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1 Department of Surgery and 2 Institute of Pharmacology, University of Vienna, and 3 Igeneon AG, Vienna, Austria
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LeY antigen belongs to the A, B, H, Lewis blood group family with the chemical structure Fuc
1
2Galß14[Fuc13]GlcNAcß1R and is expressed predominately during embryogenesis. Under physiologic conditions, expression on adult tissue is restricted to granulocytes and epithelial surfaces (6)
. However, LeY is overexpressed in the majority of carcinomas, including breast, ovary, pancreas, prostate, colon, and non-small cell lung cancers (7)
. In these cases, LeY occurs either at the plasma membrane as a glycolipid or linked to surface receptors (e.g., ErbB family; Ref. 8
). Therefore, we surmised that it should be possible to perturb signal transduction originating from LeY-modified surface receptors using antibodies directed against the LeY antigen. In this study, we verified this hypothesis by investigating whether the murine anti-LeY antibody ABL364 and its humanized version IGN311 interfere with signaling originating from ErbB receptors (9)
. We used two cellular models: (a) the human breast cancer cell line SKBR-3 that overexpresses ErbB2; and (b) the epidermoid carcinoma line A431 found originally to contain abundant amounts of LeY-modified ErbB1 (8)
. We focused on MAPK because stimulation of the MAPK cascade is a means by which growth factors promote cell proliferation (10)
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Assay for MAPK Stimulation.
SKBR-3 and A431 cells were propagated in McCoy’s modified medium and DMEM, respectively, containing 10% FCS, antibiotics, and glutamine. Subconfluent cell layers were rendered quiescent by serum starvation for 12 h; if cells were transfected with plasmid DNA, serum was withdrawn 24 h after transfection. Cells were stimulated subsequently by addition of medium containing or lacking agonists and maintained at 37°C for 5 min; antibodies were added 15 min before stimulation with agonists. The MAPK assay was done as by Klinger et al. (11)
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Preparing Cell Lysates for Immunoprecipitation of ErbB.
Cell lysates were prepared in a manner similar to that described for MAPK assay using a different lysis buffer [in mM: 20 Tris, 150 NaCl, 1 EDTA, 1 EGTA, 1 Na3VO4, 40 ß-glycerophosphate, 1 phenylmethylsulfonyl fluoride, 10 NaF (pH adjusted to 7.5 with HCl), 1% Triton X-100, 250 units/ml aprotinin, and 40 µg/µl leupeptin]. For immunoprecipitation, cellular lysates (500 µg) were incubated with ABL364 or IGN311 (each at 20 µg per individual sample) precoupled to protein G-Sepharose. Immunoblotting for ErbB receptors was performed as outlined by Klinger et al. (11)
using the appropriate antibodies.
[125I]EGF Binding to Membrane Preparations.
Confluent monolayers (in 15-cm tissue culture dishes) were washed once with ice-cold PBS and scraped from their plastic support in hypotonic (HME) buffer [in mM: 25 HEPES.NaOH (pH 7.5), 2 MgCl2, and 1 EDTA]. After centrifugation at 20,000 x g for 10 min, the cell pellet was resuspended in HME buffer and subjected to a freeze/thaw cycle with liquid nitrogen and further homogenized by brief sonication. Membranes were sedimented by centrifugation (38,000 x g for 10 min), washed twice in HME buffer, resuspended in HME buffer at a protein concentration of 8–10 mg/ml, and stored in aliquots at -80°C. For the assay, membranes were diluted in HME buffer (30 µg/assay) containing 0.1% BSA; subsequently, 0.5–1 nM [125I]EGF (specific activity, 900 cpm/fmol) and the appropriate agonist were added. The reaction was allowed to proceed for 1–90 min at 25°C. The reaction was terminated by filtrating over glass fiber filters (presoaked in 1% BSA in HME buffer) with ice-cold HME buffer containing 0.1% BSA.
[125I]EGF Binding to Intact Cells, Internalization Assay.
Cells were resuspended in culture medium containing 0.1% FCS and 10 mM HEPES-Na, pH 7.5 (5 x 105-106 cells/assay). Appropriate antibody was added subsequently, and after 15 min the reaction was started by adding 1 nM [125I]EGF (specific activity, 900 cpm/fmol). The experimental approach is based on the assay described by Yarden et al. (12)
. Briefly, to prevent internalization, the reaction was incubated at 4°C; after the [125I]EGF reached equilibrium (i.e., 60 min), the reaction was split into the individual samples, which were incubated for the indicated time at 37°C. Internalization was stopped by sedimenting the cells through ice-cold FCS at 500 x g for 5 min. The cell pellets then were resuspended in the acidic stripping buffer [150 mM acetic acid (pH 2.7) and 150 mM NaCl] and incubated on ice for 10 min. After centrifugation at 500 x g for 5 min, the radioligand released into the supernatant by the acidic strip was defined as the cell surface-associated ligand; radioactivity remaining within the cells after the acidic strip was defined as the internalized ligand. The cell pellets were solubilized in 100 mM NaOH solution containing 0.1% SDS, and their radioactivity was counted. When only [125I]EGF binding to intact cells was to be determined, cells were maintained at 4°C for 60 min, and the reaction was terminated by sedimenting the cells through ice-cold FCS at 500 x g for 5 min.
[125I]EGF Recycling Assay.
The preparation of cells, the composition of binding buffer, and the concentration of [125I]EGF were as outlined previously. The binding reaction was performed at 37°C for 60 min to allow for internalization. Cells then were sedimented twice through ice-cold FCS at 500 x g for 5 min to remove unbound [125I]EGF. The cell pellets were resuspended in binding buffer devoid of [125I]EGF in the absence or presence of the individual antibody, split into the individual samples, and incubated at 37°C for the time indicated. Recycling of ErbBs was stopped by sedimenting the cells through ice-cold FCS at 500 x g for 5 min. The radioligand appearing in the supernatant represented the recycled [125I]EGF. The acidic strip and subsequent steps were performed as described previously.
Fluorescence Microscopy Imaging of CFP-Tagged Ras and Oregon Green-Modified EGF in SKBR-3 Cells.
SKBR-3 cells were seeded onto poly-D-lysine-coated glass coverslips. After 24 h, cells were maintained under starving conditions for 12 h. If CFP-Ras was to be visualized, the cells were treated with 1 nM EGF for 15 min at 37°C. For imaging Oregon Green-modified EGF, the medium was changed against PBS lacking or containing 100 nM ABL364, IGN311, or herceptin as indicated, and after a 15-min incubation at 37°C, 20 nM Oregon Green-modified EGF was added. Cells then were maintained at 37°C or 4°C, if indicated, for 15 min, and the unbound fluorescent EGF was removed in three wash steps with PBS. If indicated, surface-bound Oregon Green-modified EGF was removed by incubating twice in acidic stripping buffer for 5 min as described previously. Imaging was done with a 63x oil immersion objective and a filter set allowing for excitation at 500 nm and recording of emission at 535 nm. The pictures were captured with a CCD camera and stored in and processed with MetaSeries software (release 4.6, MetaFluor and MetaMorph; Universal Imaging).
Immunocytochemistry of EEA-1 and Lamp-2 in SKBR-3 Cells.
SKBR-3 cells were seeded onto poly-D-lysine-coated glass coverslips and maintained subsequently under starving conditions for 24 h. Cells then were incubated in medium containing 100 nM ABL364 or IGN311, if indicated, for 15 min at 37°C and thereafter incubated with 1 nM EGF for 15 min. Cells were fixed subsequently with 4% paraformaldehyde in PBS at 37°C for 30 min. The following steps were done at room temperature, with PBS used for washes. The cells were permeabilized with 0.2% Triton X-100. The coverslips then were blocked with 1% BSA diluted in PBS for 30 min. Primary antibodies, diluted in 1% BSA/PBS, were added to the cells and left for 1 h. The coverslips were washed four times before incubation with secondary antibodies. We used the following concentrations of antibodies: anti-Lamp-2, 1 µg/ml; anti-EEA-1, 1 µg/ml; and secondary antibody, 1:1000 dilution. The coverslips were mounted in Mowiol solution, and the pictures were captured as described previously for Oregon Green-modified EGF.
Each experiment was carried out at least three times.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Lanes 1 and 2) or IGN311 (Fig. 1A
, Lanes 3 and 4). The amount of immunoprecipitated ErbB2 and ErbB1 was assessed by immunoblotting for ErbB2 and ErbB1, respectively. Pretreatment with tunicamycin did not decrease the amount of ErbB1 and ErbB2 in cellular lysates (data not shown) but reduced greatly the ability of the antibodies to immunoprecipitate ErbB2 and ErbB1 (Fig. 1A
, Lanes 2 and 4). This indicates that IGN311 and ABL364 recognize ErbB1 and ErbB2 only if the protein is glycosylated and modified by LeY antigen.
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and A431 cells (not shown). Fig. 1B
shows the association kinetics of [125I]EGF in the absence or presence of IGN311 or 2C225 antibody. The values for equilibrium binding Beq and the pseudo-first order rate constant kapp were comparable under control conditions and in the presence of IGN311 (kapp = 0.18 ± 0.02 min-1; Beq = 41.7 ± 1.6 fmol/mg and kapp = 0.26 ± 0.07 min-1; Beq = 41.5 ± 2.8 fmol/mg, respectively). In contrast, the kinetic parameters were altered in the presence of the 2C225 antibody, which was used as a positive control (kapp = 0.09 ± 0.03 min-1; Beq = 13.7 ± 0.1 fmol/mg). This suggests that binding of EGF and the 2C225 antibody are mutually exclusive; in contrast, EGF and IGN311 can be bound to the receptor simultaneously. This interpretation also was confirmed by measuring [125I]EGF binding to intact cells at 4°C (i.e., under conditions where receptor internalization is blocked and does not confound the analysis; see also below). As can be seen in Fig. 1C
, neither ABL364 nor IGN311 reduced binding of [125I]EGF to intact SKBR-3 cells (filled bar; acidic strip = surface bound). As expected, the same results were obtained for trastuzumab because trastuzumab binds to a domain of ErbB2 in the vicinity of the plasma membrane (14)
. The binding reaction was allowed to proceed at 4°C to prevent activation of the endocytotic machinery. Accordingly, the amount of internalized [125I]EGF (open bar; acid resistant = internalized) was negligible. If membrane preparations of SKBR-3 and A431 cells were used in [125I]EGF binding assays, again IGN311, ABL364, and trastuzumab did not decrease [125I]EGF binding (not shown). Consistent with its ability to bind to the EGF-binding domain of ErbB1, the anti-ErbB1 antibody 2C225 substantially reduces [125I]EGF binding to cells (Fig. 1C
, filled bar labeled C). Of note, the concentration of [125I]EGF was 
1 nM, which is in the range of the EGF concentration used in the MAPK assays wherein IGN311 or trastuzumab blunted the response to EGF (see below).
IGN311 Decreases EGF-Dependent MAPK Phosphorylation.
ErbB receptors control a signaling cascade that leads to the stimulation of MAPK (1)
. Activation of MAPK is achieved via dual phosphorylation by the upstream kinase MAPK-extracellular signal- related kinase kinase 1. This activity can be monitored using an antiserum specific against the dually phosphorylated MAPK. SKBR-3 cells were rendered quiescent by serum starvation and maintained in the absence of serum. Under these conditions, MAPK was phosphorylated only minimally (Fig. 2A and B
, Lanes U). The cells were stimulated for 5 min by EGF (Fig. 2A
, Lanes E) or by heregulin (Fig. 2A
, Lanes H) in the absence or presence of IGN311, trastuzumab, and the anti-ErbB1 antibody 2C225 (Fig. 2A
, Lanes I, T, and C in the bottom row). Addition of EGF and heregulin increased MAPK phosphorylation (Fig. 2A
, Lanes E and H, respectively). IGN311 decreased the response to EGF and heregulin; this also was demonstrated for ABL364 (data not shown; see also Fig. 3C
), trastuzumab, and the 2C225 antibody. Thus, the ErbB1-specific antibody was capable of interfering with the response to the ErbB3-specific ligand heregulin. Trastuzumab similarly inhibited signaling in response to heregulin and EGF (the ErbB1-specific agonist). These findings suggest that in SKBR-3-cells, agonist stimulation induces the formation of heteromeric complexes of ErbB receptors. More importantly, these data support strongly the conjecture that the humanized anti-LeY antibody IGN311 and its murine counterpart ABL364 are capable of inhibiting signaling via ErbB family members.
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, right blot) and ErbB2 (not shown) were not recognized by IGN311; thus, the receptors lacked LeY antigen. Accordingly, neither ABL364 nor IGN311 inhibited EGF-dependent MAPK stimulation (Fig. 2B
, left blot) in MCF-7 cells. An additional control consisted of SKBR3 cells that were treated with heat-denatured IGN311. Addition of boiled IGN311 (indicated by I* in Fig. 2C
) did not blunt the stimulatory effect of EGF (compare Lanes E+I and E+I* in Fig. 2C
). Furthermore, if SKBR3 cells were stimulated with the phorbol ester ß-phorbol 12,13-dibutyrate, no difference between MAPK phosphorylation in the presence of active or boiled IGN (Fig. 2C
, Lanes P+I and P+I*) was observed. Phorbol esters activate protein kinase C isoforms, which also stimulate MAPK phosphorylation by an action on the upstream kinases (presumably raf-1). Because phorbol esters have an intracellular site of action, IGN311 is not expected to affect stimulation by the phorbol ester, regardless of whether it is added in the active or inactive (heat-denatured) form.
IGN311 and ABL364 Antagonize EGF-Dependent MAPK Stimulation in a Noncompetitive Manner.
ABL364 and IGN311 did not compete for [125I]EGF binding (see Fig. 1C
). Nevertheless, these antibodies inhibited EGF-dependent MAPK stimulation; therefore, the action of the antibodies ought to be caused by noncompetitive inhibition. To verify this interpretation, the concentration-dependent effect of EGF was determined in the absence or presence of the appropriate antibodies. IGN311 predominantly reduced the maximum effect of EGF (Fig. 3A
, bottom row). This indicates a noncompetitive inhibition of EGF stimulated MAPK phosphorylation. The anti-EGF-receptor antibody 2C225 prevents binding of EGF to ErbB1 (see also Fig. 1, B and C
); because the mechanism of action of this antibody is well understood, it was used as a reference. Addition of antibody 2C225 shifted the concentration-response curve for EGF-induced stimulation of MAPK phosphorylation to the right, and a maximum effect was not reached at the highest concentration of EGF used (Fig. 3A
, middle row).
In Fig. 3, B and C
, the cells were stimulated with a fixed concentration of EGF or heregulin, and the concentration of ABL364 (Fig. 3B
, second and third row) and trastuzumab (Fig. 3C)
was varied. It is evident that ABL364 inhibited (Fig. 3B
, second and third row) the EGF- and heregulin- induced MAPK phosphorylation at concentrations that were in the low nanomolar range, thus consistent with the high affinity for its cognate epitope. At concentrations of [ge]1 µM, however, the inhibitory action was lost, and the stimulation actually exceeded that elicited by sole addition of EGF. If cells were challenged only by the addition of ABL364 (Fig. 3B
, top row), the antibody per se was capable of eliciting a response at concentrations [ge]1 µM. This may be because ABL364 is an IgG3 isotype, which tends to aggregate; at higher concentrations, ABL364 thereby may cross-link ErbB receptors and thus favor receptor activation. In contrast, IGN311 did not cause stimulation even at high concentrations (up to 5 µM; not shown), and this may be related to the fact that IGN311 is an IgG1 isotype.
Trastuzumab served as a useful comparison; its mechanism of action is distinct to that of ABL364 or IGN311: trastuzumab specifically targets ErbB2, and it does not block binding of EGF (see above). Trastuzumab blocked MAPK stimulation by EGF in a concentration range that was not lower than that of ABL364; at 10 nM, the extent of inhibition by any of the two antibodies was comparable (compare Fig. 3, B and C
).
No major difference in the affinity of ABL364 and IGN311 for their cognate epitope exists (9)
. If their inhibitory action were related to binding to LeY antigen-modified growth factor receptors, half-maximum inhibition of MAPK stimulation should occur over a similar concentration range. This was the case; for both antibodies the IC50 was estimated to lie in the range of 3–10 nM (Fig. 3D)
. Of note, the experiment shown in Fig. 3D
was performed on A431 cells, in which ErbB1 is overexpressed and represents the predominant ErbB isoform. Thus, the action of ABL364 and IGN311 is not restricted to breast cancer cells.
IGN311 and ABL364 Alter the Recycling Kinetics of EGF Receptors.
In many instances, receptor-dependent stimulation of MAPK depends on endocytosis of the agonist-liganded receptor, and this reaction requires the GTPase dynamin. We verified that endocytosis is important for stimulation of MAPK by cotransfecting SKBR-3 cells with plasmids encoding wild-type dynamin or the dominant-negative version of dynamin (dynamin K44A = GTPase deficient) and an epitope (GFP)-tagged reporter MAPK. EGF failed to stimulate the reporter MAPK if cells expressed dynamin K44A, but a robust stimulation was seen in cells expressing wild-type dynamin (Fig. 4A)
. As an internal control (for cell viability and loading), we determined EGF-stimulated phosphorylation of endogenous MAPK. The response did not differ in lysates prepared from cells exposed to the plasmids encoding wild-type or mutated dynamin (Fig. 4A
, bottom blot); this was to be expected based on the low transfection efficiency in SKBR-3 cells (estimated at 5%).
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revealed dynamin-dependent endocytosis as a prerequisite for MAPK stimulation in SKBR-3 cells. This finding also was confirmed using a fluorescent-tagged version of H-Ras (CFP-Ras = H-Ras tagged on its amino terminus with the cyan fluorescent protein); EGF induced a redistribution of CFP-Ras such that fluorescence for the plasma membrane disappeared and accumulated a punctate intracellular staining (Fig. 4B)
. Thus, it was conceivable that the anti-LeY antibodies exerted their inhibitory action on MAPK by affecting the intracellular routing of EGF receptors. This was addressed by measuring the rates of internalization and recycling in the absence and presence of anti-LeY antibodies. SKBR-3 cells were incubated with [125I]EGF at 4°C. Unbound ligand was removed, and internalization was initiated by warming the samples to 37°C. We measured the time-dependent change in surface-bound (i.e., acid-released) and internalized (i.e., acid-resistant) radioligand binding. Surface-bound ligand disappeared with a rapid monoexponential decay (kint = 0.39 ± 0.07 min-1), which was approximately similar to the rate determined for the appearance of internalized receptors (0.24 ± 0.06 min-1); however, the monoclonal antibodies did not cause any appreciable change in these rates (Fig. 5, A and B)
. Therefore, we characterized recycling by allowing SKBR-3 cells to internalize [125I]EGF during a preincubation at 37°C. The unbound ligand was removed subsequently, and the cells were held at 4°C. The internalized [125I]EGF then was allowed to recycle to the surface in fresh medium at 37°C. We determined the time course for the reappearance of [125I]EGF in the medium, which is indicative of receptors recycling to the cell surface. When compared with control conditions (
and 
in Fig. 6A
) and trastuzumab (
in Fig. 6C
), ABL364 ( in Fig. 6B
and 
in Fig. 6C
) and IGN311 ( in Fig. 6C
) caused an increase in a readily exchanging pool. As expected for steady-state conditions (see also Fig. 1B
), the membrane-bound fraction of [125I]EGF (i.e., the acid-sensitive fraction) remained constant and was comparable in ABL364-treated cells and control cells (Fig. 6, A and B
). The data displayed in Fig. 6
suggest that in the presence of ABL364 or IGN311, EGF receptor-containing endosomes are shifted into a compartment that allows for more rapid recycling. To visualize this shift, we studied the distribution pattern of fluorescent EGF. SKBR-3 cells were incubated with 20 nM Oregon Green-labeled EGF in the absence or presence of ABL364, IGN311, or trastuzumab. It is clear that in the presence of ABL364 (Fig. 7B)
or IGN311 (Fig. 7C)
, the distribution pattern of the fluorescent EGF differs from that under control conditions (Fig. 7A)
. Under control conditions, the fluorescence displays a disperse, punctate pattern, whereas in the presence of ABL364 or IGN311, the fluorescent EGF is clustered in a submembrane compartment. We confirmed that this fluorescence staining was intracellular by determining the effect of an acidic strip on the distribution of Oregon Green-labeled EGF. If the cells were incubated at 4°C to block internalization of ErbB receptors, they displayed surface staining with Oregon Green-labeled EGF, including the microvillar protrusion (Fig. 7E)
. Accordingly, this surface-bound Oregon Green-labeled EGF completely disappeared on incubation in acidic strip buffer. At 37°C, the internalized Oregon Green-labeled EGF was protected against acidic stripping under control conditions (Fig. 7G)
. Finally, acidic stripping did not change the staining pattern of Oregon Green-labeled EGF in the presence of IGN311 (Fig. 7H)
. As an additional control, we incubated cells with trastuzumab. This did not alter the distribution pattern of fluorescent EGF (Fig. 7D)
. This is consistent with the observations that neither the internalization (data not shown) nor the recycling kinetics (Fig. 6C
, ) was altered by the addition of trastuzumab. Furthermore, we visualized the distribution pattern of EEA-1 (a marker for early endosomes) and Lamp-2 (a marker for late endosomes). In SKBR-3 cells treated with EGF, EGF receptors colocalized with EEA-1 and Lamp-2 (data not shown). In the presence of EGF and either ABL364 (Fig. 7, K and L)
or IGN311 (Fig. 7, M and N)
, immunostaining for EEA-1 and Lamp-2 revealed a submembranous ringlike distribution similar to that seen with Oregon Green-labeled EGF (compare Fig. 7, B, C, and K–N
). In contrast, immunoreactivity for EEA-1 and for Lamp-2 was distributed as punctate pattern (Fig. 7, I and J)
in a manner similar to Oregon Green-labeled EGF (Fig. 7A)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the current experiments, we have focused on ErbB-dependent signaling, an area in which there is a precedent against which to compare our approach. However, our preliminary data suggest that the action of IGN311 and ABL364 is not confined to the ErbB family of growth factor receptors. Because cancer cells are unstable genetically, the emergence of resistance is an inherent problem, and cancer treatment is an evolutionary arms race. Thus, a large array of antibodies, signal transduction inhibitors, and cytotoxic drugs will be needed. The resistance to Herceptin that emerges after prolonged treatment exemplifies this (22) . The mechanism involves selection for cancer cells that overexpress other growth factor receptors (23) . It is clear that IGN311 offers the advantage that it also will target these receptors in cancer cells that (over-)express the LeY antigen. Therefore, IGN311 may have a broader efficacy than antibodies that target only one particular receptor.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Michael Freissmuth, Institute of Pharmacology, Vienna University, Wahringer Str. 13a, A-1090 Vienna, Austria. Phone: 43-1-4277-64171; Fax: 43-1-4277-9641; E-mail: michael.freissmuth{at}univie.ac.at
Received 8/ 6/03. Revised 11/ 1/03. Accepted 11/10/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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