.JPG?ad=18481&adview=true)
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics, Preclinical Pharmacology |
Division of Medicine, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [F. X., Y. Y., X-F. L., G. B. M., R. C. B.], and Duke University Medical Center, Durham, North Carolina 27710 [C. B.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
HRG was initially isolated during the search for a ligand that binds to the HER-2 receptor. The several isoforms of HRG bind to cells that express HER-3 or HER-4 alone, but not to cells that express only HER-2 (5, 6, 7) . However, HRG can bind to heterodimers of HER-2 and HER-3 or HER-2 and HER-4 (8, 9, 10) . Interactions between HER-2 and HER-3 may be important for HRG-induced signaling in that HER-3 lacks tyrosine kinase activity but contains multiple SH2 binding sites. HER-2 and HER-4 are capable of phosphorylating multiple substrates but contain a limited repertoire of SH2 binding sites.
Our previous studies have demonstrated that HRG and anti-HER-2 antibodies inhibit proliferation, increase invasiveness, and enhance tyrosine autophosphorylation of breast cancer cells that overexpress HER-2, such as SKBr3 cells (11 , 12) . Other investigators have reported that HRG stimulates the growth of breast cancer cells that express low levels of the HER-2 receptor (13) , but that ovarian cancer cells may be refractory to growth stimulation by the ligand (14) . The present study documents that HRG can, in fact, stimulate anchorage-independent growth of ovarian cancer cells. The ratio of HER-2:HER-3 appears to be important in determining whether cells that express both receptors are stimulated or inhibited by HRG. In cells that express only HER-2 and HER-4, HRG stimulates cell proliferation independent of HER-2 levels. When all three receptors are present, the ratio of HER-2:HER-3 appears to be a critical determinant in regulating clonogenic growth.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
Murine monoclonal antibodies to the extracellular domain of p185 were obtained from Applied BioTechnology/Oncogene Science (TA1, ID5, RC1, RC6, NB3, BD5, PB3, and OD3) and from Chiron, Inc. (Emeryville, CA; 454C11, 741F8, and 736 G9). All antibodies were of the IgG1 isotype except PB3 (IgG2a) and OD3 (IgM). MOPC21 (IgG1) was used as an isotype-matched control that did not bind to p185. The H3.105 anti-HER-3 antibody was purchased from NeoMarker (Fremont, CA) and the 10-4 and 6-4-11 anti-HER-4 antibodies were generously provided by Dr. B. D. Cohen. Antibodies against HRG (including 
and 
types) and phosphotyrosine were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Upstate Biotechnology (Lake Placid, NY), respectively.
Recombinant HRG was obtained from Genentech (South San Francisco, CA).
Antibody and HRG Binding to EGFR and HER-2, HER-3, and HER-4 Receptors.
A live cell radioimmunoassay was used to determine the binding of monoclonal antibodies and HRG to different cell lines and transfectants. Cells were trypsinized and seeded at a density of 2 x 104 cells/well in 96-well removable plates. After overnight incubation, monolayers were washed with 1% FBS in TCM supplemented with 0.1% sodium azide. For indirect binding assays, different monoclonal antibodies (10 µg/ml) were added in volumes of 50 µl to cell monolayers. After incubation at 4°C for 2 h, 125I-labeled sheep antimouse antibody F(ab')2 fragment (200,000 cpm/well) was added in a volume of 100 µl, and incubation was continued for another 2 h. Nonspecific binding was determined by adding 125I-labeled sheep antimouse antibody F(ab')2 fragment (50 µl) to cells without monoclonal antibodies or to empty wells. After incubation on ice for 4 h, unbound antibodies were removed by washing the wells four times with ice-cold TCM containing 5% FBS with 0.1% sodium azide. Individual wells were then detached, and radioactivity was determined in a Packard gamma counter. For direct binding assays, TA1, H3-105, 10-4 monoclonal antibodies, and HRG were directly labeled with 125I using the Iodogen technique and incubated with different cell lines. The EBDA program was used to calculate the number of binding sites/cell (16
, 17)
.
Transfection of HER-2, HER-3, and HER-4 cDNA.
Plasmid 9002, containing the full-length human HER-2 gene, was obtained from Applied BioTechnology/Oncogene Science, Inc. Plasmid CHER-3x, containing the full-length human HER-3 gene, was a gift from Dr. Greg Plowman (SUGEN, Inc., Redwood City, CA). Plasmid H4y, containing the full-length human HER-4 gene, was provided by Dr. B. D. Cohen. NIH 3T3 cells and OVCAR-3 cells were transfected by Lipofectamin as directed by the manufacturer (Life Technologies, Inc.). At least seven independent transfectants were cloned and cultured continuously with G418 or hygromycin. Transfected cells were selected for their ability to bind antibodies directed against HER-2, HER-3, or HER-4.
Assays of Anchorage-independent Growth.
Anchorage-independent cell growth was measured in 35-mm tissue culture dishes (Nunc, Inc., Naperville, IL). A 1-ml layer of 0.6% agar (Difco, Detroit, MI) in TCM was solidified in the bottom of each dish. Cells to be assayed were suspended in 1 ml of 0.3% agar in TCM supplemented with antibody, control medium, or different concentrations of HRG or diluant. Cells (2 x 104) were seeded in each dish. MOPC21, a monoclonal antibody that did not bind to p185HER-2 or to other cell surface determinants, was used as a control. Cells were incubated for 10–14 days at 37°C in 5% CO2 and 95% humidified air. Colonies containing more than 30 cells were counted using inverted phase microscopy.
Preparation of Total Cell Lysate and Western Immunoblot Analysis.
Cells were treated with or without 10 ng/ml HRG for different intervals and solubilized in lysis buffer. The lysates were cleared by centrifugation at 14,000 rpm for 10 min. Protein concentration of the lysates was measured by the BCA assay (Pierce Chemical Company, Rockford, IL). Equal amounts of protein were boiled in Laemmli SDS sample buffer and resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with specific antibodies. The signals were visualized with peroxidase-conjugated second antibodies and the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
Immunoprecipitation.
Aliquots of total cell lysates containing equal amounts of protein in lysis buffer [137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1 mM DTT, 1% NP40, 10% glycerol, and protease inhibitors] were precleared with 2 µg of normal mouse or rabbit IgG (Santa Cruz Biotechnology) together with 20 µl of protein A/G-agarose conjugate. In this study, 250, 500, and 750 µg of protein from total cell lysates of the six ovarian cancer cell lines were used for immunoprecipitation. Lysates were then immunoprecipitated overnight at 4°C with 3 µg each of antibodies reactive with HER-2, HER-3, and HER-4 and 20 µl of protein A/G-agarose conjugate.
Statistical Analysis.
Statistical analysis was performed using the two-sided Student’s t test. WinSTAT 3.1 software was used for this statistical analysis.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
. The number of binding sites/cell was taken as an estimate of the receptor level. All six cell lines express intermediate levels of EGFR (104–105 receptors/cell) and low levels of HER-3 and HER-4 (103–104 receptors/cell). One of the six cell lines (OVCAR-3) had low levels of HER-2, with <103 receptors/cell; four of the six cell lines (OVCA420, OVCA429, OVCA432, and OVCA433) had intermediate expression of HER-2, with 104–105 receptors/cell; whereas SKOv3 had 10–100-fold higher levels of HER-2, with 105–106 receptors/cell (Fig. 1)
.
|
. Similar binding of HRG was observed in each of the six cell lines, although p185HER-2 levels varied from 104–106 receptors/cell, consistent with the possibility that HER-2 did not contribute substantially to HRG binding.
Reactivity of Monoclonal Antibodies with EGFR and HER-2, HER-3, and HER-4 Receptors.
Our previous studies defined the reactivity of 11 monoclonal antibodies with the extracellular domain of p185HER-2 and measured their ability to inhibit the growth of SKBr3 breast cancer cells (11)
. To further investigate all four EGFR family members, these 11 monoclonal antibodies and one additional anti-HER-2 antibody, 736G9, were incubated with NIH 3T3 cell lines that had been transfected individually with EGFR, HER-2, HER-3, or HER-4. Binding of antibodies was measured indirectly with 125I-labeled sheep antimouse F(ab')2. Each of these 12 monoclonal antibodies bound strongly to the HER-2 receptor, but not to the EGF, HER-3, or HER-4 receptors. The 225 anti-EGFR, H3.105 anti-HER-3, and 10-4 anti-HER-4 antibodies bound strongly to the relevant receptors, but not to other receptors in this family. HRG bound to cells that expressed HER-3 or HER-4, but not to cells that expressed EGFR or HER-2 alone, consistent with previous reports (18)
.
Stimulation or Inhibition and Anchorage-independent Growth with HRG.
The effect of HRG on anchorage-independent growth was measured in each of the six ovarian cancer cell lines. HRG stimulated growth in five of the six cell lines but inhibited the growth of SKOv3 cells that overexpressed HER-2 (Figs. 2
and 3)
. Stimulation or inhibition was dependent on the concentration of HRG. As much as 10 ng/ml HRG was required to produce significant growth inhibition in SKOv3 cells. To evaluate the possible contribution of endogenous HRG expression to growth regulation, we have measured relative HRG expression in six ovarian cancer cell lines by Western blotting (Fig. 4)
. Each of the six cell lines expressed the 44-kDa HRG protein. OVCAR-3 cells exhibited the highest level of HRG expression, and OVCA432 had the lowest level among these six cell lines. Consequently, no correlation was found between the response to HRG treatment and the endogenous expression of HRG protein.
|
|
|
, increased phosphorylation of HER-2 after HRG treatment was observed in every cell line. Increased phosphorylation of HER-3 after HRG treatment was observed in OVCAR-3, OVCA420, OVCA429, and OVCA432, whereas little, if any, phosphorylation of HER-3 was observed in SKOv3 and OVCA433. Increased phosphorylation of HER-4 after HRG treatment was observed only in OVCAR-3 cells (Fig. 5)
. Thus, no clear correlation was observed between the impact of HRG on clonogenic growth and tyrosine phosphorylation of HER-2, HER-3, and HER-4.
|
. Incubation with HRG and ID5 did not prevent HRG-induced stimulation of anchorage-independent growth of the five cell lines with low levels of HER-2 expression (Figs. 2
and 3)
, and the ID5 antibody did not block HRG binding (data not shown).
Effects of HRG on Transfected Ovarian Cancer Cells with Different Levels of HER-2 Expression.
Because the ovarian cancer cell lines were derived from different patients, an apparent correlation between p185HER-2 expression and the effect of HRG on anchorage-independent growth might relate to other abnormalities in the cells. Consequently, we have examined ovarian cancer cells and transfectants in which HER-2 expression could be up-regulated or down-regulated on the same background. OVCAR-3 cells express 103 HER-2 receptors/cell and are stimulated by HRG. Transfection of OVCAR-3 cells with full-length HER-2 cDNA produced the OVU15 clone that expressed 105 p185HER-2 sites/cell. Expression of HER-3 and HER-4 was similar in the transfectants and in parental cells. HRG stimulated the growth of the parental OVCAR-3 cells 2–4-fold but inhibited the growth of the OVU15 cells with greater p185HER-2 expression (Fig. 6)
.
|
. Similar levels of HER-3 and HER-4 were found once again in the parental and transfected cells. HRG at high concentrations inhibited the growth of parental SKOv3 cells but stimulated the anchorage-independent growth of transfectants with decreased HER-2 expression.
|
.
|
105) and low levels of HER-3 (clone E21), HRG and ID5 inhibited anchorage-independent growth. When similar levels of HER-2 and HER-3 were expressed (clone E22), HRG, but not ID5, stimulated cell growth (Fig. 9)
. Transfectants with intermediate levels of HER-2 (clone E6) exhibited a lower level of growth stimulation by HRG (Fig. 9)
. Consequently, the inhibition or stimulation of anchorage-independent growth by HRG appeared to depend on the ratio of HER-2:HER-3 expression in cells that bore only these two receptors. Transfectants that contained combinations of HER-2 and HER-4 were stimulated regardless of the ratio of HER-2:HER-4 (Fig. 8)
. The anti-HER-2 ID5 antibody regularly inhibited clonogenic growth in cells that expressed 106 HER-2 receptors/cell.
|
In the clinic, the adverse prognostic significance of HER-2 overexpression appears greatest in node-positive breast cancer (1 , 3) and in late-stage ovarian cancer (4) . Although it may seem paradoxical that clonogenic growth can be inhibited by ligand and by agonistic anti-HER antibodies in cells that overexpress the receptor, recent data suggest that agonists increase the invasiveness of cells that overexpress HER-2 (12) . Consequently, enhanced potential for invasion and metastasis, rather than an increased rate of growth or clonogenicity, may be associated with HER-2 overexpression. However, HRG might exert autocrine growth stimulation in cells with lower levels of HER-2, and the prognostic significance of HRG expression in cells with normal HER-2 levels deserves further study.
Overexpression of HER-2 has been found in approximately 30% of ovarian cancer (4) , whereas overexpression of HER-3 and HER-4 has not been reported to date. Consistent with these studies of cancer tissues, HER-2 levels in the six ovarian cancer cell lines varied over 3 orders of magnitude, whereas the levels of EGFR, HER-3, and HER-4 varied by only 1 order of magnitude. Thus, marked heterogeneity of expression was observed with only one of the four members of the HER family of receptors. A corollary of this observation is that different levels of HER-2 expression will produce markedly different ratios of HER-2:HER-3 and HER-2:HER-4. When HRG was incubated with each of the six cell lines, growth inhibition appeared to correlate with high levels of HER-2 that produced high HER-2:HER-3 and HER-2:HER-4 ratios. Inhibition or stimulation of growth with HRG did not correlate with endogenous HRG expression or with HRG-induced phosphorylation of HER-2, HER-3, or HER-4. Consequently, downstream effects of HER-2 overexpression must contribute to the growth inhibition observed.
The effect of HRG in NIH 3T3 cells transfected with HER-2 and HER-3 or HER-2 and HER-4 suggest that the ratio of HER-2:HER-3 is important for cell growth regulation, whereas the ratio of HER-2:HER-4 is not. In OVCAR-3 cells and SKOV-3 cells that express all three receptors, modulation of HER-2 levels altered the response to HRG and to agonistic anti-HER-2 antibody. Our data suggest that the interaction of HER-2 and HER-3 may be particularly important for HRG or antibody-induced growth inhibition, overriding the stimulation produced by HER-4 homodimers or HER-2/HER-4 heterodimers. However, our data do not permit us to distinguish between the importance of the ratio of HER-2:HER-3 and the absolute level of HER-2 in determining response to HRG.
Anti-HER-2 antibodies inhibited the growth of cells with high levels of HER-2, regardless of HER-3 or HER-4 expression. Antibody-mediated growth stimulation was not observed. The ID5 antibody binds to HER-2 alone and presumably signals predominantly though this receptor. HRG can signal through HER-4 homodimers, through HER-2/HER-3 heterodimers, or through HER-2/HER-4 heterodimers. Our results pose the interesting possibility that signaling through HER-2 alone inhibits clonogenic growth, and that this is recognized most readily in cells that overexpress HER-2. Additional studies are underway to delineate signaling pathways in cells treated with HRG or with agonistic HER-2 antibodies.4
Both agents can induce autophosphorylation of HER-2 and activate mitogen-activated protein kinase. The ID5 anti-HER-2 antibody, but not HRG, increases activity of phospholipase-C 
and perturbs diacylglycerol levels. HRG, but not ID5, increases phosphatidylinositol 3'-kinase activity, AKT kinase activity, p70S6 kinase activity, and c-jun-NH2-kinase activity as well as inducing differentiation. Thus, HRG and ID5 signal by different pathways.
|
FOOTNOTES |
|---|
1 Supported by Grant RO1 CA 39930 from the NIH, Department of Health and Human Services. 
2 To whom requests for reprints should be addressed, at Division of Medicine, University of Texas M. D. Anderson Cancer Center, Box 092, 1515 Holcombe Boulevard, Houston, TX 77030.
3 The abbreviations used are: EGFR, epidermal growth factor receptor; HRG, heregulin; FBS, fetal bovine serum; TCM, tissue culture medium.
4 Xiao-Feng Le et al., unpublished data.
Received 1/28/99; revised 8/10/99; accepted 8/17/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
|---|
-Heregulin: a novel heregulin isoform that is an autocrine growth factor for the human breast cancer cell line, MDA-MB-175. Oncogene, 15: 1385-1394, 1997.[Medline]
This article has been cited by other articles:
|
|
 |
|
  D. E. Milenic, K. Garmestani, E. D. Brady, K. E. Baidoo, P. S. Albert, K. J. Wong, J. Flynn, and M. W. Brechbiel Multimodality Therapy: Potentiation of High Linear Energy Transfer Radiation with Paclitaxel for the Treatment of Disseminated Peritoneal Disease Clin. Cancer Res., August 15, 2008; 14(16): 5108 - 5115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. B. Carter, G. Niu, M. D. Ward, G. Small, J. E. Hahn, and B. J. Muffly Mechanisms of HER2-Induced Endothelial Cell Retraction Ann. Surg. Oncol., October 1, 2007; 14(10): 2971 - 2978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Milenic, K. Garmestani, E. D. Brady, P. S. Albert, A. Abdulla, J. Flynn, and M. W. Brechbiel Potentiation of High-LET Radiation by Gemcitabine: Targeting HER2 with Trastuzumab to Treat Disseminated Peritoneal Disease Clin. Cancer Res., March 15, 2007; 13(6): 1926 - 1935. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Mullen, D. A. Cameron, M. Hasmann, J. F. Smyth, and S. P. Langdon Sensitivity to pertuzumab (2C4) in ovarian cancer models: cross-talk with estrogen receptor signaling Mol. Cancer Ther., January 1, 2007; 6(1): 93 - 100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Gordon, D. Matei, C. Aghajanian, U. A. Matulonis, M. Brewer, G. F. Fleming, J. D. Hainsworth, A. A. Garcia, M. D. Pegram, R. J. Schilder, et al. Clinical Activity of Pertuzumab (rhuMAb 2C4), a HER Dimerization Inhibitor, in Advanced Ovarian Cancer: Potential Predictive Relationship With Tumor HER2 Activation Status J. Clin. Oncol., September 10, 2006; 24(26): 4324 - 4332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sain, B. Krishnan, M. G. Ormerod, A. De Rienzo, W. M. Liu, S. B. Kaye, P. Workman, and A. L. Jackman Potentiation of paclitaxel activity by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin in human ovarian carcinoma cell lines with high levels of activated AKT Mol. Cancer Ther., May 1, 2006; 5(5): 1197 - 1208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Delord, C. Allal, M. Canal, E. Mery, P. Rochaix, I. Hennebelle, A. Pradines, E. Chatelut, R. Bugat, S. Guichard, et al. Selective inhibition of HER2 inhibits AKT signal transduction and prolongs disease-free survival in a micrometastasis model of ovarian carcinoma Ann. Onc., December 1, 2005; 16(12): 1889 - 1897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Macleod, P. Mullen, J. Sewell, G. Rabiasz, S. Lawrie, E. Miller, J. F. Smyth, and S. P. Langdon Altered ErbB Receptor Signaling and Gene Expression in Cisplatin-Resistant Ovarian Cancer Cancer Res., August 1, 2005; 65(15): 6789 - 6800. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Holbro, R. R. Beerli, F. Maurer, M. Koziczak, C. F. Barbas III, and N. E. Hynes The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation PNAS, July 22, 2003; 100(15): 8933 - 8938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. R. Gilmour, K. G. Macleod, A. McCaig, J. M. Sewell, W. J. Gullick, J. F. Smyth, and S. P. Langdon Neuregulin Expression, Function, and Signaling in Human Ovarian Cancer Cells Clin. Cancer Res., December 1, 2002; 8(12): 3933 - 3942. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. Drake and J. L. Becker Aspirin-Induced Inhibition of Ovarian Tumor Cell Growth Obstet. Gynecol., October 1, 2002; 100(4): 677 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mendoza, G. L. Phillips, J. Silva, R. Schwall, and D. Wickramasinghe Inhibition of Ligand-mediated HER2 Activation in Androgen-independent Prostate Cancer Cancer Res., October 1, 2002; 62(19): 5485 - 5488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, D.-Y. Jin, R. W. M. Ng, H. Feng, Y. C. Wong, A. L. M. Cheung, and S. W. Tsao Significance of MAD2 Expression to Mitotic Checkpoint Control in Ovarian Cancer Cells Cancer Res., March 1, 2002; 62(6): 1662 - 1668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. R. Gilmour, K. G. Macleod, A. McCaig, W. J. Gullick, J. F. Smyth, and S. P. Langdon Expression of erbB-4/HER-4 Growth Factor Receptor Isoforms in Ovarian Cancer Cancer Res., March 1, 2001; 61(5): 2169 - 2176. [Abstract] [Full Text]
|
|
|
|
|
|
X.-F. Le, R. Vadlamudi, A. McWatters, D. S. Bae, G. B. Mills, R. Kumar, and R. C. Bast Jr. Differential Signaling by an Anti-p185HER2 Antibody and Heregulin Cancer Res., July 1, 2000; 60(13): 3522 - 3531. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 |
