Overview of tyrosine kinase inhibitors in clinical breast cancer


Studies of cell models and profiling of clinical breast cancer material to reveal the mechanisms of resistance to anti-oestrogen therapy, and to tamoxifen in particular, have reported that this phenomenon can be associated with increased expression and signalling through erbB Type 1 growth factor receptors, notably the epidermal growth factor receptor (EGFR) and HER2. Further molecular studies have revealed an intricate interlinking between such growth factor receptor pathways and oestrogen receptor (ER) signalling. Inhibition of receptor tyrosine kinase activity involved in the EGFR signalling cascade forms the basis for the use of EGFR specific tyrosine kinase inhibitors exemplified by gefitinib (ZD1839, Iressa) and erlotinib (OSI-774, Tarceva). Such agents have proved promising in pre-clinical studies and are currently in clinical trials in breast cancer, where gefitinib has been studied more extensively to date. Here, we present an overview of the current development of gefitinib in clinical breast cancer. This includes results from our clinical breast cancer trial 1839IL/0057 that demonstrate the efficacy of gefitinib within ER-positive, tamoxifen-resistant patients with locally advanced/metastatic disease, where parallel decreases in EGFR signal transduction and the Ki67 (MIB1) proliferation marker can be detected as predicted from model system studies. We also consider trials examining combination treatment with gefitinib and anti-hormonal strategies that will begin to address the clinically important question of whether gefitinib can delay/prevent onset of anti-hormone resistance.
Overview of tyrosine kinase inhibitors
in clinical breast cancer
A Agrawal, E Gutteridge, J M W Gee
, R I Nicholson
and J F R Robertson
Professorial Unit of Surgery, City Hospital, Nottingham, UK
Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Redwood Building, Cardiff University, Cardiff, UK
(Requests for offprints should be addressed to J F R Robertson; Email:
Studies of cell models and profiling of clinical breast cancer material to reveal the mechanisms of
resistance to anti-oestrogen therapy, and to tamoxifen in particular, have reported that this
phenomenon can be associated with increased expression and signalling through erbB Type 1
growth factor receptors, notably the epidermal growth factor receptor (EGFR) and HER2. Further
molecular studies have revealed an intricate interlinking between such growth factor receptor
pathways and oestrogen receptor (ER) signalling. Inhibition of receptor tyrosine kinase activity
involved in the EGFR signalling cascade forms the basis for the use of EGFR specific tyrosine
kinase inhibitors exemplified by gefitinib (ZD1839, Iressa) and erlotinib (OSI-774, Tarceva). Such
agents have proved promising in pre-clinical studies and are currently in clinical trials in breast
cancer, where gefitinib has been studied more extensively to date. Here, we present an overview of
the current development of gefitinib in clinical breast cancer. This includes results from our clinical
breast cancer trial 1839IL/0057 that demonstrate the efficacy of gefitinib within ER-positive,
tamoxifen-resistant patients with locally advanced/metastatic disease, where parallel decreases in
EGFR signal transduction and the Ki67 (MIB1) proliferation marker can be detected as predicted
from model system studies. We also consider trials examining combination treatment with gefitinib
and anti-hormonal strategies that will begin to address the clinically important question of whether
gefitinib can delay/prevent onset of anti-hormone resistance.
Endocrine-Related Cancer (2005) 12 S135–S144
The erbB (HER/neu 1–4) receptors are a family of
Type 1 transmembrane receptors frequently expressed
at high levels in human tumours. erbB receptors are
activated by various growth factor ligands triggering a
network of intracellular signalling pathways leading to
uncontrolled growth of cancer cells. Signalling through
this receptor family invariably involves heterodimer-
ization between the family members.
HER2/neu is a transmembrane tyrosine kinase re-
ceptor which forms part of this erbB family signalling
network. Aberrant signalling by this network is
reportedly present in a cohort of breast carcinomas
(Arteaga et al. 2002). Slamon and colleagues reported
in 1987 that HER2 gene amplification independently
predicted overall survival (OS) and disease-free survival
(DFS) in a multivariate analysis in node-positive
patients. Subsequent studies have confirmed this finding
in node-positive breast cancer (Ross & Fletcher 1998),
although there is as yet no consensus on its prognostic
value in node-negative disease (Paik et al. 1990, Clark
& McGuire 1991, Dati et al. 1991). The epidermal
growth factor receptor (EGFR/HER1), a further erbB
receptor, is expressed in a large number of breast
cancers, with a positivity rate of 14–91% reported.
Overexpression of EGFR has again been linked to a
more aggressive breast tumour phenotype, involving
This paper was presented at the 1st Tenovus/AstraZeneca
Workshop, Cardiff (2005). AstraZeneca has supported
the publication of these proceedings.
Endocrine-Related Cancer (2005) 12 S135–S144
Endocrine-Related Cancer (2005) 12 S135–S144 DOI:10.1677/erc.1.01059
2005 Society for Endocrinology Printed in Great Britain Online version via
increased potential for invasiveness and metastasis.
This has been linked to poorer patient prognosis, as
extensively reviewed by Klijn and colleagues (1992).
More recently, Tsutsui and colleagues (2002) reported
in a large series of patients that EGFR expression was
a significant prognostic factor, although this remains
controversial; Ferrero et al. (2001) and Rampaul et al.
(2004), for example, both report that EGFR is not
While the prognostic significance of EGFR and
HER2 expression in breast cancer remains some matter
of debate, there is strong pre-clinical evidence to link
an importance for EGFR expression and its signalling
to de novo and acquired anti-hormone resistance in
oestrogen receptor-positive (ER+), as well as ER-
negative (ER -), breast tumour growth. erbB receptor
signal transduction has invariably been associated
with resistance to anti-oestrogens such as tamoxifen
(Knowlden et al. 2003), as well as to long-term
oestrogen deprivation in model systems (Martin et al.
2003). For example, our in vitro studies of resistant
sublines of MCF-7 breast cancer cells developed by
prolonged exposure to the anti-oestrogen tamoxifen
(TAM-R) or Faslodex (ICI 182, 780, fulvestrant,
AstraZeneca, Macclesfield, UK; FASR) to mimic the
development of acquired resistance in the clinic have
revealed increases in expression of EGFR and HER2
mRNA and protein (McClelland et al. 2001, Nicholson
et al. 2002, 2004, Knowlden et al. 2003). Immunopre-
cipitation studies revealed increased heterodimeriza-
tion between these receptors in the TAM-R cell line.
Western blotting also revealed increased phosphoryla-
tion (and hence activity) of these receptors compared
with the parental anti-hormone responsive MCF-7
cells. Increased basal levels of ERK1/2 MAP kinase
phosphorylation (pMAPK) were also observed in
TAM-R cells. TAM-R cells were capable of generating
a range of EGFR-specific ligands and increased ex-
pression of transforming growth factor-alpha (TGFa)
or amphiregulin were observed in these cells. Immuno-
cytochemical analysis confirmed these various signal-
ling data (Knowlden et al. 2003). Interestingly, patients
expressing increased HER2 or EGFR, together with
ligands for the EGFR such as TGFa, or increased
downstream signalling through pMAPK in their breast
tumours as measured immunohistochemically, exhibit
an increased likelihood of being resistant to anti-
hormonal agents, notably tamoxifen (Nicholson et al.
1993, 1994a,b, Giai et al. 1994, Archer et al. 1995,
Elledge et al. 1998, Gee et al. 2001).
Given the emerging importance of erbB receptor
signalling in anti-hormone-resistant breast cancer, it
is encouraging that several therapeutic strategies are
being developed to target such receptors. First, there
are extracellular approaches i.e. monoclonal antibodies
to the extracellular domain of particular erbB recep-
tors. For example, monoclonal antibodies have been
developed such as trastuzumab (rhuMAbHER2,
Herceptin, Genentech, San Francisco, US), cetuximab
(Erbitux, Merck KGaA, Darmstadt, Germany) and
pertuzumab (rhuMAb 2C4, Omnitarg, Roche, Basel,
Switzerland) that act to inhibit HER2, EGFR and
HER2 dimerization respectively. Secondly, there are
intracellular approaches i.e. small molecule tyrosine
kinase inhibitors (TKIs), notably those which selectively
inhibit the EGFR tyrosine kinase (Ciardiello 2000,
Mendelsohn & Baselga 2000, Solignac 2000). Of note in
this regard are gefitinib (ZD1839, Iressa) and erlotinib
(OSI-774, Tarceva). There are also now further TKIs
that block activity of both EGFR and HER2, such as
lapatinib (GW572016; Rusnak et al. 2001), while CI-
1033, which is in the pre-clinical stage of development,
is an agent purported to inhibit all four erbB receptors.
Therapeutic approaches such as anti-sense, anti-growth
factor targeted therapies, and toxin conjugated mono-
clonal antibodies are also of some interest. To date, the
Nottingham-Tenovus group have been involved pri-
marily in the pre-clinical and clinical development of
the EGFR TKI, gefitinib, in anti-oestrogen-resistant
breast cancer, and this agent will therefore comprise
the principal focus of this overview.
EGFR TKI, gefitinib, in pre-clinical
breast cancer studies
Gefitinib is a low molecular weight inhibitor with
highly selective and reversible tyrosine kinase inhibi-
tion properties directed at the EGFR, being a
competitive inhibitor of adenosine triphosphate bind-
ing to this receptor. It is a potent inhibitor of pro-
liferation not only in cells overexpressing EGFR but
also in those that additionally overexpress HER2,
possibly mediated by gefitinib reduction of EGFR/
HER2 heterodimer phosphorylation (Anderson et al.
2001). Pre-clinically, our studies showed that gefitinib
treatment (1 mM) was associated with a loss of phos-
phorylated EGFR in the acquired tamoxifen-resistant
TAM-R cell line, shown both by Western blotting and
immunocytochemistry, with an additional partial
inhibition of phosphorylation of the principle EGFR
heterodimerization partner in these cells, HER2.
Downstream, pMAPK was markedly depleted as a
consequence of this agent in TAM-R cells. Impor-
tantly, gefitinib subsequently induced a concentration-
dependent inhibition of TAM-R cell growth, where
1 mM gefitinib reduced proliferation significantly by
Agrawal et al.: Tyrosine kinase inhibitors in breast cancer
approximately 60% (Nicholson et al. 2002, 2004,
Knowlden et al. 2003). While resistance to gefitinib
ultimately emerged in culture (Jones et al. 2004), the
duration of response was long-lasting. Treatment with
trastuzumab to block HER2 heterodimerization with
EGFR was also able to reduce pMAPK activity in
TAM-R cells and inhibit growth (Knowlden et al.
2003). Obvious inhibitory effects of gefitinib were
similarly noted in acquired tamoxifen-resistant
T47D breast cancer cells (approximately 70% growth
inhibition), and also in cells that had acquired
resistance to Faslodex (approximately 50% growth
inhibition, McClelland et al. 2001, Nicholson et al.
2004). Gefitinib has also been reported to be inhibitory
on growth of de novo tamoxifen-resistant HER2-
overexpressing MCF-7/HER2-18 breast cancer cells
(Shou et al. 2004). However, in marked contrast to its
effects in tamoxifen-resistant cells, gefitinib exerted
only a small inhibition of the anti-hormone responsive
MCF-7 cells that exhibit minimal levels of EGFR
signalling. In such cells there was only approximately
15% reduction in growth at 1 mM gefitinib (Nicholson
et al . 2002, 2004, Knowlden et al. 2003).
In total, the model system data evaluating EGFR
signal transduction and gefitinib response in various
anti-hormone resistant cell lines, coupled with avail-
able immunohistochemical profiling of the EGFR
pathway in clinical tamoxifen resistance, indicate the
considerable potential for the EGFR-selective TKI,
gefitinib, to efficiently treat anti-hormone resistant
breast cancer (Nicholson et al. 2004). EGFR TKIs are
thus being studied intensively through various clinical
trials in this disease.
EGFR TKI, gefitinib, in clinical breast
cancer trials
An open label study, 1839IL/0156, of the EGFR TKI,
gefitinib, in actively progressive, measurable, meta-
static breast carcinoma in 63 patients with no limit
on prior chemotherapy or anti-hormonal therapy
has revealed clinical benefit (CB=complete response
(CR)+partial response (PR)+stable disease (SD)
6 months) in 3 patients (4.8%), and a further 6 patients
(9.5%) with stable disease up to 6 months (Albain et al.
2002). A phase II multi-centre study of gefitinib
monotherapy at 500 mg/day did not appear to be
efficacious in taxane and anthracycline metastatic pre-
treated breast cancer patients (57 patients, with 98.3%
non-responders) (von Minckwitz et al. 2005).
The efficacy of gefitinib in these breast cancer studies
was thus relatively low, although these studies did
focus on heavily pre-treated groups. This observation
also appears to extend to another EGFR TKI, namely
erlotinib. A phase II trial of erlotinib (as monotherapy
of 150 mg/day) in locally advanced or metastatic breast
cancer (n=69) was conducted with 2 arms, both of
which contained heavily pretreated patients. Cohort 1
(n=47) examining disease progression on or after treat-
ment with an anthracycline, a taxane, and capecitabine
revealed 1 PR (23 weeks) and 6 SD (all > 12 weeks).
Cohort 2 (n=22) examining disease progression on or
after at least one chemotherapy regimen revealed 1 PR
(16+ weeks) and 2 SD (both > 8 weeks) (Winer et al.
2002). Again, the efficacy in these study groups was at
best modest and the most common side effects were
grade 1 and 2 skin rashes (78%) and diarrhoea (59%).
Another phase II trial based on pre-clinical data of
additional efficacy of combination of capecitabine with
erlotinib had 24 patients in the trial. It found that
erlotinib (100 mg/day), capecitabine and docetaxel
were generally well tolerated and that there was no
pharmacokinetic interaction between erlotinib, capeci-
tabine and docetaxel or the metabolites of these drugs
(Jones et al. 2003).
Iressa trial 57
To date, the use of EGFR TKIs in metastatic disease
refractory to multiple previous treatments has proved
relatively disappointing, despite demonstrating accept-
able tolerability. However, a recent study by our group
has revealed that there is a cohort of breast cancer
patients that appear to obtain more substantial benefit
from EGFR TKI treatment.
Our previous immunohistochemical study of primary
human tumours had reported that increased EGFR
expression was significantly associated with lack of
anti-hormone sensitivity de novo in both ER+ and
ER - breast cancers (Nicholson et al. 1994a). Exam-
ination of the in vivo breast tumour tissue showed
obvious overexpression of several erbB signalling
components in the ER+ de novo resistant tumours
i.e. an increase in EGFR, HER2, TGFa and pMAPK
(and also the Ki67 proliferation marker), approaching
levels seen in the ER - tumours (Nicholson et al. 1993,
1994a,b, Gee et al. 2001). Furthermore, ER+ breast
cancer patients who developed acquired tamoxifen
resistance showed more modest increases in expression
of EGFR, TGFa, HER2 and activation of ERK1/2
MAPK at the time of relapse (Gee et al. 2002,
Nicholson et al. 2004). These observations, coupled
with pre-clinical studies showing the importance of
EGFR signalling and anti-tumour effects of gefitinib in
various anti-oestrogen-resistant models, formed the
Endocrine-Related Cancer (2005) 12 S135–S144 S137
basis for the design of the gefitinib breast cancer trial
57 (1839IL/0057). This comprised two stratified arms
of locally advanced/metastatic disease patients with
either ER+ tamoxifen-resistant disease or ER-
tumours. The target recruitment was 54 patients, with
27 patients in each arm. The early results of this study
were presented when 33 patients had been recruited: 13
in the ER+ arm and 20 in the ER - arm. The common
side effects with gefitinib treatment were again skin
rash and diarrhoea. Six patients discontinued gefitinib
500 mg/day within 3 months owing to these side effects.
A minority of patients was also reported to have a
degree of alopecia. Interestingly, of the 27 evaluable
patients, clinical benefit was seen in 6 out of 9 patients
in the ER+ arm (66%, comprising PR=1 and SD=5).
There was a lower efficacy in the ER - arm, although 2
out of 18 patients in this group gained clinical benefit
with gefitinib (11%, comprising PR=1 and SD=1)
(Robertson et al. 2003). Median time to progression
was also longer in the ER+ tamoxifen-resistant group
compared with the ER - group (Gutteridge et al.
2004). Since then, further recruitment continued up to
53 patients by January 2005: 26 patients in the ER+
arm and all 27 patients in the ER - arm. The initial
difference in therapeutic efficacy seen between the ER+
tamoxifen-resistant group and ER - patients has been
As part of trial 57, patients with palpable disease
were asked for consent to carry out sequential biopsies
which were taken before commencement of treatment
(T0), at 8 weeks (T1) and 6 months (T3) on treatment,
and on progression of disease. Patients who obtained a
CB with gefitinib expressed lower pre-treatment levels
of EGFR than patients with progressive disease (PD)
(Robertson et al. 2003, Gee et al. 2004, Gutteridge et al.
2004). Some degree of EGFR expression was, how-
ever, detectable in all CB patients. It thus appears that
it is largely patients with ER+ acquired tamoxifen-
resistant disease whose tumours express relatively
modest EGFR that obtain clinical benefit from this
EGFR TKI, rather than a predominance in the ER - ,
EGFR overexpressing cohort. These data directly
parallel the successes of gefitinib in ER+ acquired
anti-oestrogen-resistant cell models, including our
TAM-R cells. The somewhat paradoxical lack of direct
association between high EGFR expression and the
gefitinib response has previously been noted within
in vitro and xenograft models (Wakeling et al. 2002).
Efficacy of gefitinib was also independent of EGFR
overexpression in the IDEAL non-small cell lung
carcinoma (NSCLC) trials. It was reported that the
percentage of membrane staining for EGFR was ap-
proximately 20% higher in 69 non-responsive subjects
compared with 18 responders in IDEAL 1 (although
the opposite was seen in IDEAL 2, where staining was
approximately 25% lower in 62 non-responders
compared with 8 responders). There was thus no con-
sistent association of relevance of EGFR membrane
staining and response to classify NSCLC patients for
gefitinib treatment (Bailey et al. 2003).
Our immunohistochemical analysis of 15 of the
breast cancers in trial 57 performed to date has studied
erbB signalling elements and the Ki67 (MIB1) pro-
liferation marker in paired pre-treatment and 8 weeks
(T1) after gefitinib (500 mg/day) samples. Patients
studied were categorized as having CB (n=9; all ER+)
or PD (n=6; all ER -) at 6 months. These studies
revealed maintenance of a significant association be-
tween CB and ER (and progesterone receptor, PgR)
positivity in the pre-treatment and post-treatment
samples. Change in the Ki67 proliferation marker
paralleled subsequent outcome, with a significant
decline between the pre-treatment and post-treatment
levels in patients with CB only, and as a consequence a
significantly lower post-treatment proliferation level in
patients with CB compared with PD (Gee et al. 2004).
Phosphorylated (activated) EGFR (pEGFR) was
generally low at pre-treatment and unrelated to out-
come with gefitinib in trial 57. However, 5/9 patients
with CB showed falls in pEGFR during treatment,
with parallel decreases in pMAPK and proliferation.
In pharmacodynamic immunohistochemical studies of
16 paired pre-treatment and post-treatment normal
skin biopsies in patients participating in gefitinib phase
I clinical trials receiving escalating daily doses of
gefitinib for solid tumours (Albanell et al. 2001, 2002,
Baselga et al. 2002), there was similarly a statistically
significant decrease in pEGFR, pMAPK and Ki67
post gefitinib treatment.
CB is thus achievable with gefitinib in ER+/PgR+
tamoxifen-resistant breast cancer patients expressing
relatively low EGFR, with proliferation changes at T1
paralleling the subsequent response. Patients with CB
can exhibit reduced pEGFR and pERK1/2 MAPK
during treatment, in accordance with the inhibitory
effects of gefitinib on EGFR signal transduction and
proliferation observed in pre-clinical studies in TAM-R
cells. However, decreases in pEGFR were not universal
in patients with CB in trial 57, suggesting that a non-
classical gefitinib response mechanism does exist in
some patients.
In breast cancer patients exhibiting PD with
gefitinib, some increase in EGFR expression was noted
following treatment, while the pEGFR level was
unchanged and could even increase. Thus, significantly
elevated EGFR expression and pEGFR were recorded
Agrawal et al.: Tyrosine kinase inhibitors in breast cancer
post-treatment in patients with PD versus CB (Gee
et al. 2004). While increases in EGFR expression could
perhaps underlie the absence of pEGFR decline in
de novo gefitinib resistance, EGFR phosphorylation
mediated in an EGFR kinase-independent manner by
other receptors (perhaps additional erbB receptors or
the insulin-like growth factor receptor (IGF-1R)) may
be contributory. (Gee et al. 2004, Gutteridge et al.
2004). Interestingly, tumours from PD patients in this
study also commonly exhibited increased activity of
the signalling molecule AKT, an element that has been
linked pre-clinically both to IGF-1R signalling and to
gefitinib resistance (Jones et al. 2004).
Iressa trial 225
Acquired tamoxifen resistance in MCF-7 cells and in
clinical breast cancer has been reported to be
associated with some increases in EGFR signalling
through ERK1/2 MAPK, where gefitinib blocks such
signalling activity in tamoxifen-resistant models and,
as stated above, in some ER+ tamoxifen-resistant
patients. Further pre-clinical mechanistic studies have
revealed an intricate interlinking between such signal-
ling and the oestrogen receptor signalling pathway at
the level of ER phosphorylation and ER co-activator
recruitment (Nicholson et al. 2004). In contrast,
however, EGFR/MAPK signalling is minimal in the
anti-hormone responsive MCF-7 cells and treatment
with gefitinib alone has no anti-proliferative effect.
This ‘switch’ to the use of EGFR/MAPK signalling
that appears integral to the evolution of anti-oestrogen
resistance can be blocked by the combination of
tamoxifen and gefitinib during the anti-hormone
responsive phase in vitro (Gee et al. 2003). Such a
strategy improves the growth inhibitory effect of the
anti-oestrogen and, moreover, delays/prevents devel-
opment of acquired resistance in ER+ breast cancer
models (Gee et al. 2003, Shou et al. 2004).
Cumulatively, these data not only supported the
potential clinical utility of gefitinib in anti-oestrogen-
resistant breast cancer but also suggested the possibility
of delaying or preventing resistance by the early use of a
combination of gefitinib with anti-oestrogens such as
tamoxifen (Wakeling et al. 2001). This led to the
designing of a trial known as Iressa 225 (1839IL/0225).
This is a randomized phase II trial intended to recruit
274 women with ER+ metastatic breast cancer derived
from 2 strata of patients: strata 1 comprises patients
who have newly diagnosed disease or who have
completed adjuvant tamoxifen at least one year prior
to recruitment, while strata 2 comprises patients with
recurrent disease during or after adjuvant aromatase
inhibitor (AI) or failing first-line treatment with AI.
Patients are then equally randomized to two arms:
tamoxifen (20 mg/day) plus gefitinib (250 mg/day) or
tamoxifen plus placebo. The primary endpoint of the
study is comparative time to progression in the two arms
and the secondary endpoints are the recurrence rate
(RR) and clinical benefit rate. This study should begin
to answer the clinically important question of whether
gefitinib can delay/prevent acquired resistance to
tamoxifen. Further immunohistochemical exploration
would examine downstream effectors of the erbB family,
and also key co-activators such as AIB-1 that may enable
interplay between erbB and ER signalling. No results
are thus far available for this study. A very similar trial in
the United States plans to recruit 174 patients, with the
only difference being that it will assess acquired
resistance to anastrozole instead of tamoxifen.
Iressa trials 0219 and 0223
There are also two studies assessing changes induced
by gefitinib in tumour samples obtained pre-surgically
that should prove mechanistically informative. The
pre-surgical biological trial 1839IL/0219 intends to
recruit about 50 patients (stages I, II, IIIa) and will
compare immunohistochemical biomarker changes in
pre-gefitinib tissue samples (tumour and skin biopsy)
with those taken after 14 days of gefitinib (250 mg/day)
(McKillop et al. 2004). A second trial combines the
principles of the Iressa 225 study into a neoadjuvant
protocol using anastrozole rather than tamoxifen
(1839IL/0223). This is a randomized blinded parallel
group, placebo-controlled, multi-centre trial. It intends
to recruit 175 evaluable patients with stage II–IIIb
treatment-naive ER+ tumours. In a neoadjuvant study
of 3–4 months there is usually insufficient time for
tumours to respond and then establish acquired
resistance. The hypothesis therefore being tested in
such a trial is whether gefitinib further sensitizes ER+
tumours to anastrozole (both given as a neoadjuvant)
and can reverse de novo resistance. The patients are
randomized into three arms: arm A (n=29) comprising
patients who would receive anastrozole (1 mg four
times a day)+gefitinib (250 mg four times a day) for
16 weeks prior to surgery, arm B (n=73) comprising
patients receiving anastrozole+placebo for 2 weeks
and then placebo replaced by gefitinib for the
remaining 14 weeks, and arm C (n=73) comprising
patients receiving anastrozole+placebo for the full
16 weeks. The study would assess biomarker changes
(including Ki67, pMAPK, pEGFR) and measure
mRNA expression in tumour biopsy, plasma and urine
Endocrine-Related Cancer (2005) 12 S135–S144 S139
Iressa trial 004
Our in vivo study of the effect of Faslodex (fulvestrant),
a pure anti-oestrogen with no partial agonist activity
on the ER, given for 7 days pre-operatively in clinical
breast cancer did not show any short-term alteration of
EGFR or TGFa protein levels even though ER
protein was significantly suppressed (McClelland et al.
1996). However, our in vitro studies that established an
acquired fulvestrant-resistant breast cancer cell line
(FASR) by maintenance of the anti-hormone responsive
parental MCF-7 cells in steroid-depleted, fulvestrant-
supplemented medium showed that EGFR protein
and mRNA, growth responses to TGFa, and pMAPK
were all increased by 3-months exposure to this anti-
oestrogen. Unlike the parental MCF-7 cells, FASR cells
were sensitive to growth inhibition by gefitinib or by an
inhibitor of the activation of MEK1 (MAPKK) and
hence pMAPK, PD098059. Furthermore, the parental
MCF-7 cells were markedly inhibited by combination
fulvestrant+gefitinib treatment, with prominent cell
death and reduced rates of cellular proliferation
(McClelland et al. 2001), a strategy consequently abro-
gating emergence of resistance. Thus, gefitinib may be a
viable option after resistance to fulvestrant, and may
again delay development of resistance to this agent. The
Nottingham-Tenovus group have built on these promis-
ing pre-clinical findings and initiated a phase II trial,
1839IL/0004, of gefitinib (250 mg daily) second-line
therapy in metastatic breast cancer patients who have
developed acquired resistance to the pure anti-oestrogen
fulvestrant that they received as first-line anti-hormonal
therapy. The exploratory objectives in this study are
correlation between EGFR expression and response,
and evaluation of changes in tissue and serum biomar-
kers, the main clinical endpoints being RR and CB.
Other TKI trials
There are two further phase II studies of gefitinib+
fulvestrant in patients with advanced disease. One is
being carried out in Spain, where 60 metastatic breast
cancer patients are to be recruited to first-line therapy.
Along with clinical end-points, this study will examine
EGFR and HER2 signalling components. The second
is an Eastern Co-operative Oncology Group (ECOG)
trial comparing gefitinib (250 mg/day)+fulvestrant
(250 mg i.m. monthly) with gefitinib (250 mg/day)-
+anastrozole (1 mg/day). The number of patients to
be recruited to the latter study is 204, to be divided
equally between the two arms. Again, the endpoints
will be biomarkers and the correlation of the two arms
in terms of efficacy. As yet no data are reported for
either trial.
Initial phase II studies have suggested that the EGFR
TKIs, gefitinib and erlotinib, do not have a high
efficacy in a heavily pre-treated population of patients
with metastatic breast cancer, particularly post che-
motherapy. Surprisingly, there is also evidence of only
minimal efficacy in ER - , EGFR overexpressing
breast cancer. Importantly, however, in patients with
tamoxifen-resistant ER+ tumours, gefitinib does
appear to have a significant therapeutic effect.
While such studies indicate that EGFR overexpres-
sion is not related to response to gefitinib in breast
cancer, the available data suggest that the EGFR is
required to be present at detectable levels before
patients’ tumours can respond to this agent. Indeed,
previous clinical profiling has detected modest levels of
various EGFR signalling elements in tamoxifen-
resistant material, suggesting EGFR pathway func-
tionality in such tumours. However, the presence of
ER was the most important biological marker in study
57 indicating an increased chance of responding to
gefitinib this significant relationship with ER
positivity reflects the gefitinib-sensitive population of
acquired tamoxifen-resistant tumours in this study.
Equally important findings of this study were the
decreases in the Ki67 proliferation marker and in some
of the downstream markers of EGFR signal transduc-
tion during the gefitinib response. In total, these
findings closely parallel the pre-clinical data demon-
strating the effectiveness of gefitinib in blocking the
EGFR signalling pathway and the growth of ER+
tamoxifen-resistant breast cancer cell models. Such
data should be a valuable guide in determining which
patient populations to target with EGFR TKIs in
future studies. However, future biomarker studies
examining the impact of erbB signalling, additional
growth factor receptors and the ER pathway on the
gefitinib response are likely to be of key importance in
further discriminating EGFR TKI-sensitive/resistant
patients. Microarray studies may also be important in
this regard to reveal EGFR TKI-associated gene
changes (Yang et al. 2004). Alongside these various
profiling studies, of potential interest may be the study
of EGFR mutational status. It has been observed that
in patients with NSCLC, specific mutations in the
EGFR gene, mostly those in exons 19, 21 and also 18,
have a direct correlation with the response to gefitinib
(Lynch et al . 2004, Paez et al. 2004). EGFR mutations
were also found in a lung adenocarcinoma cell line
that was hypersensitive to gefitinib inhibition (Paez
et al. 2004). In gefitinib-responsive NSCLC primary
tumours, somatic mutations in the tyrosine kinase
Agrawal et al.: Tyrosine kinase inhibitors in breast cancer
domain of the EGFR gene comprise small, in-frame
deletions or amino acid substitutions clustered around
the ATP binding pocket of the tyrosine kinase domain
(Lynch et al. 2004). In vitro, such EGFR mutants
demonstrated enhanced tyrosine kinase activity in
response to EGF and increased sensitivity to inhibition
by gefitinib (Lynch et al . 2004). The EGFR mutations
have similarly been linked to erlotinib sensitivity in
lung cancer (Pao et al. 2004). In contrast, however,
there have been reports of patients with further EGFR
mutation developing resistance to gefitinib (Kobayashi
et al. 2005). A second point mutation was found in an
EGFR-mutant, gefitinib-responsive advanced NSCLC
patient who relapsed after two years of complete
remission with gefitinib. This mutation resulted in a
threonine to methionine change at position 790 in the
EGFR (Kobayashi et al. 2005). Clearly, there is an
equivalent requirement to examine the mutational
status of EGFR in breast cancers, and such studies
are ongoing.
The pre-clinical studies and available clinical trial
data indicate that the use of EGFR TKIs as mono-
therapies, of which gefitinib is the most investigated to
date in breast cancer, is likely to be most successful
when applied to specific sub-groups of patients (i.e.
ER+ tamoxifen-resistant disease). However, pre-clin-
ical studies also suggest that a particularly rewarding
avenue could be to use EGFR TKIs in combination
with anti-hormonal agents, where anti-tumour effects
are significantly improved and emergence of resistance
is delayed or even prevented. Trial 225 builds upon
these promising pre-clinical data, and is looking to
establish if gefitinib can prevent or delay the onset of
tamoxifen resistance in the clinic. Other studies are
looking to answer similar questions with respect to the
aromatase inhibitor, anastrozole, although the pre-
clinical supportive data in this field is much more
limited. There is also some pre-clinical data showing
the potential for gefitinib to treat, and in combination,
prevent resistance to fulvestrant, and studies assessing
the clinical efficacy of gefitinib to subvert resistance to
fulvestrant are also emerging.
Finally, it should be remembered that EGFR is only
one member of the erbB family, and in addition to
homodimerization it can be activated following hetero-
dimerization with other erbB receptors (Deb et al.
2001). Additional erbB receptors might therefore be
able to activate the EGFR in a manner independent of
EGFR kinase activity and to generate signalling that is
insensitive to inhibitors of the EGFR tyrosine kinase
domain. Furthermore, there is evidence of erbB
interplay with alternative growth factor pathways,
including IGF-1R signalling that contributes to breast
cancer growth and is able to promote EGFR activation
(Nicholson et al. 2004). Emerging experimental data
suggest these complexities may be able to promote
resistance to EGFR TKIs, and, as such, strategies
targeting multiple erbB interactions are worthy of
therapeutic investigation and may improve anti-
tumour effects of the EGFR TKIs. The recently
developed agent, pertuzumab (rhuMAb 2C4), a
recombinant humanized monoclonal antibody, is of
some interest in this regard. It binds to the extracellular
domain II of the HER2 receptor and blocks its ability
to dimerize with other erbB receptors. This disrupts
ligand-dependent HER2 signalling. Pertuzumab thus
represents a new class of targeted therapeutics known
as HER2 ‘dimerization inhibitors’ that block both
homo- and heterodimerization of HER2 (Agus et al.
2005). In pre-clinical studies, pertuzumab proved
inhibitory to breast, prostate and NSCLC tumour
models, comprising those overexpressing HER2 as
well as non-overexpressing cells. A pre-clinical study
evaluated combining pertuzumab with the EGFR TKI
erlotinib in the HER2/EGFR overexpressing human
breast cancer cell line, MDA175. Sub-optimal doses of
the combination treatment revealed superior 66%
growth inhibition of the cell line compared with 6%
and 34% for pertuzumab or erlotinib alone (Totpal
et al. 2002). Another pre-clinical study examined the
effect of combining pertuzumab with the anti-HER2
antibody trastuzumab in the HER2-overexpressing
BT474 breast cancer cell line. This study revealed
synergistic inhibition of cell growth, in part because of
increased apoptosis. Combination drug treatment
reduced levels of total and phosphorylated HER-2
protein and blocked subsequent signalling through Akt
(Nahta et al. 2004). In phase I clinical trials,
pertuzumab has shown activity in a number of human
cancers, and a phase II programme is in progress
(Badache & Hynes 2004, Bianco 2004). With further
regard to inhibitors of multiple erbB receptors, clinical
activity of lapatinib that blocks activity of both EGFR
and HER2 has also been reported in a phase 1 trial
(Spector et al. 2003). In total, these recent results and
developments suggest that combining one HER-
targeting agent with another or with an EGFR-specific
TKI may be a more effective and futuristic therapeutic
strategy in breast cancer.
J F R Robertson, R I Nicholson and J M W Gee are
in receipt of research grants from AstraZeneca. R I
Nicholson also has research funding from Roche and
Merck. The Iressa study 57 was supported by a
Endocrine-Related Cancer (2005) 12 S135–S144 S141
research grant from AstraZeneca. J F R Robertson
and R I Nicholson have been members of Advisory
Boards for AstraZeneca.
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... It blocks the highly tumorigenic and inhibits downstream signaling through both the Ras/ MAP kinase and PI-3 kinase/AKT pathways [94]. It targets the overexpression of ErbB in different types of cancer and inhibits all the members of the EGFR family [95][96][97][98]. It is also active against ErbB3 and ErbB4 but has no effect on other types of tyrosine kinases [77]. ...
Human EGFR (Epidermal Growth Factor Receptor) family of tyrosine kinase receptors consists of four members, ErbB1-4. Abnormalities in the ErbB family characterize a variety of human cancers, including breast cancer. Tyrosine kinase is recruited by the activated EGFR cell surface receptor, which transmits signals from the receptor to interact with intracellular signaling pathways and regulates cellular functions and biological processes. Targeting the intracellular signaling pathways has been aided in the drug development that was already in use and more continually being developed. This review article highlights the function of ErbB receptors/ligands, their role in signaling pathways, effective targeted drugs, and a combination of targeted drug strategies in breast cancer treatment that could lead to the novel combination of anticancer drug delivery systems.
... Tyrosine kinase receptors play an essential role in tumor development, pathologic angiogenesis, and the metastatic progression of breast cancer via the phosphorylation of target proteins [4]. It has been established that the inhibition of the function of the tyrosine kinase receptors involved in the EGFR signaling cascade forms the foundation for the usage of EGFR-specific inhibitors of tyrosine kinase, exemplified by gefitinib kinase [5]. Thus, the structurally similar drug imatinib is also expected to inhibit breast cancer. ...
Full-text available
Breast cancer is the most widespread malignancy in women worldwide. Nanostructured lipid carriers (NLCs) have proven effective in the treatment of cancer. NLCs loaded with imatinib (IMA) (NANIMA) were prepared and evaluated for their in vitro efficacy in MCF-7 breast cancer cells. The hot homogenization method was used for the preparation of NANIMAs. An aqueous solution of surfactants (hot) was mixed with a molten mixture of stearic acid and sesame oil (hot) under homogenization. The prepared NANIMAs were characterized and evaluated for size, polydispersity index, zeta potential, encapsulation efficiency, release studies, stability studies, and MTT assay (cytotoxicity studies). The optimized NANIMAs revealed a particle size of 104.63 ± 9.55 d.nm, PdI of 0.227 ± 0.06, and EE of 99.79 ± 0.03. All of the NANIMAs revealed slow and sustained release behavior. The surfactants used in the preparation of the NANIMAs exhibited their effects on particle size, zeta potential, encapsulation efficiency, stability studies, and release studies. The cytotoxicity studies unveiled an 8.75 times increase in cytotoxicity for the optimized NANIMAs (IC50 = 6 µM) when compared to IMA alone (IC50 = 52.5 µM) on MCF-7 breast cancer cells. In the future, NLCs containing IMA will possibly be employed to cure breast cancer. A small amount of IMA loaded into the NLCs will be better than IMA alone for the treatment of breast cancer. Moreover, patients will likely exhibit less adverse effects than in the case of IMA alone. Consequently, NANIMAs could prove to be useful for effective breast cancer treatment.
... However, the effectiveness of EGFR tyrosine kinase inhibitors (TKI) s in the clinic has been greatly impaired by the development of de novo or acquired resistance [9][10][11]. Specifically, trials with gefitinib in breast cancer resulted in poor clinical response indicating that intrinsic resistance to gefitinib, and therefore, to TKIs, is common in breast cancer [12,13]. Similarly, the initial success of lapatinib, which was developed as an ATP-competitive reversible EGFR/HER2 inhibitor, has also been marred by intrinsic and acquired therapy resistance [14,15]. ...
Full-text available
Background Even though targeted therapies are available for cancers expressing oncogenic epidermal growth receptor (EGFR) and (or) human EGFR2 (HER2), acquired or intrinsic resistance often confounds therapy success. Common mechanisms of therapy resistance involve activating receptor point mutations and (or) upregulation of signaling downstream of EGFR/HER2 to Akt and (or) mitogen activated protein kinase (MAPK) pathways. However, additional pathways of resistance may exist thus, confounding successful therapy. Methods To determine novel mechanisms of EGFR/HER2 therapy resistance in breast cancer, gefitinib or lapatinib resistant variants were created from SKBR3 breast cancer cells. Syngenic therapy sensitive and resistant SKBR3 variants were characterized for mechanisms of resistance by mammosphere assays, viability assays, and western blotting for total and phospho proteins. Results Gefitinib and lapatinib treatments reduced mammosphere formation in the sensitive cells, but not in the therapy resistant variants, indicating enhanced mesenchymal and cancer stem cell-like characteristics in therapy resistant cells. The therapy resistant variants did not show significant changes in known therapy resistant pathways of AKT and MAPK activities downstream of EGFR/HER2. However, these cells exhibited elevated expression and activation of the small GTPase Rac, which is a pivotal intermediate of GFR signaling in EMT and metastasis. Therefore, the potential of the Rac inhibitors EHop-016 and MBQ-167 to overcome therapy resistance was tested, and found to inhibit viability and induce apoptosis of therapy resistant cells. Conclusions Rac inhibition may represent a viable strategy for treatment of EGFR/HER2 targeted therapy resistant breast cancer.
... Tyrosine kinase receptors that play an essential role in tumour development, pathologic angiogenesis, and metastatic progression of breast cancer via the phosphorylation of target proteins [4]. It has been established that the inhibition of the function of the receptor tyrosine kinase involved in the EGFR signaling cascade forms the foundation for the usage of EGFR specific inhibitors of tyrosine kinase, exemplified by gefitinib kinase [5]. Thus, the structurally similar drug imatinib also expected to perform with breast cancer inhibition. ...
Breast cancer is the most widespread malignancy in women from corner to corner of the world. Nanostructured lipid carrier (NLC) proved its efficacy in the treatment of cancer. NLC loaded with Imatinib (IMA) (NANIMA) was prepared and evaluated for in vitro efficacy in MCF-7 breast cancer cells. Hot homogenization method was used for the preparation of NANIMAs. Aqueous solution of surfactants (hot) was mixed with molten mixture of stearic acid and sesame oil (hot) under homogenization. The prepared NANIMAs were characterized and evaluated for size, polydispersity index, zeta potential, encapsulation efficiency, release studies, stability studies and MTT assay (cytotoxicity studies). The optimized NANIMA revealed particle size of 104.63 ± 9.55 d.nm, PdI of 0.227 ± 0.06 and EE of 99.79 ± 0.03. All NANIMAs revealed slow and sustained release behaviour. The surfactants used in the preparation of NANIMAs exhibited their effects on particle size, zeta potential, encapsulation efficiency, stability studies and release studies. The cytotoxicity studies unveiled 8.75 times increase in cytotoxicity for optimized NANIMA (IC50 = 6 µM) when compared with IMA alone (IC50 = 52.5 µM) on MCF-7 breast cancer cells. After this, NLC containing IMA possibly will be employed for the cure of breast cancer. A lesser amount of IMA loaded NLC will be sufficient than IMA alone for the treatment of breast cancer. Moreover, the patient will exhibit not as much of adverse effects as in case of IMA alone. Consequently, NANIMA could prove to be useful for effective therapeutic outcome in breast cancer treatment.
... However, hereditarily and acquired resistance to this therapy develops within one year [4]. Thereafter, the use of therapeutics with tyrosine kinase inhibition (TKI) activity, including LAP, neratinib, and ge tinib has been more developed [5]. Though, a number of proofs about acquired resistance to these medications through modulation of their different downstream signaling proteins have been documented [6]. ...
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We examined the effect of Metformin (MET) on Lapatinib (LAP)-induced apoptosis in SK-BR3 cells. The cell viability and cell death were measured by MTT assay and Annexin V-FITC/PI staining, respectively. The mRNA expression of Bax, Bcl2, and P21 was determined by real-time PCR. The activity of caspase 3 and 9 was measured using an ELISA kit. The MET-LAP combination index (CI) was calculated using CompuSyn software. The molecular docking studies of LAP with Akt, AMPK, and HER2 were performed using Auto Dock The strongest synergistic apoptotic death [combination index (CI) = 0.51] was obvious in MET 100 mM plus LAP 100 nM. The MET-LAP combination causes a more powerful apoptotic death (46%) compared to LAP 100 nM individually. The combination could significantly increase the expression of Bax and P21, as well as the activity of caspase 3 and 9, but decreased the expression of Bcl2. Moreover, the combination could significantly increase the level of AMPK in LAP 100 nM plus MET 30 mM and decreased the level of Akt in LAP 100 nM plus MET 40 mM. Molecular docking studies showed that LAP interacts with the active site amino acids of Akt, AMPK, and HER2 by hydrogen and hydrophobic interactions. MET-LAP combination induces more extensive apoptotic death than either drug alone in SKBR3 cells. MET-LAP combination may be considered as a valuable therapeutic intervention in breast cancer patients. However, further in-vivo studies are needed to assess the MET-LAP combination's practical value for induction of apoptosis and an anti-cancer drug.
... [12,13] Although such inhibitors have been used with success in nonsmall cell lung cancer (NSCLC) treatment, clinical trials in breast cancer have shown poor results, even in combination with chemotherapy. [14,15] It has been reported that NSCLCresistant cells enhance EGFR levels due to dysregulated degradation following loss of binding to its E3 ubiquitin ligase Cbl. [16] In addition, it has been hypothesized that mAb and TKI resistance are partly due to activation of alternative downstream pathways of EGFR. ...
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Breast cancer stem cells (BCSCs) are responsible for resistance to chemotherapy, high degree of metastasis, and poor prognosis, especially in triple‐negative breast cancer (TNBC). The CD24lowCD44high and high aldehyde dehydrogenase 1 (ALDH1) cell subpopulation (CD24lowCD44high ALDH1⁺) exhibit very high tumor initiating capacity. In the current study, the upregulated genes are analyzed in both CD24lowCD44high and ALDH1⁺ cell populations at single‐cell resolution, and a highly expressed membrane protein, SGCE, is identified in both BCSC populations. Further results show that SGCE depletion reduces BCSC self‐renewal, chemoresistance, and metastasis both in vitro and in vivo, partially through affecting the accumulation of extracellular matrix (ECM). For the underlying mechanism, SGCE functions as a sponge molecule for the interaction between epidermal growth factor receptor (EGFR) and its E3 ubiquitination ligase (c‐Cbl), and thus inhibits EGFR lysosomal degradation to stabilize the EGFR protein. SGCE knockdown promotes sensitivity to EGFR tyrosine kinase inhibitors (TKIs), providing new clues for deciphering the current failure of targeting EGFR in clinical trials and highlighting a novel candidate for BCSC stemness regulation.
... EGFR, also referred to as HER1, belongs to the HER family of transmembrane receptor tyrosine kinases, and plays important roles in the proliferation and metastasis of tumor cells (73)(74)(75). Dysregulation and the aberrant activation of EGFR induce uncontrolled tumor cell proliferation and invasiveness, decreased apoptosis and cell differentiation, and increased survival, angiogenesis, cell migration and metastasis (76,77). TNBC is associated with a high frequency of EGFR dysregulation (78,79), and EGFR expression is reported in >50% of TNBC cases. ...
Effective treatment regimens for triple-negative breast cancer (TNBC) are relatively scarce due to a lack of specific therapeutic targets. Epidermal growth factor receptor (EGFR) signaling is highly active in TNBC and is associated with poor prognosis. Most EGFR antagonists, which significantly improve outcome in lung and colon cancer, have shown limited clinical effects in breast cancer. However, limiting EGFR expression in TNBC is a potential strategy for improving the clinical efficacy of EGFR antagonists. Here, we found that the gamma-aminobutyric acid type A receptor π subunit (GABRP), as a membrane protein enriched in TNBC stem cells, interacted with EGFR and significantly sustained its expression, resulting in stemness maintenance and chemotherapy resistance. Silencing GABRP induced down-regulation of EGFR signaling, which hindered cell stemness and enhanced sensitivity to chemotherapies, including paclitaxel, doxorubicin, and cisplatin. We also identified that retigabine, an FDA-approved drug for adjunctive treatment of seizures, increased the sensitivity of EGFR to gefitinib in gefitinib-resistant cells. Our findings show that GABRP can sustain the stemness of TNBC via modulating EGFR expression, suggesting that GABRP may be a potential therapeutic target that can address EGFR inhibitor resistance in TNBC.
Aims and background Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks the expression of hormone receptors and human epidermal growth factor receptor 2 (HER2). Although TNBC represents only 15% of all types of breast cancer, it accounts for a large number of metastatic cases and deaths. Because of the high metastatic rate and both local and systemic recurrence associated with TNBC, extensive research efforts are actively looking for target therapies to effectively treat this aggressive disease. Accordingly, this study has been initiated to investigate the differential expression of biological markers in TNBC and non-TNBC Saudi women that might be utilized as potential targeted therapy and/or predict the sensitivity to currently available therapeutic regimens. Methods and study design Two hundred formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues were selected and divided into 3 groups: benign breast tissues (20), TNBC tissues (80) and non-TNBC tissues (100). Expression of mRNA in FFPE tissues was analyzed using real-time polymerase chain reaction (RT-PCR) for the following genes: poly (ADP-ribose) polymerase 1 (PARP-1), topoisomerase 2A (TOPO-2A), vascular endothelial growth factor (VEGF), C-MYC, basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMP-2 and MMP-9), human epidermal growth factor 1 (HER1) and multidrug resistance (MDR) genes. Results In the TNBC group, expression of PARP-1, TOPO-2A, HER1, C-MYC, VEGF, bFGF and MMP-2 showed a highly significant increase compared to the non-TNBC group. Conclusions The results of this study suggest that (1) TNBC patients will benefit more from TOPO-2A inhibitors as well as antiangiogenic and antimetastatic therapies; (2) inhibition of these target genes is emerging as one of the most exciting and promising targeted therapeutic strategies to treat TNBC in which the intended targets are DNA repair, tumor angiogenesis and metastasis.
Suppressors of Cytokine Signaling (SOCS) proteins are negative regulators of JAK proteins that are receptor-associated tyrosine kinases, which play key roles in the phosphorylation and subsequent activation of several transcription factors named STATs. Unlike the other SOCS proteins, SOCS1 and 3 show, in the N-terminal portion, a small kinase inhibitory region (KIR) involved in the inhibition of JAK kinases. Drug discovery processes of compounds based on KIR sequence demonstrated promising in functional in vitro and in inflammatory animal models and we recently developed a peptidomimetic called PS5, as lead compound. Here, we investigated the cellular ability of PS5 to mimic SOCS1 biological functions in vascular smooth muscle cells and simultaneously we set up a new binding assay for the screening and identification of JAK2 binders based on a SPR experiment that revealed more robust with respect to previous ELISAs. On this basis, we designed several peptidomimetics bearing new structural constraints that were analyzed in both affinities toward JAK2 and conformational features through Circular Dichroism and NMR spectroscopies. Introduced chemical modifications provided an enhancement of serum stabilities of new sequences that could aid the design of future mimetic molecules of SOCS1 as novel anti-inflammatory compounds.
577 Background: We previously described changes in EGFR kinase activity and downstream signaling pathways using IHC in tumor biopsies in 15 patients with metastatic breast cancer pre and post treatment with erlotinib (150 mg daily for 1 month). There were no clinical responses and no changes in Ki67 in tumors after erlotinib. One tumor expressed significant levels of EGFR which decreased after treatment. Aims of this study are to delineate erlotinib induced changes in gene expression profiles in EGFR positive (+) and negative (-) tumors. Methods: RNA was isolated from frozen tumor cores and gene expression profiles in 9 paired pre-post treatment tumors were measured with Affymetrix U133A GeneChip containing 22283 human genes. Results were analyzed using Affymetrix MS5.0 comparison analysis algorithm and principle components analysis. EGFR expression was validated by IHC. Results: Only 1/9 tumor expressed EGFR by IHC. cDNA array data were 100% concordant with IHC for EGFR expression. In EGFR + tumor, EGFR ...
In order to investigate the prognostic significance of erbB-2 overexpression, immunohistochemical staining for the erbB-2 protein was performed on sections from paraffin blocks of 292 primary invasive breast cancers obtained from women enrolled in the National Surgical Adjuvant Breast and Bowel Project (NSABP) protocol B-06. Positive reaction indicative of erbB-2 overexpression was observed on tumor cells in 62 (21%) samples. Women whose cancers were judged to have erbB-2 overexpression had a significantly worse overall survival (P = .0012) with twice the mortality rate of women without detectable erbB-2 expression. No statistically significant effect was evident for disease-free survival (P = .22). In multivariate analysis, detection of erbB-2 overexpression was the second most predictive independent variable for survival after nodal status. Overexpression of erbB-2 was more common among tumors of poor nuclear grade (29%) than those of good nuclear grade (12%). The association of erbB-2 overexpression with decreased survival was evident only among women with tumors of good nuclear grade. In this subgroup, erbB-2 overexpression was associated with an approximately fivefold increase in mortality rate (P = .00001). The combined predictive value of erbB-2 overexpression and nuclear grade was evident regardless of their lymph node status. These results provide evidence that detection of erbB-2 overexpression may be an independent prognostic variable for patient survival. Moreover, when combined with evaluation of nuclear grade, it may be possible to use immunostaining for erbB-2 protein to identify patients at increased risk from within a relatively low-risk group.
This paper describes the establishment of an antiestrogen-resistant MCF7 breast cancer cell subline (FASMCF) by continuous culture of the estrogen-responsive parental line in steroid-depleted, ICI 182,780 (Faslodex; 10⁻⁷m)-supplemented medium. After a 3-month period of growth suppression, cells began to proliferate in ICI 182,780 at rates similar to those of untreated wild-type cells. Immunocytochemistry showed these cells to have reduced estrogen receptor and an absence of progesterone receptor proteins. RT-PCR and transient transfection studies with estrogen response element-reporter constructs confirmed that ICI 182,780-suppressed estrogen response element-mediated signaling. FASMCF cells show increased dependence upon epidermal growth factor receptor (EgfR)/mitogen-activated protein kinase (MAPK)-mediated signaling. Thus, EgfR protein and messenger RNA, growth responses to transforming growth factor-α, and extracellular signal-regulated kinase 1/2 MAPK activation levels are all increased. Unlike wild-type cells, FASMCF cells are highly sensitive to growth inhibition by an EgfR-specific tyrosine-kinase inhibitor (TKI), ZD1839 (Iressa), and an inhibitor of the activation of MEK1 (MAPKK), PD098059. Short-term (∼3 weeks) withdrawal of cells from antiestrogen had no effect on growth or phenotype, whereas longer withdrawal (>10 weeks) appeared to partially reverse the cellular phenotype with increasing estrogen receptor and decreasing EgfR levels. In subsequent studies FASMCF cells were maintained in TKI, where their growth was again suppressed and secondary TKI resistance failed to develop within the 3-month period in which initial ICI 182,780 resistance arose. Furthermore, wild-type cells similarly maintained in combination ICI 182,780 and TKI treatment conditions remained growth arrested (>6 months), with notable cell loss through both reduced rates of cellular proliferation and increased cell death.