| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
Departments of Medicine [S. L. M., F. M. Y., R. B., C. L. A.], Cancer Biology [C. L. A.], Pathology [J. F. S.], and Vanderbilt-Ingram Cancer Center [C. L. A.], Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6307, and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 [S. K. M.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-32P]ATP in vitro onto HER2 receptors isolated from BT-474 cells was unaffected by 1 µM ZD1839. EGFR is expressed by BT-474, SKBR-3, and MDA-361 but not by MDA-453 cells, suggesting that ZD1839-mediated inhibition of the EGFR kinase explained the inhibition of HER2 phosphorylation in vivo. In SKBR-3 cells, ZD1839 exhibited a greater growth-inhibitory effect than Herceptin, a monoclonal antibody against the HER2 ectodomain. In both SKBR-3 and BT-474 cells, treatment with ZD1839 plus Herceptin induced a greater apoptotic effect than either inhibitor alone. Finally, ZD1839 completely prevented growth of BT-474 xenografts established in nude mice and enhanced the antitumor effect of Herceptin. These data imply that EGFR tyrosine kinase inhibitors will be effective against HER2-overexpressing breast tumor cells that also express EGFR and support their use in combination with HER2 antibodies, such as Herceptin, against mammary carcinomas with high levels of the HER2 proto-oncogene. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Studies with breast cancer cell lines and human tumors have demonstrated constitutive phosphorylation of HER2 (17
, 18)
. The biochemical basis for this constitutive activation is not clear, but it is consistent with the reported ability of wild-type neu, the rat homologue of human HER2, to multimerize and become activated when present at high concentrations in cells (19)
. It is unclear, however, whether this spontaneous dimerization and activation of HER2 occurs in human tumors. Another possible mechanism for activation of the HER2 tyrosine kinase in human breast cancers is the coexpression of ligand-activated EGFR, resulting in transactivation of the HER2 tyrosine kinase. This is supported by studies in which EGF was able to activate HER2 only when EGFR was also present in the same cell, suggesting transmodulation of HER2 as a result of its hetero- oligomerization with EGFR in response to EGF (20, 21, 22)
. Indeed, cooperation between EGFR and HER2 has been shown to induce accelerated transformation in studies with fibroblasts and transgenic mice (23
, 24)
. In cells that coexpress HER2, ligand-activated EGFR preferentially recruits HER2 into a heterodimeric complex that exhibits an increased rate of recycling, stability, and signaling potency compared with EGFR homodimers (2
, 5)
. In a recent report, the anti-EGFR quinazoline AG1478 suppressed mammary tumors and neu phosphorylation in MMTV/TGF
x MMTV/neu (the mouse homologue of human HER2) bigenic mice and mammary tumors, respectively (25)
, suggesting that blockade of the EGFR kinase can prevent EGFR-HER2/neu cooperation. On the other hand, inactivation of HER2/neu with single-chain HER2 antibodies or vectors encoding kinase-dead neu has been shown to impair EGFR-mediated transformation (26
, 27)
. Moreover, breast tumors that co-overexpress EGFR and HER2 exhibited a worse outcome than tumors that overexpressed either receptor alone (12)
. If receptor synergy is operational in breast cancers, interruption of EGFR function with EGFR-specific small molecular weight tyrosine kinase inhibitors may disrupt EGFR-HER2 cross-talk and result in HER2 inactivation as well. This has clinical implications because the inactivation of HER2 through the inhibition of EGFR may also increase the antitumor effect of Herceptin.
In this study, we present evidence that blockade of EGFR function with the EGFR-specific tyrosine kinase inhibitor ZD1839 (Iressa; Ref. 28 ) inhibits phosphorylation of the HER2 receptor and growth and survival of HER2-overexpressing breast carcinoma cells. We also demonstrate that in these cells, targeting the HER family signaling network at two separate molecular sites, the catalytic domain of the EGFR tyrosine kinase with ZD1839 and the ectodomain of HER2 with Herceptin, results in a greater antitumor effect both in vitro and in vivo than either agent alone. This is the first demonstration of a combined molecular approach to inhibit the HER network in HER2-overexpressing breast tumor cells, thus providing a basis for further combinations of rational anti-signaling strategies in human breast cancer.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
was from R&D Systems, Minneapolis, MN). The quinazoline ZD1839 (Iressa) was provided by Steven Averbuch (AstraZeneca Pharmaceuticals, Wilmington, DE), and Herceptin was purchased from the Vanderbilt University Hospital Pharmacy.
For immunoblot analysis and/or immunoprecipitations, the following antibodies were used: GSK-3ß and p27 (Transduction Laboratories, Lexington, KY); HER2/neu and HER3 (Neomarkers, Freemont, CA); P-Tyr (Upstate Biotechnology, Lake Placid, NY); Rb and cyclin D1 (PharMingen, San Diego, CA); MAPK, P-GSK3ß, Akt, and P-Akt (New England Biolabs, Beverly, MA); P-MAPK (Promega Corp., Madison, WI); and p85, Cdk2, and Cdk4 (Santa Cruz Biotechnology, Santa Cruz, CA). Protein in cell lysates was measured by bicinchoninic acid (Pierce, Rockford, IL) or Bio-Rad (Bio-Rad Laboratories, Hercules, CA) assays. The HA antibody HA.11 was from BabCo (Richmond, VA).
Monolayer Growth and Anchorage-independent Growth Assays.
For monolayer growth, cells were seeded at a density of 3–4 x 104 cells in 12-well plates. Twenty-four h later, ZD1839 was added to the cells. Fresh medium ± ZD1839 was replaced on day 3. On day 5, cells were harvested by trypsinization and counted with a Zeiss Coulter Counter (Beckman Coulter, Miami, FL). Colony-forming assays in soft agarose were performed as described previously (30)
. Tumor cell colonies measuring 
50 µm were counted after 7 days using an Omnicon 3800 colony counter and Tumor Colony Analysis V2.IIA software (Imaging Products International, Inc.).
Flow Cytometric Analysis of EGFR Cell Surface Expression.
Breast cancer cells were plated in 100-mm dishes. WT3 cells, containing approximately 2 x 105 EGF binding sites/cell, were used as positive controls. Cells were lifted with 0.02% EDTA in Ca2+- and Mg2+-free PBS, aliquoted into two sets of Eppendorf tubes (5 x 105 cells/tube), and washed three times with staining buffer (5% heat-inactivated FCS in PBS). One set of cells was treated with 200 µg/ml of the 528 EGFR monoclonal antibody (Santa Cruz Biotechnology) dissolved in 200 µl of staining buffer. This antibody recognizes an epitope in the extracellular portion of the EGFR. A second set of cells was treated with 1 mg/ml of a nonspecific IgG2A (Sigma Chemical Co.), incubated for 20 min on ice, and washed three times with staining buffer. This was followed by a 20-min incubation with 0.5 mg/ml of a FITC-labeled goat antimouse IgG2A (Southern Biotechnology) and 5 µl of 7-amino-actinomycin D (2 mg/ml) dissolved in 200 µl of staining buffer. All incubations were done in the dark on ice to avoid antibody and EGFR internalization. Flow cytometry of FITC-labeled cells was performed using a FACS/Calibur Flow Cytometer (Becton Dickinson, Mansfield, MA).
TUNEL Assays.
BT-474 and SKBR-3 cells in IMEM/10% FCS were treated with ZD1839, Herceptin or a combination of the two drugs. Adherent cells were harvested by scraping and pooled with floating cells. TUNEL assay was performed using the APO-BrdU kit (Phoenix Flow Systems, San Diego, CA). Flow cytometric detection of FITC-positive cells was performed using a FACS/Calibur flow cytometer.
Immunoblot Analysis and Immunoprecipitation.
Cells were washed twice with ice-cold PBS, scraped in EBC lysis buffer [50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% NP40, 100 mM NaF, 200 µM Na3VO4, and 10 µg/ml each aprotinin, leupeptin, PMSF, and pepstatin], and incubated for 20 min at 4°C while rocking. Lysates were cleared by centrifugation (10 min at 12,000 rpm, 4°C). Fifty µg protein were resolved by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblot analysis as described previously (30)
. Immunoreactive bands were detected using chemiluminescence (Roche Molecular Biochemicals, Indianapolis, IN). For immunoprecipitations, 1 mg of total protein from cell lysates was incubated overnight with primary antibody at 4°C; protein A-Sepharose (Sigma Chemical Co.) or protein G-Sepharose (Pharmacia) was then added for 2 h at 4°C while rocking. The precipitates were washed four times with ice-cold PBS, resuspended in 6x Laemmli sample buffer, and resolved using SDS-PAGE followed by immunoblot analysis.
In Vitro Kinase Assays.
The HER2 in vitro kinase reaction was performed as described previously (31)
with modifications. BT-474 cells were lysed in EBC buffer. One mg of total protein was precipitated with the HER2 antibody Ab-7 (Neomarkers) and protein A-Sepharose beads. Immunoprecipitates were washed three times with 20 mM HEPES (pH 7.5), 0.5 M LiCl and resuspended in kinase assay buffer [20 mM HEPES (pH 7.5), 10 mM MgCl, and 2 mM MnCl]. Kinase activity in the immune complexes was tested by adding 5 µCi [-32P]ATP (specific activity, 3000 Ci/mmol; Amersham Pharmacia) and 10 µM cold ATP (Sigma Chemical Co.). After a 10-min incubation at room temperature, the reaction was stopped by heating and adding Laemmli buffer; kinase products were resolved by SDS-PAGE, transferred to nitrocellulose, and exposed to X-ray film. The same nitrocellulose membrane was rinsed and then subjected to a HER2 immunoblot procedure (above).
For determination of PI3K activity, BT-474 cells were seeded at a density of 5 x 105 cells/100-mm dish 24 h before treatment with ZD1839. After treatment, the cells were washed twice with 137 mM NaCl, 20 mM Tris (pH 7.5), 1 mM CaCl2, 1 mM MgCl2, and lysed in the wash buffer supplemented with 10% glycerol, 1% NP40, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM NaF, and 200 µM Na3VO4. Cell extracts precleared by centrifugation were precipitated overnight with a P-Tyr monoclonal antibody (Upstate Biotechnology) and protein A-Sepharose. Immune complexes were washed three times with 1% NP40 in PBS; two times with 100 mM Tris (pH 7.5), 0.5 M LiCl; two times with 10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA; and two times with kinase assay buffer [10 mM Tris (pH 7.5), 100 mM NaCl, 4 mM MgCl2, 1 mM EDTA, and 0.5 mM EGTA]. All wash buffers contained 200 µM Na3VO4. The beads were suspended in 40 µl of kinase buffer, followed by addition of 10 µCi of [-32P]ATP and sonication in 0.2 mg/ml phosphatidylinositol 4,5-bisphosphate (Avanti). The kinase reaction proceeded for 10 min at room temperature and was terminated with stop buffer (1:1 methanol:HCl), followed by extraction with chloroform. The reaction products were separated by TLC on 1% oxalate pretreated TLC with chloroform:methanol:acetone:glacial acetic acid:water (60:20:23:18:11) and detected by autoradiography.
Immunofluorescent Localization of p27KIP1.
BT-474 cells were seeded on coverslips in six-well plates at a density of 4 x 104 cells/well. After a 12-h incubation with ZD1839, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 10 min, washed, and stored overnight at 4°C. The cells were permeabilized with 0.1% Triton X-100/PBS for 15 min, washed, and then incubated for 1 h with a p27 monoclonal antibody (Transduction Laboratories) diluted 1:250 in 0.05% Triton X-100/PBS. After extensive washes, the cells were incubated for 45 min with antimouse Cy3 IgG (Jackson Immunoresearch Labs., West Grove, PA) diluted 1:500 in 0.05% Triton X-100/PBS. The cells were then washed six times with 0.05% Triton X-100/PBS, stained with 1 mg/ml Hoechst, and mounted in AquaPoly Mount (PolySciences, Inc.). Cy3 immunofluorescence was recorded with a Princeton Instruments cooled digital CCD camera on a Zeiss Axiophot upright microscope.
Xenograft Studies in Athymic Mice.
Five-week-old female Balb/C athymic nude mice (Harlan Sprague Dawley, Madison, WI) were implanted with 0.72-mg, 60-day release, 17ß-estradiol pellets (Innovative Research, Sarasota, FL). The next day, 2.5 x 107 BT-474 cells suspended in (300 µl) Growth Factor Reduced Matrigel (BD Biosciences, Bedford, MA) were injected s.c. in the right flank. Once tumors reached a volume 200 mm3, seven to eight mice/group were randomly allocated to treatment with: (a) vehicles: 0.05% Tween 80 by oral gavage daily and sterile PBS by i.p. injection twice a week; (b) 10 mg/kg Herceptin in sterile PBS, administered by i.p injection twice a week, and Tween 80 vehicle daily; (c) ZD1839 in 0.05% Tween 80, administered at 200 mg/kg/day by oral gavage, and sterile PBS i.p. twice a week; and (d) Herceptin and ZD1839. Tumor diameters were serially measured with calipers, and tumor volumes were calculated by the formula: volume = width2 x length/2. After 4 weeks of treatment, 2 mice/group were injected with a single dose of BrdUrd (100 mg/kg), and their tumors were harvested 2 h later. Other tumors were homogenized using a Polytron homogenizer (Brinkmann, Westbury, NY) in TNE lysis buffer [50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 1 µg/ml pepstatin A, 2 µg/ml aprotinin, and 0.5 µg/ml leupeptin]. After homogenization, 1% (v/v) NP40 was added. Equivalent amounts of protein from the tumor lysates were next subjected to immunoblot analysis. For histological analysis, excised tumors were embedded in paraffin, sectioned, and stained with H&E. BrdUrd labeling of tumor cell nuclei was visualized by staining the sections with BrdUrd antibody (Biogenics, San Ramon, CA) using a streptavidin-biotin stain (Histo-mouse kit; Zymed, South San Francisco, CA). Additional sections were used to stain for apoptotic cells using the DNA in situ nick end-labeling (TUNEL) immunohistochemical method (Intergen, Purchase, NY) according to the manufacturer’s instructions.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, Lanes 1 and 2). Consistent with the in vitro IC50 for EGFR, addition of 
0.1 µM ZD1839 markedly inhibited ligand-induced EGFR phosphorylation, whereas >1 µM was required to block HER2 phosphorylation, thus confirming that ZD1839 is specific for EGFR in vivo.
|
. In contrast, treatment with 10 µM ZD1839 was required to moderately reduce HER2 phosphorylation in the EGFR-negative MDA-453 cells. The ability of low concentrations of ZD1839 to inhibit HER2 phosphorylation in MDA-361, SKBR-3, and BT-474 cells implied a dissociation between the effects of the inhibitor on the HER2 kinase in vitro versus in vivo. Therefore, we next performed a more detailed dose response of ZD1839 on HER2 receptors in intact BT-474 cells versus HER2 receptors isolated from the same cell line. Treatment of BT-474 cells with 1 µM ZD1839 resulted in near complete inhibition of HER2 phosphorylation in vivo (Fig. 2B)
. In contrast, increasing the concentrations of ZD1839 to 2–5 µM only modestly reduced the phosphorylation of isolated HER2 receptors in an in vitro kinase reaction (Fig. 2C)
, suggesting that ZD1839 was a far better inhibitor of the HER2 kinase in intact cells. This result suggests that ZD1839 uses an indirect mechanism for HER2 inhibition in intact cells but does not directly inhibit the catalytic activity of HER2.
|
1 µM concentrations of ZD1839 for 5 days substantially impaired cell proliferation compared with controls. Higher concentrations of ZD1839 (10 µM) were required to inhibit MDA-361 cells but had no effect on MDA-453 cells (Fig. 3A)
. Similarly, 1 µM ZD1839 markedly inhibited SKBR-3 and BT-474 colony formation, whereas 10-fold higher concentrations were required to reduce MDA-361 and MDA-453 colony growth. However, in this assay the IC50 of ZD1839 was 2 µM and >5 µM for MDA-361 and MDA-453 cells, respectively (data not shown).
|
. In addition to altering p27 levels, treatment with ZD1839 induced redistribution of p27 from the cytosol to the nucleus as detected by immunofluorescence (Fig. 4B)
. These effects on Akt, MAPK, and p27 were not seen in the EGFR-negative MDA-453 cells (Fig. 4C)
.
|
, left), indicating the presence of constitutive HER2/HER3 complexes. HER3 exhibited a low level of basal tyrosine phosphorylation and was constitutively associated with p85, the regulatory subunit of PI3K (Fig. 5A
, right). Treatment with 1 µM ZD1839 for 20 h eliminated the association of HER3 with HER2, the association of HER3 with p85, and the low level of HER3 basal phosphorylation. Consistent with the loss of association of HER3 with PI3K, ZD1839 eliminated PI3K activity in the HER2-overexpressing cells as measured by the ability of P-Tyr precipitates from BT-474 cell lysates to induce the formation of phosphatidylinositol 3,4,5 trisphosphate from phosphatidylinositol 4,5 bisphosphate in vitro (Fig. 5B)
.
|
1 µM) inhibited SKBR-3 colony formation in soft agarose. Herceptin also inhibited colony formation but less than ZD1839, and the combination was not more potent than ZD1839 alone (Fig. 6A)
. In this assay, BT-474 cells were exquisitely sensitive to either ZD1839 or Herceptin with an IC50 of 0.2 µM and 0.1 µg/ml, respectively (data not shown). Because of the exquisite sensitivity of BT-474 colonies to either inhibitor, we were unable to demonstrate a synergistic or supra-additive effect of the combination against these cells in this assay. Because the colony-forming assay measures both anchorage-independent proliferation and survival, we next focused on an effect of the combination on tumor cell apoptosis as measured by DNA double-strand breaks in the presence of terminal deoxynucleotidyl transferase. In SKBR-3, treatment with either Herceptin or ZD1839 for 72 h induced a 3-fold increase in the basal level of apoptosis with the combination inducing a 6–7-fold supra-additive effect. On the other hand, neither inhibitor induced apoptosis per se of BT-474 cells. However, BT-474 cells treated with both Herceptin and ZD1839 exhibited a 5-fold increase in apoptosis compared with untreated controls (Fig. 6B)
.
|
-induced BT-474 Cell Proliferation Better Than Herceptin.-induced proliferation of BT-474 cells. Addition of 20 ng/ml (3 nM) TGF- resulted in a 2.5-fold increase in cell number over 5 days. This increase was completely abrogated by 1 µM ZD1839 but not by a saturating concentration (20 µg/ml) of Herceptin (Fig. 7)
.
|
200 mm3, tumor-bearing nude mice were randomly allocated to either no treatment, ZD1839, Herceptin, or the combination. ZD1839 completely prevented tumor growth but did not induce complete remissions (n = 8). Herceptin alone induced complete remission in two of seven, whereas the combination resulted in three of eight complete responses (P = 0.06, Student’s unpaired t test; Fig. 8
). No mice exhibited treatment-related toxicity. Three mice treated with ZD1839 plus Herceptin, in which tumors regressed completely, remained tumor free for >6 months after discontinuation of therapy and had no detectable tumor at necropsy.
|
, but this decrease was more marked in the tumors treated with ZD1839. The antiproliferative effect of the combination was statistically greater than that induced by Herceptin alone (10.4 versus 22% BrdUrd-positive tumor cells, respectively; P = 0.001, Student’s unpaired t test). The total number of apoptotic bodies in 10 high-power fields as detected by TUNEL analysis was not different among tumors from any of the treatment groups (data not shown). Finally, we examined for evidence of inhibition of HER2 signaling as a result of treatment. HER2 content as measured by immunoblot analysis of tumor lysates was roughly equal in all groups. In all treatment groups, Rb was hypophosphorylated, and both active MAPK and Akt were inhibited with no change in total MAPK and Akt tumor levels (Fig. 8C)
. These data suggest that, in addition to arresting or eliminating xenografts, all three treatments interfered with HER2 signaling in situ. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1 µM ZD1839 to inhibit the catalytic activity of HER2 in an in vitro kinase reaction using HER2 receptors isolated from BT-474 cells (Fig. 2C)
; (b) the requirement of 10 µM ZD1839 to inhibit the phosphorylation of HER2-FKBP chimeric receptors in response to AP1510 (Fig. 1)
; and (c) the inability of 1 µM ZD1839 to inhibit HER2 phosphorylation and downstream signaling pathways in the EGFR-negative MDA-453 cells (Figs. 2A
and 4C
). Although growth inhibition was seen in all cell lines with 10 µM ZD1839, it is possible that at these concentrations, ZD1839 cross-reacts nonspecifically with HER2 and other EGFR-homologous kinases. Of note, maximal steady-state (trough) plasma levels of ZD1839 of 
1 µM have been achieved in patients at the recommended clinical dose of (0.5 mg/day) ZD1839.7
This suggests that concentrations >1 µM, at which ZD1839 could interact with other targets nonspecifically, may not be practically achievable. Therefore, at concentrations that would not have been predicted from an in vitro EGFR kinase reaction, EGFR-specific inhibitors also block, albeit indirectly, the HER2 kinase in cells that also coexpress EGFR. These results also suggest that, in addition to EGFR levels, the coexpression of HER receptors and their functional interaction with EGFR in different tumor cell backgrounds might dictate the true antitumor effect of some ATP-competitive inhibitors of the EGFR tyrosine kinase.
Interestingly, ZD1839 also disrupted the previously reported constitutive association of HER2 with HER3 in cells that overexpress HER2 (17
, 43)
. Treatment with ZD1839 resulted in the uncoupling of HER3 from p85 and loss of the basal PI3K activity in HER2-overexpressing cells (Fig. 5)
. This result is somewhat surprising in that there is no strong rationale to suspect an interaction of the quinazoline with the less EGFR-homologous, kinase-impaired HER3 receptor. It has been reported, however, that the anti-EGFR quinazolines AG1478 and AG1517, highly similar in structure to ZD1839, can induce the formation of inactive EGFR homodimers as well as inactive EGFR/HER2 heterodimers (44)
. Lichtner et al. (45)
reported recently that ZD1839 was also capable of inducing the formation of signaling-inactive EGFR complexes. In SKBR-3 cells, the inactive heterodimerization of EGFR/HER2 mediated by AG1517 was temporally associated with loss of the growth response to the HER3 ligand heregulin (44)
. In another study, treatment of PC12 rat cells with EGF induced primary EGFR/Neu heterodimers and secondary Neu/ErbB3 (HER2/HER3) heterodimers (46)
. The formation of EGF-induced secondary receptor heterodimers was eliminated by the EGFR-specific kinase inhibitor AG1478 (47)
but modestly inhibited by the Neu kinase inhibitor AG879 (46)
, suggesting that EGFR-mediated transmodulation of Neu (the rat homologue of human HER2) is essential for the formation of Neu/ErbB3 secondary dimers. This result raises the speculation that in cells with high levels of constitutively active HER2 that also express EGFR and HER3, the inhibition of EGFR/HER2 cross-talk with ZD1839 may lead secondarily to the uncoupling of HER2/HER3, as implied by the result shown in Fig. 5
. Taken together, these data suggest that some EGFR kinase inhibitors, such as ZD1839, may have the ability to prevent HER2 from heterodimerizing with other HER receptors in a dominant-negative manner and thus widely affect signaling by the HER (erbB) network.
Blockade of the EGFR kinase with ZD1839 was markedly more effective than Herceptin in inhibiting TGF--induced cell proliferation in BT-474 cells (Fig. 7)
. This result is also consistent with the report by Gamett et al. (46)
; the HER1-specific kinase inhibitor AG1478 almost completely eliminated EGF-induced (trans) phosphorylation of Neu, whereas AG879, a direct inhibitor of Neu autophosphorylation with no activity against EGFR (47
, 48) , had a modest effect. In another study, treatment of OVCA420 ovarian cancer cells with EGF induced phosphorylation of HER2 that was not blocked by mAb 4D5, the mouse homologue of Herceptin (49)
. In this same study, mAb C225, an anti-EGFR humanized IgG1 that binds to the ectodomain of the receptor, induces receptor homodimerization, blocks ligand binding to EGFR, and was unable to block basal or EGF-induced tyrosine phosphorylation of HER2 (49)
. In addition, a constitutively active mutant form of EGFR, lacking the extracellular domain and the hydrophobic leader sequence, associated strongly with HER2, despite its inability to insert into the plasma membrane (39)
. Finally, EGF induces efficient homodimerization of purified EGFR extracellular domains but is unable to stabilize EGFR/HER2 hetero-oligomers of receptor extracellular domains (40)
. Taken together, these data and the results shown in Fig. 7
point to the role of the EGFR kinase on HER2 transmodulation. If so, targeting the kinase domain with therapeutic inhibitors might be a more effective strategy to rapidly disable lateral signaling within the HER network than the use of bivalent antibodies against the ectodomain of the receptor.
Combined treatment with ZD1839 and Herceptin resulted in enhanced tumor cell apoptosis (Fig. 6)
and larger tumor reduction than either inhibitor alone (Fig. 8)
. It is conceivable that the combination exerts a more effective or sustained inhibition of survival signals up-regulated by the HER signaling network. Elucidation of these pathways to explain the supra-additive antitumor effect will require further investigation. Nonetheless, these results suggest that simultaneous blockade of different molecular sites within the HER network may diminish potential compensatory mechanisms by tumor cells compared with more limited targeted approaches against this signaling network. Of note, ZD1839 arrested tumor growth (Fig. 8)
and markedly inhibited BrdUrd incorporation in tumor cells but failed to completely eradicate eight of eight BT-474 xenografts (Fig. 9). On the other hand and as published previously (41)
, Herceptin induced two of seven complete tumor regressions but induced less detectable inhibition of tumor cell proliferation as measured by BrdUrd incorporation. This result suggests the possibility that Herceptin may induce cancer regressions by tumor cell nonautonomous mechanisms as suggested by Clynes et al. (50)
. In this stimulating report, BT-474 xenografts were established in mice deficient in the Fc receptor FcRIII, which is involved in the recruitment of immune cells that mediate antibody-dependent cell-mediated cytotoxicity. In these mice, the antitumor effect of Herceptin against BT-474 xenografts was drastically reduced. Moreover, HER2 antibodies engineered to disrupt Fc binding did not arrest tumor growth in vivo (50)
, suggesting that Fc receptor-dependent mechanisms contribute substantially to the cytotoxic effect of Herceptin. Nonetheless, the data presented in Fig. 9 clearly demonstrate that Herceptin and ZD1839 inhibited signaling molecules downstream HER2 in the BT-474 xenografts. This is the first demonstration of an anti-signaling effect of Herceptin in vivo. This tumor cell-autonomous effect may complement the potential Fc receptor-mediated mechanism of tumor elimination proposed by Clynes et al. (50)
.
In summary, we have presented data to support the use of EGFR-specific tyrosine kinase inhibitors against EGFR-positive, HER2-overexpressing human breast tumor cells. ZD1839, the ATP-competitive inhibitor of the EGFR tyosine kinase used in these studies, effectively disrupted HER2/HER3 interactions, completely prevented TGF--induced mitogenesis of tumor cells with high HER2 levels, and synergized with the HER2 antibody Herceptin against SKBR-3 and BT-474 cells. Taken together, these data support the use of EGFR kinase inhibitors in combination with Herceptin in patients bearing breast cancers with HER2 overexpression. Whether the combination will be more effective against tumors that also express a threshold level of EGFR, as implied by the studies herein, will require prospective investigation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by NIH Grant R01 CA80195 (to C. L. A.), a Department of Veteran Affairs Clinical Investigator Award (to C. L. A.), a Massachusetts State Breast Cancer Grant (to S. K. M.), and Vanderbilt-Ingram Cancer Center Support Grant CA68485. S. L. M. is the recipient of a Translational Research Fellowship Award from the American Association for Cancer Research. 
3 Present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724.
4 To whom requests for reprints should be addressed, at Division of Oncology, Vanderbilt University, 777 Preston Research Building, Nashville, TN 37232-6307. Phone: (615) 936-3524; Fax: (615) 936-1790; E-mail: carlos.arteaga{at}mcmail.vanderbilt.edu
5 The abbreviations used are: EGFR, epidermal growth factor receptor; TGF, transforming growth factor; GSK, glycogen synthase kinase; TUNEL, terminal deoxynucleotide transferase dUTP nick end labeling; PMSF, phenylmethylsulfonyl fluoride; PI3K, phosphatidylinositol-3 kinase; BrdUrd, bromodeoxyuridine; HA, hemagglutinin antigen; FKBP, FK506-binding protein; Cdk, cyclin-dependent kinase; mAb, monoclonal antibody.
6 Alan Wakeling, AstraZeneca Pharmaceuticals, personal communication.
7 Steven Averbuch, personal communication.
Received 8/30/01. Accepted 11/ 1/01.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in the mammary epithelium of transgenic mice. Mol. Cell. Biol., 16: 5726-5736, 1996.[Abstract]
bigenic mice. Proc. Natl. Acad. Sci. USA, 97: 9609-9614, 2000.-Pseudomonas exotoxin 40 fusion protein. Cancer Res., 54: 4703-4709, 1994. or GTPase-activating protein, distinguishes ErbB-3 signaling from that of other ErbB/EGFR family members. Mol. Cell. Biol., 14: 492-500, 1994.This article has been cited by other articles:
|
|
 |
|
  D. Lesniak, Y. Xu, J. Deschenes, R. Lai, J. Thoms, D. Murray, S. Gosh, J. R. Mackey, S. Sabri, and B. Abdulkarim {beta}1-Integrin Circumvents the Antiproliferative Effects of Trastuzumab in Human Epidermal Growth Factor Receptor-2-Positive Breast Cancer Cancer Res., November 15, 2009; 69(22): 8620 - 8628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Normanno, A. Morabito, A. De Luca, M. C. Piccirillo, M. Gallo, M. R Maiello, and F. Perrone Target-based therapies in breast cancer: current status and future perspectives Endocr. Relat. Cancer, September 1, 2009; 16(3): 675 - 702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Lu, Q. Wang, G. Hu, C. Van Poznak, M. Fleisher, M. Reiss, J. Massague, and Y. Kang ADAMTS1 and MMP1 proteolytically engage EGF-like ligands in an osteolytic signaling cascade for bone metastasis Genes & Dev., August 15, 2009; 23(16): 1882 - 1894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Kedrin, J. Wyckoff, P. J. Boimel, S. J. Coniglio, N. E. Hynes, C. L. Arteaga, and J. E. Segall ERBB1 and ERBB2 Have Distinct Functions in Tumor Cell Invasion and Intravasation Clin. Cancer Res., June 1, 2009; 15(11): 3733 - 3739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Liu, M. Abdelrahim, A. Abudayyeh, P. Lei, and S. Safe The nonsteroidal anti-inflammatory drug tolfenamic acid inhibits BT474 and SKBR3 breast cancer cell and tumor growth by repressing erbB2 expression Mol. Cancer Ther., May 1, 2009; 8(5): 1207 - 1217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Abba, Y. Hu, C. C. Levy, S. Gaddis, F. S. Kittrell, J. Hill, R. P. Bissonnette, P. H. Brown, D. Medina, and C. M. Aldaz Identification of Modulated Genes by Three Classes of Chemopreventive Agents at Preneoplastic Stages in a p53-Null Mouse Mammary Tumor Model Cancer Prevention Research, February 1, 2009; 2(2): 175 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Strecker, Q. Shen, Y. Zhang, J. L. Hill, Y. Li, C. Wang, H.-T. Kim, T. M. Gilmer, K. R. Sexton, S. G. Hilsenbeck, et al. Effect of Lapatinib on the Development of Estrogen Receptor-Negative Mammary Tumors in Mice J Natl Cancer Inst, January 21, 2009; 101(2): 107 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Li, H. Wang, E. S. Yang, C. L. Arteaga, and F. Xia Erlotinib Attenuates Homologous Recombinational Repair of Chromosomal Breaks in Human Breast Cancer Cells Cancer Res., November 15, 2008; 68(22): 9141 - 9146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Arteaga, A. O'Neill, S. L. Moulder, M. Pins, J. A. Sparano, G. W. Sledge, and N. E. Davidson A Phase I-II Study of Combined Blockade of the ErbB Receptor Network with Trastuzumab and Gefitinib in Patients with HER2 (ErbB2)-Overexpressing Metastatic Breast Cancer Clin. Cancer Res., October 1, 2008; 14(19): 6277 - 6283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Whittington, M. P. Piechocki, H. H. Heng, J. B. Jacob, R. F. Jones, J. B. Back, and W.-Z. Wei DNA Vaccination Controls Her-2+ Tumors that Are Refractory to Targeted Therapies Cancer Res., September 15, 2008; 68(18): 7502 - 7511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. T. Lee-Hoeflich, L. Crocker, E. Yao, T. Pham, X. Munroe, K. P. Hoeflich, M. X. Sliwkowski, and H. M. Stern A Central Role for HER3 in HER2-Amplified Breast Cancer: Implications for Targeted Therapy Cancer Res., July 15, 2008; 68(14): 5878 - 5887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Rowe, T. Ozbay, L. M. Bender, and R. Nahta Nordihydroguaiaretic acid, a cytotoxic insulin-like growth factor-I receptor/HER2 inhibitor in trastuzumab-resistant breast cancer Mol. Cancer Ther., July 1, 2008; 7(7): 1900 - 1908. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. L. Gomez, D. C. Doval, M. A. Chavez, P. C.-S. Ang, Z. Aziz, S. Nag, C. Ng, S. X. Franco, L. W.C. Chow, M. C. Arbushites, et al. Efficacy and Safety of Lapatinib As First-Line Therapy for ErbB2-Amplified Locally Advanced or Metastatic Breast Cancer J. Clin. Oncol., June 20, 2008; 26(18): 2999 - 3005. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Burstein, A. M. Storniolo, S. Franco, J. Forster, S. Stein, S. Rubin, V. M. Salazar, and K. L. Blackwell A phase II study of lapatinib monotherapy in chemotherapy-refractory HER2-positive and HER2-negative advanced or metastatic breast cancer Ann. Onc., June 1, 2008; 19(6): 1068 - 1074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Guix, N. de Matos Granja, I. Meszoely, T. B. Adkins, B. M. Wieman, K. E. Frierson, V. Sanchez, M. E. Sanders, A. M. Grau, I. A. Mayer, et al. Short Preoperative Treatment With Erlotinib Inhibits Tumor Cell Proliferation in Hormone Receptor-Positive Breast Cancers J. Clin. Oncol., February 20, 2008; 26(6): 897 - 906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Massarweh, C. K. Osborne, C. J. Creighton, L. Qin, A. Tsimelzon, S. Huang, H. Weiss, M. Rimawi, and R. Schiff Tamoxifen Resistance in Breast Tumors Is Driven by Growth Factor Receptor Signaling with Repression of Classic Estrogen Receptor Genomic Function Cancer Res., February 1, 2008; 68(3): 826 - 833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Emanuel, T. V. Hughes, M. Adams, C. A. Rugg, A. Fuentes-Pesquera, P. J. Connolly, N. Pandey, S. Moreno-Mazza, J. Butler, V. Borowski, et al. Cellular and in Vivo Activity of JNJ-28871063, A Nonquinazoline Pan-ErbB Kinase Inhibitor That Crosses the Blood-Brain Barrier and Displays Efficacy against Intracranial Tumors Mol. Pharmacol., February 1, 2008; 73(2): 338 - 348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Harari, G. W. Allen, and J. A. Bonner Biology of Interactions: Antiepidermal Growth Factor Receptor Agents J. Clin. Oncol., September 10, 2007; 25(26): 4057 - 4065. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Ritter, M. Perez-Torres, C. Rinehart, M. Guix, T. Dugger, J. A. Engelman, and C. L. Arteaga Human Breast Cancer Cells Selected for Resistance to Trastuzumab In vivo Overexpress Epidermal Growth Factor Receptor and ErbB Ligands and Remain Dependent on the ErbB Receptor Network Clin. Cancer Res., August 15, 2007; 13(16): 4909 - 4919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Lahusen, M. Fereshteh, A. Oh, A. Wellstein, and A. T. Riegel Epidermal Growth Factor Receptor Tyrosine Phosphorylation and Signaling Controlled by a Nuclear Receptor Coactivator, Amplified in Breast Cancer 1 Cancer Res., August 1, 2007; 67(15): 7256 - 7265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Yamasaki, D. Zhang, C. Bartholomeusz, T. Sudo, G. N. Hortobagyi, K. Kurisu, and N. T. Ueno Sensitivity of breast cancer cells to erlotinib depends on cyclin-dependent kinase 2 activity Mol. Cancer Ther., August 1, 2007; 6(8): 2168 - 2177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Piechocki, G. H. Yoo, S. K. Dibbley, and F. Lonardo Breast Cancer Expressing the Activated HER2/neu Is Sensitive to Gefitinib In vitro and In vivo and Acquires Resistance through a Novel Point Mutation in the HER2/neu Cancer Res., July 15, 2007; 67(14): 6825 - 6843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Jimeno, B. Rubio-Viqueira, M. L. Amador, V. Grunwald, A. Maitra, C. Iacobuzio-Donahue, and M. Hidalgo Dual mitogen-activated protein kinase and epidermal growth factor receptor inhibition in biliary and pancreatic cancer Mol. Cancer Ther., March 1, 2007; 6(3): 1079 - 1088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Schaefer, L. Shao, K. Totpal, and R. W. Akita Erlotinib Directly Inhibits HER2 Kinase Activation and Downstream Signaling Events in Intact Cells Lacking Epidermal Growth Factor Receptor Expression Cancer Res., February 1, 2007; 67(3): 1228 - 1238. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Araujo, R. Ribeiro, I. Azevedo, A. Coelho, M. Soares, B. Sousa, D. Pinto, C. Lopes, R. Medeiros, and G. V. Scagliotti Genetic Polymorphisms of the Epidermal Growth Factor and Related Receptor in Non-Small Cell Lung Cancer--A Review of the Literature Oncologist, February 1, 2007; 12(2): 201 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Toschi and F. Cappuzzo Understanding the New Genetics of Responsiveness to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Oncologist, February 1, 2007; 12(2): 211 - 220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Ariazi, R. J. Kraus, M. L. Farrell, V. C. Jordan, and J. E. Mertz Estrogen-Related Receptor {alpha}1 Transcriptional Activities Are Regulated in Part via the ErbB2/HER2 Signaling Pathway Mol. Cancer Res., January 1, 2007; 5(1): 71 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Perez-Torres, M. Guix, A. Gonzalez, and C. L. Arteaga Epidermal Growth Factor Receptor (EGFR) Antibody Down-regulates Mutant Receptors and Inhibits Tumors Expressing EGFR Mutations J. Biol. Chem., December 29, 2006; 281(52): 40183 - 40192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ono and M. Kuwano Molecular Mechanisms of Epidermal Growth Factor Receptor (EGFR) Activation and Response to Gefitinib and Other EGFR-Targeting Drugs Clin. Cancer Res., December 15, 2006; 12(24): 7242 - 7251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yan, Y. Lu, M. Wang, H. Vikis, R. Yao, Y. Wang, R. A. Lubet, and M. You Effect of an Epidermal Growth Factor Receptor Inhibitor in Mouse Models of Lung Cancer Mol. Cancer Res., December 1, 2006; 4(12): 971 - 981. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.-H. Tseng, Y.-C. Wang, S.-C. Weng, J.-R. Weng, C.-S. Chen, R. W. Brueggemeier, C. L. Shapiro, C.-Y. Chen, S. E. Dunn, M. Pollak, et al. Overcoming Trastuzumab Resistance in HER2-Overexpressing Breast Cancer Cells by Using a Novel Celecoxib-Derived Phosphoinositide-Dependent Kinase-1 Inhibitor Mol. Pharmacol., November 1, 2006; 70(5): 1534 - 1541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lanza-Jacoby, R. Burd, F. E. Rosato Jr., K. McGuire, J. Little, N. Nougbilly, and S. Miller Effect of Simultaneous Inhibition of Epidermal Growth Factor Receptor and Cyclooxygenase-2 in HER-2/Neu-Positive Breast Cancer. Clin. Cancer Res., October 15, 2006; 12(20): 6161 - 6169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Wang, I. Shin, F. Y. Wu, D. B. Friedman, and C. L. Arteaga HER2/Neu (ErbB2) Signaling to Rac1-Pak1 Is Temporally and Spatially Modulated by Transforming Growth Factor {beta} Cancer Res., October 1, 2006; 66(19): 9591 - 9600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Massarweh, C. K. Osborne, S. Jiang, A. E. Wakeling, M. Rimawi, S. K. Mohsin, S. Hilsenbeck, and R. Schiff Mechanisms of Tumor Regression and Resistance to Estrogen Deprivation and Fulvestrant in a Model of Estrogen Receptor-Positive, HER-2/neu-Positive Breast Cancer Cancer Res., August 15, 2006; 66(16): 8266 - 8273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Arteaga Can Trastuzumab Be Effective Against Tumors With Low HER2/Neu (ErbB2) Receptors? J. Clin. Oncol., August 10, 2006; 24(23): 3722 - 3725. [Full Text] [PDF]
|
|
|
|
|
|
L. Jerome, N. Alami, S. Belanger, V. Page, Q. Yu, J. Paterson, L. Shiry, M. Pegram, and B. Leyland-Jones Recombinant Human Insulin-like Growth Factor Binding Protein 3 Inhibits Growth of Human Epidermal Growth Factor Receptor-2-Overexpressing Breast Tumors and Potentiates Herceptin Activity In vivo. Cancer Res., July 15, 2006; 66(14): 7245 - 7252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Engelman and L. C. Cantley The Role of the ErbB Family Members in Non-Small Cell "Lung Cancers Sensitive to Epidermal Growth Factor Receptor Kinase Inhibitors". Clin. Cancer Res., July 15, 2006; 12(14): 4372s - 4376s. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-C. Wen, S.-A. Cheng, S.-P. Hsuen, Y.-L. Huang, Z.-K. Kuo, H.-F. Lee, C.-H. Kuo, J.-L. Du, and W.-B. Wang SV40 T/t-Common Polypeptide Specifically Induces Apoptosis in Human Cancer Cells that Overexpress HER2/neu Cancer Res., June 1, 2006; 66(11): 5847 - 5857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhan, B. Xiang, and S. K. Muthuswamy Controlled Activation of ErbB1/ErbB2 Heterodimers Promote Invasion of Three-Dimensional Organized Epithelia in an ErbB1-Dependent Manner: Implications for Progression of ErbB2-Overexpressing Tumors. Cancer Res., May 15, 2006; 66(10): 5201 - 5208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Pino, M. Shrader, C. H. Baker, F. Cognetti, H. Q. Xiong, J. L. Abbruzzese, and D. J. McConkey Transforming Growth Factor {alpha} Expression Drives Constitutive Epidermal Growth Factor Receptor Pathway Activation and Sensitivity to Gefitinib (Iressa) in Human Pancreatic Cancer Cell Lines. Cancer Res., April 1, 2006; 66(7): 3802 - 3812. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Yang, Y. Liu, M. A. Lemmon, and M. G. Kazanietz Essential Role for Rac in Heregulin {beta}1 Mitogenic Signaling: a Mechanism That Involves Epidermal Growth Factor Receptor and Is Independent of ErbB4 Mol. Cell. Biol., February 1, 2006; 26(3): 831 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. E. Konecny, M. D. Pegram, N. Venkatesan, R. Finn, G. Yang, M. Rahmeh, M. Untch, D. W. Rusnak, G. Spehar, R. J. Mullin, et al. Activity of the Dual Kinase Inhibitor Lapatinib (GW572016) against HER-2-Overexpressing and Trastuzumab-Treated Breast Cancer Cells Cancer Res., February 1, 2006; 66(3): 1630 - 1639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Xia, J. Bisi, J. Strum, L. Liu, K. Carrick, K. M. Graham, A. L. Treece, M. A. Hardwicke, M. Dush, Q. Liao, et al. Regulation of Survivin by ErbB2 Signaling: Therapeutic Implications for ErbB2-Overexpressing Breast Cancers Cancer Res., February 1, 2006; 66(3): 1640 - 1647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Shin, T. Miller, and C. L. Arteaga ErbB Receptor Signaling and Therapeutic Resistance to Aromatase Inhibitors Clin. Cancer Res., February 1, 2006; 12(3): 1008s - 1012s. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Normanno, M. Di Maio, E. De Maio, A. De Luca, A. de Matteis, A. Giordano, F. Perrone, and on behalf of the NCI-Naples Breast Cancer Group Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer Endocr. Relat. Cancer, December 1, 2005; 12(4): 721 - 747. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Kurokawa, Y Arimura, H Yamamoto, Y Adachi, T Endo, T Sato, T Suga, M Hosokawa, Y Shinomura, and K Imai Tumour matrilysin expression predicts metastatic potential of stage I (pT1) colon and rectal cancers Gut, December 1, 2005; 54(12): 1751 - 1758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Arora and E. M. Scholar Role of Tyrosine Kinase Inhibitors in Cancer Therapy J. Pharmacol. Exp. Ther., December 1, 2005; 315(3): 971 - 979. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Albitar, L. L. Laidler, R. Abdallah, and K. K. Leslie Regulation of signaling phosphoproteins by epidermal growth factor and Iressa (ZD1839) in human endometrial cancer cells that model type I and II tumors Mol. Cancer Ther., December 1, 2005; 4(12): 1891 - 1899. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Stegmaier, S. M. Corsello, K. N. Ross, J. S. Wong, D. J. DeAngelo, and T. R. Golub Gefitinib induces myeloid differentiation of acute myeloid leukemia Blood, October 15, 2005; 106(8): 2841 - 2848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Normanno, M. Campiglio, F. Perrone, A. De Luca, and S. Menard Is the gefitinib plus trastuzumab combination feasible in breast cancer patients? Ann. Onc., October 1, 2005; 16(10): 1709 - 1709. [Full Text] [PDF]
|
|
|
|
|
|
J. Baselga, J. Albanell, A. Ruiz, A. Lluch, P. Gascon, V. Guillem, S. Gonzalez, S. Sauleda, I. Marimon, J. M. Tabernero, et al. Phase II and Tumor Pharmacodynamic Study of Gefitinib in Patients with Advanced Breast Cancer J. Clin. Oncol., August 10, 2005; 23(23): 5323 - 5333. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. Burris III, H. I. Hurwitz, E. C. Dees, A. Dowlati, K. L. Blackwell, B. O'Neil, P. K. Marcom, M. J. Ellis, B. Overmoyer, S. F. Jones, et al. Phase I Safety, Pharmacokinetics, and Clinical Activity Study of Lapatinib (GW572016), a Reversible Dual Inhibitor of Epidermal Growth Factor Receptor Tyrosine Kinases, in Heavily Pretreated Patients With Metastatic Carcinomas J. Clin. Oncol., August 10, 2005; 23(23): 5305 - 5313. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Cappuzzo, M. Varella-Garcia, H. Shigematsu, I. Domenichini, S. Bartolini, G. L. Ceresoli, E. Rossi, V. Ludovini, V. Gregorc, L. Toschi, et al. Increased HER2 Gene Copy Number Is Associated With Response to Gefitinib Therapy in Epidermal Growth Factor Receptor-Positive Non-Small-Cell Lung Cancer Patients J. Clin. Oncol., August 1, 2005; 23(22): 5007 - 5018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. R. Fantin, M. J. Berardi, H. Babbe, M. V. Michelman, C. M. Manning, and P. Leder A Bifunctional Targeted Peptide that Blocks HER-2 Tyrosine Kinase and Disables Mitochondrial Function in HER-2-Positive Carcinoma Cells Cancer Res., August 1, 2005; 65(15): 6891 - 6900. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Schilder, M. W. Sill, X. Chen, K. M. Darcy, S. L. Decesare, G. Lewandowski, R. B. Lee, C. A. Arciero, H. Wu, and A. K. Godwin Phase II Study of Gefitinib in Patients with Relapsed or Persistent Ovarian or Primary Peritoneal Carcinoma and Evaluation of Epidermal Growth Factor Receptor Mutations and Immunohistochemical Expression: A Gynecologic Oncology Group Study Clin. Cancer Res., August 1, 2005; 11(15): 5539 - 5548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kiguchi, L. Ruffino, T. Kawamoto, T. Ajiki, and J. DiGiovanni Chemopreventive and Therapeutic Efficacy of Orally Active Tyrosine Kinase Inhibitors in a Transgenic Mouse Model of Gallbladder Carcinoma Clin. Cancer Res., August 1, 2005; 11(15): 5572 - 5580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhou and M. G. Brattain Synergy of Epidermal Growth Factor Receptor Kinase Inhibitor AG1478 and ErbB2 Kinase Inhibitor AG879 in Human Colon Carcinoma Cells Is Associated with Induction of Apoptosis Cancer Res., July 1, 2005; 65(13): 5848 - 5856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S R D Johnston Clinical trials of intracellular signal transductions inhibitors for breast cancer -- a strategy to overcome endocrine resistance Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S145 - S157. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Haas-Kogan, M. D. Prados, T. Tihan, D. A. Eberhard, N. Jelluma, N. D. Arvold, R. Baumber, K. R. Lamborn, A. Kapadia, M. Malec, et al. Epidermal Growth Factor Receptor, Protein Kinase B/Akt, and Glioma Response to Erlotinib J Natl Cancer Inst, June 15, 2005; 97(12): 880 - 887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N Normanno, A De Luca, D Aldinucci, M R Maiello, M Mancino, A D'Antonio, R De Filippi, and A Pinto Gefitinib inhibits the ability of human bone marrow stromal cells to induce osteoclast differentiation: implications for the pathogenesis and treatment of bone metastasis Endocr. Relat. Cancer, June 1, 2005; 12(2): 471 - 482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Wang, F. Y. Wu, I. Shin, S. Qu, and C. L. Arteaga Transforming Growth Factor {beta} (TGF-{beta})-Smad Target Gene Protein Tyrosine Phosphatase Receptor Type Kappa Is Required for TGF-{beta} Function Mol. Cell. Biol., June 1, 2005; 25(11): 4703 - 4715. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Hirata, F. Hosoi, M. Miyagawa, S.-i. Ueda, S. Naito, T. Fujii, M. Kuwano, and M. Ono HER2 Overexpression Increases Sensitivity to Gefitinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, through Inhibition of HER2/HER3 Heterodimer Formation in Lung Cancer Cells Cancer Res., May 15, 2005; 65(10): 4253 - 4260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Miller, J. M. Trigo, C. Wheeler, A. Barge, J. Rowbottom, G. Sledge, and J. Baselga A Multicenter Phase II Trial of ZD6474, a Vascular Endothelial Growth Factor Receptor-2 and Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, in Patients with Previously Treated Metastatic Breast Cancer Clin. Cancer Res., May 1, 2005; 11(9): 3369 - 3376. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Baselga and C. L. Arteaga Critical Update and Emerging Trends in Epidermal Growth Factor Receptor Targeting in Cancer J. Clin. Oncol., April 10, 2005; 23(11): 2445 - 2459. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Shin, J. Edl, S. Biswas, P. C. Lin, R. Mernaugh, and C. L. Arteaga Proapoptotic Activity of Cell-Permeable Anti-Akt Single-Chain Antibodies Cancer Res., April 1, 2005; 65(7): 2815 - 2824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Fabian and B. F. Kimler Selective Estrogen-Receptor Modulators for Primary Prevention of Breast Cancer J. Clin. Oncol., March 10, 2005; 23(8): 1644 - 1655. [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Engelman, P. A. Janne, C. Mermel, J. Pearlberg, T. Mukohara, C. Fleet, K. Cichowski, B. E. Johnson, and L. C. Cantley ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines PNAS, March 8, 2005; 102(10): 3788 - 3793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Solit, Y. She, J. Lobo, M. G. Kris, H. I. Scher, N. Rosen, and F. M. Sirotnak Pulsatile Administration of the Epidermal Growth Factor Receptor Inhibitor Gefitinib Is Significantly More Effective than Continuous Dosing for Sensitizing Tumors to Paclitaxel Clin. Cancer Res., March 1, 2005; 11(5): 1983 - 1989. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. DiGiovanna, D. F. Stern, S. M. Edgerton, S. G. Whalen, D. Moore II, and A. D. Thor Relationship of Epidermal Growth Factor Receptor Expression to ErbB-2 Signaling Activity and Prognosis in Breast Cancer Patients J. Clin. Oncol., February 20, 2005; 23(6): 1152 - 1160. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tuccillo, M. Romano, T. Troiani, E. Martinelli, F. Morgillo, F. De Vita, R. Bianco, G. Fontanini, R. A. Bianco, G. Tortora, et al. Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor-2 and Epidermal Growth Factor Receptor Small Molecule Tyrosine Kinase Inhibitor, in Combination with SC-236, a Cyclooxygenase-2 Inhibitor Clin. Cancer Res., February 1, 2005; 11(3): 1268 - 1276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Johnston Combinations of Endocrine and Biological Agents: Present Status of Therapeutic and Presurgical Investigations Clin. Cancer Res., January 15, 2005; 11(2): 889s - 899s. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Reyzer, R. L. Caldwell, T. C. Dugger, J. T. Forbes, C. A. Ritter, M. Guix, C. L. Arteaga, and R. M. Caprioli Early Changes in Protein Expression Detected by Mass Spectrometry Predict Tumor Response to Molecular Therapeutics Cancer Res., December 15, 2004; 64(24): 9093 - 9100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P M Harari Epidermal growth factor receptor inhibition strategies in oncology Endocr. Relat. Cancer, December 1, 2004; 11(4): 689 - 708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Milella, D. Trisciuoglio, T. Bruno, L. Ciuffreda, M. Mottolese, A. Cianciulli, F. Cognetti, U. Zangemeister-Wittke, D. Del Bufalo, and G. Zupi Trastuzumab Down-Regulates Bcl-2 Expression and Potentiates Apoptosis Induction by Bcl-2/Bcl-XL Bispecific Antisense Oligonucleotides in HER-2Gene-Amplified Breast Cancer Cells Clin. Cancer Res., November 15, 2004; 10(22): 7747 - 7756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. H. Kalish, R. A. Kwong, I. E. Cole, R. M. Gallagher, R. L. Sutherland, and E. A. Musgrove Deregulated Cyclin D1 Expression Is Associated with Decreased Efficacy of the Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Gefitinib in Head and Neck Squamous Cell Carcinoma Cell Lines Clin. Cancer Res., November 15, 2004; 10(22): 7764 - 7774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tracy, T. Mukohara, M. Hansen, M. Meyerson, B. E. Johnson, and P. A. Janne Gefitinib Induces Apoptosis in the EGFRL858R Non-Small-Cell Lung Cancer Cell Line H3255 Cancer Res., October 15, 2004; 64(20): 7241 - 7244. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Britten Targeting ErbB receptor signaling: A pan-ErbB approach to cancer Mol. Cancer Ther., October 1, 2004; 3(10): 1335 - 1342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Matar, F. Rojo, R. Cassia, G. Moreno-Bueno, S. Di Cosimo, J. Tabernero, M. Guzman, S. Rodriguez, J. Arribas, J. Palacios, et al. Combined Epidermal Growth Factor Receptor Targeting with the Tyrosine Kinase Inhibitor Gefitinib (ZD1839) and the Monoclonal Antibody Cetuximab (IMC-C225): Superiority Over Single-Agent Receptor Targeting Clin. Cancer Res., October 1, 2004; 10(19): 6487 - 6501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Tan, X. Yang, S. M. Hewitt, A. Berman, E. R. Lepper, A. Sparreboom, A. L. Parr, W. D. Figg, C. Chow, S. M. Steinberg, et al. Evaluation of Biologic End Points and Pharmacokinetics in Patients With Metastatic Breast Cancer After Treatment With Erlotinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor J. Clin. Oncol., August 1, 2004; 22(15): 3080 - 3090. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Huang, E. A. Armstrong, S. Benavente, P. Chinnaiyan, and P. M. Harari Dual-Agent Molecular Targeting of the Epidermal Growth Factor Receptor (EGFR): Combining Anti-EGFR Antibody with Tyrosine Kinase Inhibitor Cancer Res., August 1, 2004; 64(15): 5355 - 5362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Brown, M. Lun, J. W. Prichard, T. M. Blasick, and P. L. Zhang Morphoproteomic and Pharmacoproteomic Correlates in Hormone-Receptor-Negative Breast Carcinoma Cell Lines Ann. Clin. Lab. Sci., July 1, 2004; 34(3): 251 - 262. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ueda, S. Wang, N. Dumont, J. Y. Yi, Y. Koh, and C. L. Arteaga Overexpression of HER2 (erbB2) in Human Breast Epithelial Cells Unmasks Transforming Growth Factor {beta}-induced Cell Motility J. Biol. Chem., June 4, 2004; 279(23): 24505 - 24513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. Burris III Dual Kinase Inhibition in the Treatment of Breast Cancer: Initial Experience with the EGFR/ErbB-2 Inhibitor Lapatinib Oncologist, June 3, 2004; 9(suppl_3): 10 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Miller The Role of ErbB Inhibitors in Trastuzumab Resistance Oncologist, June 3, 2004; 9(suppl_3): 16 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Rabindran, C. M. Discafani, E. C. Rosfjord, M. Baxter, M. B. Floyd, J. Golas, W. A. Hallett, B. D. Johnson, R. Nilakantan, E. Overbeek, et al. Antitumor Activity of HKI-272, an Orally Active, Irreversible Inhibitor of the HER-2 Tyrosine Kinase Cancer Res., June 1, 2004; 64(11): 3958 - 3965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Normanno, M. Di Maio, F. Perrone, and M. Campiglio Molecular Markers to Predict Response to Gefitinib: EGFR, ErbB-2, or More? J. Clin. Oncol., May 15, 2004; 22(10): 2035 - 2036. [Full Text] [PDF]
|
|
|
|
|
|
M. Ono, A. Hirata, T. Kometani, M. Miyagawa, S.-i. Ueda, H. Kinoshita, T. Fujii, and M. Kuwano Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signal-regulated kinase 1/2 and EGF receptor/Akt pathway for proliferation Mol. Cancer Ther., April 1, 2004; 3(4): 465 - 472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Warburton, W. H. Dragowska, K. Gelmon, S. Chia, H. Yan, D. Masin, T. Denyssevych, A. E. Wallis, and M. B. Bally Treatment of HER-2/neu Overexpressing Breast Cancer Xenograft Models with Trastuzumab (Herceptin) and Gefitinib (ZD1839): Drug Combination Effects on Tumor Growth, HER-2/neu and Epidermal Growth Factor Receptor Expression, and Viable Hypoxic Cell Fraction Clin. Cancer Res., April 1, 2004; 10(7): 2512 - 2524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Yang, K. Q. Cai, J. A. Thompson-Lanza, R. C. Bast Jr., and J. Liu Inhibition of Breast and Ovarian Tumor Growth through Multiple Signaling Pathways by Using Retrovirus-mediated Small Interfering RNA against Her-2/neu Gene Expression J. Biol. Chem., February 6, 2004; 279(6): 4339 - 4345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Pack, O. M. Alper, K. Stromberg, M. Augustus, M. Ozdemirli, A. M. Miermont, G. Klus, M. Rusin, R. Slack, N. F. Hacker, et al. Simultaneous Suppression of Epidermal Growth Factor Receptor and c-erbB-2 Reverses Aneuploidy and Malignant Phenotype of a Human Ovarian Carcinoma Cell Line Cancer Res., February 1, 2004; 64(3): 789 - 794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Perez-Soler HER1/EGFR Targeting: Refining the Strategy Oncologist, February 1, 2004; 9(1): 58 - 67. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Yang, R. Bagheri-Yarmand, R.-A. Wang, L. Adam, V. V. Papadimitrakopoulou, G. L. Clayman, A. El-Naggar, R. Lotan, C. J. Barnes, W. K. Hong, et al. The Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor ZD1839 (Iressa) Suppresses c-Src and Pak1 Pathways and Invasiveness of Human Cancer Cells Clin. Cancer Res., January 15, 2004; 10(2): 658 - 667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Nyati, D. Maheshwari, S. Hanasoge, A. Sreekumar, S. D. Rynkiewicz, A. M. Chinnaiyan, W. R. Leopold, S. P. Ethier, and T. S. Lawrence Radiosensitization by Pan ErbB Inhibitor CI-1033 in Vitro and in Vivo Clin. Cancer Res., January 15, 2004; 10(2): 691 - 700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Ciardiello, R. Bianco, R. Caputo, R. Caputo, V. Damiano, T. Troiani, D. Melisi, F. De Vita, S. De Placido, A. R. Bianco, et al. Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Human Cancer Cells with Acquired Resistance to Antiepidermal Growth Factor Receptor Therapy Clin. Cancer Res., January 15, 2004; 10(2): 784 - 793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sumitomo, T. Asano, J. Asakuma, T. Asano, A. Horiguchi, and M. Hayakawa ZD1839 Modulates Paclitaxel Response in Renal Cancer by Blocking Paclitaxel-Induced Activation of the Epidermal Growth Factor Receptor-Extracellular Signal-Regulated Kinase Pathway Clin. Cancer Res., January 15, 2004; 10(2): 794 - 801. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. E. Konecny, C. A. Wilson, and D. J. Slamon Is There a Role for Epidermal Growth Factor Receptor Inhibitors in Breast Cancer Prevention? J Natl Cancer Inst, December 17, 2003; 95(24): 1813 - 1815. [Full Text] [PDF]
|
|
|
|
|
|
C. Lu, C. Speers, Y. Zhang, X. Xu, J. Hill, E. Steinbis, J. Celestino, Q. Shen, H. Kim, S. Hilsenbeck, et al. Effect of Epidermal Growth Factor Receptor Inhibitor on Development of Estrogen Receptor-Negative Mammary Tumors J Natl Cancer Inst, December 17, 2003; 95(24): 1825 - 1833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R Shah, T. L Walsh, C. B Williams, and S. A Soefje Gefitinib (ZD1839, Iressa(R)): a selective epidermal growth factor receptor-tyrosine kinase inhibitor Journal of Oncology Pharmacy Practice, December 1, 2003; 9(4): 151 - 160. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), ZD1839 (
), Herceptin (
), or the combination (x) for the next 28 days. Tumor volumes were measured serially as indicated in "Materials and Methods." Each data point represents the mean tumor volume of seven to eight mice; bars, SD. B, inhibition of tumor cell proliferation. On day 28 of treatment, two mice/group were randomly selected and pulsed with BrdUrd before tumor harvesting. Tumor sections were stained with a BrdUrd antibody, and BrdUrd-positive nuclei were counted in 10 high-power microscopic fields. Numbers in insets denote the percentage of BrdUrd-positive nuclei relative to the total number of tumor cell nuclei in the same fields. The total number of nuclei counted in each group were 1214 (controls), 1014 (ZD1839), 1810 (Herceptin), and 975 (ZD1839 plus Herceptin). P = 0.001, Student’s unpaired t test. C, inhibition of HER2 signaling in situ. On day 28, some tumors were harvested, snap-frozen in liquid nitrogen, and later homogenized in a buffer containing protease and phosphatase inhibitors as described in "Materials and Methods." An equivalent amount of protein from the tumor lysates were subjected to HER2, P-Tyr, Rb, P-Atk, total Akt, P-MAPK, and total MAPK immunoblot analyses. Left, molecular weights in thousands.