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Optimizing Outcomes in HER2-Positive Breast Cancer: The Molecular Rationale

Optimizing Outcomes in HER2-Positive Breast Cancer: The Molecular Rationale

ABSTRACT: The epidermal growth factor (EGF) receptor HER2 is a transmembrane receptor tyrosine kinase that plays a crucial role in the regulation of cell proliferation and survival. The overexpression of HER2 correlates strongly with prognosis in breast cancer. The targeted blockade of HER2 activity with monoclonal antibodies (eg, trastuzumab [Herceptin]) and small-molecule tyrosine kinase inhibitors (eg, lapatinib) results in the inhibition of tumor growth in HER2-positive cancers. Anti-HER2 therapies have also shown efficacy in combination with chemotherapy in clinical trials in patients with HER2- positive breast cancer. Their efficacy may, however, be limited by molecular mechanisms that compensate for HER2 suppression (eg, activity of EGF receptor) or mechanisms of resistance (eg, loss of PTEN). HER2 continues, however, to be overexpressed by the cancer cells, and the continued suppression of HER2 may be required for maximum antitumor effect. It should be noted that in the absence of definitive data from randomized trials showing an absence or presence of benefit, the use of anti-HER2 agents such as trastuzumab in multiple sequential regimens has become the standard of care. Combining HER2 blockers with agents that overcome the compensatory or resistance mechanisms may increase the efficacy of anti-HER2 therapies. In addition, anti-HER2 therapies can have synergy with common chemotherapy regimens and remain effective through multiple lines of therapy. Optimizing the use of therapies that target HER2 signaling will lead to further advances in the treatment of breast cancer.

The epidermal growth factor
(EGF) receptor family of tyrosine
kinases regulates a complex
signaling cascade that controls
the proliferation, survival, adhesion,
migration, and differentiation of
cells.[1,2] The dysregulation of EGF
receptor signaling by mechanisms
such as receptor or ligand overexpression
and constitutive activation of
receptors can lead to greater cell proliferation
and other tumor-promoting activities.[
3-5] This pathway is therefore
tightly regulated in normal cells.

The EGF receptor family consists
of four distinct receptors: EGFR
(ErbB-1), HER2 (HER2/neu, ErbB-
2), HER3 (ErbB-3), and HER4 (ErbB-
4),[2] and their abnormal activation is
associated with human cancers of various
origins.[6,7] The HER2 gene was
cloned in the early 1980s by investigators
at Massachusetts Institute of
Technology[8,9] and the National Institutes
of Health[10] and in Japan.[11]
Overexpression of HER2, which has
been reported in approximately a third
of breast cancers, was found to correlate
with tumor resistance to chemotherapy
and poor prognosis.[12-15]
A meta-analysis that related the expression
of HER2 with outcome found
that HER2 gene amplification or overexpression
of HER2 protein predicted
outcome in breast cancer on either
univariate or multivariate analysis in
92% of the studies evaluated.[16]

Three cellular mechanisms underlie
the poor prognosis in patients with
HER2-overexpressing tumors.[16-18]
First, HER2 overexpression increases
the metastatic properties of cancer
cells, such as invasion, angiogenesis,
and greater survival. Second, HER2
overexpression also confers greater
resistance to therapeutic agents (eg,
chemotherapy and hormone therapy),
which can result in a poor response to
treatment. This may correlate with the
absence of steroid hormone receptors
on HER2-positive cells. Third, HER2
overexpression confers a strong proliferative
advantage to tumor cells that
are characterized by a high percentage
of S-phase cells. In addition,
HER2 overexpression has been correlated
with larger tumor size and aneuploidy.[
16,18]

This article discusses the molecular
mechanisms of the oncogenicity
of HER2 overexpression, reviews approaches
that use molecular targeting
for treating HER2-positive cancers,
and describes strategies for optimizing
outcomes by using agents that target
HER2.

Molecular Mechanism:
Oncogenicity of HER2
Overexpression

All members of the EGF receptor
family share a similar structure: they
consist of an extracellular ligand-binding
domain, a single membrane-spanning
region, and an intracellular
domain with tyrosine kinase activity.
On ligand binding to the extracellular
domain, EGF receptors form
heterodimers or homodimers, resulting
in the activation of intracellular
tyrosine kinase and autophosphorylation
of specific tyrosine residues.[
2,19] The phosphotyrosine
residues in turn recruit adaptor proteins
or enzymes, which initiate signaling
cascades to produce a
physiologic outcome.[2] Receptor signaling
is terminated primarily by endocytosis
of the receptor-ligand
complex, followed by its recycling to
the cell surface or degradation.

In normal cells, HER2 does not
bind to any known ligand with high
affinity, but can signal only by recruiting
another activated EGF
receptor.[20] The subsequent transactivation
and autophosphorylation of
HER2 generates intracellular signals
that are significantly stronger and of
substantially longer duration than signals
that emanate from other receptor
pairs.[21] There are several molecular
reasons for the strength of the signal
generated by HER2-containing
heterodimers (Table 1).

First, HER2 is the preferred heterodimerization
partner of all other EGF
receptors in normal cells, as well as
tumor cells.[22] Overexpression of
HER2 in tumor cells may further drive
its dimerization by increasing its availability
to pair with ligand-activated
receptors.[23]

HER2 does not bind to any known
ligand with high affinity, but on dimerization
it increases the affinity of
ligands to their receptors by decreasing
the rate at which ligands dissociate
from the active dimers. In addition,
HER2 makes its dimerization partner
more promiscuous, allowing it to bind
to a broader spectrum of EGF-like
ligands.[24] As a result, HER2-
containing heterodimers can respond
to more ligands with a prolonged and
stronger signal.

Cancers do not necessarily result
from an increased rate of cell proliferation;
a disruption of the balance
between cell division and cell survival
is also a crucial factor. Another
basis for the oncogenicity of HER2
stems from the potent activation of
both the cell-proliferative Ras-MAPK
pathway and the cell-survival pathway
that is mediated by PI3K/Akt
(Figure 1). Because it does not bind
any ligand, HER2 cannot signal directly
through these pathways, but it
can gain control of the pathways by
dimerizing with EGFR and HER3.

Epidermal growth factor receptor
signaling is terminated by the internalization
of cell surface receptors,
followed by their degradation. HER2
evades this process of signal attenuation
by two mechanisms, resulting in
its prolonged signaling.[25] Heterodimers
that contain HER2 are internalized
more slowly than other
heterodimers, resulting in impaired
signal attenuation. In addition, HER2-
containing heterodimers are not targeted
to a degradative pathway;
instead, they are recycled to the cell
surface. By defective internalization
and greater recycling, HER2 heterodimers
remain at the cell surface
longer, increasing the strength and
duration of the intracellular signal.

It should be noted that the overexpression
of HER2 has been associated
with its homodimerization and
ligand-independent activation (Figure
1).[26-28] Signaling through the
MAPK pathway is also significantly
enhanced and prolonged in cells that
overexpress HER2 when compared
with cells that express low levels.[24]
This constitutive activity may play a
crucial role in the transformation and
proliferation of HER2-positive breast
cancer cells.

Targeting HER2
in Breast Cancer

Because the overexpression of
HER2 correlates with the pathogenesis
of and prognosis in breast cancer,
it is an important therapeutic target.
Anti-HER2 therapies (Table 2) reduce
the proliferation and survival of tumors
that overexpress HER2.

Immunologic Therapies
Trastuzumab (Herceptin), a recombinant
humanized monoclonal antibody
to the extracellular domain of
HER2, is the only anti-HER2 agent
that has been approved by the US
Food and Drug Administration to treat
patients with metastatic breast cancer
whose tumors overexpress HER2.
Trastuzumab has been shown to have
both cytostatic and cytotoxic effects
in vitro (Table 3).

Trastuzumab disrupts receptor
signaling through the downstream
proapoptotic PI3K/Akt cell-survival
pathway (Figure 2).[29-31] Trastuzumab
has also been shown to activate
the phosphatase activity of the
tumor suppressor PTEN, which reverses
the activation of PI3K and
Akt.[32] Early studies have shown that
trastuzumab induces HER2 internalization
and degradation in HER2-
overexpressing cells[29,33]; however,
recent findings suggest that this may
not be true.[34]

Cells treated with trastuzumab also
undergo growth arrest in the G1 phase,
accompanied by the induction of the
cyclin-dependent kinase inhibitor
p27.[33,35,36] Trastuzumab has been
shown to suppress angiogenesis in
vivo by inducing antiangiogenic factors,
such as thrombospondin 1, and
suppressing proangiogenic factors,
such as vascular endothelial growth
factor, transforming growth factor,
angiopoietin 1, and plasminogen
activator inhibitor 1.[37,38] In addition,
trastuzumab can block the process
of metalloproteinase-mediated
HER2 ectodomain shedding, which
has been shown to cause constitutive
HER2 signaling.[39]

By virtue of being an antibody, trastuzumab
can harness immune-mediated
responses to cause tumor cell
toxicity. For example, trastuzumab
has been shown to initiate antibodydependent
cell-mediated cytotoxicity
(ADCC).[40]

Trastuzumab has also been shown
to potentiate the effects of chemotherapy
by multiple mechanisms of
action in vitro, as well as in vivo.[41]
Pietras and colleagues have shown
that treatment with trastuzumab can
prevent DNA repair after treatment
with DNA-damaging agents.[42] Another
molecular explanation for this
synergy may be the suppression of
the Akt-mediated survival pathway;
trastuzumab can therefore induce apoptosis.
In fact, a recent trial of trastuzumab
in the neoadjuvant setting in
primary breast cancers showed that
trastuzumab induced apoptosis, confirming
that the antibody exerts a cytotoxic
effect in vivo.[43]

Pertuzumab, another humanized
monoclonal antibody to HER2, is currently
in phase III trials in patients
with breast cancer. In contrast to trastuzumab,
pertuzumab binds HER2
near the center of the dimerization
arm[44] and can prevent the formation
of ligand-induced HER2-containing
dimers.[45] As a dimerization inhibitor,
pertuzumab diminishes ligandactivated
HER2 signaling, including
HER2 phosphorylation and activation
of MAPK and Akt.[45,46] Pertuzumab
would also be expected to recruit effector
cells such as macrophages and
monocytes to the tumor through the
binding of the antibody constant Fc
domain to specific receptors on those
immune cells.

Other antibody-based strategies to
attenuate HER2 signaling are in various
stages of development. These
strategies involve the use of intracellular
single-chain Fv antibody fragments
as well as armed antibodies.
Examples of the latter are toxinlabeled antibodies to HER2[47,48]
and antibodies to HER2 labeled with
radionuclides such as yttrium-90 and
iodine-131.[49,50]

Small-Molecule Tyrosine
Kinase Inhibitors

The inhibition of tyrosine kinase
activity is another strategy for targeting
EGF receptor pathways in the treatment
of cancer. Small-molecule
tyrosine kinase inhibitors (TKIs) have
a range of activity, with some specific
for a single receptor kinase and others
equally active against several receptors.[
51] Tyrosine kinase inhibitors that
are specific for EGFR, eg, gefitinib
(Iressa) and erlotinib (Tarceva), have
shown only limited efficacy as monotherapies
for breast cancer in the preclinical
and clinical settings, suggesting
that EGFR does not drive tumor
growth.[52-54]

Dual-kinase inhibitors are a new
generation of TKIs that can block signal
transduction through both EGFR
and HER2 (Table 4). These TKIs inhibit
the growth and survival of tumor
cells by reducing both MAPK
and PI3K signaling (Figure 2).[55,56]
Lapatinib, a member of this class of
TKIs, has shown promising activity
in preclinical and early clinical investigations.
In clinical trials lapatinib
induced apoptosis and caused growth
arrest of tumors that overexpressed
HER2 or EGFR.[57] Lapatinib is a reversible
inhibitor, and EKB-569 is another
dual-kinase inhibitor that
irreversibly inhibits the kinase activity
of EGFR and HER2; however, the clinical
significance of irreversible inhibition
has not yet been determined.[51]

The potential advantages of dualkinase
inhibitors are that they inhibit
both ligand-dependent and ligand-independent
signaling, they can potentially
overcome resistance to
trastuzumab in tumors that develop
compensatory mechanisms, and they
appear to have synergy with chemotherapy.

Heat Shock Protein 90 Inhibitors
Another class of small-molecule
inhibitors influences EGF signaling
by increasing receptor degradation.
These inhibitors (eg, geldanamycin)
block heat shock protein 90 (HSP90),
a chaperone protein that is crucial in
maintaining EGF receptors in a signaling-
competent form.[58,59] As a
consequence, HSP90 inhibitors prevent
the stabilization of EGF receptors
at the membrane and target these
receptors for degradation.[60,61]

Geldanamycin, in particular, binds
to members of the HSP90 family,
blocking the assembly of HSP90 heterocomplexes
and destabilizing existing
heterocomplexes.[62] As a
result, geldanamycin downregulates
surface HER2 through greater degradative
sorting in endosomes.[34]
Treatment with geldanamycin has
been shown to result in decreased Akt
activity and is correlated with a loss
of Akt phosphorylation in breast cancer
cells that overexpress HER2.[63]
The major drawback of HSP90 inhibitors,
however, is their relatively low
specificity for EGF receptors; their
use can affect the function of many
other cellular proteins that require
HSP90 for structural stability. Their
specificity may be increased by using
them in combination with specific
anti-EGF receptor TKIs.

Other Strategies
Gene therapy strategies for targeting
EGF receptor activity aim at blocking
the transcription, translation, and
maturation of members of EGF receptor
transcripts or proteins.[2]
Among the agents in development are
the adenovirus type 5 early region 1A
gene product,[64] triplex-forming oligonucleotides,
antisense oligonucleotides,
and ribozymes.[65-67] Further
experience with these agents may lead
to novel strategies that significantly
reduce HER2 signaling.

Determinants of Clinical
Response to Anti-HER2 Agents

The clinical benefits of anti-HER2
agents may not be observed in all
HER2-positive patients. For example,
trastuzumab monotherapy produces an
objective response in about a third of
patients with HER2-positive disease and
clinical benefits in almost half the patients
who overexpress HER2.[68]
These responses are higher when trastuzumab
is used in combination with
chemotherapy; objective responses
were observed in 50% of patients. Recent
studies suggest that there may be a
correlation between the decrease in serum
concentration of HER2 between 2
and 4 weeks after the start of trastuzumab-
based treatment and progression-
free survival.[69] The predictive
value of serum HER2 levels should be
investigated further.

It is important to understand the
molecular mechanisms that confer resistance
to trastuzumab so that patients
with disease that will not
respond to the therapy are not exposed
to its potential adverse effects.[32]
Some HER2-positive tumors
may have intrinsic resistance to
trastuzumab and other anti-HER2
agents, and others may have acquired
resistance, ie, they may have developed
compensatory mechanisms (discussed
in greater detail in the next
section).

Ongoing research indicates that the
intrinsic resistance of tumor cells to
trastuzumab may have a number of
causes. An in vitro study showed that
primary resistance to trastuzumab
could stem from a masking of membrane
proteins by a membraneassociated
mucin.[70] This masking
is thought to result in decreased
accessibility to and lack of activation
of HER2.[70] Evaluation of a HER2-
overexpressing breast cancer cell line
after exposure to trastuzumab showed
an association between downregulation
of p27kip1 levels and secondary
resistance.[71] Loss or mutation of
the tumor suppressor gene PTEN is
another important cause of tumor-cell
resistance to trastuzumab.[32,72] A
retrospective analysis of breast carcinomas
found that the responses to
trastuzumab-based therapy were significantly
poorer in patients with
PTEN-deficient tumors than in those
with normal PTEN expression.[32]

A recent study has elucidated the
role of PTEN in increasing sensitivity
to HER2 blockers such as trastuzumab.
On binding to HER2, trastuzumab
stabilizes and activates the PTEN
tumor suppressor, thereby downregulating
the proapoptotic PI3K/Akt signaling
pathway. Thus, PTEN
sensitizes tumor cells to trastuzumab,
and in the absence of PTEN the antitumor
effects of trastuzumab are impaired.[
32]

These findings suggest that drugs
that augment PTEN activity may sensitize
tumors to trastuzumab. Combinations
of HER2 blockers and PI3K
inhibitors (once these are commercially
available) may have greater efficacy.
The mammalian target of
rapamycin (mTOR) kinase, which is
an important downstream mediator of
the PI3K/Akt pathway, is another potential
target for overcoming resistance
to trastuzumab. The greater
proapoptotic activity of PI3K in the
absence of PTEN can be attenuated
by inhibiting the downstream mediator,
mTOR. The addition of mTOR
inhibitors, such as sirolimus (rapamycin
[Rapamune]) and temsirolimus,
may help overcome resistance to trastuzumab
in tumors that lack
PTEN.[73]

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