FGFRS IN BREAST CANCER:
EXPRESSION,
DOWNSTREAM EFFECTS
AND POSSIBLE DRUG TARGETS
MASTER THESIS BY MILOU TENHAGEN
DECEMBER 2010
SUPERVISOR: DR. PETRA VAN DE GROEP
STUDENT NUMBER: 3158136
M.TENHAGEN@STUDENTS.UU.NL
INDEX
INDEX ........................................................................................................................................................ 2
ABSTRACT .................................................................................................................................................. 3
INTRODUCTION TO BREAST CANCER ................................................................................................................ 4
CHAPTER 1: FIBROBLAST GROWTH FACTOR RECEPTORS: .................................................................................... 6
CHAPTER 2: GENETIC ABERRATIONS IN FGFRS IN BREAST CANCER PATIENTS ........................................................ 8
CHAPTER 3: MOLECULAR IMPLICATIONS OF GENETIC ABERRATIONS IN FGFRS ON TUMOR CHARACTERISTICS .......... 16
CHAPTER 4: DRUGS IN DEVELOPMENT AND POTENTIAL DRUG TARGETS ............................................................. 22
CONCLUSION & DISCUSSION ....................................................................................................................... 26
REFERENCES.............................................................................................................................................. 29
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
2
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
ABSTRACT
In the search of new drugs to treat cancer patients, many targeted therapies are being developed.
Fibroblast growth factor receptors (FGFRs) are one of the many molecules that are currently under
investigation for their potential as a drug target in breast cancer patients. These receptor tyrosine
kinases play a major role in several processes including proliferation, angiogenesis, and migration.
Alterations in these basal processes can contribute to the development and progression of tumors. In
breast cancer patients, several subgroups of patients have been identified to harbor genetic
aberrations in FGFRs. These genetic aberrations include amplifications of FGFR1, -2 and -4, and
mutations in FGFR2 and -4. Multiple in vitro and in vivo models have partly elucidated the molecular
implications of these different genetic aberrations on the characteristics of the tumors. Based on
these results several drug targets have been suggested. For some of these targets drugs have already
been developed, which are currently being tested in in vitro and in vivo settings. Future experiments
will have to be performed in order to further clarify the molecular implications of the genetic
aberrations, to develop and test drugs but also to identify the subgroup of patients that will benefit
from these newly developed therapies.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
3
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
INTRODUCTION TO BREAST CANCER
In the Netherlands 12.000 women are diagnosed with breast cancer every year and with 1.4 million
newly diagnosed cases yearly worldwide, breast cancer is the most common cancer among
women1,2. Even though the prognosis is quite good, 458.000 women died from breast cancer in 2008,
making it an important cause of death due to cancer3.
Histopathological types of breast cancer
Table 1: WHO histopathological classification
Considering the pathological features, breast
of breast cancer and their incidence4
cancers can be divided into invasive and nonSubtypes
Incidence
invasive carcinomas, which are less severe. Table 1
Non-invasive Carcinomas
gives an overview of the types of breast cancers
Intraductal carcinoma (DCIS)
15%
and their incidence4. Non-invasive carcinomas are
Lobular carcinoma in situ (LCIS)
2%
confined to either the ductal basement membrane
Invasive Adenocarcinomas
of the breast or the terminal breast lobules and are Infiltrating ductal carcinoma
60%
called ductal carcinoma in situ (DCIS) and lobular
(not otherwise specified)
carcinoma in situ (LCIS) respectively. LCIS is usually
Infiltrating lobular carcinoma
5-10%
not treated, but it is considered a risk factor for
Mucinous (colloid) carcinoma
2-5%
developing an invasive carcinoma (which can be
Medullary
carcinoma
1-5%
both lobular and ductal)4. In contrast to DCIS and
Tubular carcinoma
2-5%
LCIS, invasive carcinomas spread into the
Papillary carcinoma
2-5%
surrounding tissues. Because of this invasive
Other
types
1-5%
character, these carcinomas are able to metastasize
resulting in a worse prognosis for the patient4. Infiltrating ductal and lobular carcinomas have their
origin in the ducts or lobules of the breast respectively and have invaded into the surrounding breast
tissue. Mucinous, medullary, tubular, and papillary carcinomas are all subtypes of infiltrating ductal
carcinomas. These subtypes can be distinguished by the shape, size, or surrounding structures of the
tumor cells. The tumor cells of mucinous carcinomas are surrounded by mucin, whereas medullary
carcinomas can be recognized by the shape of the tumor which resembles the medulla of the brain.
Tube-shaped cells are characteristic for tubular carcinomas and the tumor cells of papillary
carcinomas are shaped like threads5. Other types of invasive breast cancers include inflammatory
carcinoma and Paget’s disease. Inflammatory carcinoma is an invasive ductal carcinoma which
invades into the lymph system very early, whereas Paget’s disease is a DCIS that invades into the
nipple4.
Next to these pathological types, the development of breast cancer can also be divided in stages that
describe the progress of the tumor growth. This classification is based on the size and spread of the
tumor, the spreading of tumor cells into the lymph nodes, and further metastasis into other organs4.
Finally, several histological grading systems have been developed that asses the grade of
differentiation by scoring the mitotic rate, the formation of tubules, and the presence of nuclear
pleomorphisms6. The combination of the histopathological type, the stage, and the grade of the
tumor determine the prognosis for the patient. In general, the higher the stage and grade, the poorer
the prognosis is4, 6.
Treatment options
The current therapies for breast cancer are very diverse. First of all, breast cancer treatments can be
divided into local and systemic therapy. Local therapy includes breast surgery and radiation which
both focus on the malignant cells in the breast. The surgery can be breast-conserving (lumpectomy)
or includes the removal of the entire breast (mastectomy). This depends on the size and spreading of
the tumor in the breast. Systemic therapy intends to target cancer cells throughout the body by
chemotherapy, hormone therapy and/or targeted therapy4. What type of treatment is applied, is not
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
4
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
only based on the histopathological type, stage, and grade, but is also based on the genetic
aberrations present in the patient. These genetic aberrations can be small mutations in oncogenes or
tumor suppressor genes, but also amplifications or losses of genes or translocations. Since these
genetic aberrations are highly heterogenic among different patients, different hormone therapies
and other targeted therapies have been developed in order to achieve a personalized treatment.
Most genetic aberrations in cancer patients accumulate during life. Breast cancer however can also
be partly inherited, resulting in a much higher risk of developing breast cancer for people who have
two or more first-degree relatives with breast cancer4. Two genes that play a role in the inheritance
of breast cancer are mutations in the tumor suppressor genes BRCA17, and BRCA28. Because of a
genetic predisposition in one of these genes, most familiar breast cancers occur earlier in life
whereas most late-onset breast cancer patients do not have a known family history of breast cancer9.
The presence of inherited and somatic mutations in cancer patients can be seen as markers that play
a role in the development, progression, and/or the resistance to therapy. Because of the increasing
knowledge about the involvement of these specific genotypes in breast cancer, the therapies that are
currently being developed are more and more focusing on the targeting of these genotypes by the
use of hormone therapies and other targeted therapies10.
In order to develop these therapies, it is important to fully understand the molecular basis of the
oncogenic pathways. One of the experiments performed to unravel this, is the expression profiling of
over 100 unselected breast cancer samples. This has led to the identification of five distinct
molecular subtypes: basal-like, luminal A, luminal B, ERBB2-overexpression and normal breast like11,
12
. These subtypes have different survival rates, but more importantly they show a different response
to treatment13. This proves the importance of analyzing the molecular profile of each patient
separately in order to determine the most effective treatment plan. As a result, much research is
currently ongoing to identify markers and how to determine the right therapy for each single patient.
An example of a hormonal therapy that is only affective in a subgroup of patients is tamoxifen. This
estrogen-agonist blocks the estrogen receptors expressed by breast cancer cells. This treatment is
only effective for hormone positive carcinomas: estrogen receptor-positive (ER+) and/or
progesterone receptor-positive (PR+) carcinomas. The malignant cells in these tumors express a
certain threshold level of these receptors, making them dependent on estrogen for their survival and
are therefore called hormone sensitive14.
One of the targeted therapies currently used for a subset of breast cancer patients is the monoclonal
antibody Herceptin. This antibody is directed against the extracellular domain of the Her2 receptor
(also known as the ERBB2 receptor). Again, this only works for breast cancers that are Her2
positive15. The target of Herceptin is the Her2 receptor, a member of the receptor tyrosine kinase
(RTK) family. This receptor family plays an important role in important biological processes like
proliferation, differentiation, and apoptosis. Since these processes are often deregulated in cancer,
the members of the RTK family have been an interesting drug target over the last couple of years,
resulting in a large amount of tyrosine kinase inhibitors such as Herceptin and Gleevec16.
One of the RTK-subgroups that is currently under investigation for its involvement in breast cancer is
the family of fibroblast growth factor receptors (FGFRs). In this thesis, the signaling network and the
involvement of FGFRs in normal cells are described first. The second chapter will focus on the
findings of genetic alterations in FGFRs in breast cancer patients. Also, the association of these
alterations with other clinicopathological parameters is discussed. In chapter three, the effects of
these genetic aberrations on signaling and characteristics of the malignant cells will be explained by
in vitro and in vivo experiments performed. Chapter four will focus on possible drugs, targeting the
abnormal FGFR signaling in breast cancer patients. Finally, future goals concerning the identification
of mutations in patients and drug design will be discussed.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
5
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
CHAPTER 1: FIBROBLAST GROWTH FACTOR RECEPTORS:
EXPRESSION AND SIGNALING
Fibroblast growth factor receptors form a subgroup within the
RTK family consisting of four members in mammalians: FGFR1, 2, -3, and -4 17. Because genetic aberrations in these receptors
have been associated with breast cancer in several studies
(Chapter 2), they are good candidates to develop drug therapy
against. FGFRs consist of an extracellular ligand-binding
domain, a transmembrane domain and a cytoplasmic domain.
Next to a protein tyrosine kinase domain, the intracellular part
of the FGFR contains several regulatory regions18. The
extracellular domain can be divided into three immunoglobulinlike domains (Ig1-3, figure 1). IgII and -III are responsible for the
binding to ligands: fibroblast growth factors (FGFs)19. Until now,
Figure 1: FGF-FGFR structure
18 different secreted FGFs are known to be expressed in a
tissue specific way, just as their receptors. This tissue-specific
The extracellular domain consists of
expression and the differences in binding affinity contribute the
three Ig domains to which the ligand
specificity of the ligand-receptor interaction17. This specificity is
binds. This binding is stabilized by
also achieved by splice variation: FGFR1, -2, and -3 express two
HPSG. Intracellular, a tyrosine kinase
splice variants of the IgIII domain, resulting in a IIIb and IIIc
domain is present17.
isoform. The IIIb isoform is present on epithelial cells and the
IIIc is being expressed by mesenchymal cells19. In order for the FGF’s to bind firmly to their receptors,
stabilization by heparin sulphate proteoclycan (HSPG) is needed. HPSG is present on the surface of
every cell and binds to FGF’s with a very high affinity. Because of this, FGFs can only bind to receptors
present on nearby cells explaining the short-range signaling20.
After binding of the ligand, FGF receptors
dimerize, which results in the activation of the
intracellular kinase domain leading to crossphosphorylation of tyrosine residues present
on the intracellular tail17. Activated FGFR’s also
phosphorylate FGFR substrate 2 (Frs2) which
induces recruitment of growth factor receptorbound 2 (GRB2) and son of sevenless (SOS) that
activate the Ras-MAPK pathway, resulting in
proliferation and/or differentiation17, 21.
Furthermore, recruitment of Grb2-associated
binding protein 1 (GAB1) and subsequent
recruitment of phospho-inositol 3 kinase (PI3K)
activates AKT, resulting in the inhibition of
apoptosis. In addition, the phosphorylated
tyrosine residues at the C-terminus of the FGF
receptor create a binding site for phospholipase
C- y1 (PLCy) which is then phosphorylated by
the FGF receptor resulting in activation of
protein kinase C (PKC) reinforcing the MAPK
pathway. Finally, downstream signaling of FGF
receptors also includes activation of STAT
signaling (Figure 2)17.
Figure 2: Fgfr downstream signaling
Upon binding of the ligand, the receptors dimerize,
resulting in cross-phosphorylation. This leads to the
attraction of several docking proteins which can also
be phosphorylated. Downstream signaling occurs
through four main pathways: PLCy, STATs, AKT, and
MAPK17.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
6
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
All of these pathways play an important role in several basic cell processes including proliferation,
differentiation, migration and survival. In addition, FGFR signaling is known to be involved in
angiogenesis and wound repair17, 18. Which downstream pathway is activated and what the effect on
the cells eventually will be, depends on the cell context including the presence of the pathway
players, and crosstalk with other signaling pathways22. Moreover, the type of FGFR is also important,
for example: downstream activation by FGFR4 is less strong than FGFR123, and FGFR1 signaling
sustains longer than FGFR2 because of faster degradation of FGFR2 after activation24. So far, no
evidence has been found for an influence of the type of ligand on downstream signaling22.
Altogether, if you consider the basic cell processes in which FGFR signaling is involved, perturbation
of FGFR could very well contribute to cancer development by an increase in proliferation, infiltration
of blood vessels, and by avoiding apoptosis. The genetic aberrations in FGF receptors contributing to
breast cancer, their downstream signaling effects, and the possibility to drug these disturbances will
be described in the following chapters.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
7
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
CHAPTER 2: GENETIC ABERRATIONS IN FGFRS IN BREAST CANCER PATIENTS
As described in the introduction, the development, progression and/or response to therapy of breast
tumors is influenced by the genetic aberrations present in the malignant cells. These genetic
aberrations can influence the expression levels of the receptors, the functioning of the receptors, or
both. In general, the aberrations can be divided into mutations and copy number changes. If a
mutation is located in the coding region of the gene, it can influence the functioning of the receptor,
for example by changing its subcellular localization, its kinase activity, or its binding properties to
ligands or adaptor proteins. If a mutation is located in one of the introns of the gene, this can
influence the functioning of the protein by inducing alternative splicing. In addition, a mutation in
one of the regulatory gene regions can change the expression of the receptor for example by altering
the binding affinity of a transcription factor to this regulatory region. On the other side, copy number
alterations can also change the expression level of the receptors. These copy number alterations are
a feature of advanced cancers, since the genetic instability increases during the development of
cancer25. The paragraphs below describe for each FGFR-gene, the genetic aberrations that have been
identified in breast cancer patients.
FGFR1
FGFR1 amplifications in breast cancer patients
Several studies have identified amplifications of FGFR1 in breast cancer patients 13, 25-30. Other genetic
aberrations like somatic or germ-line mutations have not been identified for FGFR1 in breast cancer
patients. The chromosomal band 8p11-12, the region in which the FGFR1 gene is located, has been
found to be frequently amplified in a group of 145 early-stage breast tumor samples13.
Unfortunately, the resolution of the technique that was used (array comparative genomic
hybridization: aCGH) was 1Mb, making it unable to identify amplifications at a single gene level.
However, RNA microarrays showed that the expression level of FGFR1 was significantly associated
with the 8p11-12 copy number, suggesting that amplification of this region resulted in the
overexpression of FGFR1 in these breast cancer patients13.
Table 1: Copy number alterations in FGFR1 identified in breast cancer patients
Type of BC (number of Technique, whole
Samples)
genome /gene specific
Stage II-III BC’s (106)
aCGH: 70 kb interval,
whole genome
Primary BC (161)
SNP DNA microarray,
whole genome
Invasive BC (319)
FISH, gene specific
Unselected BC (496)
CISH, gene specific
Invasive BC (104)
MLPA, gene specific
MPC (micropapillary
carcinoma, 12)
CISH, gene specific
Results: FGFR1
amplification/loss
Highly amplified in 10%
Reference
Amplified in 7,5%
26
Amplified in 9.4%
Amplified in 8.7%
Amplified in 17% (7%: highly
amplified)
Lost in 10%
Amplified in 16.6%
27
28
29
25
30
Table 1 gives an overview of several studies analyzing the copy number alterations at a single gene
resolution. As can be seen, the percentage of FGFR1 amplifications ranges from 7.5-17%. A possible
explanation for this big range is that most of these studies included tumor samples of patients
without any selection on histopathological type or clinical parameters. Furthermore, each study used
a different technique to assess the copy number alterations which can also account for the different
percentages. In general, the techniques used can be divided into two subgroups: 1; genome wide
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
8
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
analysis and 2; gene specific analysis of gene amplification and/or loss. High-resolution aCGH can be
used to identify copy number alterations in the entire genome. When analyzing 106 stage 2-3 breast
tumor samples using this technique, FGFR1 was found to be amplified in 10 samples. Again, RNA
microarrays proved that the amplification of FGFR1 resulted in significant overexpression of FGFR125.
Another genome-wide approach to map amplifications on gene level is by the use of a SNP
microarray. By calculating the average increase or decrease for all the probes mapped within a gene,
copy number alterations of all genes can be identified. Using this approach, FGFR1 was found to be
amplified in 7.4% of 161 breast tumor samples26.
Next to genome-wide approaches, copy number gains and losses can be analyzed for specific regions.
Two similar techniques are fluorescent in situ hybridization (FISH) and chromogenic in situ
hybridization (CISH). Using these techniques FGFR1 was found to be amplified in 9.4% of 319 invasive
breast cancer samples27 and in 8.7% of 496 unselected breast cancer samples respectively28. In a
smaller study containing 24 pure invasive micropapillary carcinomas, CISH identified FGFR1
amplification in 4 cases (16.6%)30.
With multiplex ligation-dependent probe amplification (MLPA) primers specific for FGFR1 are being
used resulting in a reliable measurement of the gene copy number. Using this technique on 104
invasive breast cancer samples, amplification of FGFR1 was identified in 17% of the patients29. In
addition, loss of FGFR1 was also identified in 10% of the patients. With the exception of Chin et al.
describing the deletion of 8p11-12 in breast cancer tumors, no other records are known describing
the loss of FGFR1. The identified loss of 8p11-12 was shown to be associated with a poor outcome13.
However, since this region contains many genes, it is not sure whether this poor outcome can be
contributed to the loss of FGFR1.
Association of FGFR1 amplifications with clinical and pathological patient characteristics
As described above, amplification of FGFR1 occurs in 7.4-17% of all breast cancer types. Some of the
studies mentioned above did not only characterize the copy number alterations of the tumor
samples but also analyzed the association of these alterations to clinical characteristics currently
used to determine the prognosis and treatment. All of the parameters tested for association with
FGFR1 amplifications are described in table 2.
Table 2: association of FGFR1 amplification in unselected cohorts with clinical and pathological
parameters
Parameter
Association: yes/no
Reference
Development of distant metastasis
Yes: positive association
28
Grade
No
27-29
Tumor size
No
26-29
Histopathologic type
No
26, 27, 29
Lymph node invasion
No
26-28
Vascular invasion
No
27, 29
Tumor stage
No
26, 28
Molecular subtypes (basal-like, luminal
No
27, 28
A/B, ERBB2-positive, normal-like)
P53 status
No
27
EGFR status
No
28
Expression of low- and high-molecular
No
28
weight cytokeratins
Androgen receptor status
No
28
Proliferation
Yes: positive association
27
No
29
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
9
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
ER status: positive or negative
PR status: positive or negative
Age (<50/≥50)
Her2 status
No
Yes: associated with a positive status
No
Yes: trend with a negative status
No
Yes: if amplified, more likely to be older
than 50
No
Yes: inverse correlation with Her2
overexpression
Yes: amplification was positively
associated with Her2+ status
26-28
29
26, 27, 29
28
27, 29
28
27, 29
28
26
Most of the parameters are not significantly associated with amplification of FGFR1. However,
patients with an amplification of FGFR1 are more likely to develop distant metastasis28.
Unfortunately, this parameter has not been tested in other large-scale studies. Finally, for several
parameters the associations found are inconsistent between different studies. This can be the
consequence of a difference in characterization of the parameters, for example: Letessier et al. used
the expression of Ki67 as a measurement of proliferation27, whereas Moelans et al. assessed the
mitotic activity index to quantify the proliferation29. Also, the Her2 status is assessed differently:
Elbauomy Elsheikh et al. and Kadota et al. only discriminate between Her2-positive and Her2negative tumor samples26, 28, whereas Moelans et al. and Letessier et al. subdivide the tumor samples
in 0-1+, 2+, and 3+ Her2 expression levels based on IHC27,29. However, for the parameters that were
assessed in the same way in different studies other clinical or pathological parameters could act as
confounders. In order to exclude these factors, multifactorial analyses are needed.
Relationship of FGFR1 amplifications with the patients’ survival
Several studies have not only correlated amplification of FGFR1 with the expression of the markers
and clinical characteristics described above, but have also performed association analysis of FGFR1
amplification with the clinical outcome. The clinical outcome can be described in different ways. First
of all, the disease free survival time (DFS) is used to annotate the length of time after treatment that
the patient survives and does not have any signs of relapse. Next to this, the overall survival (OS) is
used to determine the time between the treatment and death of the patient. Finally, metastasis free
survival (MFS) describes the period between the treatment and the diagnosis of metastasis.
In a cohort of 496 unselected breast cancer samples, amplification of FGFR1 was significantly
associated with a shorter overall survival and has a trend with a shorter disease-free survival (Figure
1 A,B)28. This prediction of poor overall survival was independent of parameters known to be
involved in survival (such as grade, tumor size, lymph node invasion, and ER status). Interestingly,
when grouping these patients into ER+ and ER- tumors, FGFR1 amplification remained a significant
independent risk factor for poor DFS and OS in ER+ patients (Figure 1 C,D), even being a stronger
predictor than grade, size and lymph node invasion. However, in ER- tumors, FGFR1 amplification
was not associated with the DFS and OS of the patients28. So maybe there is an interaction between
FGFR1 signaling and ER signaling resulting in a poor prognosis. The observation that FGFR1 is
associated with a poor prognosis in ER+ breast cancer patients is confirmed in a group of 87 ER+
tumor samples from patients which were treated with adjuvant endocrine therapy (tamoxifen). In
this group, FGFR1 overexpression was significantly associated with a poor metastasis free survival
compared to samples with normal FGFR1 expression levels31. Finally, two studies show that there is
an association between amplification of 8p(11-)12 and a poor disease outcome13, 27. Even though
these analyses are not specific for FGFR1, it supports the findings in the previous studies.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
10
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
Figure 1: Kaplan-Meier
survival analysis of
patients with and
without amplification
of FGFR1
A: Entire cohort,
disease free survival.
B: Entire cohort, overall
survival.
C: ER+ cases, disease
free survival.
D: ER+ cases, overall
survival28.
FGFR2
FGFR2 amplifications in breast cancer patients
In the genome wide screen identifying copy number alterations using a SNP microarray, not only
FGFR1, but also FGFR2 was found to be amplified. Given the fact that it was only amplified in 2 out of
the 161 primary breast cancer samples (1.2%), it was considered to be infrequently amplified26.
Other genome wide screens analyzing large groups of unselected BC samples also identified FGFR2 to
be rarely amplified32, but others did not find any amplifications or losses of FGFR225. The
identification of only rare or no amplification of FGFR2 can be due to the fact that all of these studies
analyzed a large number of unselected breast cancer samples without subgrouping them based on
histological and/or clinical features. It could very well be possible that FGFR2 is only amplified in a
subgroup of breast cancers. Supporting prove for this idea comes from an analysis of 165 triple
negative breast cancer samples (meaning ER-, Her2-, and PR-). In this group of patients, FGFR2 was
found to be amplified in 4%, whereas no amplification events were found in 214 samples belonging
to other subtypes. In addition, the mRNA expression levels of FGFR2 were significantly increased in
amplified tumor samples vs. non-amplified tumor samples33, suggesting a potential role for FGFR2 in
this subtype of breast cancer. Also in familial breast cancer, FGFR2 is suggested to play a role in a
subgroup of patients. When comparing the expression profiles of 7 breast cancer patients with
mutations in BRCA1 and 6 breast cancer patients with mutations in BRCA2, FGFR2 was found to be
significantly higher expressed in BRCA2 mutation carriers34.
SNPs in FGFR2 in breast cancer patients
So far, no somatic mutations in FGFR2 have been identified in breast cancer patients35, but there are
several germ-line mutations in FGFR2 that are associated with breast cancer risks.
A genome-wide study analyzing 1145 invasive breast cancer patients and 1142 controls identified
several SNPs to be highly associated with breast cancer risks36, 37. Four of these SNPs were located in
intron 2 of FGFR2. An independent study comprising 21860 invasive breast cancer patients and
22578 healthy controls also identified a SNP in intron 2 of FGFR2 to be associated with breast
cancer37. The risk allele of this SNP was strongly associated with a positive ER and PR-status, and a
lower grade38. Further analysis of the SNPs located in the second intron of FGFR2 has resulted in the
identification of a haplotype block of 8 SNPs to be the minor disease-predisposing allele39.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
11
Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
Several ideas on how the SNPs located in the second intron of FGFR2 could contribute to breast
cancer have been proposed. When correlating the expression levels of FGFR2 in 170 invasive breast
cancer samples with the haplotype of the 8 SNPS, RNA levels of FGFR2 were significantly increased in
tumor samples that were homozygous for the minor alleles when compared to samples homozygous
for the common alleles39. This indicates that the risk genotype results in a change in the expression of
FGFR2. Since the sequence of intron 2 contains several possible binding sites for transcription factors
it could be that the SNPs influence the binding of these transcription factors, thereby regulating the
expression levels of FGFR237.
An alternative hypothesis is that the SNPs influence the functioning of the FGFR2 protein. Since the
haplotype block of the 8 SNPs is not in linkage disequilibrium with any coding regions of FGFR239, a
change in the protein sequence of FGFR2 resulting in a functional difference is excluded. However,
the SNPs could induce alternative splicing, resulting in impaired functioning of FGFR2 or a change in
the stability of the protein. When studying the two most common splicing variations (including or
excluding exon 3) no significant difference was observed between minor homozygotes and common
homozygotes39. In vitro evidence for the influence of the SNPs in the expression and functioning of
FGFR2 will be further discussed in the chapter 3.
FGFR3
Genetic aberrations in FGFR3 in breast cancer patients
Even though activating mutations in FGFR3 have been identified in several cancer types40-43
mutations in breast cancer seem to be absent35, 40, 44. There is one breast cancer patient known to
carry a heterozygous missense mutation in exon 7 of FGFR3 (P250R)40. This mutation is located in the
ligand-binding domain of FGFR3 which leads to activation of the kinase-domain45. Moreover, this
mutation is located next to the mutational hotspots found in bladder and cervical carcinomas
(R248C, S249C)40. This patient however also suffers from the Saethre-Chotzen syndrome, a disease
that is caused by a mutation in FGFR3 in a subset of patients45. Therefore, it is unclear whether the
FGFR3 mutation in this patient has a role in the development of breast cancer. To clarify this and to
identify additional breast cancer patients carrying an activating mutation in FGFR3, more studies
should be performed.
FGFR4
Overexpression of FGFR4 in breast cancer patients
Next to amplifications of FGFR1 and FGFR2, also amplification of FGFR4 is occasionally found in
breast cancer patients. In a small scale study including 30 unselected primary breast cancer samples,
10% of the breast tumors had an amplification of FGFR4. This amplification was associated with an
ER+ and PR+ status and lymph node invasion46. Furthermore, in 103 breast tumors the mRNA levels
of FGFR4 were found to be elevated in 32% of the samples47 indicating that high levels of FGFR4
could play a role in breast cancer. One of these roles could be the resistance to therapy, as indicated
by a retrospective study analyzing 285 ER+ breast cancer samples that were treated with tamoxifen
as first-line therapy. In this group of patients, high levels of FGFR4 resulted in a worse outcome as
quantified by the tumor size and post-relapse overall survival after treatment. This role of FGFR4 as a
predictive factor in tamoxifen treatment was independent of known parameters that predict the
clinical outcome48. A molecular explanation why increased levels of FGFR4 are associated with a high
chance of failure of tamoxifen treatment will be explained in chapter 3.
FGFR4: Gly388Arg polymorphism in breast cancer patients
In addition to alterations in expression of FGFR4, mutations in FGFR4 are also present in several
cancer types49-52. A mutation of FGFR4 was found after expression and sequence analyses of different
RTK’s in several breast cancer cell lines. This led to the identification of a novel missense mutation in
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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the transmembrane domain of FGFR4: Gly388Arg16 now known as SNP rs351855 and further denoted
as Arg388. Immunohistochemical analysis of 372 breast cancer samples did not result in any
correlation of the SNP genotype with the expression levels53 excluding the possibility of the SNP
having influence on the expression level.
Sequence analysis of a total number of 1103 breast cancer patients revealed that the Arg388
genotype, either heterozygous or homozygous, was present in 37-43% and 8-11% respectively (Table
3)16, 53-55. When comparing the distribution of the Arg388 allele in healthy controls and breast cancer
patients, no significant difference could be identified. This indicates that this polymorphism is not
associated with the initiation of breast cancer16.
Table 3: FGFR4 Gly388Arg allele distribution in breast cancer patients and controls
Group
Gly/Gly
Gly/Arg
Arg/Arg
Reference
Healthy controls (n=123)
Breast cancer patients (n=145)
Breast cancer patients (n=234)
Breast cancer patients (n=372)
Breast cancer patients (n=352)
45%
46%
52%
49%
46%
49%
43%
37%
43%
43%
6%
11%
11%
8%
11%
16
16
54
53
55
Since the minor allele does not seem to be associated with the development of breast cancer, it
could be that it influences the progression in the tumor, making this genomic variation a prognostic
marker. First of all, when analyzing the coincidence of Arg388 with known prognostic markers, no
significant correlation was found for age, Her2 status, ER status, PR status, grade, and triple negative
breast cancers16, 53-55. Correlations were found for tumor stage53, 55, tumor size53, and axillary lymph
node involvement16, 55, but these associations were not confirmed by other studies16, 53, 54. Finally,
when analyzing the disease free survival period, no correlation was found with the Arg388 allele16.
However, in 46 unselected primary breast cancer samples from patients positive for lymph node
metastasis (N+), but not in N- patients, the disease free survival period was significantly shorter in
patients carrying the minor Arg388 allele, than carriers of the common allele Gly388 (Figure 2)16. This
suggests that the Arg388 is a marker for increased tumor aggressiveness in advanced breast cancer.
Figure 2: Kaplan-Meier survival analysis of breast cancer patients for Fgfr4-388 alleles
A: Probability of disease free survival for patients with axillary lymph node involvement
B: Probability of disease free survival for patients without axillary lymph node involvement.
For both plots, the Gly/Arg carriers were combined with the Arg/Arg carriers into the Fgfr4
Arg388 group16.
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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Even though the involvement of the Arg388 allele in the survival of N+ patients is confirmed by
Thussbas et al.53, Jézéquel et al. does not find a significant association of the allele with the DFS of
139 N+ unselected primary breast cancer cases54. An explanation for this discrepancy could be that
other clinicopathologic parameters known to influence the patients’ survival act as confounding
factors. A multivariate analysis of a large group of N+ patients could exclude this possibility.
Association of the Arg388 allele with response to therapy
A second explanation for the conflicting statements about the involvement of the Arg388 allele in the
survival of N+ patients could be that the type of therapy acts as a confounding factor. Thussbas et al.
tried to clarify this by examining the DFS in patient groups that did or did not receive adjuvant
systemic therapy, one of the therapies having a great impact on the patients’ survival. If Arg388 is
involved in the progression of breast cancer, this influence should be apparent in the DFS of patients
not receiving therapy. This however was not the case: the genotype was not associated with the DFS
for patients that did not receive adjuvant systemic therapy. However, when adjuvant systemic
therapy was applied, the DFS and OS were shorter in patients carrying one or two Arg388 alleles. This
result could be directly linked back to the involvement of Arg388 in N+ patients, but not in N- patients,
since the administration of adjuvant systemic therapy greatly depends on the nodal status53.
Interestingly, when splitting up this patient group into chemotherapy treated patients and patients
that receive endocrine therapy, this significant effect of the FGFR4 genotype on DFS only remained
significant for the patient group treated with chemotherapy, making it a possible marker for therapy
resistance in this patient group (Figure 3)53.
A
B
Figure 3: Kaplan-Meier survival analysis of breast cancer patients for Fgfr4-388 alleles after adjuvant
systemic therapy
A: Probability of disease free survival for patients treated with adjuvant endocrine therapy
B: Probability of disease free survival for patients treated with adjuvant chemotherapy. For both plots,
the Gly/Arg carriers were combined with the Arg/Arg carriers into the Fgfr4 Arg388 group53.
This dependence on the type of therapy for the FGFR4-388 allele to be a prognostic marker can be a
possible explanation for the fact that Jézéquel et al.54 did not find a significant effect of Arg388 on the
survival of N+ patients: their patient group could have received different adjuvant systemic therapy.
On the other side, Marmé et al. did not find an association between the FGFR4 genotype and DFS in
patients treated with adjuvant chemotherapy. More surprisingly, they found that in patients that
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
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Milou Tenhagen, Master Thesis, December 2010
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received chemotherapy before operation (neoadjuvant chemotherapy), the Arg388 allele was
significantly associated with a better clinical and pathological response. A multivariate analysis even
showed that Arg388 was an independent predictor of a complete pathological response which means
that all invasive cancer cells in the breast have disappeared55. Again, the differences in study design
and type of therapy could be an explanation for these different results. This shows once again that
large-scale studies, analyzing many parameters are needed to confirm the ideas about the
involvement of the Arg388 allele in the response to therapy.
In summary, amplifications of FGFR1, -2, and -4 are present in breast cancer patients. On top of that,
sequence variations have been identified in the FGFR2 and -4 genes. The involvement of genetically
aberrant FGFR3 in breast cancer has not been demonstrated. The genetic aberrations in the other
FGFRs however, have been shown to contribute to the risk of breast cancer, but also to the
progression of the tumor resulting in a worse prognosis, and to the response to therapy. Much
research is ongoing to find out the molecular basis for these effects, and the results of these
experiments will be described in the next chapter.
FGFRs in breast cancer: expression, downstream
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CHAPTER 3: MOLECULAR IMPLICATIONS OF GENETIC ABERRATIONS IN
FGFRS ON TUMOR CHARACTERISTICS
Since FGFR’s have so many downstream signaling pathways whose activation depends on several
factors, the genetic aberrations identified in patients all influence breast tumors in a different way. In
order to unravel the underlying molecular mechanisms, several in vitro and in vivo studies have been
performed. The experiments described below give more insight into the effects of abnormal FGFR
signaling.
FGFR1
iFGFR1 as a model for overactive FGFR1
signaling
In order to find out the effects of
overactivation of FGFR1, an inducible FGFR1construct was made (iFGFR1) which can be
activated by administration of a drug:
AP20187, resulting in ligand-independent
dimerization of the receptor. In transgenic
mice, activation of iFGFR1 resulted in
increased phosphorylation of iFGFR1 and
downstream targets such as Akt, and MAPK.
Furthermore, administration of BrdU showed
increased proliferation in the lateral buds and
ductal cells in AP20187-treated mice
compared to their non-treated littermates.
Phenotypically, induction of iFGFR1 resulted
in increased lateral budding and ductal
branching (Figure 1) of the mammary
epithelium56.
Figure 1: activated
iFgfr1 induces lateral
budding and ductal
branching
A: Normal mammary
epithelium of
transgenic mice
treated with diluent
B: Increased lateral
budding (arrowhead)
and ductal branching
(arrows) in transgenic
mice after 2 weeks of
AP20187 treatment56.
When AP20187 was administered for a sustained period of time, the epithelial cells further
proliferated, becoming multicellular and eventually forming invasive lesions (Figure 2A). The cells in
these invasive lesions have lost their cell polarity, and detach from the basement membrane without
anoikis being induced. The invasive character of the lesions is established by several factors. First of
all, the myoepithelial cell barrier normally located between the luminal epithelial and stromal cells, is
partly absent in transgenic mice treated with AP20187. The absence of these cells is correlated with a
reduced and disorganized extracellular matrix (ECM)56, which can be explained by the fact that the
myoepithelial cells are responsible for the formation and maintenance of the ECM57. Finally, the
invasive lesions in iFGFR1 transgenic mice are surrounded by an increased number of highly
branched small vessels56. Again, the loss of myoepithelial cells can play a role in this process, since
they secrete anti-angiogenic factors58.
Another explanation for the loss of the ECM and increased angiogenesis comes from an iFGFR1 in
vitro model made up of a 3D culture of iFGFR1 transfected mouse mammary epithelial cells (HC11
cells) that faithfully recapitulates the morphological changes found in vivo upon iFGFR1 activation59.
In these cells, the activity of MMP-2 and -9 is increased upon activation of iFGFR156. Furthermore, the
expression of MMP-3 and -9 is induced after 24 hours of administration of AP2018738, 59. This
induction of MMPs by FGFR1 signaling is known to be mediated through the Mapk-Erk-pathway 38.
These matrix metalloproteases are involved in the breakdown of the ECM, but also induce
angiogenesis by increasing the availability of angiogenic growth factors, thereby contributing to the
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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invasive character of the lesions56, 59. In addition to the induction of MMPs, also several indications
for irreversible epithelial to mesenchymal transistion (EMT) became apparent in the 3D HC11 iFGFR1
in vitro culture system. This included elevated expression of mesenchymal markers and a loss of cellcell junctions59. These established factors of EMT in addition to loss of polarity and absence of
anoikis have also been observed in a 3D culture of iFGFR1 transformed MCF10A cells60, making both
iFGFR1 transfected HC11 and MCF10A 3D cultures a reliable model to study the effects of overactive
FGFR1 signaling.
Figure 2: iFgfr1 induced lesions in the
mammary epithelium of transgenic
mice
A: After administration of AP20187,
lateral buds start to appear. This is
followed by the multicellularization of
the epithelium, eventually resulting in
invasive lesions.
B: This is accomplished by increased
proliferation and remodeling of the
ECM. Increased expression of MMPs (●)
disrupts the ECM and can induce
vascular branching by increasing the
availability of growth factors ( ). This
disruption of the ECM and increased
vascularity is also achieved by the loss of
the myoepithelium. Finally, epithelial
cell polarity is lost due to misslocalization of E-cadherin and loss of ZO1, also leading to a more invasive
phenotype56.
How all these processes described above are regulated on a molecular level, either by direct or
indirect downstream signaling of FGFR1, is not exactly known yet. It has been shown that several
downstream targets are activated upon induction of iFGFR1 including RSK, the PI3K-Akt-, and the
MAPK-ERK-pathway56, 59, 60. In order to find out the initial target genes of these pathways after
activation of iFGFR1, mammary tissue of induced iFGFR1 mice was analyzed at early time points
(from 0 to 24 hours), around which lateral budding starts. Microarray analysis revealed several genes
whose expression was altered at any time point compared to non-treated transgenic mice. Most of
these genes (88%) are already upregulated after 8 hours, indicating that these genes are directly
regulated by FGFR1. The function of several clusters of genes, whose expression is changed upon
induction of iFGFR1, is linked to tumor formation. These tumorigenic processes include angiogenesis,
cell cycle regulation, chemotaxis, and the response to inflammation61. Inflammation in breast cancer
has been linked to a more invasive phenotype and poor prognosis62. When investigating the
infiltration of immune cells into the induced iFGFR1 mammary epithelium, it became apparent that
the number of macrophages was strongly increased. This is a consequence of the increased
expression of osteopontin, a macrophage chemoattractant, by the epithelial cells after activation of
iFGFR1. These macrophages have been shown to be required for the formation of the lateral buds
and to be involved in the formation of small blood vessels in the induced iFGFR1 mice61. These
processes following the presence of macrophages are mediated by the production of IL-1β63.
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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In conclusion, iFGFR1 induction results in the formation of multicellular invasive lesions according to
the model that is presented in figure 2. This is not only accomplished by increased proliferation,
inhibition of anoikis, loss of cell polarity, and EMT, but also includes remodeling of the ECM and
stroma, the induction of angiogenesis, and the attraction of macrophages. This implies that
overactivation of FGFR1 does not only affect the characteristics of the epithelial cells, but also
interacts with the microenvironment. Even though the genetic aberrations in FGFR1 found in
patients are not the same as the induction of FGFR1 signaling in iFGFR1 transgenic mice, this model
can still be very useful. It can provide more insight into the molecular mechanisms by which FGFR1
contributes to the progression of the tumor, resulting in a worse prognosis in patients harboring
FGFR1 amplifications. It has to be taken into account that breast cancer patients harbor additional
genetic aberrations that could interact with FGFR1 signaling, making the model a less good
representation of the human situation. An indication for interacting pathways comes from the
MMTV-Wnt1 mouse model. Crossing this mouse model with iFGFR1 transgenic mice and subsequent
activation of iFGFR1 dramatically increases and accelerates tumor formation, suggesting an
interaction between FGFR1 and Wnt signaling64. Crossing the iFGFR1 transgenic mice with other
breast cancer mouse models could provide more insight into the interaction of iFGFR1 signaling with
other oncogenic pathways. Finally, the interaction of FGFR1 signaling with aberrant signaling in other
pathways might even be necessary for the invasive carcinomas to metastasize. A possible way to find
out whether overactive FGFR1 signaling is sufficient to form malignant carcinomas that are capable
of metastasizing, prolonged administration of AP20187 to iFGFR1 transgenic mice could be tested.
Cell lines harboring FGFR1 amplifications as an in vitro model
To simulate the FGFR1 amplification found in breast cancer patients more accurately, cell lines
having endogenous amplifications and subsequent overexpression of the FGFR1 gene are used:
MDA-MB-134, CAL120, JIMT-1, MFM223, S68 and SUM4431, and CMA, MDA-MB-361 and HCC3865.
Induction of FGFR1 signaling in these cell lines using a small amount of Fgf2 resulted in downstream
activation of multiple pathways, including Frs2, Erk1/2, and RSK phosphorylation, whereas this was
not observed in control cell lines31. Furthermore, overexpression of FGFR1 induces a basal level of
downstream signaling without the presence of a ligand31. Finally, cells harboring FGFR1
amplifications seem to have an oncogenic addiction to FGFR1 signaling. This can be seen by the
impaired proliferation of MDA-MB-134 cells, but not of cell lines without FGFR1 amplification, upon
inhibition of FGFR1 signaling via administration of either FGFR1-siRNA or an FGFR inhibitor
(SU5402)66.
Furthermore, FGFR1 amplified cell lines were resistant to treatment with 4-hydroxytamoxifen (4OHT), one of the drugs used as an endocrine therapy. Additionally, inhibition of FGFR1 by siRNA
increased their sensitivity to this treatment. The initial resistance of the cells to 4-OHT is mediated by
activation of the MAPK-pathway leading to the expression of Cyclin D1 and a subsequent increased Sphase fraction. These results suggest that amplification of FGFR1 is involved in resistance to
endocrine therapy. This is supported by the observation that amplification of FGFR1 is associated
with a poor prognosis in ER+ patients that were treated with tamoxifen as an adjuvant endocrine
therapy31.
FGFR2
Effects of FGFR2 amplifications in vitro
The amplification of FGFR2 found in breast cancer patients is also observed in several breast cancer
cell lines, making it possible to elucidate the effects of FGFR2 amplification in vitro. Two cell lines
containing amplifications and increased expression levels of the FGFR2 gene are the triple negative
lines SUM52PE and MFM223. Both cell lines depend on FGFR2 signaling for survival, indicated by a
significant decrease in survival upon inhibition of FGFR2 compared to cell lines without FGFR2
amplification. This addiction to FGFR2 is mediated by the activation of the PI3K-Akt signaling pathway
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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that results in inhibition of apoptosis33. Furthermore, downstream targets of FGFR signaling like Frs2,
Akt and Erk, are activated in serum starved conditions and inactivated when an inhibitor of FGFR’s
(PD173074) is added. This indicates that the phosphorylation of these targets occurs independent of
ligand-binding, but is dependent on FGFR-kinase activity33. Whether this is also the case in patients
harboring FGFR2 amplifications should be elucidated in order to identify therapeutic targets, which
will be further discussed in chapter 4.
Signaling effects of SNPs in intron 2 of FGFR2
As stated in the second chapter, a haplotype block of 8 SNPs in intron 2 of FGFR2 was strongly linked
to an increased risk of breast cancer (Figure 3). This could be due to an altered binding capacity of
transcription factors resulting in changed expression of FGFR2. Indeed a number of the 8 SNPs
forming the minor disease-predisposing allele are in the vicinity of transcription factor binding sites:
rs35054928 and rs2981578 are located next to an Oct-binding site, while the risk allele of rs2981578
also creates a binding site for Runx. Furthermore, the risk allele of rs10736303 creates a binding site
for ER and the common allele of rs7895676 is part of a binding site for C/EBP-β67 (Figure 3).
Figure 3: Genetic linkage of
the FGFR2 gene (HapMap)
The 8 SNPs most strongly
associated with breast
cancer risk which are in
strong linkage
disequilibrium and form the
minor disease-predisposing
allele. The two alleles
shown in green have
differential binding
affinities for transcription
factors (blue). Adapted
from Meyer et al.39
Only two of these SNPs show different protein-binding affinities compared to the common allele39.
The common allele of rs7895676 displays a stronger binding to C/EBP-β, compared to the minor
allele. At the same time, rs2981578 showed equal affinity for Oct-1, but a much higher affinity for
Runx2 by the minor allele39. This difference in affinity is thought to be affected by differences in
H3/H4 acetylation of the SNP sites68. Overall, these differences in binding affinity result in a higher
expression of FGFR2 in cell lines carrying the minor haplotype. This is in accordance with the
increased expression levels of FGFR2 in breast cancer samples that were homozygous for the minor
alleles39.
FGFR4
The role of FGFR4 overexpression in a poor prognosis
As stated in the second chapter, the poor response to chemotherapy is associated with
overexpression of FGFR448. This is recapitulated in several breast cancer cell lines showing increased
expression of FGFR4 in case of resistance to doxorubicin and cyclophosphamide69. FGFR4 contributes
to this resistance by activating anti-apoptotic signaling via activation of MAPK and subsequent
increase the Bcl-xl levels69. In accordance with this, inhibition of FGFR4 led to a reduction of ERKphosphorylation and decreased levels of Bcl-xl, ultimately leading to increased sensitivity of the cells
to chemotherapeutic drugs69.
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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FGFR4-Arg388 as a marker of poor prognosis: possible underlying mechanisms
The SNP in FGFR4 identified in patients by Bange et al.16 results in the substitution of a glycine
residue, being a neutral amino acid, into the hydrophilic amino acid arginine in the transmembrane
region of FGFR4. This affects the structure and could alter the regulation of the receptor activity.
However, so far, no sign of enhanced tyrosine kinase activity was found in breast cancer cells
expressing the Arg388 allele16. This suggests that a different mechanism must be involved in the effect
of the Arg388 allele on decreased patient survival16, 53, and resistance to chemotherapy53. To identify
this mechanism, MDA-MB-231 cells, a breast cancer cell line lacking endogenous FGFR4, was
transfected with the Gly388 or Arg388 allele. With these cell lines, no difference in proliferation was
found. The Gly388-expressing cells however, were less invasive compared to the control and Arg388expressing cells70. This indicates that the Gly388 allele protects patients from tumor invasion which is
in accordance with a better prognosis16. For this protective effect, the kinase domain was essential70,
suggesting that downstream signaling plays an important role. Gene expression analysis revealed a
differential regulation of several genes including upregulation of the LPA receptor gene Edg-2 in
Arg388-expressing cells. Edg-2 induces cell movement by activation of PI3 and Akt upon binding of the
ligand LPA, explaining the increased cell motility in the cells expressing Arg388. The observed
upregulation of MMP-1 in the Arg388-expressing cells possibly contribute to an increase of the
invasion capacities of the cells as a result of increased degradation of the ECM. Finally, Pai-1 is
downregulated in cells expressing the Arg388 allele70. This gene has been shown to downregulate uPA
resulting in a decrease in migration and invasion. In addition, upregulation of uPA sensitizes the
Arg388-expressing cells to apoptosis induced by chemotherapy71. All of these (indirect) downstream
targets of the Arg388 allele possibly increases tumor invasion and decreases sensitivity to
chemotherapy, explaining the worse prognosis of Arg388 carriers and the prognostic value of this
allele for failure of adjuvant chemotherapy.
The decreased invasion capacities but unchanged proliferative capacity of Gly388 transfected breast
cancer cells is also observed in the WAP-TGFα breast cancer mouse model in which either the Arg385
or Gly385 allele (the mouse equivalents of the human 388 alleles) was knocked-in: the tumors of the
mice carrying the Arg385 allele were more invasive than in their Gly385 littermates due to increased
migration. This promotion of tumor invasion by Arg385 however, depends on the oncogenes that drive
tumor formation since no difference could be identified in a breast cancer mouse model having the
constitutive activation of Src as the trigger of malignant transformation. This observation is
supported by in vitro experiments testing the influence of the FGFR4-388-alleles in different
oncogenic backgrounds72. These differences in oncogenes can very well be an explanation for the
contradictory results about the influence of the Arg388 allele on patients’ prognosis (Chapter 2).
In addition to increased invasion, WAP-TGFα mice carrying the Arg385 allele also suffer from fast
forming metastases in the lungs and an increase of growth of these metastatic tumors, compared to
the metastatic tumors in Gly385-expressing WAP-TGF α mice. Based on RT-PCR, several genes that
could have a function in faster tumor progression and metastasis have been identified to be
differentially expressed in the Gly385 and Arg385-expressing mice72. For example, p21 is downregulated
in Arg385 tumor samples and since p21 acts as a tumor suppressor this could contribute to a more
aggressive tumor progression. One of the upregulated genes in Arg385 tumors is CDK1, which is
associated with migration. The expression of Flk-1, CD44, and MMP-13 and -14 is also increased in
Arg385 tumors. These genes are all involved in increased cell invasion72. The involvement of MMPs in
Arg388-mediated tumor progression is also found in breast cancer patients: FGFR4-Arg388 is strongly
co-expressed with MT1-MMP (membrane-type MMP)73. The Arg388 allele stabilizes the MT1MMP/FGFR4 complex, resulting in enhancement of both tyrosine kinase and proteolytic activity of
FGFR4 and the MMP respectively74.
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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Overall it can be said that for several genetic aberrations in FGFRs, the molecular mechanism
influencing the characteristics of the tumor has been (partly) unraveled. However, since there is not
much known about genetic aberrations in FGFR3 in breast cancer patients, no data on the
involvement of FGFR3 in breast cancer on a molecular level have been obtained yet. Several
experiments should still be performed in order to elucidate the exact role of FGFRs in breast cancer.
The results of these future experiments and the results of experiments described above lead to the
identification of possible drug targets. In addition, several model systems for each FGFR are
mentioned in this chapter. Next to their usefulness in finding out the changes in signal transduction,
these models can also be used to perform preclinical drug tests on. Chapter 4 will discuss several
ways of interfering with the abnormal FGFR signaling, and the advances made in drugs targeting this
abnormal signaling.
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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CHAPTER 4: DRUGS IN DEVELOPMENT AND POTENTIAL DRUG TARGETS
Based on the genetic aberrations in FGFRs identified in breast cancer patients and their
consequences on a molecular level, several approaches can be used to target FGFR signaling. For this
goal, three points of interference are possible: upstream, downstream, or the receptor itself. For
each of these points, two levels of interference can be discriminated being either on expression level
or on the functioning of the proteins. In vitro and in vivo results of drugs tested for each FGFR in
breast cancer are discussed in the following sections, which are split up according to their point of
interference.
Upstream intervention
Interference upstream of the receptor will have to be on the level of the ligand. As explained in
chapter 3, cell lines with FGFR1 amplifications and overexpression of FGFR1 have a low activity of
downstream signaling without the presence of a ligand. Addition of a ligand however, greatly induces
the downstream activation, making interference of ligand binding a useful approach to inhibit the
overactive FGFR1 signaling. One of the possible methods is by designing FGF ligand traps using the
extracellular part of any of the FGFRs linked to the Fc region of human IgG17. In this way, FP-1039 was
designed: a fusion protein existing of the extracellular domain of FGFR1 and the Fc region of IgG. FP1039 was shown to have antiangiogenic effects in vivo. Moreover, FP-1039 was able to block tumor
formation of human breast tumor xenografts, depending on their expression of FGFs and FGFRs75.
FP-1039 is currently being tested in a phase I clinical trial76. Furthermore, since the FGFRs have
several ligands in common, not only FGFR1 activation but also activation of FGFR3 and FGFR4 is
blocked by binding of FP-1039 to FGFs75, making it possible to also use FP-1039 for the treatment of
ligand-dependent overactive FGFR3 or -4 signaling.
A second method to interfere with the binding of the ligand is the use of peptide mimics. At present,
agonistic peptide mimics have been designed for FGFR1c and FGFR2b77. However, in order to inhibit
the overactive FGFR-signaling found in breast cancer, the design of antagonistic peptide mimics is
necessary. Upon binding of these antagonists to the receptor downstream activation of the receptor
will not take place, resulting in a diminished FGFR-signal. Just like the FGF ligand traps, these
antagonistic peptide mimics could be an effective drug in patients harboring FGFR1 amplifications.
Experiments in FGFR2-amplified cell lines have shown that downstream signaling in these cells takes
place independent of ligand-binding33, suggesting that interference on the level of the ligand will not
be an effective treatment for patients having FGFR2 amplifications. It is however not clear whether
this ligand-independent induction of downstream signaling is only a low level of activation, and
whether addition of a ligand strongly increases the downstream signal. If this would be the case,
peptide mimics of FGFs acting as antagonist or FGF ligand traps could be beneficial for patients
harboring FGFR2 amplifications. No experiments testing this hypothesis have been performed yet.
Finally, also for patients overexpressing FGFR4 and their concomitant poor response to
chemotherapy, the dependence on ligand binding to the receptor will have to be investigated before
designing and testing antagonistic peptide mimics and/or ligand traps.
FGF-receptor inhibition
To target the FGF-receptors themselves, several methods can be used. First of all, numerous tyrosine
kinase inhibitors (TKIs) targeting FGFRs have been developed. These small molecules compete with
ATP to bind to the receptor resulting in reduced activity of the kinase domain17, 67. However, since
the kinase domain of receptor tyrosine kinases are very similar, most TKIs are not specific for one
FGFR and also inhibit the activity of Vegfrs and/or Pdgfrs, making them less potent inhibitors of
FGFRs17. Brivanib alaninate is an example of a TKI targeting both FGFRs and VEGFRs. This drug is
currently being tested in clinical trials including liver and colon cancer patients. Its effect on breast
cancer has so far only been tested in vitro. It has been shown that cell lines having FGFR1
amplification and overexpression are more sensitive to the drug than non-amplified cell lines55,
FGFRs in breast cancer: expression, downstream
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Milou Tenhagen, Master Thesis, December 2010
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suggesting that this drug could be effective in patients harboring FGFR1 amplifications. However,
since brivanib alaninate is not specific for FGFR1, its effects on other receptor kinases and possible
side effects remain to be tested.
The downside of using TKIs that target multiple tyrosine kinase receptors is that it can lead to many
side effects, making it very important to develop FGFR-specific TKIs. So far only PD173074 and
SU5402 have been designed as FGFR-specific TKIs17. Both compounds have not been tested in clinical
trials of breast cancer patients yet17, and since these FGFR-inhibitors are not specific for one of the
FGF-receptors, side effects cannot be ruled out. SU5402 has been tested on several breast cancer cell
lines having different FGFR1 copy numbers. Interestingly, administration of SU5402 resulted in
decreased proliferation exclusively in cell lines having FGFR1 amplification and overexpression.
Therefore, SU5402 might be a promising drug for patients having FGFR1 amplifications and
overexpression31, 66. As stated in chapter 3, MDA-MB-134 cells depend on FGFR1 for their survival and
inhibition of FGFR1 by siRNA or SU5402 treatment reduces their proliferation66. A different MDA-MB134 cell line however was found to be insensitive for siRNA treatment. This was found to be the
result of a KRAS mutation which was absent in the first cell line31. This indicates that the genetic
background plays a role in the effect of FGFR1 inhibition. Since breast cancer tumors also have
different oncogenic backgrounds, the response to FGFR1 inhibition can differ between patients.
Further in vitro and in vivo studies will have to be performed in order to identify the subgroup of
patients within the patient group harboring FGFR1 amplification and overexpression to be sensitive
for SU5402, or for possible other drugs targeting FGFR1 or downstream targets.
The effect of PD173074 has been tested in triple negative cell lines. Administration of this TKI turned
out to be much more effective in cells harboring FGFR2 amplification and overexpression compared
to control cell lines33.Therefore, inhibition of FGFR2 signaling by TKIs might also be beneficial in the
treatment of triple negative breast cancer patients harboring FGFR2 amplifications and
overexpression. To elucidate if this is the case, several in vitro and in vivo experiments will have to be
performed using different TKIs. In addition, the effect of PD173074 could be tested in cell lines
having amplifications of other FGFRs.
In order to circumvent the side effects of unspecific TKIs, monoclonal antibodies can be a good
solution. For FGFR1-IIIc and FGFR3 an antibody has already been made, but these have not been
tested in any breast cancer model yet17. For FGFR4 an antibody has been developed (10F10) and
tested in cell lines naturally expressing FGFR4 at a high level. The administration of 10F10 strongly
increased their sensitivity to doxorubicin treatment69, suggesting that the use of this antibody could
also be beneficial for patients overexpressing FGFR4 in order to increase their response to
chemotherapy.
Another highly specific way to target the FGFRs is by RNA aptamers. These short RNA
oligonucleotides are selected for their high affinity and specificity to bind to the target proteins,
thereby acting as a synthetic antibody78. Currently, RNA aptamers that target the FGFR2 kinase
domain, the FRS2 interacting domain or the extracellular domain of FGFR2 are being developed.
Their effectiveness remains to be tested67.
The TKIs and (synthetic) antibodies described above are all aimed at the inhibition of the functioning
of the FGFRs. But also the expression of the receptors can be altered in several ways, for example by
the use of siRNA or miRNA. These therapeutic strategies are however still in a very early stage in
which off-target effects and the delivery of these molecules to the tumor are still under
investigation67.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
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Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
Downstream drug targets
To target downstream signal molecules of FGFRs it is very important to identify the key pathway
players that lead to the addiction of tumor cells to FGFR-signaling. For several genetic aberrations
found in FGFRs in patients the molecular implications are already extensively studied, but many
molecular mechanisms remain to be investigated. If these molecular implications are unraveled, the
ways to inhibit or enhance the expression or function of these proteins are almost unlimited, using
any of the techniques described above. It is however very important to bear in mind that many of the
proteins that are involved in aberrant FGFR signaling also play a role in other signaling pathways, and
in healthy cells. This makes it very difficult to target these proteins without having many side effects.
Targeting of downstream targets of FGFR1 as a monotherapy or as an additional therapy could be
very effective as indicated by several studies inhibiting downstream targets. One of these proposed
drug targets is RSK60. In iFGFR1 transfected MCF10A cells, inhibition of RSK by administration of CMK,
an irreversible small molecule inhibitor specific for RSK79, reduced the proliferation and re-induced
anoikis of cells in which iFGFR1 was activated by exposure to AP2018760. Administration of CMK also
reduced the proliferation MDA-MB-134 cells, a mouse lobular carcinoma cell line overexpressing
FGFR1, whereas administration of CMK to breast cancer cell lines lacking FGFR1 amplification did not
affect their proliferation. Overall, RSK seems to be a good drug target in patients having FGFR1
amplification and overexpression, especially since CMK only exerts its inhibitory effect on cells
overexpressing FGFR1, but not on wildtype cells. In addition to CMK, several other drugs are
available to target different domains of RSK60. Further in vivo studies will have to be performed to
analyze the effectiveness of these RSK-inhibitors and whether RSK-inhibitors can be used in all types
of breast cancers having FGFR1 amplification and overexpression.
MMPs could be another drug target downstream of FGFR1 signaling, since these have been shown to
play a role in invasion and blood vessel formation 56, 59. Inhibition of MMP-3 in the 3D iFGFR1 HC11
model by treatment with a pan MMP inhibitor (GM6001) resulted in decreased invasion of the
lesions which is mediated by the re-establishment of E-cadherin at cell-cell junctions. In addition,
expression of the mesenchymal marker SMA was reduced after GM6001 treatment, suggesting that
MMPs also play a role in EMT. Proliferation, apoptosis, and cell polarity however was not changed by
addition of the MMP inhibitor59. This indicates that inhibition of MMP activity in patients will not be
sufficient as a monotreatment.
For patients having FGFR2 amplifications several downstream molecules have been identified as
possible drug targets. Since FGFR2-amplified cell lines rely on FGFR2 signaling for their survival via
the PI3K pathway inhibiting apoptosis, several molecules in this pathway could be possible drug
targets. This idea is supported by a study in FGFR2-amplified cell lines which pointed out that dual
targeting of FGFR2 and downstream PI3-kinase was very effective and could be very useful in
targeting FGFR2-amplified tumors33.
In patients overexpressing FGFR4, one can consider targeting ERK, Bcl-xl and other players in this
anti-apoptotic pathway. Since these proteins have been shown to mediate the resistance to
chemotherapy in this group of patients, drugs targeting these proteins could increase the response
rate of this group of patients. No studies in this field have been performed so far.
As for patients harboring SNPs in the second intron of the FGFR2-gene, very little insight into the
contribution of these SNPs to tumor progression has been obtained. Since the expression levels of
FGFR2 are increased in patients carrying the disease-allele39, it would be reasonable that this leads to
increased downstream signaling. This still remains to be investigated, but if this would be the case,
several upstream, downstream and drugs targeting the receptor could be tested as therapeutic
strategies for this group of patients.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
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Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
Finally, several drug targets can be suggested to treat patients with the FGFR4-Arg388 allele. This
polymorphism does not result in increased tyrosine kinase activity16, making drugs interfering with
the ligand binding or the activity of the receptor unsuitable for treating FGFR4-Arg388 breast cancer
patients. Several downstream molecules have been identified to play a role in the increased
migration capacity of Arg388 expressing cells. For example, Edg-2 expression is increased in cells
transfected with the Arg388 allele, so inhibition of Edg-2 could be a possible way to inhibit the
migration of Arg388 cells. Indeed, transfection of cells carrying the Arg388 FGFR4 allele with siRNA
targeting Edg-2 significantly reduced their migration70, proving its function as a drug target. Since the
inhibition of cell movement in Gly388 cells by decreased expression levels of Edg-2 is mediated by
decreased PI3-kinase and Akt activity, PI3-K and Akt could also be possible drug targets. MMP-1
expression is also decreased in Gly388 expressing cells70 whereas MMP-13 and -14 are overexpressed
in Arg385 mouse tumors72, making inhibitors of MMPs a possible way to inhibit migration of Arg388
expressing cells. Finally, since Cdk-1, Flk-1, and CD44 are upregulated in Arg385 mouse tumors,
inhibition of either of these molecules could diminish the aggressive phenotype of these tumors.
Again, it has to be noted that these proteins are involved in many processes. Therefore, the use of
drugs targeting these proteins possibly results in many side effects. No studies regarding these drug
targets have been performed yet.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
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Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
CONCLUSION & DISCUSSION
In the past decade several research areas have contributed to our understanding of the role of FGFRs
in breast cancer. Depending on the type of receptor, different genetic aberrations have been
identified in subgroups of breast cancer patients. Mutations have been identified in the FGFR2 and -4
genes in breast cancer patients. Mutations in the other two FGFR genes have not been linked to
breast cancer. Mutations in FGFR3 are however present in other cancers and a similar mutation has
been identified in a Saethre-Chotzen patient having breast cancer. Whether these mutations also
have a role in breast cancer is to be investigated. The SNPs identified in intron 2 of FGFR2 have been
shown to be associated with an increased breast cancer risk in several studies. It is however not
exactly clear yet how these SNPs lead to this increased risk on a molecular level. Especially the role of
ER in the minor disease-predisposing allele remains to be investigated. Clarifying this and other
molecular implications of the FGFR2 allele will contribute to designing a possible drug for this group
of patients. For the Gly388Arg polymorphism identified in the FGFR4 gene, some contradicting
results regarding the involvement of the Arg388 allele in the prognosis and therapy resistance of
breast cancer patients are described. To clarify this, large-scale studies are needed in which the
clinical and pathological parameters are monitored closely.
Next to mutations in the FGFRs, also the amplification of these genes has been studied in breast
cancer patients. Many studies have been focusing on the amplification status of FGFR1 resulting in
different percentages of patients harboring FGFR1 amplification. This can be contributed to the large
variation of tumor samples, all having different clinical and pathological characteristics. For future
screens trying to identify copy number variations in any gene in (breast) cancer patients, several
important factors will have to be taken into account. First of all, the group of patients will have to be
large to prevent rare amplification events from being overlooked. In addition, it is important to
monitor the clinical and pathologic characteristics of the patients like disease progression,
treatments, tumor size, stage and grade, molecular subtype, hormonal status, and other
characteristics mentioned in the second chapter. All these factors are important in order to find out
associations with the amplifications or losses, but also to be able to discard confounding factors.
A second important parameter influencing the identification of copy number variations is the type of
technique that is applied. Depending on the goal of the study, genome-wide or single-gene analysis
of amplification and losses can be studied. Currently, several techniques are available to monitor the
copy number variation on a very high resolution, and in a quantitative manner. The importance of a
high resolution is illustrated by the amplification of the 8p11-12 region in which the FGFR1 region is
located. At first, FGFR1 was thought to be the target oncogene of this region, but amplification of this
region does not always lead to overexpression of FGFR1, suggesting that other oncogenes might be
involved28, 30. Analysis of the genome and expression at a higher resolution led to the identification of
four amplicon cores within the 8p11-12 region, with FGFR1 located in the second core80, proving that
low-resolution techniques are not ideal to study the amplification of a single gene. Next to the highresolution techniques that are available it is also possible to combine techniques having a low
resolution with expression data obtained by RNA-microarray analysis or qPCR. Using one of these
possibilities will not only enable the amplification and overexpression on the level of single genes,
but will hopefully also help to resolve the contradicting results about the association of FGFR1
amplification and overexpression with clinical and pathological parameters.
As described in chapter 3, the next step after identifying genetic aberrations in the patients is to find
out what the molecular mechanisms are by which these genetic aberrations contribute to the
development, and progression of breast cancer, or to resistance to therapy. For FGFR1 an extensive
model has been set up using an iFGFR1 construct that can be constitutively activated upon
administration of a drug. It has to be noted that this model of overactive FGFR1 signaling is not
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
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Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
exactly the same as endogenous overactivity of FGFR1: natural FGFR1 contains several regulatory
regions in the extracellular domain and the transmembrane domain, regulating the length, intensity
and type of the downstream signals. Since the iFGFR1 lacks these domains, not only the membrane
localization is altered, but the activation kinetics could also be altered56. It is however a useful
method to identify the molecular pathways involved in tumorigenesis induced by FGFR1, which
ultimately contributes to the identification of drugable proteins. For this reason, it would be a good
idea to also make iFGFR constructs for the other FGFRs, in order to find out whether these exert the
same effects on the mammary epithelium. Such a construct has already been made for FGFR2, and
has been transfected into prostate cells. Interestingly, in contrast to the induced iFGFR1 construct in
these cells, the activated iFGFR2 construct was not able to induce neoplasia in the prostate81. This
suggests that FGFR1 is more oncogenic than FGFR2. However, whether this is also the case in
mammary tissue and other organs remains to be investigated.
Not only the iFGFR1 construct, but also the other genetic aberrations identified in breast cancer
patients have been tested both in in vitro and in in vivo models. It is very important to realize that the
in vitro models are easy to manipulate, but these models also have a disadvantage: they are unable
to simulate the microenvironment including stromal cells, ECM, blood vessels, and the immune
response. All of the in vivo models described in chapter 3 have shown that all of these factors play a
major role in the development of breast cancer. The culture conditions of cell lines have shown to
also play a role in their response to drug administration: several FGFR1 amplified cell lines were
insensitive to siRNA treatment in a 2D culture, whereas a 3D culture of CAL120 cells did show
dependence on FGFR-activity to avoid anoikis31. This highlights that it is always important to check
not only the molecular effects of a genetic aberration but also the efficacy of a drug in an in vivo
model.
As already pointed out in chapter 3, the in vivo models are still not an exact representation of the
situation in human breast cancer patients. Not only because mouse tissue is different from human
tissue, but also because human breast cancer tissue harbors multiple oncogenic mutations which can
interact with the altered FGFR signaling. Therefore, it is very useful to cross the mice harboring the
genetic aberrations in one of the FGFRs with other breast cancer mouse models harboring mutations
in oncogenes that frequently occur in breast cancer patients. Finally, this thesis has only focused on
genetic aberrations in one single FGFR. It might also be possible that breast cancer patients harbor
genetic aberrations in multiple FGFR genes. If this is the case, these multiple aberrations can also be
introduced in in vitro and in vivo models in order to find out whether there exists an interaction or a
combinatorial effect of multiple genetic aberrations in FGFRs.
The ultimate goal of identifying genetic aberrations in FGFRs in patients, and of identifying their
molecular implications is of course to identify drug targets. For the development of future drugs,
several points will have to be taken into account. First of all it is important to know that these
targeted drugs are never to be used as a monotreatment, but always in combination with surgery,
radiation, or chemotherapy in order to destroy the malignant cells. Second of all, the targeted
therapies will have to act very specifically in order to constrain the side effects. As can be read in
chapter 1, FGFR signaling also plays an important role in healthy tissue, which emphasizes the
importance of preferably only targeting the cancer cells and not healthy cells. Furthermore, as can be
seen for TKIs, it is difficult to design drugs that target only one specific protein. The use of
monoclonal antibodies might therefore be a better alternative. Unfortunately however, as could be
seen for Herceptin, a monoclonal antibody targeting Her2, these highly specific drugs often result in
the development of drug resistance. The reasons for this resistance are very diverse: there can be
mutations present downstream of the Her2 receptor, there is an increase in signaling of other
receptors that have the same downstream targets, or mutations can be present in the extracellular
domain of Her2 resulting in a less stable interaction with the drug15. These ways to circumvent the
action of a drug can potentially also take place in patients having genetic aberrations in FGFRs. For
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
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Milou Tenhagen, Master Thesis, December 2010
3158136, M.Tenhagen@students.uu.nl
the first problem, downstream mutations, it is important to also design drugs for proteins
downstream of these mutations. Secondly, in order to prevent other receptors from taking over, it
has been suggested that it is better to design drugs that target multiple RTKs at the same time, in
order to inhibit multiple pathways82. Also the design of drugs targeting proteins that are downstream
targets of both receptors could be a possible way to prevent relapse. For the final problem,
mutations in the protein that is drugged preventing the drug from binding, again drugs targeting
downstream molecules can be a solution.
Finally, it is very important to identify the subgroup of breast cancer patients that will respond to this
therapy. In order to do this, it is important that the genetic aberrations of the genes targeted by the
drugs are identified in a quick, easy, and cheap way. If this is done in a proper way and the targeted
drugs are combined with conventional therapies, this personalized treatment will evolve to be the
standard treatment of breast cancer patients.
FGFRs in breast cancer: expression, downstream
effects, and possible drug targets
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3158136, M.Tenhagen@students.uu.nl
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