| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
Tumor Biology Program, Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905 [H. L., N. J. M.], and Department of Molecular Oncology, Genentech, Inc., South San Francisco, California 94080 [R. W. A., M. X. S.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
HRG, ErbB3, and its preferred heterodimerization partner, ErbB2, are of particular medical interest because they have become important new targets in breast cancer therapy (8) . Whereas several naturally occurring secreted inhibitors of EGFR/ErbB family members have been reported (i.e., herstatin, avian p95 sErbB1, and Argos), these proteins all target receptors with active tyrosine kinases (9, 10, 11) . As mentioned above, p85-sErbB3 and the other naturally occurring soluble/secreted isoforms of ErbB3 do not fall into this category. However, the wide distribution of expression of these alternate receptor isoforms in human tissues suggests that they may also have regulatory functions. We have therefore studied the role of p85-sErbB3 in modulating ErbB3-mediated signaling. ErbB3-mediated signal transduction has been implicated in the regulation of diverse biological events including Schwann cell differentiation, neural regulation of skeletal muscle differentiation, heart development, and proliferation and differentiation of normal and malignant breast epithelial cells (7 , 8) . Here we report that p85-sErbB3 can bind to HRG with high affinity and can effectively block HRG binding to cell surface receptors, thereby inhibiting HRG-stimulated activation of ErbB2, ErbB3, and ErbB4, as well as activation of downstream effectors such as MAPK, Akt, and PI3K. Treatment of breast carcinoma cells with p85-sErbB3 also results in efficient inhibition of cell proliferation. The mechanism of p85-sErbB3-mediated growth inhibition is compared with the mechanism of other recently described secreted receptor inhibitors, and the physiological significance of the expression of these inhibitors in both normal and malignant tissues is discussed.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
EGF domain177–241 (HRG) and HRGß1176–246 (HRGß), R&D Systems; and (b) HRGß11–241, NeoMarkers. HRGß177–244 was prepared as described previously (12
, 13)
. The three recombinant HRGß1 sequences have a region consisting of aa 177–226 sufficient for both binding and stimulation of receptor phosphorylation (14)
.
A Ba/F3 cell line derivative expressing exogenous human ErbB2 and ErbB3 [Ba/F3 (ErbB2+ErbB3)] was provided by David F. Stern of Yale University (New Haven, CT). Cells were grown as described by Riese et al. (15)
. T47D and MCF7 cells were maintained in DMEM:Ham’s F-12 supplemented with 10% FCS. Stable clones were prepared by transfecting a quail fibroblast cell line, QT6 (16)
, with the pCDM8 (Invitrogen)-based eukaryotic expression vector containing alternate c-erbB3 cDNA clones encoding either p45-sErbB3 (clone R2F) or p85-sErbB3 (clone R31F; Fig. 1
; Ref. 2
, followed by selection using the drug G418. The clones were maintained in DMEM supplemented with 10% FCS.
|
Immunoprecipitation and Western Blots.
For analysis of tyrosine phosphorylation of ErbB receptors, cells were serum-starved for 24 h before the addition of HRG and/or sErbB3. After HRG treatment with or without sErbB3 for 10 min at room temperature, cells were washed once and lysed in radioimmunoprecipitation assay lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM NaF, 15 mM sodium molybdate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 3 µg/ml pepstatin, 10 µg/ml aprotinin, and 2 mM Na3VO4]. Equal amounts of protein were incubated with the indicated antibodies for 2 h at 4°C and precipitated with protein A/G-agarose by incubation at 4°C for 2 h. The immunoprecipitates were washed three times with lysis buffer and eluted by boiling for 5 min in sample buffer before separation using a 7.5% SDS-PAGE gel. After separation, proteins were transferred to a nitrocellulose membrane. Western blotting was performed using the enhanced chemiluminescence system by probing with the anti-phosphotyrosine antibody, and then a peroxidase-conjugated antibody against mouse IgG was used as the secondary antibody. Filters were stripped and reprobed with anti-ErbB antibodies.
For analysis of activation of MAPK and Akt, 100 µg of cell lysates were subjected to Western blotting using a set of anti-p44/42 extracellular signal-regulated kinase and anti-phospho-specific p44/42 extracellular signal-regulated kinase antibodies and a set of anti-Akt and anti-phospho-specific Akt antibodies (New England BioLabs), respectively. For detection of ErbB3 association with PI3K (p85), cell lysates were subjected to immunoprecipitation using an anti-ErbB3 antibody (Santa Cruz Biotechnology) followed by Western blotting using an anti-PI3K (p85) antibody as the primary antibody.
Purification of p85-sErbB3.
p85-sErbB3 was isolated from the concentrated conditioned medium of cells stably transfected with clone R31F and was purified in two steps. The first step was lectin affinity chromatography with a Concanavalin A column (Sigma Chemical Co.). The bound p85-sErbB3 was washed with column buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MnCl2, and 1 mM CaCl2], eluted using column buffer containing 1 M -methyl D-mannoside, and then dialyzed against 20 mM Tris-HCl (pH 7.5) overnight. The second step of purification was accomplished using a Mono Q ion exchange fast protein liquid chromatography column (Pharmacia). The bound p85-sErbB3 was eluted from the column with a 0–500 mM NaCl gradient containing 20 mM Tris-HCl (pH 7.5). Samples taken from each step were subjected to SDS-PAGE in duplicate and analyzed by Coomassie Brilliant Blue staining and by Western blot using an anti-ErbB3 236 antibody recognizing the ECD of ErbB3 (2)
.
HRG-p85-sErbB3 Cross-Linking.
HRGß (5 µg in PBS) was labeled using IODO-BEADS (Pierce) with 400 µCi of Na125I. The reaction mixture was separated on a D-Salt Excellulose column (Pierce). The specific activity was approximately 60,000 cpm/ng. p85-sErbB3 (25 nM) was incubated with 50 nM
125I-HRGß with or without the indicated concentrations of cold HRGß or insulin for 2 h at room temperature in PBS in a volume of 17.5 µl. Cross-linking was initiated by adding BS3 to a final concentration of 2 mM and incubating for 30 min at room temperature. Reactions were stopped by adding 2 µl of 1 M Tris-HCl (pH 7.4) and analyzed by SDS-PAGE.
Affinity Measurements of p85-sErbB3 to HRG.
HRGß177–244 was radioiodinated using lactoperoxidase as described previously (13)
. Binding assays were performed in Nunc break-apart immunomodule plates in quadruplicate. Plate wells were coated overnight with 100 µl of 2 µg/ml p85-sErbB3 or ErbB3-IgG (17)
in 50 mM carbonate buffer (pH 9.6) at 4°C. Wells were rinsed three times with wash buffer (PBS/0.05% Tween 20) followed by incubation with 100 µl of PBS/0.05% Tween 20/0.2% BSA for 1 h at room temperature. After three rinses with wash buffer, 125I-HRGß177–244 at a final concentration of 230 pM and varying concentrations of cold HRGß177–244 in 100 µl of binding buffer [0.2% BSA in RPMI 1640 buffered with 20 mM HEPES (pH 7.2)] were added to wells and incubated at room temperature for 2 h. Wells were then rinsed three times with binding buffer and drained, and individual wells were broken apart and counted using a gamma counter.
HRG Binding Assays on Cells.
T47D cells were plated in 24-well plates at 1.5 x 105 cells/well in the culture medium. After 2 days, the cultures were washed twice with binding buffer. Varying concentrations of p85-sErbB3 were added to each well in triplicate and preincubated for 30 min at room temperature. 125I-HRGß177–244 was added to each well for a final concentration of 100 pM, and the incubations were continued for 2 h at room temperature. Competition experiments with monoclonal antibody 2C4 (17)
were performed simultaneously. After the incubation, cells were washed twice with binding buffer and lysed in lysis buffer of 8 M urea and 3 M acetic acid. Radioactivity was determined using a gamma counter.
Cell Growth Assays.
MCF7 cells were trypsinized and washed in assay medium (DMEM:Ham’s F-12 supplemented with 0.02% BSA and 10 µg/ml transferrin), and cell numbers were counted. Twenty µl of the assay medium with or without HRGß were added to each well. Varying concentrations of p85-sErbB3 were preincubated with the washed cells for 30 min and transferred into 96-well plates in 100 µl. Growth was monitored after 3 days using the Cell Titer AQ kit (Promega). A similar degree of growth inhibition by p85-sErbB3 was consistently observed regardless of whether BSA was present or absent in the assay medium or whether cells were seeded before the addition of p85-sErbB3 and HRGß.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. To examine whether p45-sErbB3 and p85-sErbB3 can modulate HRG receptor activation, we isolated cells stably transfected with these corresponding cDNA clones. These cells secrete p45-sErbB3 and p85-sErbB3 into the culture medium (Fig. 2A)
. The conditioned medium from these cells was used as the source of p45-sErbB3 or p85-sErbB3 in a series of preliminary experiments described below.
|
EGF domain177–241 (HRG) and HRGß1176–246 (HRGß) in the absence or presence of concentrated conditioned media containing p45-sErbB3 and p85-sErbB3. As shown in Fig. 2
, HRGß was at least 20-fold more effective than HRG in stimulating ErbB3 tyrosine phosphorylation. Conditioned media containing sErbB3 inhibited HRG-stimulated ErbB3 activation by 40% (p45-sErbB3) and 80% (p85-sErbB3) at 1 µg/ml HRG, as determined by densitometric analysis. However, at a higher concentration (2 µg/ml), conditioned media containing p85-sErbB3 decreased activation by 30%, whereas inhibition by conditioned media containing p45-sErbB3 was negligible (Fig. 2B)
. In the presence of conditioned medium containing either p45-sErbB3 or p85-sErbB3, ligand stimulation of ErbB3 tyrosine phosphorylation was decreased by 60% and 90%, respectively, at both 50 and 100 ng/ml HRGß (Fig. 2C)
. These data indicate that p85-sErbB3 inhibited ErbB3 phosphorylation in response to both HRG and HRGß more effectively than p45-sErbB3, although the concentration of p85-sErbB3 used in these studies was lower than that of p45-sErbB3 (Fig. 2A)
. Hence, we focused the remainder of our studies on p85-sErbB3. To eliminate the possibility that the use of conditioned media might introduce experimental complications, p85-sErbB3 was isolated in a two-step process using Concanavalin A affinity chromatography and Mono Q ion exchange chromatography. The final p85-sErbB3 pool was homogeneous on SDS-PAGE, and the identity of the purified protein was confirmed by Western blot analysis (data not shown). Purified preparations of p85-sErbB3 were used in all subsequent experiments.
p85-sErbB3 Binds to HRG with High Affinity.
In a previous report, we based our assignation of the subdomain boundaries of the ErbB3 ECD on the subdomain boundaries of EGFR (2)
as defined by the genomic structure of avian ErbB1 (18)
. Accordingly, p85-sErbB3 is composed of subdomains I through III and includes the first 45 aa of subdomain IV (aa 1–519), as well as a unique 24-aa sequence at the COOH terminus. Binding studies using EGF indicate that EGFR subdomains I and III are low and high affinity binding sites for EGF binding, respectively (19)
. Because p85-sErbB3 contains both subdomains I and III, we hypothesized that p85-sErbB3 should be able to bind to HRG.
Direct binding between p85-sErbB3 and radiolabeled HRGß was examined using the chemical cross-linker BS3. As shown in Fig. 3A
, a protein complex of Mr 90,000 was formed between p85-sErbB3 and 125I-HRGß. Formation of this complex could be inhibited by the addition of excess cold HRGß but not by the addition of excess insulin, indicating that p85-sErbB3 binding to HRGß is specific and that our purified preparations of p85-sErbB3 are biologically active. We then performed an analysis of 125I-HRGß177–244 binding to immobilized p85-sErbB3 using an ErbB3-IgG homodimer (17)
as a positive control. As shown in Fig. 3
, p85-sErbB3 binds to HRGß177–244 with a KD of 3.0 ± 0.2 nM. In comparison, ErbB3-IgG binds to HRGß177–244 with a KD of 4.7 ± 0.2 nM. These results demonstrate that p85-sErbB3 binds to HRGß177–244 with an affinity similar to that of the ECD of ErbB3. Based on the results of these two complementary experimental approaches, we concluded that p85-sErbB3 binds to HRG with an affinity equivalent to the affinity of HRG for the full-length ECD of ErbB3.
|
, p85-sErbB3 and 2C4 inhibit HRGß177–244 binding to cell surface receptors with similar IC50 values (0.45 ± 0.03 and 0.55 ± 0.03 nM, respectively), although the mechanism of inhibition by these two molecules is distinct. Whereas 2C4 inhibits HRG binding to cell surface receptors by blocking ErbB2-ErbB3 heterodimerization via binding to the ErbB2 ECD (17)
, p85-sErbB3 appears to inhibit receptor activation, at least in part, by competing for HRG binding to the cell surface.
|
, when Ba/F3 (ErbB2+ErbB3) cells were treated with p85-sErbB3 at a 10-fold molar excess over HRGß1–241, ErbB3 phosphorylation levels were reduced to basal levels. A similar level of receptor inhibition also was apparent when either a 2.5- or 5-fold molar excess of p85-sErbB3 was used in these experiments. Interestingly, exogenous addition of p85-sErbB3 also inhibited HRG-induced ErbB2 activation. p85-sErbB3 blocked HRG stimulation whether the cells were treated with the EGF-like domain of HRG (HRG or HRGß), as shown in Fig. 2
, or with HRGß1–241 (Fig. 5)
, suggesting that inhibition by p85-sErbB3 occurs, at least in part, through a direct interaction between p85-sErbB3 and the EGF-like domain of HRG. Cells treated with the same concentration of p85-sErbB3 but not stimulated with HRG did not exhibit altered ErbB2 or ErbB3 tyrosine phosphorylation or show any change in the level of either ErbB2 or ErbB3 expression, suggesting that p85-sErbB3 does not function as a "ligand" for these receptors.
|
, addition of p85-sErbB3 (at a 6-fold molar excess relative to HRGß) inhibited HRG-induced activation of ErbB2, ErbB3, and ErbB4 in both the T47D and MCF7 cell lines. In contrast, at least in these two cell lines that express low EGFR levels, EGFR phosphorylation remained at the basal level in cells treated with HRGß, regardless of whether p85-sErbB3 was present or not (data not shown). Similarly, EGF-induced phosphorylation of EGFR or ErbB2 or, to a lesser degree, EGF-induced phosphorylation of ErbB3 was not decreased by p85-sErbB3 (data not shown). These results demonstrate that inhibition by p85-sErbB3 is specific for HRG-induced activation of ErbB2, ErbB3, and ErbB4.
|
. In this regard, it has been reported that the polyclonal ErbB3 antibody used in this study preferentially recognizes nonphosphorylated ErbB3 on Western blots (21)
. Hence, when T47D or MCF7 cells are stimulated with HRG, a significant fraction of ErbB3 is probably phosphorylated and therefore undetectable with this particular ErbB3 antibody. The anti-ErbB antibodies used in these experiments recognize the COOH-terminal 17-aa (ErbB3) and 18-aa (ErbB2 and ErbB4) sequences of these receptors. Each of these sequences contains one tyrosine residue. Immunoblot detection by the anti-ErbB2 and anti-ErbB4 antibodies used in this study may therefore reflect either the level of receptor expression or the unphosphorylated fraction of these receptors, and we are currently unable to distinguish between these two possibilities.
p85-sErbB3 Inhibits Activation of Downstream Effectors of HRG.
HRG-stimulated activation of ErbB2, ErbB3, and ErbB4 leads to activation of two major signal transduction pathways: (a) the PI3K pathway; and (b) the MAPK pathway (22)
. We tested whether p85-sErbB3 also could inhibit activation of these two downstream effector pathways in T47D cells. Specifically, we examined activation of MAPK and Akt by analyzing the phosphorylation levels of these proteins, and we also examined the ability of p85 PI3K to interact with ErbB3 after HRGß treatment. In the presence of p85-sErbB3 (10-fold molar excess relative to HRGß), tyrosine phosphorylation of ErbB3 was reduced to basal levels. In the same cell population, addition of exogenous p85-sErbB3 abrogated the phosphorylation of both MAPK and Akt as determined by Western blot analysis and also inhibited the association of ErbB3 with p85 PI3K (Fig. 6B)
. These results further demonstrate that p85-sErbB3 can inhibit the activation of ErbB2, ErbB3, and ErbB4 and that this inhibition affects the activation of downstream signaling molecules such as MAPK, Akt, and PI3K.
p85-sErbB3 Inhibits HRG-stimulated Cell Growth.
We next examined whether the inhibition of HRG-induced phosphorylation of ErbB receptors by p85-sErbB3 could be correlated with the modulation of the biological effects of HRG. Specifically, we performed a cell growth assay using MCF7 cells stimulated with HRGß and showed that, within the concentration range tested, the growth of this cell line was dose dependent (Fig. 7)
. We observed that at a concentration of 0.4 nM HRGß, the cell growth rate was half of the rate observed at saturating levels of HRGß. In cell cultures grown in the presence of 0.4 nM HRGß and p85-sErbB3 (a 100-fold molar excess relative to HRGß), p85-sErbB3 inhibited cell growth by 75% and 90% at densities of 5000 and 8000 cells/well, respectively, whereas the same concentration of p85-sErbB3 did not affect cell growth in the absence of HRGß (Fig. 7)
. Based on these results, we conclude that p85-sErbB3 is a potent inhibitor of HRG-dependent breast carcinoma cell growth in vitro.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although much is known about the structural regions of the ligands required for binding of EGF to its receptor and for HRG binding to ErbB3 and ErbB4 (23 , 24) , little is known about the structural features of the receptors that are necessary for ligand recognition. In this regard, interactions between HRGs and ErbB3 have been predicted to be dependent on contact sites that are analogous to those important for EGF binding to EGFR. This prediction is based on the following observations: (a) the EGF-like domains of the HRGs are necessary for binding to ErbB3 or ErbB4 (12 , 14) ; and (b) all four ErbB receptors, including ErbB3, share similar ECD structures (25) . However, the biochemical properties and biological functions of these four ErbB receptors are not identical. Studies using various soluble forms of the EGFR ECD have shown that the isolated ECD of EGFR binds to EGF with a KD in the range of 10–500 nM and that the ECD of the receptor forms homodimers on EGF binding (26) . Whereas the ECD of ErbB4 can also form homodimers and heterodimers with the ECD of ErbB2 in the presence of HRG, the ECD of ErbB3 does not form ligand-dependent homo- or heterodimers with any of the other ErbB receptor ECDs (27) . Various recombinant proteins corresponding to the ErbB3 ECD have been reported to bind to HRG as a monomer; however, the KD values of these ErbB3 ECDs vary (27, 28, 29) . Although these variations in ligand-binding affinities may arise from differences between protein preparations and/or the methods used in measuring affinities, they may also reflect subtle differences in the COOH-terminal sequences of these proteins. In support of this suggestion, a recombinant ErbB3 ECD containing the first 620 aa of the receptor followed by a COOH-terminal His tag binds to HRGß with a KD of 250 nM (27) . In contrast, another recombinant ErbB3 ECD, which contains four additional COOH-terminal aa residues (aa 1–624) and has a COOH-terminal V5-His tag, binds to a thioredoxin-HRGß with a KD of 1.7 nM (28) . This latter ErbB3 ECD oligomerizes in the absence of HRG, a unique property among ErbB3 ECDs. Hence, oligomeric forms of the ErbB3 ECD may render it in a higher affinity HRG binding state. In this regard, p85-sErbB3 contains subdomains I through III and the first 45 aa of subdomain IV (i.e., aa 1–519) but is missing the remaining 101 aa of subdomain IV. Although the unique 24-aa sequence at the COOH terminus of p85-sErbB3 may contribute to high affinity ligand binding, the high affinity of this interaction might also indicate that sequences in subdomain IV of ErbB3 exert a negative constraint on ligand binding. In any event, the results presented in this study provide new insight into the regions of the ErbB3 ECD that are required for HRG binding.
Using various recombinant truncated forms of EGFR, it has been shown that efficient inhibition of full-length EGFR activation by dominant-negative heterodimerization occurs only when these deletion mutants retain the transmembrane domain in addition to the ECD (30)
. Similarly, a recombinant dominant negative ErbB3 mutant with a deleted cytoplasmic domain that retains its transmembrane domain can inhibit full-length ErbB2 and ErbB3 activation (31)
. In contrast, however, in avian tissues, expression of a naturally occurring soluble EGFR/ErbB1 inhibits transforming growth factor -dependent transformation (10)
. Soluble EGFR secreted by the A431 human carcinoma cell line has also been reported to inhibit the kinase activity of purified full-length EGFR in a ligand-independent manner (32)
. In no case do these soluble EGF/ErbB1 receptors function as antagonists through high affinity ligand binding. Similarly, herstatin, a naturally occurring sErbB2 protein that inhibits ErbB2 activation, appears to function by blocking ErbB2 dimerization; this inhibition is thought to be mediated via ligand-independent binding of herstatin to ErbB2 (9)
. The unique COOH-terminal sequence of herstatin contributes to this binding to cell surface ErbB2. In contrast, p85-sErbB3 appears to inhibit HRG-induced stimulation of ErbB2, ErbB3, and ErbB4, at least in part, by neutralizing ligand activity through competitive binding. Our preliminary evidence further suggests that p85-sErbB3 may also be capable of binding to the cell surface, suggesting that p85-sErbB3 may undergo ligand-induced hetero-oligomerization with cell surface receptors.4
Additional studies will be necessary to determine how much, if any, this cell surface interaction contributes to the mechanism of inhibition by p85-sErbB3.
Another important area of investigation is the physiological role of p85-sErbB3 in normal tissues. In this regard, our results indicate that whereas a much higher concentration (100-fold) was required to inhibit cell growth, a 10-fold molar excess of p85-sErbB3 was sufficient for inhibition of phosphorylation of ErbB receptors. Perhaps this is because, at this ratio, a small fraction of receptors are still activated, which may be enough for growth stimulation (33) . We know that the 2.1-kb transcript encoding p85-sErbB3 is expressed at low levels in comparison to the full-length c-erbB3 transcript in all cell lines and tissues examined to date (2) ; however, local expression of this transcript has not yet been studied in detail. It is therefore plausible that p85-sErbB3 acts as an HRG antagonist locally in a tissue-specific and/or stage-specific manner, and related studies to examine the distribution of p85-sErbB3 in selected tissues are currently under way. Evidence recently has been presented suggesting that local concentrations of autocrine growth factors such as EGF are exquisitely regulated and do not travel far from the cell surface from which they are released (34) . In this context, tightly regulated levels of local p85-sErbB3 expression may have important consequences for HRG-mediated cell growth. These consequences might be even more dramatic in cancer cells in which cell polarity is typically lost, resulting in deregulation of normal spatial and temporal control of growth factor-receptor interactions.
In this regard, transgenic mice that have been engineered to overexpress HRG in mammary glands have been reported to exhibit persistent terminal end buds and, over time, to develop mammary adenocarcinomas (35) . ErbB3 expression studies on tumor tissues as well as on cell lines show frequent coexpression of ErbB2 and ErbB3 receptors (36, 37, 38, 39) . In addition, both ErbB2 and ErbB3 are activated in mammary tumors formed in transgenic mice harboring only the activated form of ErbB2 (39) . Studies to determine whether p85-sErbB3 has the potential to block activation of the HRG-related signal transduction pathways in relevant tumor models such as breast cancer and prostate cancer in vivo and to determine whether p85-sErbB3 alone or in combination with monoclonal antibodies such as 2C4 has therapeutic potential should therefore be possible.
In this study we have defined the biochemical and biological characteristics of a novel sErbB3 isoform designated p85-sErbB3. We conclude that as a naturally occurring HRG inhibitor, p85-sErbB3 is unique in that it can block HRG binding to cell surface receptors via binding to HRG with high affinity, thereby inhibiting HRG-induced stimulation of ErbB2, ErbB3, and ErbB4. This inhibition is sufficient to effectively block HRG-stimulated cell growth. This novel ErbB3 receptor isoform therefore has the potential to be a potent modulator of HRG-regulated cell proliferation and differentiation in normal human tissues and may be an ideal candidate for the development of novel cancer therapeutics.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by a scholarship from the Prospect Creek Foundation and National Cancer Institute Grant CA85133. 
2 To whom requests for reprints should be addressed, at Tumor Biology Program, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284-0279; Fax: (507) 284-1767; E-mail: maihle{at}mayo.edu
3 The abbreviations used are: sErbB, soluble ErbB; EGF, epidermal growth factor; EGFR, EGF receptor; HRG, heregulin; BS3, bis[sulfosuccinimidyl]suberate; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3'-kinase; ECD, extracellular domain; aa, amino acid(s).
4 H. Lee and N. J. Maihle, unpublished observations.
Received 12/21/00. Accepted 3/28/01.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Narayan, J. A. Wilken, L. N. Harris, A. T. Baron, K. D. Kimbler, and N. J. Maihle Trastuzumab-Induced HER Reprogramming in "Resistant" Breast Carcinoma Cells Cancer Res., March 15, 2009; 69(6): 2191 - 2194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-H. Lin, Y.-C. Lee, M. B. Choueiri, S. Wen, P. Mathew, X. Ye, K.-A. Do, N. M. Navone, J. Kim, S.-M. Tu, et al. Soluble ErbB3 Levels in Bone Marrow and Plasma of Men with Prostate Cancer Clin. Cancer Res., June 15, 2008; 14(12): 3729 - 3736. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Reiter, N. J. Maihle, and G. M. Clinton Therapeutic potential of epidermal growth factor receptor-related protein. Mol. Cancer Ther., November 1, 2006; 5(11): 2954 - 2954. [Full Text] [PDF]
|
|
|
|
|
|
K.-L. Schaefer, K. Brachwitz, D. H. Wai, Y. Braun, R. Diallo, E. Korsching, M. Eisenacher, R. Voss, F. van Valen, C. Baer, et al. Expression Profiling of t(12;22) Positive Clear Cell Sarcoma of Soft Tissue Cell Lines Reveals Characteristic Up-Regulation of Potential New Marker Genes Including ERBB3 Cancer Res., May 15, 2004; 64(10): 3395 - 3405. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. A. Justman and G. M. Clinton Herstatin, an Autoinhibitor of the Human Epidermal Growth Factor Receptor 2 Tyrosine Kinase, Modulates Epidermal Growth Factor Signaling Pathways Resulting in Growth Arrest J. Biol. Chem., May 31, 2002; 277(23): 20618 - 20624. [Abstract] [Full Text] [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 |
) or 8000 cells/well (
) in 96-well plates with increasing concentrations of HRGß in serum-free medium, and growth was measured after 3 days (inset). MCF7 cells were trypsinized, washed, incubated with p85-sErbB3 for 30 min, and plated with or without 0.4 nM HRGß in serum-free medium. At 40 nM (a 100-fold molar excess to HRGß) in the presence of HRGß, p85-sErbB3 inhibited cell growth by 75% and 90% at densities of 5000 and 8000 cells/well, respectively, whereas the same concentration of p85-sErbB3 did not affect cell growth in the absence of HRGß. The data presented are the mean ± SD of six replicates. This experiment was repeated three times, and the results shown represent all three trials.