR2 is a receptor tyrosine kinase (RTK) belonging to the Fibroblast Growth Factor Receptor (FGFR) family, an important family of signaling proteins in eukaryotic cells. In a major advance two years ago, several genome-wide association studies (GWAS) independently identified FGFR2 as a major susceptibility locus for postmenopausal invasive breast cancer. These studies identified single nucleotide polymorphisms (SNPs) within Intron 2 of FGFR2 as highly significant, and postulated that these SNPs may generate a putative estrogen receptor (ER) binding site. The experiments proposed to examine the hypothesis that FGFR2 expression is altered in an estradiol-dependent manner as a result of these polymorphisms.

The recent genetic linkage studies both identified single nucleotide polymorphisms (SNPs) within FGFR2 as highly significant for increased risk. Our original proposal was based on the hypothesis that these novel disease-associated polymorphisms create a binding site for an estradiol-responsive transcription factor, resulting in altered FGFR2 expression or activity.

In Aim 1 of our research, we characterized 5 different human cells lines for the presence or absence of the “at-risk” haplotype (i.e., a combination of alleles at multiple loci that are inherited together on the same chromosome). These experiments revealed the following:
1) For all 6 SNPs examined, there was conservation of the entire haplotype: i.e. the “at-risk” haplotype was present at all SNPs, or at none of the SNPs;
2) The breast cancer cell line MDA-MB-453 exhibits the “at-risk” A-A-G-T haplotype defined by GWAS studies, as does the cell line HEK293;
3) The breast cancer cell lines MCF-7 and T47D do not possess the “at-risk” A-A-G-T haplotype;
4) Similarly, the cell line U2OS is also negative.

In Aim 2, we examined FGFR2 expression in human breast cancer cells carrying disease-associated polymorphisms for either qualitative or quantitative changes in expression that are estradiol-dependent. We characterized different human cell lines for alterations in the size of FGFR2 expressed, following treatment with estradiol and/or FGF. Several cell lines were chosen to examine FGFR2 under the following conditions: i) mock; ii) estradiol stimulated; iii) estradiol + FGF1; iv) FGF1 alone. Results for cell lines that are ER-negative were uninformative. Results obtained for MCF-7 cells, which are ER-positive, revealed an estradiol+FGF dependent change in the size of FGFR2 expressed. Thus, we were able to demonstrate qualitative changes in FGFR2 expression in response to estradiol + FGF treatment.

The central issues for future research are twofold:
1) What is the difference in the FGFR2 that is expressed under these conditions?
2) Are there global changes in the estradiol-dependent expression of other proteins by these cells, in the absence or presence of the “at-risk” polymorphisms that are relevant to the proliferation, apoptosis, or adherence properties of these cells? With reliance on global proteomic approaches having been significantly increased from the time of our original 2008 application submission, these are questions that are now experimentally within reach of being answered.



The atypical CDK activator Spy1 regulates the intrinsic DNA damage response and is dependent upon p53 to inhibit apoptosis.
Periodical:Cell Cycle
Index Medicus: Cell Cycle
Authors: McAndrew CW, Gastwirt RF, Donoghue DJ
Yr: 2009 Vol: 8 Nbr: 1 Abs: Pg:66-75