Clinical activities of the epidermal growth factor receptor family inhibitors in breast cancer.

Abstract

The epidermal growth factor (EGF) receptors play an important role in epithelial cell function. Upon stimulation of these receptors, an extensive network of signal transduction pathways is activated, including the PI3K/AKT and Ras/Erk pathways. This activation leads to cellular proliferation and survival. In breast cancer, the EGF receptor, ErbB2 (HER2/neu), can be amplified and over-expressed and this is associated with poor prognosis and drug resistance. Trastuzumab is a monoclonal antibody against ErbB2 and has demonstrated activity in the therapy of breast cancer patients with over-expression of ErbB2, both in the metastatic and adjuvant setting. Recently, a tyrosine kinase inhibitor, lapatinib, that targets both ErbB1 and ErbB2, has also shown activity in metastatic breast cancer. In this review, we will discuss the ErbB receptors and their signaling networks in breast cancer, as well as the clinical activities of trastuzumab and lapatinib in this disease.

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© 2007 Dove Medical Press Limited. All rights reserved
Biologics: Targets & Therapy 2007:1(3) 229–239
229
R E V I E W
Clinical activities of the epidermal growth factor
receptor family inhibitors in breast cancer
Elizabeth S Henson1
James B Johnston1,2
Marek Los1
Spencer B Gibson1
1Manitoba Institute of Cell Biology,
CancerCare Manitoba, University of
Manitoba, Winnipeg, MB, Canada
2 Department of Internal Medicine,
University of Manitoba, Winnipeg,
Manitoba, Canada
Correspondence: Spencer B Gibson
Manitoba Institute of Cell Biology,
CancerCare Mantioba, 675 McDermot
Ave. Winnipeg, MB R3E 0V9, Canada
Email gibsonsb@cc.umanitoba.ca
Abstract: The epidermal growth factor (EGF) receptors play an important role in epithelial
cell function. Upon stimulation of these receptors, an extensive network of signal transduction
pathways is activated, including the PI3K/AKT and Ras/Erk pathways. This activation leads
to cellular proliferation and survival. In breast cancer, the EGF receptor, ErbB2 (HER2/neu),
can be amplifi ed and over-expressed and this is associated with poor prognosis and drug resis-
tance. Trastuzumab is a monoclonal antibody against ErbB2 and has demonstrated activity in
the therapy of breast cancer patients with over-expression of ErbB2, both in the metastatic and
adjuvant setting. Recently, a tyrosine kinase inhibitor, lapatinib, that targets both ErbB1 and
ErbB2, has also shown activity in metastatic breast cancer. In this review, we will discuss the
ErbB receptors and their signaling networks in breast cancer, as well as the clinical activities
of trastuzumab and lapatinib in this disease.
Keywords: trastuzumab, lapatinib, ErbB receptors, breast cancer and tyrosine kinases
Introduction
Some of the most important signaling molecules that maintain cell survival and proliferation
are the growth factor receptors. Epidermal growth factor (EGF) receptors are key regulatory
factors in promoting both cell survival and proliferation in epithelial cells. EGF receptors
(ErbB receptor family) transduces specifi c cellular signals to the cell leading to specifi c
cellular responses through their intracellular tyrosine kinase domain (Burgess et al 2003;
Herbst 2004). In breast cancer, these ErbB receptors are up-regulated, their tyrosine kinase
activity is increased and their signaling pathways are constitutively activated making inhib-
iting ErbB receptor tyrosine kinase activity an attractive target in this disease (Hynes and
Lane 2005). The development of antagonists to one of these ErbB receptors, ErbB2, is an
example of how molecular-targeted therapy is utilized in breast cancer therapy (Nahta and
Esteva 2007). In this review, we will discuss how ErbB receptors are activated and how using
ErbB receptor inhibitors are being developed as effective treatments for breast cancer.
EGF family of receptors (Figure 1)
The EGF receptor (ErbB) family consists of four closely related tyrosine kinase trans-
membrane receptors: ErbB1 (EGFR/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and
ErbB4 (HER4) that bind to an array of ligands (Normanno et al 2005). These ligands can
be classifi ed in two categories, ligands that predominantly bind to only ErbB1 and ligands
that bind to ErbB3 and/or ErbB4. EGF binds to only ErbB1 whereas other ligands such as
neuregulin bind to ErbB3 and ErbB4. Upon binding to their corresponding ligands, the ErbB
receptors form homo- or hetero-dimers. Upon EGF ligation with ErbB1, the receptor either
homo-dimerizes or hetero-dimerizes with the other three ErbB receptors. ErbB2 fails to bind
to ErbB receptor ligands, indicating ErbB2 is a coreceptor contributing to the activation of
the other ErbB receptors through dimerization. ErbB3 has an inactive kinase domain which
indicates that is also acts as a co-receptor for activation of other ErbB receptor members

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Henson et al
(Normanno et al 2005). After dimerization, the tyrosine kinase
domain in the receptors phosphorylates tyrosine residues in
the neighboring receptor. This allows recruitment of adaptor
proteins, such as Grb2, to receptors and the initiation of signal
transduction pathways. The receptors are then often endocytozed
and bind to an adaptor protein, c-cbl, that targets ErbB receptors
for degradation (Muthuswamy et al 1999). Other receptors are
recycled back to the plasma membrane where they are available
to bind to other ligands (Burgess et al 2003).
ErbB receptors are expressed in a variety of human tissues
of epithelial, mesenchymal and neuronal origin. The biologi-
cal function of these receptors has been demonstrated in a
number of different mouse models. Mice lacking ErbB1 have
abnormal eyes and epidermal tissues and die due to defects
in epithelial organ development. In mice lacking ErbB2, a
malformation of the heart is found and this contributes to
death of the mice at midgestation. ErbB3 knockout mice die
from defects in the heart and neural crest, and lack Schwann
cell precursors (Olayioye et al 2000).
ErbB receptor signaling pathways
(Figure 2)
Upon ligation, ErbB receptors are activated. The activated
receptor kinase phosphorylates tyrosine residues on the
C-terminal tail of the ErbB receptors (Muthuswamy et al
1999; Belsches-Jablonski et al 2001). These tyrosine phos-
phorylated sites allow the binding of proteins containing the
Src Homology 2 (SH2) domain. These proteins consist of
intracellular docking proteins or adaptor proteins, such as
Grb2 and Shc. Upon binding to the ErbB receptors, these
protein associate with other proteins leading to the activation
of serine threonine kinases that phosphorylate serine or threo-
nine residues on other protein kinases and/or transcription
factors (Olayioye et al 2000). This kinase cascade leads to
amplifi cation of a network of signaling pathways resulting in
changes in protein functions and activation of gene transcrip-
tion. Two of the most prominent signaling pathways found
after ErbB receptor activation are the Ras/Erk and the PI3K/
AKT pathways. Mitogen activated protein kinases (MAPKs)
are a superfamily of protein serine-threonine kinases in
which Erk1/2 are members. ErbB receptors activate Erks
through the binding of adaptor protein Grb2 to ErbB recep-
tor recruiting son of sevenless (SOS) protein to the receptor
(Wu et al 1993). SOS is a guanyl nucleotide-release protein
(GNRP) that upon recruitment to the plasma membrane by
the activated cell surface receptor causes the small G protein
RAS to release GDP and exchange it for GTP. When Ras
has GTP bound to it, it becomes active. Activation of Ras
ErbB1/ErbB2
ErbB2/ErbB2
ErbB1/ErbB3
ErbB1/ErbB4
ErbB2/ErbB4
ErbB2/ErbB3
ErbB1/ErbB1
ErbB3/ErbB4
ErbB4/ErbB4
Homo-dimer
Hetero-dimer
Figure 1 ErbB receptors form homo- and hetero-dimers upon activation. The ErbB receptor family consists of ErbB1 (EGFR), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4
(HER4). These receptors form homo- or hetero-dimers upon activation. The only exception is ErbB3 homo-dimer since ErbB3 has a non-functional tyrosine kinase domain.
ErbB2 homo-dimers occur independent of ligand whereas the other dimers depend on binding of ligands for activation.

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ErbB receptor inhibitors in breast cancer
leads to the activation of the MKKK Raf-1, Raf-1 kinase then
phosphorylates and activates the MKK Mek1/2. Activated
Mek1/2 then phosphorylates and activates Erk1/2 (Johnson
et al 2005). This increased expression results in phosphoryla-
tion of a variety of substrates including 90 kDa ribosomal S6
protein kinase (Rsk), Msk1, cytosolic phospholipase A2, and
transcription factors c-Myc, NF-IL6, Tal-1, Ets-2, and Elk
(Widmann et al 1999). As a result, there is enhanced gene
transcription with increased cell survival and proliferation
(Lin et al 2002).
ErbB receptors also activate the PI3K/AKT pathway
through recruitment of PI3K to the plasma membrane and
this is mediated by binding of its SH2 domain to tyrosine
phosphorylated proteins. PI3K activation catalyzes the trans-
fer of a phosphate group from ATP to phosphatidylinositol
generating a 3'-phosphatidylinositol phosphate (PIP). PIPs
act as binding sites for proteins with pleckstrin homology
(PH) domains. PI3K signaling is negatively regulated by
PTEN that dephosphorylates PIPs. AKT is a serine/threonine
kinase that contains a PH domain. AKT’s PH domain binds to
phosphatidylinositols generated by PI3K translocating AKT
to the plasma membrane where it becomes phosphorylated
and activated (Song et al 2005). Activated AKT can either
activate or inhibit a number of downstream targets that
participate in cellular survival. AKT activates transcription
factor NFκB, HIF-1α and CREB resulting in increased tran-
scription of anti-apoptotic genes such as growth factors and
Bcl-2 family members that prevent cell death. In contrast,
AKT inactivates transcription factors Foxo (forkhead family)
and p53, either directly phosphorylating Foxo proteins or by
phosphorylating and activating Mdm2, a negative regulator
of p53 (Brunet et al 1999; Zhou et al 2001). In both cases,
pro-apoptotic gene expression is decreased and cell sur-
vival is increased. AKT also phosphorylates and activates
mTOR, a serine/threonine kinase. mTOR activation lead to
phosphorylation and activation of ribosomal S6 kinase and
the eukaryotic initiation factor 4E (eIF4E) – binding protein
1 (4E-BP1). This increases the translational capacity within
Bcl-2
family
EGF
Grb2
Raf-1
TF
PI3K
mTOR
CREB
HIF-1α
Src
MEK1/2
RAS
ErbB
receptors
AKT
Erk1/2
SOS
IkB
P
BAD
IAPs
Rsk
TF
Casp. 9
BAD
NFkB
IkB
IKK
ErbB2
NFkB
FOXO
PTEN
Survival genes
Figure 2 ErbB receptor signaling. EGF binds to its ErbB receptors leading to activation of a network of signaling pathways, including the PI3K/AKT and RAS/ERK pathways.
These pathways lead to the activation of anti-apoptotic proteins (white) and the inactivation of pro-apoptotic proteins (black). ErbB2 homodimers activate the same
pathways but do not require EGF for activation and ErbB receptors can also be activated through tyrosine phosphorylation of the ErbB receptors mediated by non-tyrosine
kinase Src.

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Biologics: Targets & Therapy 2007:1(3)
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Henson et al
cells and has been implicated in cell survival (Chan 2004;
Sun et al 2005).
ErbB receptor family and breast
cancer
ErbB receptor family members were fi rst implicated in cancer
in the early 1980s when the avian erythroblastosis tumor virus
was found to encode an aberrant form of the receptor ErbB1
(Herbst 2004). Since then, activation of ErbB receptor family
members has been implicated in the pathogenesis of gliomas
and lung, head and neck and renal carcinomas. However, it
is the activation of ErbB receptors in breast cancer that has
been the focus of the most research and clinical investiga-
tion (Janmaat and Giaccone 2003). In breast cancer, ErbB2
expression is increased in approximately 25% of tumors,
as a result of gene amplifi cation (Normanno et al 2005).
Since ErbB2 does not require a ligand to become activated,
increased expression in breast cancer causes increased ErbB2
signaling through homo- and hetero-dimerization and this
leads to to increased resistance to cell death (apoptosis),
increased cell proliferation and enhanced invasiveness.
Breast cancer patients with with high ErbB2 expression
generally have aggressive disease and poor prognosis (Nor-
manno et al 2005). In addition, ErbB1 expression is elevated
in about 30% of breast tumors and is often complexed with
ErbB2. The ErbB1 gene is rarely mutated in breast cancer but
in lung cancer and glioblastoma multiforme, the intracellular
domain of ErbB1 is mutated rendering the tyrosine kinase
constitutively active (Pedersen et al 2001). These patients
generally have aggressive disease. The microenvironment
in breast cancer can also infl uence ErbB receptor signaling
as the stromal cells may secrete EGF which then leads to
activation of the receptors (Normanno et al 2005).
Activation of the Ras/Erk signaling pathway by ErbB is
deregulated by mutations in Ras that increase Erk signaling
(Rajagopalan et al 2002; Duursma and Agami 2003). Many
breast cancers also contain mutations in the tumor suppres-
sor PTEN, that dephosphoryates the second messenger lipid,
PtdIns (3,4,5) triphosphate, causing down-regulation of the
PI3K/AKT signaling pathway. In approximately 25% of
breast tumors, PTEN is mutated and this inhibits its lipid
phosphatase activity which increases EGF-mediated AKT
activation and cell survival (Sansal and Sellers 2004). In
addition, transcription factors are often altered in breast
cancer. NFκB and CREB transcriptional activation are
often increased whereas tumor suppressor p53 is mutated in
many breast cancers (Dolcet et al 2005; Shankar et al 2005;
Yu and Zhang 2005). As a result, there is an alteration in
the expression of the pro- and anti-apoptotic proteins. For
example, anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL
and Mcl-1 levels are often elevated in breast cancer and this
is associated with drug resistance (Kumar et al 1996; Stoll
et al 1998; Wang et al 1999; Henson et al 2003; Yu and Zhang
2005). The multi-faceted alterations in the ErbB receptor
mediated signaling pathways in breast cancer ensure that the
cells can survive in different environments and contribute
to tumor progression. As a result, these receptors, especially
ErbB2, are attractive targets for cancer therapy.
ErbB receptor inhibitors in breast
cancer therapy (Table 1)
There are two classes of ErbB receptor inhibitors. The
fi rst class of inhibitors are monoclonal antibodies which
are directed against the ErbB receptors and these inhibit
ErbB signaling and may induce immune mediated cell
killing. These monoclonals target the over-expression of
ErbB in breast cancer and the most effective antibody to
date is trastuzumab (Dassonville et al 2007; Nahta and Esteva
2007). The second class of inhibitors are tyrosine kinase
inhibitors that inhibit the tyrosine kinase activity of ErbB
receptors and thereby inhibit ErbB receptor signaling. The
most promising tyrosine kinase inhibitor in breast cancer
treatment is lapatinib that blocks the activation of ErbB1 and
ErbB2. Lapatinib has similar effects to trastuzumab and may
be particularly effective when combined with trastuzumab
(Moy and Goss 2006). The clinical responses for transtu-
zumab and lapatinib in breast cancer are discussed below.
Trastuzumab in breast cancer
Trastuzumab is the first monoclonal antibody targeting
ErbB2 that has been approved for clinical use. This antibody
binds to ErbB2, causing receptor endocytosis and/or abla-
tion of ErbB2 signaling, which include the activation of the
Ras/Erk and PI3K/AKT pathways (Johnston et al 2006). In
addition, trastuzumab contains a human IgG1Fc region that
activates antibody-dependent cellular cytotoxicity (ADCC)
when it binds to ErbB2 (Hynes and Lane 2005). The initial
clinical trials with trastuzumab in metastatic breast cancer
over-expressing ErbB2 showed response rates ranging from
12 to 34% (Baselga et al 1996; Cobleigh et al 1999; Vogel
et al 2002). Subsequent studies combined trastuzumab with
paclitaxel or docetaxel in metastatic breast cancer. Response
rates ranged from 50% to 72%, with the time to progression
ranging from 7.4 to 9 months, and overall survival from 25
to 31 months (Seidman et al 2001; Slamon et al 2001; Esteva
et al 2002). These results were signifi cantly higher than

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ErbB receptor inhibitors in breast cancer
Table 1 Clinical trials for trastuzumab and lapatinib
Treatment
Phase
Patient
Combination
Tumor type
Dose
Complete
Partial
Stable
Time to
Median
Ref.


number



response
response
disease
progression
survival
Trastuzumab
II
213
Single agent
ErbB2
Loading dose:
4%
12%

9.1 months
13 months
Cobleigh1999




overexpressing
4 mg/kg with weekly









metastatic breast
administration of









cancer with
2-mg/kg









progressive disease







III
64
Chemotherapy
ErbB2 positive,
Paclitaxel 225 mg/m2,
P+FEC



P+FEC
Buzdar 2007



alone (n = 19) or
non-infl ammatory
fl uorouracil,
54.5%



85.3%



in combination
breast cancer.
500 mg/m2,








(n = 45) with

cyclophosphamide
P+FEC+H



P+FEC+H



trastuzumab (3)

500 mg/m2, and
60%



100%





75 mg/m2 epirubicin.




36.1





trastuzumab loading




months





dose: 4 mg/kg with










weekly










administration of










2 mg/kg





Lapatinib
I
67
Single agent
Heavily
Dose range of 500,

10%
10%


Burris 2005(1)


(30)

pretreated ErbB1
650, 900, 1200 and









and/or ErbB2
1600 mg of lapatinib









positive










metastatic cancer







II
39
Single agent
ErbB2 positive
750 mg lapatinib

5% had
20% had
3.02 months
6.57
Lin 2006




breast cancer
twice daily

CNS
stable

months




with new or


partial
disease






progressing


response,
in CNS






brain metastases


25% had
at 16









non-CNS
weeks









partial










response




III
324
Capecitabine
ErbB2 positive
1250 mg lapatinib
Capecitabine
C = 14%
C = 18%
C = 4.4

Geyer 2006



alone (n = 161) or
breast cancer that
daily with
alone (C) 0%


months




in combination
progressed after
capecitabine 2000

CL = 22%
CL = 27%





with Labatinib
treatment (2).
mg/m2 two divided
Capecitabine


CL = 8.4




(n = 163)

doses days 1–14 of a
and lapatinib


months






21-day cycle. Single
(C+L) ?1%









dose capecitabine










administered at










2500 mg/m2 two










divided doses days










1–14 of a 21-day










cycle.





(Continued)